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NATIONAL EYE INSTITUTE 




Cover Photograph 



A Y-79 human retinoblastoma cell treated 
with pigment epithelium-derived factor 
(PEDF). Photograph courtesy of Dr. Gerald 
Chader, Chief, Laboratory of Retinal Cell 
and Molecular Biology, NEI. Dr. Chader's 
report begins on page 255. 



NATIONAL INSTITUTES OF 
NIH LIBRARY 



AUG 2 2 1995 



National Eye iNSTiTore 



BLDG 10, 10 CENTER DR. 
BETHESDA, MD 20892-1150 



Annual Report 
Fiscal Year 1994 



U.S. Department of Health and Human Services 

Public Health Service 

National Institutes of Health 



^t 






1 



Table of Contents 



Statement of the Institute Director i 

Carl Kupfer, M.D. 



Extramural Research 7 

Report of the Associate Director 9 

Jack A. McLaughlin, Ph.D. 

Division of Basic Vision Research 9 

Peter Dudley, Ph.D. 

Retinitis Pigmentosa 9 

Glaucoma 10 

Keratoconus of the Cornea 11 

Acquired Immunodeficiency Syndrome 12 

Retinal Neuroscience — Molecular Basis of Signaling 12 

Corneal Angiogenesis 14 

Lens Development 14 

Neural Processing of Visual Information 15 

Development 15 

Personal Guidance System for the Visually Impaired 16 

Division of Collaborative Clinical Research 17 

Richard Mowery, Ph.D. 

Retinal Diseases 17 

Glaucoma 19 

Corneal Diseases 20 

Strabismus, Amblyopia, and Visual Processing 20 



Division of Biometry and Epidemiology 23 

Report of the Acting Director 25 

Roy C. Milton, Ph.D. 

Research Highlights 25 

Research Activities 26 

Publications 29 



FY 1994 NEl Annual Report 



Office of International Program Activities 31 

Report of the Acting Assistant Director 33 

Terrence Gillen, M.A., M.B.A. 

Highlights of Recent Scientific Advances Resulting 

From International Activities 33 

Summary of International Programs and Activities 34 

Activities With International and Multinational Organizations 37 

Extramural Programs 37 

Intramural Programs and Activities 37 



Office of Science Policy and Legislation 39 

Report of the Associate Director 41 

Michael P. Davis, M.S. 

Policy, Legislation, Planning, and Evaluation Branch 41 

Carmen P. Moten, Ph.D. 

Management Information Systems Branch 43 

David Scheim, Ph.D. 



Office of Health, Education, and Communication 47 

Report of the Director 49 

Judith A. Stein, M.A. 

National Eye Health Education Program 49 

25th Anniversary Program 50 

PubUc Inquiries Program 50 

Scientific Reporting 51 



Office of the Scientific Director 53 

Office of the Scientific Director 

Francisco M. de Monasterio, M.D., D.Sc. 

Anatomical Studies of the Primate Visual System 55 

Physiological Studies of the Primate Visual System 57 

Helen H. Hess, M.D. 

Biochemistry of Retina and Pigmented Epithelium in Health and Disease .... 59 



Laboratory of Immunology 63 

Report of the Chief 65 

Robert B. Nussenblatt, M.D. 



Table of Contents 



Section on Experimental Immunology 

Charles E. Egwuagu, Ph.D., M.P.H. 

Transgenic Rat and Mouse Models for the Study of 

Intraocular Effects of IFN-y and Autoimmunity 71 

Analysis of T Lymphocytes and Cytokines Involved 

in Experimental Autoimmune Uveoretinitis 74 

Igal Gery, Ph.D. 

Immune Responses to Ocular Antigens 77 

Section on Clinical Immunology 
Frangois G. Roherge, M.D. 

Inhibition of EAU in Monkey With Humanized Anti IL-2 Receptor 82 

Study of the Effect of NPC 15669, an Inhibitor of Neutrophil 

Recruitment in Uveitis 85 

Study of the Role of Nitric Oxide in Uveitis 87 

Study of Immunosuppressants for the 

Treatment of Uveitis in Animal Models 90 

Section of Ocular Gene Therapy 
Karl G. Csaky, M.D., Ph.D. 

Ocular Gene Transfer 91 

Section on Immunopathology 
Scott M. Whitcup, M.D. 

The Diagnosis and Treatment of Human Uveitis 

and AIDS-Related Ocular Disease 94 

Chi-Chao Chan, M.D. 

Immunopathology in Eyes With Experimental and Clinical Ocular Diseases ... 99 
Scott M. Whitcup, M.D. 

Immunologic Mechanisms of Ocular Disease 105 

Ocular Toxicity of 2',3'-Dideox)dnosine(ddI) 109 

Chi-Chao Chan, M.D. 

Cytokines and Ocular Antigens in the Eye 112 

Section on Immunoregulation 
Robert B. Nussenblatt, M.D. 

Cyclosporine Therapy in Uveitis 113 

Oral Administration of Antigen and the Ocular Immime Response 116 

Rachel R. Caspi, Ph.D. 

Cellular and Immunogenetic Mecharusms in Uveitis 119 

Marc D. de Smet, M.D. 

Characterization of Immune Responses to Retinal Specific Antigens 125 

Surgical Management of Uveitis 128 

Ocular Manifestations of the Acquired Immune Deficiency Syndrome 129 

Section on Immunology and Virology 
John J. Hooks, Ph.D. 

Interferon System in Cellular Function and Disease 132 

Studies on the Bioregulatory Aspects of the Retinal Pigment EpitheHal Cell ... 135 

Virus Infections in the Eye 138 

Chandrasekharam N. Nagineni, Ph.D. 

Role of Retinal Pigment Epithelium in Retinal Disorders 143 

John J. Hooks, Ph.D. 

Toxoplasmosis Infections in the Eye 147 



¥Y 1994 NEI Annual Report 



Section of Genetics and Molecular Immunology 

Moncef Jendoubi, Ph.D. 

Gene Targeting of Invariant Chain Gene: A Tool To Study 

Immunoregulation in Autoimmune Diseases 149 

Retinal Survival in Transgenic Mice Expressing 

Human Ornithine 6-Aminotransferase 152 

Enzymatic Correction of OAT Deficiency: Progress Toward 

Gene Therapy to Ocular Genetic Disease 155 

Isolation and Characterization of the Mouse OAT Gene for Gene Targeting ... 160 

Gene Therapy for Ocular Genetic Disease 163 

Immunopathology of Ocular Diseases in Humans 164 



Laboratory of Mechanisms of Ocular Diseases i65 

Report of the Chief 167 

/. Samuel Zigler, Jr., Ph.D. 

Section on Cataracts 

/. Samuel Zigler, Jr., Ph.D. 

Structure and Composition of Lens Crystallins With 

Respect to Cataractogenesis 169 

Donita L. Garland, Ph.D. 

Oxidation of Proteins in Cataractogenesis 173 

Studies on Human Lens Proteins 176 

Deborah Carper, Ph.D. 

Structure and Expression of Polyol Pathway Enzymes 179 

Paul Russell, Ph.D. 

Characterization of the Lens 183 

Lenticular Expression of the HTV Protease 186 

Autoantibodies to Lens Crystallins 188 

James Fielding Hejtmancik, M.D., Ph.D. 

Inherited Ocular Diseases 190 

Section on Pathophysiology 
W. Gerald Robison, Jr., Ph.D. 

Ultrastructure and Function of the Cells and Tissues of the Eye 195 



Laboratory of Molecular and Developmental Biology 199 

Report of the Chief 201 

Joram Piatigorsky, Ph.D. 

Section on Cellular Differentiation 

Peggy S. Zelenka, Ph.D. 

Proto-oncogene Expression During Lens Differentiation and Development .... 204 
Section on Molecular Genetics 

Joram Piatigorsky, Ph.D. 

CrystaUin Genes: Structure, Organization, Expression, and Evolution 209 

Molecular Biology of the Cornea 217 



Table of Contents 



Section on Molecular Structure and Function 

Graeme J. Wistow, Ph.D. 

Moleciilar Biology and Functions of the Lens Proteins 220 

Section on Regulation of Gene Expression 
Ana B. Chepelinsky, Ph.D. 

Genetically Engineering the Eye With the aA-CrystaUin Promoter 224 

Regulation of Expression of Lens Fiber Membrane Genes 228 

Section on Transgenic Animal and Genome Manipulation 
Eric Wawrousek, Ph.D. 

NEl Cential Transgenic Animal Production Fadlity 231 

a-CrystaUin Gene Disruption in the Mouse 234 

Transgenic Animal Models 237 



Laboratory of Ocular Therapeutics 241 

Report of the Chief 243 

Peter F. Kador, Ph.D. 

Peter F. Kador, Ph.D. 

Pharmacology of Ocular Complications 244 

Sanai Sato, M.D., Ph.D. 

NADPH Reductases and Polyol Pathway in Ocular Complications 250 



Laboratory of Retinal Cell and Molecuur Biology 253 

Report of the Chief 255 

Gerald J. Chader, Ph.D. 

Section on Biochemistry 

Barbara YJiggert, Ph.D. 

Vitamin A and Ocular Tissues 258 

Section on Gene Regulation 
Susan Gentleman, Ph.D. 

Microtubule Stability as a Factor in Retinal Degenerations 263 

Diane E. Borst, Ph.D. 

Molecular Genetics of the Eye and Ocular Diseases 266 

Gerald J. Chader, Ph.D. 

Molecular Biology of the Retina and Pigment Epithelium 270 

Visual Control Mechanisms and Hereditary Degeneration 273 

T. Michael Redmond, Ph.D. 

Molecular Biology of Outer Retina-Specific Proteins 277 

Section on Molecular Biology 
Toshimichi Shinohara, Ph.D. 

Molecular Biology of Experimental Autoimmune Uveitis 280 

Molecular Biology of Phototransduction 283 



Laboratory of Sensorimotor Research 287 

Report of the Chief 289 

Robert H. Wurtz, Ph.D. 



FY 1994 NEI Annual Report 



Section on Visual Behavior 

David Lee Robinson, Ph.D. 

Visuomotor Properties of Neurons in the Thalamus 293 

Section on Neuro-Ophthalmologic Mechanisms 
Michael E. Goldberg, M.D. 

Cerebral Cortical Mechaiusms for Eye Movements and Visual Attention 297 

Section on Oculomotor Control 
Frederick A. Miles, D.Phil. 

Visual Motion and the Stabilization of Gaze 301 

Section on Visuomotor Integration 
Robert H. Wurtz, Ph.D. 

Visuomotor Processing in the Primate Brain 305 

Section on Neural Modeling 
Lance M. Optican, Ph.D. 

Ii\formation Processing by Visual System Neurons 310 



Ophthalmic Genetics and Clinical Services Branch 315 

Report of the Chief 317 

Muriel L Kaiser-Kupfer, M.D. 

Section on Cataract and Corneal Diseases 

Manual B. Datiles, M.D. 

The Effects of Corneal Contact Lenses on the Cornea 321 

Documentation and Monitoring of Opacities in the Human Lens 322 

Use of Human Lens Material for Determining Possible Causes 

of Cataracts 325 

Muriel I. Kaiser-Kupfer, M.D. 

Addendum to Use of Human Lens Material for 

Determining Possible Causes of Cataracts 328 

Carl Kupfer, M.D. 

Anterior Chamber Anomalies Associated With Glaucoma or 

Ocular Hjrpertension 330 

Section on Ophthalmic Genetics 

Muriel L Kaiser-Kupfer, M.D. 

Pigment Dispersion With and Without Glaucoma 332 

Visual Function and Ocular Pigmentation in Albinism 335 

Gyrate Atrophy of the Choroid and Retina and Other Retinal Degenerations . . 338 

NTH Interinstitute Genetics Program: The Genetics Clinic 342 

Usher Syndrome — Clinical and Molecular Studies 345 

A Double-Masked Controlled Randomized CUnical 

Trial of Topical Cysteamine [II] 347 

A Double-Masked Controlled Randomized Clinical 

Trial of Topical Cysteamine [I] 348 

Mark H. Scott, M.D. 

Characteristics of Macular Scotomas in Patients 

With Primary Monofixation Syndrome 350 



Table of Contents 



Section on Eye Services 

Rafael Caruso, M.D. 

Clinical Psychophysics of the Visual System 351 

Clinical Electrophysiology of the Visual System 353 

Visual Function Diagnosis Service 355 



Index 357 



Statement of the Institute Director 



statement of the Institute Director 



Carl Kupfer, M.D. 



In this first year of our new research plan 
Vision Research — A National Plan: 1994- 
1998, vision researchers have made sig- 
nificant progress in achieving the stated goals 
and objectives that are considered so impor- 
tant to improving the visual health of the 
American people. During this fiscal year, 
1,210 research grants were funded for nearly 
$235 million, and an additional 19 research 
and development contracts were funded for 
approximately $7.8 million. Another $30 mil- 
lion was expended in support of the intramu- 
ral research program. As this year's annual 
report demonstrates, the investment by Ameri- 
can taxpayers in vision research has led to 
several important breakthroughs by our intra- 
mural and extramural laboratories and clinical 
scientists and has continued to improve our 
understanding of the pathological processes 
involved in diseases of the eye and disorders 
of vision. 

As an example, the National Eye Institute 
(NEI)-supported intiamural scientists continue 
to provide new and important data on the role 
and function of the visual motor system. A 
study investigating the control of movement 
by visual input and the systems in the brain 
that perform this vital function is under way. 
Recentiy, these scientists tested the hypothesis 
that the spread of activity in the superior coUi- 
culus, a brain structure critical to rapid eye 
movement, controls the amplitude of such 
movement. Minute injections of a neurotrans- 
mitter inhibitor into the superior colliculus 
changed the direction of the eye movement. 
This suggests that not only the end-point but 
also the path taken by the eye is influenced by 
the superior colliculus; this information is pro- 
foundly important not only for understanding 



how the brain controls eye movement but also 
how it controls movements in general. This 
remarkable advance in understanding the sen- 
sory-motor system creates opportunities to 
examine new mechanisms of controlling 
movement and could lead to a better under- 
standing of how to control disease-induced 
disorders of eye movement control. 

Intramural researchers have also contin- 
ued their important studies on the role of 
aldose reductase (AR) in inhibiting ocular 
compUcations associated with diabetes. They 
have developed an animal model (galactose- 
fed dogs) that develops both the chnical and 
histological lesions associated with all stages 
of diabetic retinopathy. Using this model, 
investigators have conducted studies to image 
noninvasive cataract formation and to mea- 
sure the levels of AR in the lens in vivo. With 
this technique, they have also demonstrated a 
direct correlation between inhibition of AR in 
the lens and the prevention of the retinal peri- 
cyte degeneration, which leads to diabetic 
retinopathy. 

Intramural scientists are continuing their 
basic and clinical studies on the natural histo- 
ry and the role of the ornithine aminotiansfer- 
ase gene in the development of gyrate atrophy 
(GA), an inherited degenerative retinal disor- 
der. The research is currentiy directed at ge- 
netically altering the defective gene and cul- 
turing skin tissues that will be used as a 
tiansfer vehicle for inserting the altered gene 
in GA patients. This research may lead not 
only to an effective therapy for GA but may 
be extended to genetic interventions for other 
blinding genetic disorders. 



FY 1994 NEI Annual Report 



Scientists in NEI's intramural laboratories 
have discovered several genes that may be 
implicated in macular degeneration, a leading 
cause of bUndness in older Americans. In 
addition, a new gene has been cloned that 
could be the defective gene in Bardet-Biedl's 
syndrome, a hereditary disease of blindness 
and mental retardation. These investigators 
also have discovered a protein that promotes 
the survival of neurons within the central ner- 
vous system. This protein (PEDF) promotes 
maturation of photoreceptor-Uke cells derived 
from the retina. It also greatly lengthens the 
Ufe-span of brain neurons in tissue culture 
experiments. With these characteristics, future 
researchers will explore whether PEDF can be 
used in transplantation procedures that could 
be helpful in conditions such as Parkinson's 
disease. 

NEI intramural scientists are examining 
the role of ceU adhesion molecules and cyto- 
kines in the development of ocular inflamma- 
tory disease. CeU adhesion molecules are 
surface proteins that are important for antigen 
sensitization and leukocytes' migration to 
inflammation sites. Researchers are currentiy 
investigating compounds that block cell adhe- 
sion molecules as a treatment for uveitis. 
They have been able to inhibit significantiy the 
development of endotoxin-induced uveitis in 
mice with a single injection of anti-Mac-1 
antibody. Research is now under way to de- 
velop and test topically administered sub- 
stances that can block critical cell adhesion 
molecules. If animal studies are successful, 
scientists plan to test therapies based on block- 
ing critical cell adhesion molecules and cyto- 
kines in the cUnical trials of uveitis patients. 

Scientists in NEI's intramural program 
have been studying the trabecular meshwork, 
a tissue in the eye that has been implicated in 
the development of open-angle glaucoma. In 
a series of experiments, investigators have 
analyzed the proteins present in the four 
quadrants of the normal trabecular meshwork 
and are now examining the differences be- 
tween normal tissue and glaucomatous trabec- 
ular meshwork and the changes that precede 
glaucoma. Such results would offer important 



clues on the causes of open-angle glaucoma 
and may provide the foundation for the devel- 
opment of effective therapies. 

The most common causes of blindness in 
the United States are associated with the aging 
process. NEI epidemiologists have initiated a 
natural history study to assess the cUnical 
course, prognosis and risk factors of age-re- 
lated macular degeneration (AMD), and cata- 
ract. Further investigation of the risk factors 
in the development and progression as well as 
their pathophysiologies may lead to methods 
for preventing these debilitating ocular disor- 
ders. Additionally, the effects of pharmaco- 
logic doses of antioxidants — ^vitamins C and E 
and beta-carotene — and zinc on the incidence 
and progression of AMD and antioxidants on 
the incidence and progression of lens opacities 
wiU be assessed as part of a randomized clini- 
cal trial, using the cohort of 4,600 patients 
developed for the natural history study. 

NEI-supported extramural scientists have 
reported the loss of neuronal cells in the 
brains and retinas of acqviired immunodefi- 
ciency syndrome (AIDS) patients, which is 
thought to contribute to the neurologic and 
retinal dysfunction often associated with hu- 
man immunodefiency virus (HIV)-1 infection. 
Retinal ganglion cells involved in the trans- 
mission of information from the retina to the 
brain appear to undergo physical changes in 
these patients. These scientists have shown 
that a complex web of interactions between 
the cells of the immune system and the neu- 
rons is involved, involving a protein known as 
gpl20. This protein is found on the surface of 
the HIV virus. They have found that adding 
gpl20 to retinal ganglion cells growing under 
laboratory conditions causes injury to the cells. 
They have also found a receptor antagonist 
that may give protection from gpl20 neurotox- 
icity, which may one day lead to the develop- 
ment of the means to prevent the neurological 
manifestations of AIDS. 

An abnormal proliferation and leakage of 
retinal blood vessels leading to loss of vision 
are characteristic of a number of conditions in 
which the oxygen supply to the retina is ob- 



Statement of the Institute Director 



structed. These conditions include severe 
diabetic retinopathy, retinal vascular occlu- 
sions, and retinopathy of prematurity. Recent 
studies supported by the NEI have demon- 
stiated that a protein called vascular endothe- 
lial growth factor (VEGF) is present in greatly 
increased amounts in the ocular fluid of pa- 
tients with these conditions. Production of 
VEGF is also enhanced by experimental proce- 
dures that decrease the oxygen supply to the 
retina. As VEGF is well known to be capable 
of stimulating blood vessel growth in the eye, 
these new studies indicate that VEGF may 
play a major role in diabetic retinopathy and 
other ischemic eye conditions. Investigators 
will expand the search for ways to either 
block the production of VEGF or to block its 
ability to stimulate the growth of new blood 
vessels. 

Because several inherited macular degen- 
erative diseases share significant similarities to 
AMD, NEI-supported scientists are searching 
for defective genes in affected farrdlies. Inves- 
tigators have localized gene mutations to spe- 
cific chromosomes for three forms of inherited 
macular degeneration. These investigators 
have shown that a mutation in one of these 
genes causes macular degeneration, while a 
different mutation in the same gene causes 
retinitis pigmentosa (RP). The information 
gained from these studies should help in 
understanding the cause of the more prevalent 
AMD. 

During this last year, investigators from 
the NEI-supported Prospective Evaluation of 
Radial Keratotomy (RK) reported the results of 
their 10-year foUowup study. The data contin- 
ue to indicate that RK remains a reasonably 
safe and effective method for correcting myo- 
pia (nearsightedness). However, more than 40 
percent of RK-operated eyes continue to have 
a gradual shift toward farsightedness. This 
finding suggests that some people who have 
RK may need glasses at an earlier age for poor 
close-up vision, a common problem after age 
40, than if they had chosen not to have the 
surgery. Data on quality of life, symptoms 
related to glare and fluctuating vision, and 
subjective visual function remain to be analyzed. 



Last year NEI-supported researchers re- 
ported the localization of the gene for juvenile 
onset glaucoma, a form of the disease charac- 
terized by early adulthood onset and elevated 
intraocular pressure, to chromosome 1. These 
researchers have now mapped the gene to a 
region of chromosome 1 extensive enough to 
contain approximately 20 genes. Once the 
exact location of this gene is determined, the 
gene can be cloned and sequenced. The hope 
is that the deoxyribonucleic acid (DNA) se- 
quence of the juvenile glaucoma gene will 
reveal clues about the fundamental cause of 
glaucoma. 

A new technique, which uses microlaser 
stimulation and microelecfrodes, allowed 
researchers supported by the NEI to stimulate 
selected nerve cells in individual circuits in 
this dense meshwork of cells. Results from 
this approach show how individual nerve cells 
function together. Previously, these results 
were very difficult to obtain because of the 
inaccessibility of these functional uruts. Fur- 
ther refinement of this approach could lead to 
the development of a powerful tool for isolat- 
ing discrete networks of nerve cells and for 
learning how they function and organize in- 
formation. 

During fiscal year (FY) 1994, results from 
a NEI-supported study the Orinda Longitudi- 
nal Study of Myopia (OLSM) were published 
suggesting a possible genetic link to myopia. 
Measurements were made in more than 700 
school children ages six to 14 on each child's 
refractive error, corneal curvature, crystaUine 
lens power, and axial ocular dimensions. 
Parents reported their own vision status. The 
investigators found that school children whose 
parents are nearsighted have differently 
shaped eyeballs than children whose parents 
are not nearsighted. Children of myopic 
parents had longer eyes even before the onset 
of myopia. These results suggest that the 
premyopic eye in children with a family 
history of myopia already resembles the 
elongated eye present in niyopia. The OLSM 
will continue to provide a wealth of informa- 
tion on ocular and refractive development in 
children in the years ahead. 



FY 1994 NEI Annual Report 



These are highlights of a few of the re- reports and project descriptions contained in 
search accompUshments of the intramural and this annual report. It is a pleasure to provide 
extramural laboratory and clinical scientists this recognition of the researchers' efforts in 
supported by the NEI during FY 1994. More addressing the visual health needs of our 
detail on these and other accompUshments in Nation, 
the field of vision research can be found in the 

Carl Kupfer, M.D. 

Director 

National Eye Institute 



Extramural Research 



Report of the Associate Director for 
Extramural Research) 



Jack A. McLaughlin, Ph.D. 



Research activities supported by the 
extramural Vision Research program 
address the leading causes of blind- 
ness and unpaired vision in the United States, 
including retinal diseases, corneal diseases, 
cataract, glaucoma, strabismus, and ambly- 
opia. The program seeks to increase under- 
standing of the normal development and 
function of the visual system; to understand 
the causes of and to better diagnose, prevent, 
and treat sight-threatening conditions; and, to 
enhance the rehabilitation, training, and quaU- 
ty of life of individuals who are partially 
sighted or bUnd. 

In working to this end, the Vision Re- 
search program supports vision research 
through grants, cooperative agreements, and 
research and development contracts; encourag- 
es high quality cHnical research, including 
dinical trials and other epidemiologic studies; 
encourages research training and career devel- 
opment in the sciences related to vision; 
sponsors scientific workshops in high-priority 
research areas to encourage exchange of 
information among scientists; and carries out 
a construction, alteration, and instrumentation 
program of grants for public and private 
nonprofit vision research facilities. 

For FY 1994, an estimated total of 
$248,425,000 was expended for NEl extramural 
grants, cooperative agreements, and research 
and development contracts in the following 
categories and amounts: 



Research Grants $233,541,000 

Research Training Awards $7,294,000 
Research and Development 

Contract $7,590,000 

Total Extramural Support $248,425,000 

The following sections highlight some of 
the recent accomplishments of NEl-supported 
investigators. 



Division of Basic Vision Research 

Peter Dudley, Ph.D., Director 

Retinitis Pigmentosa 

The major diseases of the retina affect primari- 
ly the photoreceptor ceUs and the neighboring 
tissue called the pigmented epithelium. The 
photoreceptors are fragile and easily damaged 
in the face of hereditary defects, aging, toxic 
agents, overexposure to Ught, and dietary 
deficiencies. They are targets for such dis- 
eases as RP and AMD, which are leading 
causes of blindness. The basis for understand- 
ing these diseases Ues in gaining fundamental 
knowledge of the molecular machinery and 
cellular organization of these specialized ceUs. 
Structural information, combined with de- 
tailed biochemical and physiological knowl- 
edge, sets the stage for a molecular dissection 
of genetic diseases. Recent identification of 
defects in photoreceptor-specific genes in 
some inherited retinal diseases support this 
concept. 



FY 1994 NEI Annual Report 



RP is one of the most common human 
inherited eye disorders, causing bUndness 
from degeneration of the rod and cone photo- 
receptors in the retina. Patients with RP 
develop night bUndness and loss of midperi- 
pheral vision early in the disease. As the 
disease progresses, a shrinking island of 
central vision remains resulting in "tunnel 
vision." Unfortunately, many patients are 
bUnd by middle age. In the United States, RP 
affects 50,000 to 100,000 people of all races. 
RP exhibits heterogeneity, meaning that differ- 
ent mutations in a person's genetic material or 
DNA cause similar cUnical symptoms. There 
are two forms: "allelic heteogeneity," refers to 
different mutations within a single gene (rho- 
dopsin for example), and "locus heteroge- 
neity," which refers to gene defects at different 
chromosome locations. RP can be transmitted 
as an autosomal dominant, autosomal reces- 
sive, or X-Unked trait. 

The understanding of RP, currentiy an 
incurable retinal degeneration, has progressed 
recentiy with the discovery of a number of ge- 
netic mutations in important photoreceptor 
proteins. In about one-quarter of the cases of 
RP, mutations have been identified in the 
genes for rhodopsin, peripherin/RDS, rod 
phosphodiesterase-p subunit, and the cyclic 
guanosine monophosphate (cGMP)-gated 
channel. Although most cases are considered 
to be monogenic, i.e., in one particular family 
only one chromosome locus is thought to be 
defective. Dr. Dryja, from the Harvard Medi- 
cal School, has demonstrated an unusual in- 
heritance pattern in three famUies with RP 
called "digenic inheritance." Mutations in two 
genes, the unlinked photoreceptor-specific 
genes-ROMl and peripherin/RDS, were found 
to be responsible for this particular form of 
RP. The interesting feature of this unusual 
form of inheritance is that although variability 
in disease severity was observed in patients 
with other mutations {e.g., rhodopsin), the 
complete lack of clinical symptoms in some 
persons with a peripherin/RDS gene muta- 
tion, had not been observed. The explanation 
for this was revealed when a mutation in 
ROMl was discovered in patients who also 



had the peripherin/RDS mutation resulting in 
disease expression. 

Norrie's disease (ND) is a rare X-Unked 
hereditary eye disease characterized by con- 
genital bUndness. Investigations so far show 
the disease-causing gene to be located on the 
X chromosome. Genetic analysis has pinpoint- 
ed a chromosome site that may be the actual 
gene. Four separate mutations have been 
identified that cause deletions of part of the 
chromosome housing the apparent ND gene. 
But formal conclusive evidence that the ND 
gene is involved in the disease awaits identifi- 
cation of more gene mutations in ND patients. 

Dr. Wong, from Duke Uruversity, has 
discovered a new mutation in the ND gene of 
a male infant that results in loss of a small 
part of the ND protein. The normal ND 
protein, which appears to function in retinal 
and brain development, contains 11 cysteine 
amino acids. The point mutation discovered 
by Dr. Wong results in a loss of two cysteine 
residues. The discovery of another separate 
point mutation increases the Ust of mutations 
in the ND protein and appears to confirm the 
importance of the fuU complement of 11 
cysteines in its function. This discovery may 
result in a useful diagnostic test to confirm an 
initial clinical detennination of ND. 

Glaucoma 

In the United States, about two milUon people 
have glaucoma, but, because of the insidious 
nature of the disease, many are unaware of its 
presence. AdditionaUy, about five milUon 
Americans, some of whom wUl develop glau- 
coma, have elevated intraocular pressure 
(lOP). Primary open-angle glaucoma (POAG) 
is the most severe form of the disease and is 
most common in people older than age 60. 
Approximately 80,000 people with this form of 
the disease will become bUnd. African Ameri- 
cans are affected disproportionately from 
POAG with a risk factor five times that of the 
white population for people older than age 40. 
The rate for blindness due to POAG in African 
Americans is six times higher than that of the 



10 



Extramural Research 



rest of the population, reflecting a more severe 
disease. 

The mechanism by which the optic nerve 
is damaged by glaucoma is not known. The 
relative influence of genetic and environmen- 
tal factors is also unclear. However, there is 
some hope for understanding the cause of the 
disease because juvenile onset glaucoma, a 
form of the disease characterized by early 
adulthood onset and elevated lOP, displays an 
autosomal dominant pattern of inheritance. 
This mode of inheritance makes a genetic 
approach ideal for the study of this disease. 

Dr. Stone, from the University of Iowa, Dr. 
Richards, from the University of Michigan, 
and Dr. Wiggs, from the Tufts New England 
Medical Center, have identified a number of 
families with sufficient numbers of individuals 
with glaucoma so that it is now possible to 
perform genetic linkage studies. To date med- 
ical histories have been collected fiom families 
in Michigan, New England, and Iowa. The 
ultimate goal is to identify a "glaucoma gene." 
Recentiy, one disease-associated gene has been 
mapped by Unkage analysis to chromosome 1. 
Corroborating data from difterent laboratories 
using different families have confirmed this 
location. Linkage analysis has placed the gene 
to within approximately a 20 to 80 gene re- 
gion on the chromosome. 

A possible association between juvenile 
onset glaucoma and POAG may Ue in identi- 
fying a causal factor for elevated lOP, be it a 
block in the aqueous humor outflow pathway 
or some other as yet unidentified factor. 

Keratoconus of the Cornea 

Keratoconus is a progressive condition charac- 
terized by norvinflammatory thinning and 
protrusion of the cornea, leading to its cone- 
shaped appearance. Keratoconus has an inci- 
dence of about five in 10,000 in the general 
population, and approximately 100,000 kerato- 
conus patients require eye care in the United 
States annually. Most of these patients need 
multiple contact lens fittings during their hfe 



and 10 to 20 percent ultimately require a pro- 
cedure called a penetrating keratoplasty to 
correct their condition. Recent retrospective 
studies suggest that keratoconus is the leading 
cause for cornea transplantation in the United 
States. 

Dr. Rabinowitz, from the Cedar-Sinai 
Medical Center, has been examining the 
genetic basis for keratoconus. He is using a 
techruque called videokeratography to obtain 
an accurate assessment as early as possibleof 
the chances for developing this condition. 
PUot studies on patients with advanced dis- 
ease indicated three distinguishing features: 
central corneal steepness, nonsymmetric steep- 
ness when comparing the superior and inferi- 
or corneal regions, and large central refractive 
differences between the right and left corneas. 
These features were quantitatively indexed 
and appUed to broader family studies. This 
work suggests an autosomal dominant mode 
of heredity with variable expressivity. 

Dr. Rabinowdtz's group is exploring three 
independent approaches to identifying a kera- 
toconus gene(s); cytogenetic studies have been 
initiated. These have not yet been informa- 
tive, although there is a report in the Uterature 
that Angehnans' syndrome patients with kera- 
toconus as one of their ocular findings carry a 
deletion on the long arm of chromosome 15. 
Family pedigree studies have been initiated, 
using videokeratographic data. To date 200 
normal subjects, 110 keratoconic patients, and 
210 family members have been analyzed and 
have donated blood for future genetic studies. 
Enrollment is approximately 60 percent com- 
plete, and, so far, more than 70 family pedi- 
grees have been constructed. These include 
several large families with multiple aftected 
members in at least four generations. Analy- 
sis will be performed within the year to deter- 
mine the genetic mode of inheritance. Longi- 
tudinal candidate gene studies are under way 
in one of the large multigenerational families. 
Some specific genes involved in the synthesis 
of collagen have been excluded. Other colla- 
gen genes and the genes for enzymes involved 
in coUagen metaboUsm are being explored. 



11 



FY 1994 NEI Annual Report 



Acquired Immunodeficiency Syndrome 

As many as one-third of adults with AIDS 
eventually develop neurological symptoms, 
including problems with memory, coordinated 
movement, and sensation. These problems 
can occur in the face of almost complete ab- 
sence of direct infection of nerve ceUs or neu- 
rons by the HIV-1. Recently, loss of neuronal 
cells in the brains and retinas of AIDS patients 
has been observed and is thought to contrib- 
ute to the neurologic and retinal dysfunction. 
Retinal gangUon cells involved in the trans- 
mission of information from the retina to the 
brain appear to undergo physical changes in 
these patients. 

What are the mechanisms that cause the 
observed changes in retinal gangUon cells in 
AIDS patients? Dr. Lipton, from Children's 
Hospital in Boston, has been studjdng this 
problem. There appears to be growing scien- 
titic evidence that for infections like HIV, 
which lead to the injury of neurons, a complex 
web of interactions between ceUs of the im- 
mune system and neurons is involved. Dr. 
Lipton has been studjdng one particular pro- 
tein involved in this system called gpl20. 

Gpl20 is a protein present on the outer 
surface of the HTV virus and is associated 
with increased intracellular concentrations of 
the ion calcium (Ca"^*). When gpl20 is added 
to retinal ganglion ceUs growing under labora- 
tory conditions, the cells are injured. How 
does this happen? In general, when cells take 
up Ca"^"^ in an uncontrolled fashion, they die. 
Dr. Lipton has shown that there is a certain 
kind of receptor for the neurotransmitter 
glutamate that is apparentiy involved in this 
process. Glutamate is actively taken back into 
ceUs after release as a neurotransmitter so that 
it can be reused. Apparentiy gpl20 can hin- 
der this "reuptake." Recentiy, Dr. Lipton has 
discovered a novel antagonist to the glutamate 
receptor that may give protection from gpl20 
neurotoxicity. In addition to its affect on 
glutamate, gpl20 can also cause macro- 
phages — ceUs that can swallow up and de- 
stroy bacteria and foreign materials — ^to re- 
lease substances toxic to neuronal cells. 



Retinal Neuroscience— Molecular Basis 
of Signaling 

Vision begins when cells in the retina called 
rods and cones capture Ught and initiate a 
series of biochemical events that send an elec- 
trical message to the brain. Rod cells respond 
to dim light. The red-, green-, and blue-sensi- 
tive cones are sensitive to bright Ught and give 
us color vision. The orchestrated interplay of 
molecules in photoreceptors is called signaling 
or visual phototransduction and describes the 
way Ught is converted to an electrical signal 
destined for the brain. 

Light, which is focused by the cornea and 
the lens, enters the retina as energy particles 
caUed photons, which are absorbed by rho- 
dopsin. Attached to rhodopsin is a smaU 
molecule, vitamin A, which changes its molec- 
ular configuration sUghtiy when struck by a 
photon. This change in configuration or isom- 
erization is the initial event in a process caUed 
the transduction cascade, which now becomes 
rapidly amplified as rhodopsin coUides with 
many molecules of transducin. Transducin 
also has a smaller molecule attached to it. In 
this case, it is not a vitamin but a nucleotide 
called guanosine triphosphate (GTP). The 
binding of GTP to transducin is a consequence 
of the initial contact with rhodopsin. Trans- 
ducin then dissociates into separate parts, one 
of which binds to yet another molecule caUed 
phosphodiesterase (PDE), activating it. PDE, 
an enzyme, actuates the triggering event in 
phototransduction — ^the conversion of cGMP 
into an altered form caUed simply GMP to 
denote the fact that it is no longer a circular 
molecule. This hydrolysis or spUtting of 
cGMP closes a molecular gate in the mem- 
brane of the rod ceU resulting in a decreased 
flow of smaU ions Uke sodium into the ceU. 
This change in membrane potential of the ceU 
generates an electrical signal to the neighbor- 
ing retinal ceUs and then to the brain for sig- 
nal decoding. 

Current research has now turned to estab- 
Ushing the complete molecular basis of signal- 
ing in visual ceUs, signal termination, and 
adaptation. Knowledge of the structure of 



12 



Extramural Research 



each of the components of the phototransduc- 
tion cascade is necessary to understand the 
mechanisms behind the dynamics of, and 
potential sites for, regulation of signaling in 
visual cells. 

The electrical events that initiate vision 
begin with the capture of light that leads to 
the closing of cGMP-controUed membrane ion 
charmels. These ion channels directly control 
the flow of ions across the outer cell mem- 
brane. Rods and cones use similar but not 
identical molecvdar components to transduce 
Hght into neural signals to the brain, having 
their own distinct visual pigments as well as 
enzymes necessary for phototransduction. In 
general, although cones appear to have a 
similar phototransduction scheme to rods, the 
exact mechanisms underlying their low sensi- 
tivity and quick response to Ught remain un- 
clear. To study the basis for these differences 
Dr. Molday in Vancouver, British Columbia, 
has cloned the cGMP-gated channels from rod 
and cone photoreceptors of the chicken retina 
and examined their molecular and electrical 
properties. Using a special technique to engi- 
neer the cells to express specific molecules, he 
demonstrated that chicken rod and cone cells 
each synthesize different forms of cGMP-gated 
channels. 

The characterization of the rod channel 
protein is of great scientific interest because of 
its central involvement in phototransduction. 
Dr. Yau, from The Johns Hopkins School of 
Medicine, has cloned a protein from human 
retina that has about one-third similarity in 
structure to a similar protein from bovine 
retina. By itself, the human-derived protein 
does not self-associate to form a functional 
working channel. However, when combined 
with the channel protein isolated from bovine 
retina it functions characteristically like the 
native channel. What does this mean? It 
implies that the newly discovered human 
protein is but one of a group of different 
proteins that when assembled together form 
the native charmel protein. It is a clue that the 
channel protein may be made up of different 
subunits, a characteristic shared by other 



channel proteins that are controlled by small 
molecules like cGMP. 

At the end of a signaling event, the mole- 
cules involved must return to their original 
state. A key feature in the recovery and reac- 
tivation of phototransduction is the involve- 
ment of the ion calcium in Ca**-sensitive regu- 
lation of guanylyl cyclase (GC), an enzyme 
that synthesizes cGMP. During visual trans- 
duction, closure of the cGMP-gated channels 
reduces Ca** influx, while efflux by Na^ K^ 
and Ca*"^ continues, causing a decrease in Ca** 
within the cell. This fall in Ca^* stimulates GC 
and allows recovery of the sensitivity to Ught. 
Dr. Palczewski, from the University of Wash- 
ington, has shown that the regulation of GC is 
mediated by a novel photoreceptor-specific 
protein. The cloning and characterization of 
this molecule have shown there are three 
areas on the molecule that can bind Ca**, 
which suggests it has features in common 
with a large family of molecules called calci- 
um-binding proteins. In addition. Dr. Hurley, 
from the University of Washington, has shown 
that human photoreceptors use similar physio- 
logically relevant proteins in responding to 
and recovering from Ught. 

Human color vision involves three sepa- 
rate Ught-capturing molecules, caUed the red, 
green, and blue photopigments, to designate 
the wavelength of Ught to which they are 
most receptive. Although the sequence of 
amino acids for the red and green pigments 
are identical, except for 15 of their 365 amino 
acids, their Ught absorption characteristics 
differ sigruficantly. In an extensive study 
using a technique caUed mutagenesis to selec- 
tively and individuaUy alter specific amino 
adds. Dr. Oprian, from Brandeis University, 
has determined that there are only seven ami- 
no acids responsible for the Ught absorption 
differences between the red and green pig- 
ments. The mechanisms by which these ami- 
no acids determine the Ught absorbing proper- 
ties of the photopigment are unknown. How- 
ever, they do indicate how sensitive biological 
molecules can be to small changes in amino 
add composition. These changes can have 
health impUcations when the change or "muta- 



13 



FY 1994 NEI Annual Report 



tion" leads to disease. A good example is RP, 
where certain mutations in the gene coding 
for rhodopsin result in disease. On the other 
hand, some mutations in the gene coding for 
rhodopsin can manifest themselves only in an 
altered sensitivity to wavelengths of light with 
no impact on quality of life. 

The physical structure of the biologically 
active subunit of rod cell transducin was de- 
scribed recentiy by Dr. Hamm, from the Uni- 
versity of Illinois. Her research suggests how 
"nucleotide exchange" might occur in photo- 
transduction so as to induce changes in the 
surfaces of proteins to activate them and ex- 
plain a mechanism for GTPase activity not 
evident from previous studies. Dr. McKay, 
from Stanford University, has determined the 
crystal structure of recoverin, a recentiy dis- 
covered member protein that serves as a calci- 
um sensor. Ca*^ plays a critical role in the 
recovery phase of visual excitation and in 
adaptation to background Ught. The light- 
induced lowering of Ca"^* in retinal rod outer 
segments appears to function in system recov- 
ery after a Ught response. With the recent 
discovery that calcium-bound recoverin pro- 
longs the photoresponse, most likely blocking 
the phosphorylation of photoexdted rhodop- 
sin, the information obtained on the crystal 
structure will lead to further insights about 
recovery and adaptation in vision. 

Corneal Angiogenesis 

The growth of new capillary blood vessels, 
neovascularization or angiogenesis, occurs 
during many normal ocular processes such as 
wound healing and embryonic development. 
However, angiogenesis can also be a compo- 
nent of serious eye pathologies such as diabet- 
ic retinopathy, corneal clouding following 
infection or traumatic injury, and neovascular 
glaucoma. The presence of blood vessels in 
the cornea makes it much more difficult for a 
transplanted cornea to survive and remain 
clear. 

Current treatment of corneal neovasculari- 
zation relies on the use of topical steroids. 
Unfortunately, these agents have significant 



side effects, including immunosuppression, 
osteoporosis, stomach ulcers, and diabetes. 
Dr. Proia, from Duke Uruversity School of 
Medicine, and Dr. Schwartzman, from the 
New York Medical College, are developing 
new categories of angiostatic drugs to inhibit 
new vessel growth with a more favorable 
therapeutic profile. Recent advances include 
the discovery of a novel class of angiostatic 
steroids with fewer side effects. An indepen- 
dent family of angiostatic substances, Uke the 
cytochrome oxidase P450 inhibitors flurbipro- 
fen and clotrimazole, has also been recentiy 
described. These drugs decrease corneal 
inflammation and neovascularization by 
affecting the levels of prostaglandins synthe- 
sized during the inflammatory process. 

Lens Development 

Lens formation begins with lens epithelial ceU 
division and proceeds with differentiation into 
fiber cells. Fiber cells are terminally differenti- 
ated cells that are essentially "sacs" of proteins 
devoid of cellular structures. Continuous 
differentiation of epithelial cells into fiber cells 
is required to maintain lens tiansparency. 
Any disruption of the internal controls that 
balance growth and differentiation has impli- 
cations for cataract formation. 

Determining the signals that control epi- 
thelial cell division and differentiation into 
fiber cells has been an area of active investiga- 
tion. Using model systems. Dr. Paul Over- 
beek, from the Baylor College of Medicine, has 
shown the importance of growth factors in 
this process. Growth factors are a diverse 
group of small molecules that can trigger cell 
division. A challenge to lens biologists is to 
determine which of the many known growth 
factors are responsible for cell differentiation. 
Scientists have identified a number of poten- 
tially important growth factors including insu- 
Un-Uke growth factor I (IGF-I), fibroblast 
growth factor (FGF), and a novel factor found 
in the vitreous — ^lentropin. Dr. Overbeek has 
been stud5dng these growth factors by putting 
the genes for their expression into a mouse 
and creating what is called a transgenic mouse 
so that one can assign a functional role to 



14 



Extramural Research 



various growth factors. Overexpression of 
IGF-I and FGF in the lens of transgenic mice 
leads to congenital cataracts, substantiating the 
importance of these molecules in lens develop- 
ment and providing scientists with a model 
for cataract formation. 

Dr. DePinho, from the Albert Einstein 
Medical School, and Dr. Creep, from the 
University of Wisconsin, are interested in how 
lens development is controlled. Because fiber 
cell formation is essential for maintenance of 
lens transparency, preserving the balance 
between cell division and cell differentiation is 
critical. Recentiy, the retinoblastoma (Rb) and 
protein(p)53 tumor suppressor genes have 
been shown to play a role in eye development. 
Transgenic mice in which the normal function- 
ing Rb gene is knocked out demonstrate a 
condition caUed microphthalmia (small eyes). 
In these mice, lens cell DNA s5Tithesis pro- 
ceeds unimpeded while cell elongation and 
differentiation is inhibited. Continuous epi- 
thelial cell division is incompatible with nor- 
mal lens functioning. To avoid this problem 
it appears that p53 acts as a backup regulatory 
gene. Thus, in the absence of Rb activity, p53 
is expressed causing apoptosis or programmed 
cell death and resulting in the microphthalmic 
condition. 

Neural Processing of Visual Information 

Understanding visual processing and its disor- 
ders requires knowledge of the human ner- 
vous system, including molecular, genetic, 
chemical, cellular, and integrative processes 
that underlie perception and the control of eye 
movements. As this knowledge increases it 
will help research aimed at preventing or 
treating disorders such as strabismus (mis- 
alignment of the eyes), amblyopia (commonly 
known as "lazy eye"), myopia, and neuro- 
ophthalmological disorders. 

Although disorders of visual processing 
may not always cause total blindness, they 
may seriously diminish the quaUty of life of 
those they afflict. Because these conditions 
affect more than 10 percent of the population, 
they constitute serious public health problems. 



Continued advancement of clinical investiga- 
tion in this field rests upon an improved un- 
derstanding of basic visual mechanisms. 

Development 

Using a new technique to trigger activity in 
selected nerve cells in a circuit. Dr. Katz, fiom 
Duke University, has shown that developing 
visual circuits are quite different than anatom- 
ical studies alone would indicate. Katz and 
his colleagues are using an approach he devel- 
oped called "laser photostimulation" or "caged 
glutamate" to show how the wiring in the 
mammaUan visual system changes during 
development. 

The nervous system is a dense network of 
many individual nerve cells that communicate 
with each other at speciaUzed gaps called 
synapses by chemical messengers called trans- 
mitters. The sending cell releases the trans- 
mitter at the synapse and the receiving cell 
responds if it has an appropriate receptor for 
the transmitter. Currently, Dr. Katz is explor- 
ing the visual circuits in the brain that use one 
common transmitter, glutamate. Thin slices of 
the nerve tissue are bathed in glutamate, 
which is "caged" — each molecule is trapped 
within a surrounding molecule that renders it 
inactive. A tiny electrode is inserted inside a 
single nerve cell and the surrounding tissue is 
bombarded with minute spots fiom an ulfiavi- 
olet laser. This bombardment releases the 
glutamate from its chemical cage causing that 
single nerve cell to become active. This gives 
researchers information on how the surround- 
ing neurons functionally interact. 

Initial results indicate that the visual 
system does not produce large numbers of 
neurons that are subsequently pruned during 
development. Instead, there is more of a 
redistribution of synapses in a circuit rather 
than an eHmination during development. Of 
greater significance for the field of neurosci- 
ence, however, is that this method promises to 
become a powerful tool to trace how networks 
of brain ceUs function and organize informa- 
tion. 



15 



FY 1994 NEI Annual Report 



Dr. McConnell, from Stanford University, 
is investigating how the highly organized 
cellular patterns and connections among nerve 
cells in the adult visual cortex arise during 
development. When these nerve cells are 
"bom," they receive instructions to leave the 
site of their origin and migrate long distances 
through a complex environment to other sites 
in the brain where they acquire a new identi- 
ty. Dr. McConnell wants to determine if an 
individual nerve cell "knows" who it is during 
its development. This is done by transplant- 
ing young nerve cells (whose normal fate she 
can predict) from one brain to another and 
then allowing the behavior of the transplanted 
nerve cell to reveal its "commitment" to its 
normal identity. What has been found is that 
by the time they are bom, nerve cells seem to 
"know" exactly who they are. In fact the pro- 
genitor cells, or mothers of the young cells, 
actually make decisions about the fates of the 
progeny, and they do so in a way that is ex- 
quisitely sensitive to the local environment in 
which the young nerve cells are generated. 

Dr. McConnell has been tr5dng to find out 
how the progerutor cells generate different 
types of nerve cells at specific times in devel- 
opment. It appears that the progenitors can- 
not make the right choice without information 
from neighboring cells. The progenitor cells 
are either sending signals to one another or 
receiving them from other nerve cells, which 
provide instructions that are essential for nor- 
mal fate determination. Currently, studies are 
aimed at trying to identify the molecules and 
genes that are involved in this process. This 
research will provide us with insights into 
how the complex connections of the brain are 
formed and how inappropriate connections 
are established when this process is flawed. 

Personal Guidance System for the Visually 
Impaired 

Drs. Loomis and Klatzky, researchers from the 
University of California, Santa Barbara, are 
developing a "personal guidance system" that 



"shows" the environment to blind users. The 
users wear stereo earphones mounted on 
glasses through which they receive informa- 
tion that tells them how to navigate through 
an unfamiliar environment. 

The system, housed in a backpack, picks 
up signals from the global positioning system 
(GPS) that was developed for the military and 
is transmitted from satellites. The system 
integrates this positional information with a 
geographic information system (CIS) in the 
form of a computerized map to create a "virtu- 
al acoustic display" that the user perceives as 
a talking map where preprogrammed objects 
and landmarks announce themselves as words 
through the earphones at the appropriate time 
and volume to cue the user to their precise 
location as he or she navigates. This provides 
the user with information on distance and 
direction for successful navigation through an 
unfamiliar environment. The device relies on 
triangulation of signals from GPS satellites to 
determine precise location. The information is 
transmitted to the system's on-board computer 
that contains a digitized map. The user wears 
an electroiuc compass that tells the system 
exactiy where he or she is. The system then 
generates speech that the user perceives as 
coming from the location of the object or land- 
mark in the real world. This allows the user 
to preview a trip permitting a rehearsal of a 
planned walk. Other workers are developing 
similar systems that use the GPS, but this is 
the first one to use a virtual acoustic display. 
Dr. Loomis and his colleagues plan to minia- 
turize the system further to make it less bulky 
and easier to carry. A cane or a guide should 
still be used with this system to inform the 
user of any obstacles that are not on the com- 
puter map. When the personal navigation 
system is fuUy implemented, it promises to 
expand the mobility of bUnd persons for navi- 
gating through unfamiliar areas and signifi- 
cantiy improve their ability to carry out every 
day tasks. 



16 



Extramural Research 



Division of Collaborative Clinical 
Research 

Richard Mowery, Ph.D., Director 

The Division of Collaborative Clinical Re- 
search plans and directs a program of grant, 
cooperative agreement, and contract support 
for appUed cUnical vision research, including 
clinical trials, natural history studies, surveys, 
cohort studies, and case-control studies. Cur- 
rently, the division manages 19 clinical trials, 
17 epidemiology projects, three eye health 
education demonstration projects, and 10 
analysis and planning projects with an annual 
budget of $38.6 million. 

Following are highlights of some of the 
major findings from these research projects. 

Retinal Diseases 

Macular Degeneration 

AMD is the leading cause of legal blindness in 
the United States for persons older than age 
60. Despite the major public health signifi- 
cance of AMD, the cause of the disease is not 
understood and major risk factors, other than 
age, have not been determined. For instance, 
a case-control study of risk factors for AMD 
was conducted on Long Island, New York, 
among individuals ages 50 to 79. Patients 
with "wet" (exudative) AMD and patients with 
"dry" (nonexudative) AMD were compared 
with individuals without the disease. An 
increased risk for both AMD types were asso- 
ciated with current smoking, light eye-color, 
family history of AMD, poorly controlled 
hypertension, and certain nutritional factors. 
This study indicates that many causative fac- 
tors may contribute to the disease. 

Beneficial treatment effects of laser photo- 
coagulation for some of the people with the 
neovascular form of the disease have been 
reported for the Macular Photocoagulation 
Study. However, laser photocoagulation does 
not restore severe vision loss due to the dis- 
ease, and some people lose vision despite 
treatment. 



Arumal research and preUminary epidenni- 
ologic studies suggest a protective role for 
certain micronutrients in the development of 
both cataract and macular degeneration. In 
particular, vitamins and certain trace minerals 
with antioxidant capabilities are being studied 
for their role in age-related vision loss. Cur- 
rently, published studies are intriguing but are 
not conclusive in establishing a role for antiox- 
idant nutrients in the development or worsen- 
ing of AMD or cataract. 

The Age Related Eye Diseases Study 
(AREDS) has as one of its goals to evaluate 
the effect of high-dose antioxidant vitamins 
and zinc on the progression of macular dis- 
ease and lens changes. Patients with minor to 
severe drusen pathology and minimal lens 
opacities are being enrolled in the AREDS, 
randomized to a high-dose dietary supple- 
ment or placebo, and followed for a minimum 
of seven years to assess the progression of 
both eye disorders. Because patients enrolled 
will not have optically significant lens opaci- 
ties, this study will also provide information 
regarding the risk factors for cataract forma- 
tion. The prospective nature of this study, its 
focus on the progression of drusen pathology, 
and its evaluation of the effects of high-dose 
supplements make this a unique and much 
needed study. Eleven clinical centers, a coor- 
dinating center, a photographic reading center, 
a drug distribution center, a central laboratory, 
and the provider of the supplements — Ameri- 
can Cyanamid/Storz-Lederle — participate in 
the study. Patient enrollment and randomiza- 
tion are ongoing. 

Diabetic Retinopatliy 

A well-coordinated pubUc health approach to 
diabetic retinopathy requires accurate data 
regarding the incidence and associated risk 
factors for the disease. Diabetes is a major 
cause of visual impairment and blindness in 
the United States. Results from a 10-year 
population-based cohort study conducted in 
southern Wisconsin recentiy reported the 10- 
year incidence rate of blindness (visual acuity 
of 20/200 or worse) in diabetic persons was 
1.8 percent, 4.0 percent, and 4.8 percent in 



FY 1994 NEI Annual Report 



younger-onset, older-onset taking insulin, and 
older-onset not taking insulin, respectively. 
This rate of blindness increased with age and 
with duration of diabetes. Respective 10-year 
rates of visual impairment (visual acuity of 
20/40 or worse) were 9.4 percent, 37.2 percent, 
and 23.9 percent. For all three diabetic 
groups, macular edema or more severe reti- 
nopathy was associated with greater visual 
loss. Also, visual loss was associated with 
smoking (younger-onset group) and increased 
systoHc blood pressure (older onset group 
taking insuUn). These data indicate visual loss 
is common in diabetic populations and identi- 
fy several modifiable risk factors for visual 
loss due to diabetes. The data also assist in 
the estimation of costs for diabetic care. 

Retinopathy of Prematurity 

Retinopathy of prematurity (ROP) is a serious 
pubUc health problem among low-birth-weight 
infants. The NEI supported a multicenter trial 
of Cryotherapy for Retinopathy of Prematur- 
ity, which demonstrated that cryotherapy 
reduces by approximately one-half the risk of 
unfavorable retinal and functional outcome 
from threshold ROP. Cryotherapy, although 
helpful, is expensive, ablates a significant 
portion of the retina, is not always successful, 
and its long-term sequelae are unknown. 

Despite the highly significant advances in 
the treatment of ROP by crvotherapy, ROP 
continues to affect an increasing number of 
very low-birth- weight survivors. The NEI is 
currently supporting the Supplemental Thera- 
peutic Oxygen for Prethreshold Retinopathy of 
Prematurity (STOP-ROP) Study. The STOP- 
ROP is a multicenter, controlled cUnical trial 
initiated to determine if supplemental thera- 
peutic oxygen wiU reduce the proportion of 
infants with prethreshold retinopathy of pre- 
maturity who advance to threshold ROP. In 
addition to ophthalmic outcomes, this study 
wdll obtain neonatal outcome information, 
including data on growth, ventilatory stability, 
chronic lung disease, neurological maturation, 
and length of hospital stay. Recruitment for 



this study began in early 1994 and is being 
conducted at 20 clinical centers in the United 
States. In addition to the support being pro- 
vided by the NEI, the Institute has arranged 
collaboration and support for this study from 
the National Institute for Quid Health and 
Human Development (NICHD) and the Na- 
tional Institute for Nursing Research. 

Cytomegalovirus Retinitis 

C5^omegalovirus (CMV) retinitis is by far the 
most crucial ocular problem for people with 
AIDS who Uve more than one year. A poten- 
tially bUnding disease of the retina, CMV 
retinitis affects between 25 and 30 percent of 
people with AIDS. Through the Studies of the 
Ocular CompUcation of AIDS (SOCA), a net- 
work of investigators with expertise in AIDS 
clirucal research, retinal disease, and clinical 
trial methodology, the NEI has expedited the 
testing of tieatments for CMV retinitis. These 
investigators demonstiated the safety and 
efficacy of foscamet for the treatment of CMV 
retinitis. Foscamet was also shown to extend 
the life expectancy of people with AIDS by 
approximately four months compared with 
patients who took ganciclovir. AU patients 
v\dth CMV retinitis relapse when anti-CMV 
therapy is discontinued. Therefore, continu- 
ous maintenance therapy is required. Even on 
continuous maintenance therapy, all patients 
with CMV retinitis eventually relapse. 

Although relapse can often be controlled, 
with each relapse, additional retina is des- 
troyed. Therefore, treatment strategies de- 
signed to prolong the time to relapse are 
needed. The SOCA investigators are currentiy 
comparing the use of combination therapy 
using foscarnet and ganciclovir with standard 
therapy to see if it will extend the time to 
relapse and disease progression. Other stud- 
ies that are being designed wiU use newly 
developed oral compounds and implant 
devices to look at safety and efficacy issues, 
extension of relapse time, plus the improve- 
ment in quality of Ufe. 



18 



Extramural Research 



Glaucoma 

Epidemiology 

Glaucoma is a major cause of visual impair- 
ment in the African-American populations. 
African Americans have an earUer age of onset 
of the disease compared with Caucasians, 
suffer bUndness at a greater rate, and may be 
more resistant to treatment. Unfortunately, 
few well-designed epidemiologic studies have 
examined the prevalence rate of glaucoma in 
African-American populations and Umited 
information exists on risk factors for the 
disease. 

The Barbados Eye Study is a population 
based-prevalence survey that was recently 
conducted among 4,709 residents of Barbados 
ages 40 to 84. The prevalence of glaucoma 
was found to be seven percent. Male gender 
and a family history of glaucoma were associ- 
ated with the disease. Open-angle glaucoma 
was diagnosed by strict definition, including 
both visual field and optic disc criteria. This 
miivimum, conservative prevalence estimate of 
glaucoma was high, particularly among those 
older than age 50. If study results are extrap- 
olated to the population of Barbados, the 
prevalence of glaucoma is one in 11 among 
persons older than age 50, one in rune in per- 
sons older than age 60, and increases to one in 
six in persons older than age 70. Comparison 
of this study with that conducted in 2,395 
urban African Americans and 2,913 urban 
Caucasian residents of Baltimore, indicates the 
age-adjusted prevalence of glaucoma in Barba- 
dos is 1.5 times higher than that in the Balti- 
more African- American population and 7.1 
times higher than in the Baltimore Caucasian 
population. As such, a higher prevalence of 
glaucoma in African-Caribbean populations 
compared with African- American popvdations 
is found and these differences raise the ques- 
tion of possible genetic factors. 

The contribution of genetic factors to 
glaucoma are planned for investigation in a 
study designed to address familial aggregation 
of the disease. The surviving parents, sibUngs, 
spouses, and children (older than age 40) of 



100 Barbados participants with glaucoma will 
be examined for eye disorders, and risk factor 
information wiU be obtained. This informa- 
tion wiU increase our understanding of the 
inheritance of glaucoma and have direct clini- 
cal implications in identifying those family 
members at high risk for developing the dis- 



A report of family history from the Balti- 
more Eye Survey, a population-based preva- 
lence survey conducted among 5,308 African- 
American and Caucasian residents of east 
Baltimore as a risk factor for glaucomas, has 
been published. Glaucoma was diagnosed in 
161 participants in this survey. Participants in 
this survey were interviewed to determine if 
first-degree relatives (parents, siblings, and 
children) had glaucoma. Results indicated a 
higher risk of glaucoma in siblings than in 
parents. However, these estimates may be 
biased because glaucoma was self-reported, 
not diagnosed, and prior knowledge of glau- 
coma diagnosis among participants influenced 
their recall of affected individuals. 

Treatment 

Currentiy, the most common treatment for 
glaucoma is the use of medications to lower 
pressure in the eye. Although the medications 
have been demonstrated to be very effective at 
lowering pressure, the impact of lowering 
pressure on visual field loss is not completely 
understood. The economic effect of glaucoma 
treatment with medications is substantial, 
accounting for millions of doUars in yearly 
expenditures in the United States alone. Fur- 
thermore, these pressure-lowering medications 
can often have significant side effects and 
require the patient to adhere to a strict admin- 
istration schedule, thus potentially having a 
profound effect on a patient's quality of Ufe. 

The NEl is currentiy supporting three new 
cUnical trials designed to evaluate treatment 
strategies for ocular hypertension and glauco- 
ma. The Ocular Hypertension Treatment 
Study is designed to determine whether medi- 
cal reduction of intraocular pressure prevents 
or delays the onset of visual field loss and /or 



19 



FY 1994 NEI Annual Report 



optic disc damage. Recruitment of patients 
began in early 1994 at 21 clinical sites 
throughout the United States. Fifteen hun- 
dred ocular hypertensive subjects, at least 500 
of whom will be African Americans, are being 
randomly assigned to either close observation 
or to a stepped medical regimen. Participants 
will be followed for a minimum of five years. 

The Early Manifest Glaucoma Trial is a 
randomized, controlled clinical trial designed 
to determine whether and to what extent 
reduction of intraocular pressure (lOP) influ- 
ences the course of chronic open-angle glauco- 
ma. Investigators at the University of Lund in 
Makno, Sweden, collaborating with investi- 
gators from the State University of New York 
at Stony Brook, are studying 300 patients vsdth 
newly diagnosed glaucoma. Participants are 
being randomized to receive pressure lower- 
ing treatment or observation with no or de- 
layed treatment. Both groups are followed 
closely with computerized perimetry and 
fundus photography. Recruitment of patients 
began in 1993 and will continue for an esti- 
mated two to three years. FoUowup of pa- 
tients will be conducted for approximately 
four years. 

Filtration surgery can also be effective in 
controlling lOP. When successful, filtration 
surgery may provide the most effective long- 
term, consistent control of lOP with the least 
Ukehhood of requiring supplemental medica- 
tions. The Collaborative Initial Glaucoma 
Treatment Study is designed to compare the 
efficacy of a medical regimen with filtration 
surgery as the initial treatment for newly 
diagnosed glaucoma. This clinical trial will 
compare the two treatment strategies in terms 
of controUing TOP and will specifically investi- 
gate the impact of these treatment strategies 
on the participants quality of Ufe. Recruit- 
ment of the 600 patients began in November 
1993. The study is being conducted at 11 
clinical centers located throughout the Uruted 
States. 



Corneal Diseases 

Prospective Evaluation of Radial Keratotomy Study 

Approximately 25 percent of adults in the 
western world have myopia. Some of these 
people may be candidates for radial keratoto- 
my (RK), a procedure aimed at correcting or 
reducing myopia through surgical incisions 
made in the cornea. The NEI has been sup- 
porting a multicenter controlled clinical trial 
designed to evaluate the short- and long-term 
safety and efficacy of a single standardized 
technique of RK. 

The five-year followoip results from the 
Prospective Evaluation of Radial Keratotomy 
(PERK) Study indicated that RK was safe and 
that few very serious complications resulted 
from the procedure. However, it was difficult 
to predict the outcome for an individual eye. 
In addition, the refractive error continued to 
change in some patients over time. 

In 1994, the PERK investigators completed 
10-year foUowup examinations of patients 
who were enrolled in the PERK Study. Data 
from these examinations will soon be available 
and wiU be one of the few sources of informa- 
tion on the long-term stability of RK. 

Strabismus, Ambiyopia, and Visual Processing 

Optic Neuritis and Multiple Sclerosis 

More than one-half of aU people with first- 
time optic neuritis, a vision-impairing inflam- 
mation of the optic nerve that affects more 
than 25,000 Americans each year, will eventu- 
ally develop multiple sclerosis. Multiple scle- 
rosis is a debOitating disease of the central 
nervous system that affects as many as 500,000 
Americans. Based on data from two years of 
foUowup of patients enrolled in the Optic 
Neuritis Treatment Trial, researchers found 
that treating first-time optic neuritis patients 
with a combination of intravenous and oral 



20 



Extramural Research 



corticosteroids lowers their risk of developing 
miiltiple sclerosis. The results from this re- 
search, pubUshed in Tlie New England Journal 
of Medicine, offer the first scientific evidence 
that intravenous corticosteroids help to delay 
the progression of multiple sclerosis. It also 
suggests that this treatment may provide simi- 
lar benefits for people with not only optic 
neuritis but other early symptoms of multiple 
sclerosis. 

Myopia 

Results from an NEI-supported study pub- 
lished in the Journal of the American Medical 
Association suggest a genetic Unk to myopia. 
Researchers from the University of California, 
Berkeley, School of Optometry report on 716 
school children ages six to 14 who are enrolled 
in the OLSM. Measurements were made on 
each child's refractive error, corneal curvature, 
crystalline lens power, and axial ocular dimen- 
sions. Parents reported their own vision 
status. The investigators found that school 
children whose parents are myopic have 
different shaped eyeballs than children whose 
parents are not nearsighted. Children of 
myopic parents had longer eyes even before 
the onset of myopia. These results suggest 
that the premyopic eye in children with a 
family history of myopia already resembles 
the elongated eye present in myopia. 



The OLSM wiU provide a wealth of infor- 
mation on ocular and refractive development 
in children in the years ahead. Future results 
may provide the eye care provider or pediatri- 
cian with the answer to the question frequent- 
ly asked by myopic parents — "What are the 
chances that my child will develop myopia?" 

Ocular Injuries 

Ocular trauma is a major cause of monocular 
blindness in the United States and, in 1986, 
was responsible for estimated hospital costs of 
up to $200 mUlion. Self-reports of lifetime 
ocular injuries were obtained in the popula- 
tion-based Baltimore Eye Survey of adults 
older than age 40. Ocular injuries were re- 
ported by 14.4 percent of participants 
(n=5308); men reported a greater number of 
ocular injuries than women. The number of 
injuries were similar among both African- 
American and Caucasian men, however, visu- 
al consequences of injuries were more severe 
among African- American men. 



21 



Division of Biometry and Epidemiology 



Report of the Acting Director, 
Division of Biometry and Epidemiology 



Roy C. Milton, Ph.D. 



The Division of Biometry and Epidemi- 
ology (DBE) is made up of a Clinical 
Trials Branch, an Epidemiology 
Branch, and a Biometry Section. Dr. Roy 
Milton is the Acting Director for the division. 
Drs. Frederick Ferris HI and Robert Sperduto 
serve as chiefs of the two branches, respective- 
ly; Dr. Roy Milton is the head of biometry. 

The DBE has three main functions: re- 
search, education, and consultation. Research 
is the dominant function. It is the division's 
mission to plan, develop, and conduct human 
population studies concerned with the cause, 
prevention, and treatment of eye disease and 
vision disorders, with emphasis on the major 
causes of blindness. This includes studies of 
incidence and prevalence in defined popula- 
tions, prospective and retrospective studies of 
risk factors, natural history studies, clinical 
trials, genetic studies, and studies to evaluate 
diagnostic procedures. 

The DBE carries out a program of educa- 
tion in biometric and epidemiologic principles 
and methods for the vision research communi- 
ty. This program consists of courses, work- 
shops, a fellowship program for ophthalmolo- 
gists, publications, and consultation and 
collaboration on research. 

The DBE provides biometric and epidemi- 
ologic assistance to the NEI intramural and 
extramural staff and to vision research work- 
ers elsewhere. The assistance ranges from 
consultation to collaboration as coinvestigator. 



Research Highlights 

The Eye Disease Case-Control Study 

One of the diseases studied in the Eye Disease 
Case-Control Study was idiopathic macular 
holes, a disease by which women are more 
affected than men. Results from this study 
showed that higher fibrinogen levels and a 
history of glaucoma were associated with an 
increased risk of the condition. The use of 
exogenous estrogens was associated with a 
decreased risk. The fibrinogen finding was 
unexpected, and it is not clear whether this is 
a chance finding or whether higher levels of 
fibrinogen can increase susceptibility to the 
forces of vitreous traction, perhaps by compro- 
mising the macular blood supply or by some 
yet unexplained mechanism. 

Within this large case-control study, a sub- 
study was conducted to evaluate the relation- 
ships between dietary intake of carotenoids 
and vitamins A, C, and E and the risk of neo- 
vascular macular degeneration, the leading 
cause of irreversible blindness among adults. 
A higher intake of dietary carotenoids was 
associated with a lower risk. Among the 
specific carotenoids, lutein and zeaxanthin 
were most strongly associated with a reduced 
risk of this form of macular degeneration. 
Several food items rich in carotenoids were 
inversely associated with macular degenera- 
tion. In particular, a higher frequency of in- 
take of spinach or collard greens was associat- 
ed with a substantially lower risk for AMD. 



25 



FY 1994 NEI Annual Report 



The Framingham Offspring Eye Study 

Eye examination data from 1,086 parents ex- 
amined in the Framingham Eye Study and 896 
offspring examined in the Framingham Off- 
spring Eye Study (FOES) were used to study 
familial associations for nuclear, cortical, and 
posterior subcapsular lens opacities. For any 
pair of siblings, if one sibUng had a nuclear 
opacity the odds of the other sibling having 
such an opacity were estimated to be more 
than triple. Similar findings were noted for 
posterior subcapsular opacity. The strong 
associations between siblings for nuclear and 
posterior subcapsular opacities suggest there 
is clustering of lens opacities within families. 
The clustering may be due to genetic or envi- 
ronmental factors. 

The Italian-American Natural History Study of 
Age-Related Cataract 

The ItaUan- American Natural History Study of 
Age-Related Cataract has estimated the inci- 
dence and progression of cortical, nuclear, and 
posterior subcapsular opacities in a large fol- 
lowup study. The three-year cumulative inci- 
dence for persons ages 65 to 74 years (the 
largest group studied) was 18 percent, six 
percent, and six percent for cortical, nuclear, 
and posterior subcapsular opacities. Progres- 
sion was much higher than incidence for each 
type of opacity. The study suggested that 
patient age, baseUne lens status, cataract 
grading system, definition of change, and 
analytic methodology may have important 
effects on estimates of cataract incidence and 
progression. 

The Early Treatment Diabetic Retinopathy Study 

Recent publications from the Early Treatment 
Diabetic Retinopathy Study (ETDRS) multicen- 
ter, randomized clinical trial include an evalu- 
ation of the effect of aspirin versus placebo on 
mortality and morbidity from all causes and 
specifically from cardiovascular disease. This 
study is one of two primary prevention stud- 
ies of the effect of aspirin on cardiovascular 
disease and is the only large study to include 
women. The results are similar to the studies 



on males without diabetes and demonstrate 
that aspirin use reduces the risk of cardiovas- 
cular disease. 

Previous ETDRS publications had not 
reported any increase in the occurrence of vit- 
reous /preretinal hemorrhages in study pa- 
tients assigned to aspirin. With new data 
suggesting the importance of aspirin use in 
persons with diabetes who are at risk for 
cardiovascular disease, it was important to 
investigate this possible risk of aspirin use in 
more depth. Data published recentiy {ETDRS 
Report No. 20) demonstrate that the severity 
and duration of these hemorrhages were not 
significantly affected by the use of aspirin and 
that there were no ocular contiaindications to 
its use in persons with diabetes who require it 
for tieatment of cardiovascxilar disease or for 
other medical indications. 



Research Activities 

Clinical Trials 

The Early Treatment in Diabetic Retinopathy Study 

The ETDRS was designed to determine when 
to use photocoagulation for diabetic retinopa- 
thy. Patients with macular edema, preproli- 
ferative retinopathy, and mild or moderate 
proliferative retinopathy were studied. Vari- 
ous treatment strategies of focal and scatter 
(panretinal) photocoagulation were compared 
with no photocoagulation. In addition, the 
study evaluated the placebo-contioUed effects 
of daily administration of aspirin on the inci- 
dence of microvascular and macrovascular 
complications. The study also investigated 
factors associated with the progression of 
disease. 

Recruitment was completed in March 1985 
with the enrollment of 3,711 patients. In De- 
cember 1985, the study reported that focal 
photocoagulation of clinically significant dia- 
betic macular edema substantially reduces the 
risk of visual loss. It was further reported 
that focal treatment increases the chances of 



26 



Division of Biometry and Epidemiology 



visual improvement, decreases the frequency 
of persistent macular edema, and causes only 
minor visual field losses. Subsequent reports 
indicated that whether treated early with 
scatter photocoagulation or followed closely 
and treated as soon as they reached the high- 
risk stage, all eyes had low rates of severe 
visual loss. Scatter photocoagulation is not 
recommended for eyes with mUd or moderate 
nonproliferative diabetic retinopathy, provided 
careful followup can be maintained. When 
retinopathy is more severe, scatter photocoag- 
ulation should be considered and usually 
should not be delayed if the eye has reached 
the high-risk proliferative stage. 

Sixteen ETDRS reports have been pub- 
lished and additional manuscripts are being 
prepared. Drs. Lloyd Aiello and Frederick L. 
Ferris, HI serve as cochairmen. Dr. Richard L. 
Mowery is project officer, and Dr. Emily Y. 
Chew serves as a member of the analysis 
planning group. The results of effects of aspi- 
rin on vitreous hemorrhage in patients with 
diabetes mellitus have been accepted for pub- 
lication. A report of accommodation in the 
ETDRS population has been submitted for 
publication. Analyses in progress include the 
association of serum Upids and retinal hard 
exudates, risk factors for severe visual loss, 
risk factors for development of high-risk pro- 
liferative diabetic retinopathy, fibrovascular 
proliferation associated with macular edema, 
and causes of severe visual loss. In collabora- 
tion with Dr. Thomas Gardner, from Hershey 
Medical Center, results of analyses of digoxin 
and retinopathy have also been submitted for 
publication. 

In addition, patients with mild to prolifer- 
ative retinopathy are being followed with ex- 
tensive psychophysical testing in the NEI 
Clinical Center, to determine the mechanisms 
for loss of visual acuity in diabetic retinopa- 
thy. An additional study of long-term foUow- 
up of diabetic retinopathy following laser 
photocoagulation is under way at the NEI 
CUnical Center. 



The Krypton-Argon Regression of 
Neovascularization Study 

The CUrucal Trials Branch began the Krypton- 
Argon Regression of Neovascularization Study 
(KARNS) in three pUot clinics in December 
1983. The major objective of this randomized 
cUnical trial is to compare red krypton laser 
with blue-green argon laser panretinal photo- 
coagulation for treating neovascularization on 
the optic nerve head caused by diabetic reti- 
nopathy. Twenty-nine new cUrucs were en- 
rolled in KARNS starting in August 1984. At 
the termination of the study in June 1990, a 
total of 1,063 patients had been randomized. 
This study is unique for the NEI because the 
functions for both the coordinating center and 
the fundus photography reading center are 
being handled by staff of the Clinical Trials 
Branch. Another feature of this multicenter 
trial is that the participating clinics receive no 
financial reimbursement from the NEI for their 
participation. Drs. Ferris and Chew direct this 
study along with Dr. Lawrence Singerman. 
KARNS Report No. 1 {Ophthalmology, 1993) 
showed the two treatments were equally 
effective in arresting neovascularization of the 
disc; additional analyses are under way. 

The Linxian Eye Study 

The NEI joined an ongoing NCI-supported 
clinical trial of nutrition and cancer in north 
central China in 1991 to determine whether 
the vitamin /mineral dietary supplements 
administered in the Linxian Cancer Trials for 
the preceding five years have affected the risk 
of age-related cataract and AMD. Eye exami- 
nations were conducted in 1991 on 5,390 
members of the Linxian Study cohort. Dr. 
Sperduto is project officer, and the project 
team includes Drs. Milton and Chew from 
DBF and Dr. Tian-Sheng Hu, an ophthalmolo- 
gist from Beijing. Findings for cataract have 
been pubUshed. Analyses are being conduct- 
ed for the macular degeneration component. 



27 



FY 1994 NEI Annual Report 



The Italian-American Trial of Age-Related Cataract 

A new collaborative Italian-American cUnical 
trial is being planned to study the effect of a 
broad spectrum vitamin /mineral supplement 
on the risk of age-related cataract. Twelve 
hundred people will be randomized to either 
the supplement or a matching placebo and 
followed for five years. Recruitment into the 
study will begin in early 1995. The study wlU 
complement the ongoing AREDS, which is 
being conducted in the United States. Dr. 
Sperduto is the project officer and Dr. Milton 
is a member of the project team. 

Intramural Program Clinical Trials 

Drs. Ferris and Chew and Ms. Remaley are 
collaborating with Dr. Nussenblatt on four 
additional randomized cUrucal trials in the 
NEI Intramural Program of the Clinical Cen- 
ter: (a) a trial of a sustained-release intraocu- 
lar drug deUvery system for ganciclovir thera- 
py of cytomegalovirus retinitis in patients 
with AIDS; (b) a trial to evaluate the efficacy 
of a heparin-surface modified intraocular lens 
in reducing the incidence and severity of post- 
operative inflammatory episodes following 
extracapsular surgery in uveitis patients with 
cataracts; (c) a trial of anti-inflammin, a pep- 
tide, in the treatment of anterior uveitis; and 
(d) a trial of oral S-antigen or retinal extract 
versus placebo in patients with uveitis. 

Other 

Dr. Ferris now represents the NEI on the Data 
Monitoring Committee of the United Kingdom 
Prospective Diabetes Study, a clinical trial of 
alternative treatment regimens in the manage- 
ment of patients with diabetes. FoUowup is 
scheduled to continue in this study until 1997. 

Epidemiology 

The Age-Related Eye Disease Study 

The AREDS is designed to coUect natural 
history data of 4,600 patients of ages 55 to 80 
years with bilateral drusen of different types 
or with unilateral advanced AMD. This study 



will evaluate the rates of development and 
progression of AMD, the rates of visual loss 
due to retinal lesions of AMD, and the risk 
factors associated with the development and 
progression of AMD. Evaluation of lens 
change over the 10-year AREDS study period 
will provide an opportunity to evaluate factors 
associated with the development of cataracts. 
In addition, a cUnical trial wiU be performed 
to determine whether antioxidants (vitamin C, 
E, and beta-carotene) and zinc can prevent the 
development or retard the progression of 
AMD and cataract. There are 11 cUnical cen- 
ters, a photographic reading center, a central 
laboratory, and a coordinating center. Identi- 
fication of study participants began in Septem- 
ber 1990. In November 1992, participants 
were evaluated with qualifying visits, and 
participants were randomly assigned to the 
study medications begirming in February 1993. 
Dr. Ferris, chairman; Dr. Sperduto, director of 
lens project; and Dr. Chew are directing the 
scientific aspects of the AREDS; Dr. Natalie 
Kurinij is project officer. 

The Eye Disease Case-Control Study 

The Eye Disease Case-Control Study is de- 
signed to identify risk factors for neovascular 
macular degeneration, idiopathic branch reti- 
nal vein occlusion, idiopathic central retinal 
vein occlusion, rhegmatogenous retinal de- 
tachment, and idiopathic macular hole. Dr. 
Sperduto is the study chairman, Ms. Rita Hil- 
ler is director of data analysis, and Dr. Chew 
is a member of the project team. Two papers 
were published this year: risk factors for idio- 
pathic macular hole and the effect of dietary 
carotenoids and vitamins A, C, and E on neo- 
vascular age-related macular degeneration. 

The Diabetes in Early Pregnancy Study 

Dr. Chew and Ms. Remaley, in collaboration 
with Dr. James Mills of the NICHD, examined 
the effects of pregnancy on diabetic retinopa- 
thy in the Diabetes in Early Pregnancy Study. 
Data collection terminated in 1985, and a man- 
uscript has been submitted. Further analyses 
on the effects of pregnancy in proliferative 
retinopathy are planned. 



28 



Division of Biometry and Epidemiology 



The Italian-American Natural History Study of Age- 
Related Cataract 

The Italian- American Natural History Study of 
Age-Related Cataract was designed to esti- 
mate the rates of development and progres- 
sion of the different types of lens opacities and 
the associated risk factors. Dr. Sperduto is the 
project officer; Dr. Milton and Ms. Remaley 
are members of the project team. Three pa- 
pers were pubUshed this year, including one 
that provides estimates of the rates of cataract 
development and progression. Analyses of 
risk factors for cataract development and pro- 
gression are under way. 

The Framingham Offspring Eye Study 

Dr. Sperduto is the project officer. Dr. Milton 
is the alternate project officer, and Drs. Podgor 
and Freidlin and Ms. HUler are members of 
the project team for FOES. This study is de- 
signed to examine famihal relationships for 
age-related cataract and AMD among parents 
examined in the Framingham Eye Study (1973 
to 1975) and their children examined 1989 to 
1991. Dr. Podgor has used generalized esti- 
mating equation methodology in the analyses 
of these data. A manuscript describing the 
study's findings for cataract has been accepted 
for publication. A manuscript describing the 
association of opacities between and within 
eyes of individuals has been submitted. Anal- 
yses of macular degeneration are planned. 

Other 

A manuscript has been accepted for publica- 
tion on risk factors for strabismus, using data 
from the NICHD Collaborative Study and in 
collaboration with Dr. Mark Klebanoff, from 
the NICHD. The DBF project team includes 
Drs. Chew, Tamboli, Zhao, and Podgor and 
Ms. Remaley. Esotropia developed in three 
percent and exotropia in 1.2 percent of the 
children followed for seven years. Esotropia 
was more common in Caucasians than in 
African Americans. The occurrence of exotro- 
pia was similar in the two races. Maternal 
cigarette smoking during pregnancy and low 
birth weight were independent and important 



risk factors for both esotropia and exotropia. 
Analyses are under way on sibling association 
in strabismus and on risk factors for congeni- 
tal cataract. 

Drs. Valerie Freidlin and Marvin Podgor 
continued to provide consultations with NEI 
CUrucal Center investigators, especially for 
various studies of measurement of lens opaci- 
ties. Dr. Freidhn is collaborating with Dr. 
EUwein in management and analysis of Medi- 
care data on ophthalmologist services. 

Statistical Methods 

Dr. Podgor and Dr. Joseph Gastwirth, from 
the George Washington University, collaborat- 
ed in an investigation of the use of scores for 
stratified data. A paper has been accepted for 
publication. Drs. Podgor, Gastwirth, and 
Cyrus Mehta, from Cytel Corporation and 
Harvard University, have proposed methodol- 
ogy for efficiency robust tests for ordered 2xK 
contingency tables. A paper has been submit- 
ted. 

Ongoing Activities 

Members of DBE are active in consultations 
and educational and professional activities, 
including referees for professional journals, 
associate editors or members of editorial 
boards, members of data and safety monitor- 
ing committees for clinical trials, training of 
staff fellows, invited and contributed presenta- 
tions at professional society and other meet- 
ings, advisory committees for grant-supported 
cooperative agreements, and technical advi- 
sors to the World Health Organization 
(WHO). 



Publications 

Chew EY, Klein ML, Murphy RP, Remaley 
NA, Ferris FL and The Early Treatment Dia- 
betic Retinopathy Study Research Group: 
Effects of aspirin on vitreous hemorrhage in 
patients with diabetes meUitus. ETDRS Re- 
port No. 20, Arch Ophthalmol, in press. 



19 



FY 1994 NEI Annual Report 



Chew E, Remaley NA, Tamboli A, Zhao J, 
Podgor MJ, BGebanoff M: Risk factors for 
esotropia ar\d exotropia. Arch Ophthalmol, in 
press. 

Ferris FL: How effective are treatments for 
diabetic retinopathy? Commentary. JAMA 
269(10):1290-1291, 1993. 

Ferris HI FL, Freidlin V, Kassof A, Green SB, 
Milton RC: Relative letter and position diffi- 
culty on visual acuity charts from the Early 
Treatment of Diabetic Retinopathy Study. Am 
J Ophthalmol 116:735-740, 1993. 

Javitt JC, AieUo LP, Chiang Y, Ferris FL, Can- 
ner JK, Greenfield S: Preventive eye care in 
people with diabetes is cost-saving to the 
Federal Government. Implications for health- 
care reform. Diabetes Care 17(8):909-917, 1994. 

Magno BV, Freidlin V, DatHes MB: Reproduci- 
bility of the NEI Scheimpflug cataract imaging 
system. Invest Ophthalmol Vis Sci 35:3078-3084, 
1994. 

Magno BV, Freidlin V, Lasa MSM, Datiles MB: 
Comparison of linear, multilinear and mask 
microdensitometric analysis of Scheimpflug 
images of the lens nucleus. Invest Ophthalmol 
Vis Sci, in press. 

Maraini G, Rosmini F, Graziosi P, Tomba MC, 
Bonacini M, Cotichini R, Pasquini P, Sperduto 
RD, and the Italian American Cataract Study 
Group: Influence of type and severity of pure 
forms of age-related cataract on visual acuity 
and contrast sensitivity. Invest Ophthalmol Vis 
Sci 35:262-267, 1994. 

Nussenblatt RB, De Smet M, Podgor M, Lane 
C, Polls M, Pizzo P, Perry C, Beifort Jr R: The 
use of the flarephotometry in the detection of 
cytomegalic virus retinitis in AIDS patients. 
AIDS 8:135-136 [letter], 1994. 

Podgor MJ: Review of Gibbons, JD (1993) 
Nonparametric Measures of Association. J Am 
Stat Assoc 89:719 [book review], 1994. 



Podgor MJ, Gastwirth JL: On nonparametric 
and generalized tests for the two-sample prob- 
lem with location and scale change alterna- 
tives. Stat Med 13:747-758, 1994. 

Podgor MJ, Gastwirth JL: A cautionary note 
on appl5dng scores in stratified data. Biomet- 
rics, in press. 

Rosmini F, Stazi MA, Milton RC, Sperduto 
RD, Pasquini P, Maraiiu G, and the Italian- 
American Cataract Study Group: A dose-re- 
sponse effect between a sunlight index and 
age-related cataract. Ann Epidemiol 4:266-270, 
1994. 

Sastry SM, Sperduto RD, Waring GO, Remaley 
NA, Lynn MJ, Blanco PE, Miller DN: Radial 
keratotomy does not affect intraocular pres- 
sure. Refractive and Corneal Surgery 9:459-464, 
1993. 

Seddon JM, Ajani UA, Sperduto RD, HiUer R, 
Blair N, Burton TC, Farber MD, Gragoudas 
ES, Haller J, Miller DT, Yannuzzi LA, Willett 
W: Dietary carotenoids, vitamins A, C, and E 
and advanced age-related macular degenera- 
tion — a multicenter study. JAMA, in press. 

Sperduto RD: Age-related cataracts — scope of 
problem and prospects for prevention. Prev 
Med, in press. 

The Eye Disease Case-Control Study Group: 
Risk factors for idiopathic macular holes. Am 
J Ophthalmol, in press. 

The Framingham Offspring Eye Study Group: 
Familial aggregation of lens opacities: the 
Framingham Eye Study and the Framingham 
Offspring Eye Study. Am J Epidemiol, in press. 

The Italian-American Cataract Study Group: 
Incidence and progression of cortical, nuclear 
and posterior subcapsular cataracts. Am J 
Ophthalmol, in press. 



30 



Office of International Program Activities 



Report of the Acting Assistant Director for 
International Program Activities 



Terrence Gillen, M.A., M.B.A. 



The mission of the NEI is to reduce the 
prevalence of blindness, visual impair- 
ment, and eye disease worldwide 
through basic and applied research and train- 
ing. Although excellent ophthalmic proce- 
dures and eye-care delivery systems are acces- 
sible in the developed world, adequate health 
care is not readily available in aU parts of the 
developing world. This widening gap in 
visual health between developed and develop- 
ing nations threatens to have ominous conse- 
quences. If present trends continue, the num- 
ber of blind people — estimated at 24 mil- 
lion — ^wUl more than quadruple during the 
next 40 years. As many as 90 percent of these 
blind people wiU Uve in developing countries. 

This large-scale disablement caused by 
bHndness is not only a costly obstacle to 
economic development, it is also a catastroph- 
ic loss of human potential in the areas of the 
world most desperately in need of a healthy 
w^orkforce. In addition, because more than 80 
percent of aU cases of blindness can be consid- 
ered avoidable — that is, they could have been 
prevented or could be cured using available 
and locally appropriate technology — such 
deprivation is a truly needless denial of a 
basic human right for mUhons of people. 
Therefore, the NEI undertakes international 
activities to facUitate the development and 
application of effective prevention and inter- 
vention programs. These efforts are coordi- 
nated by the Institute's Office of International 
Program Activities (OIPA), which was created 
in February 1989. The OIPA enhances NEI's 
international programs by: 



• Evaluating available health technologies, 
promoting the most cost-effective inter- 
vention and prevention programs, and 
encouraging their availability for affected 
populations, especially in developing 
countries. 

• Conducting collaborative applied research 
studies to develop preventive methods for 
tieating specific eye diseases. 

• Conducting contioUed dinical evaluations 
of promising research findings. 

• Exchanging information on recent scientif- 
ic advances and their appropriate applica- 
tion to visual problems. 

The NEI supports international research 
on six blinding diseases that have a major 
worldwide effect: cataract, onchocerciasis, 
ocular toxoplasmosis, glaucoma, diabetic 
retinopathy, and vitamin A deficiency. 



Highlights of Recent Scientific 
Advances Resulting From 
International Activities 

Because cataract is responsible for about one- 
half of the developing world's curable bHnd- 
ness and is a major problem for the United 
States as well, the NEI has developed a collab- 
orative research program that includes projects 
to prevent blindness from cataract with collab- 
orating groups in Italy, India, and Latin Amer- 
ica. In addition, health services research 
expertise from the NEI is made available to 



33 



FY 1994 NEI Annual Report 



selected collaborating partners through train- 
ing activities and the conduct of joint research 
projects. 

For example, intramural scientists from 
NEl's Laboratory of Mechanisms of Ocular 
Diseases (LMOD) in collaboration with col- 
leagues at the Centre for CeUular and Molecu- 
lar Biology in Hyderabad, India, are studying 
aging-related modifications to lens crystalUns. 
These scientists have demonstrated that chem- 
icals present in smoke, either from tobacco 
products or from wood fires, can directly 
damage lenses in organ culture studies. In 
addition, molecular geneticists in the NEI's 
Section on Cataract have initiated gene linkage 
studies with scientists at Osmarua University 
and the L.V. Prasad Eye Institute in 
Hyderabad on selected families with heredi- 
tary cataract. 

The collaborative Italian-American Study 
of the Natural History of Age-Related Cataract 
has completed a four-year foUowup study of 
cataract. Objectives of the natural history 
study were to estimate the rates of develop- 
ment and progression of the various tj^es of 
lens opacities, identify risk factors associated 
with the development and progression of 
cataracts, and evaluate cataract classification 
schemes. A manuscript describing study 
results has been accepted for publication in 
the American Journal of Ophthalmology. 



Summary of International 
Programs and Activities 

Country-to-Country Activities 

Barbados 

Open-angle glaucoma is the leading cause of 
blindness in African Americans and is a major 
cause of visual impairment and disability. 
The incidence of glaucoma has not been mea- 
sured precisely in any population, and the risk 
factors related to its development are largely 
unknown. Since 1988, the Barbados Eye Study 
has examined more than 4,200 persons ages 40 



to 86 years as part of a population-based 
study to determine the prevalence and risk 
factors for glaucoma and other eye disorders 
such as diabetic retinopathy, AMD, cataract, 
and visual impairment. In 1992, the Barbados 
Incidence Study was initiated to estimate the 
incidence of glaucoma and other ocular disor- 
ders in individuals in the Barbados prevalence 
survey who were free of disease. In addition, 
risk factor analysis wiU be conducted to iden- 
tify associations with development of glauco- 
ma and to characterize those who have pro- 
gressive eye disease. (See "The Barbados Eye 
Study: Prevalence of Open-Angle Glaucoma" 
in the June 1994 issue of Archives of Ophthal- 
mology, 112 (6): 821-829.) 

Brazil 

In collaboration with the U.S. National Insti- 
tute of AUergy and Infectious Diseases 
(NIAID), NIH and three Brazilian scientific 
organizations in Sao Paulo — Escola PauUsta de 
Medicina, CUnica Erexim, and Laboratory 
Fleury — ^the NEI developed a research pro- 
gram on the immunology, basic mechanisms, 
and epidemiology of toxoplasmosis in south- 
em Brazil. The prevalence of ocular toxoplas- 
mosis in this population was found to be 
more that 30 times higher than previous esti- 
mates for the same condition elsewhere. In 
this population, ocular toxoplasmosis appears 
to be a sequela of postnatal rather than con- 
genital infection. Studies performed in 1993 
on postnatal blood from newborns in southern 
Brazil have shown a low percentage of immu- 
noglobtdin M positivity, further suggesting 
that the disease in southern Brazil is acquired. 

India 

The NEI and the Indian Council of Medical 
Research (ICMR) have developed a collabora- 
tive blindness research program under the 
1983 Indo-U.S. Science and Technology Initia- 
tive. This program includes projects to reduce 
blindness from vitamin A deficiency, cataract, 
and Eales' disease in India. Indian govern- 
ment funds for the work come through the 
ICMR, and U.S. Government funds are pro- 
vided through the National Science Founda- 



34 



International Program Activities 



tion and the NEI. In addition, the NEI collab- 
orates with Indian scientists under the U.S.- 
Indo Subcommission program. 

The NEI director, deputy director, and 
special advisor to the director participated as 
consultants to the World Bank to develop a 
proposal by the Government of India for an 
initiative in cataract blindness control. Techni- 
cal meetings have been held in New Delhi and 
Madurai to provide the knowledge base on 
which training and surgical guidelines can be 
developed for a twofold expansion of cataract 
surgery, with expUcit attention to the quaUty 
and extent of vision restoration. 

Intramural scientists from the NEI, LMOD 
are collaborating with colleagues at the Centre 
for Cellular and Molecular Biology on studies 
on aging-related modifications to lens crystal- 
Uns. Cataracts typically occur at an earHer age 
and are more heavily pigmented in the Indian 
population than in the U.S. population. In an 
attempt to elucidate the molecular mechan- 
isms underlying this difference in color, the 
scientists are comparing over a wide range of 
ages the fluorescence spectra for normal intact 
lenses from the Indian population with age- 
matched Eye Bank lenses from the U.S. popu- 
lation. The Indian population lenses have 
signiftcantiy greater amounts of pigmented 
fluorescent compounds than do the U.S. popu- 
lation lenses. These compounds may play a 
direct role in cataractogenesis through their 
ability to function as photosensitizers. 

In organ culture studies, collaborating 
investigators have demonstrated that chemi- 
cals present in smoke, either from tobacco 
products or from wood fires, can directly 
damage lenses. A primary site of damage 
appears to be the cell membrane. Epidemi- 
ological studies have indicated that smoke- 
derived compounds are probable risk factors 
for cataract. These studies will continue in an 
effort to identify the pathological mechanisms 
involved. A paper describing the studies to 
date has been submitted for publication. 

Molectdar geneticists in the NEI, Section 
on Cataract have initiated gene linkage studies 



with scientists at Osmania University and the 
L.V. Prasad Eye Institute in Hyderabad on 
selected families with hereditary cataract. The 
prevalence of consanguineous marriages in 
this region of India greatly increases the hkeU- 
hood of recessive cataract phenotypes. Blood 
samples from individuals in suitable pedigrees 
are being shipped from India to the NEI for 
linkage analysis. A geneticist from Osmania 
University, who was trained at the NEI in 
relevant techniques, has returned to Hyder- 
abad to estabUsh a laboratory so that much of 
the work can be performed in India. In one 
family the cataract trait has been Unked to a 
particular chromosome and a potential candi- 
date gene has been identified. 

In September, Dr. Prem Prakash, chief of 
the Dr. Rajendra Prasad Centre for Ophthal- 
mic Sciences, a component of the All India 
Institute of Medical Sciences, visited the NEI 
to discuss possible future research collabora- 
tion. 

Italy 

The Collaborative Italian-American Study of 
the Natural History of Age-Related Cataract 
has completed a four-year foUowup study of 
cataract. Objectives of the natural history 
study were to estimate the rates of develop- 
ment and progression of the various types of 
lens opacities, identify risk factors associated 
with the development and progression of 
cataracts, and evaluate cataract classification 
schemes. A total of 1,297 patients participated 
in the foUowup study. Data collection lasted 
from April 1989 to April 1994. Organizations 
participating in the study included the Insti- 
tute of Ophthalmology at the University of 
Parma, the Laboratory for Epidemiology and 
Biostatistics at the Istituto Superiore di Sanita 
in Rome, and the NEI in the United States. A 
manuscript describing study results has been 
accepted for publication in the American Jour- 
nal of Ophthalmology. 

Investigators at the University of Parma 
and NEI are also collaborating in a study to 
determine whether the complete or partial 
deletion of the glutathione-S-fransferase I gene 



35 



FY 1994 NEI Annual Report 



is an important risk factor in the development 
of senile cataract. Blood has been drawn from 
approximately 300 cataract patients and is 
now being analyzed to determine complete or 
partial gene deletion at the University of 
Parma and the NEFs LMOD. 

A new collaborative Italian-American 
study is being planned to evaluate the effect 
of multivitamin supplements on the risk of 
cataract development and progression. Ap- 
proximately 1,200 subjects will be randomized 
to either a multivitamin /mineral supplement 
or matching placebo and followed for five 
years. Professor Giovanni Mararni, from the 
University of Parma, and Dr. Robert Sperduto, 
from the NEI, will be the study's principal 
investigators. The Laboratory for Epidemiolo- 
gy and Biostatistics, Istituto Superiore di 
Sanita, Roma, and the DBE at the NEI serve as 
the study's Coordinating Centers. The seven- 
year study was scheduled to start in late 1994. 

Mexico 

An international collaboration has been estab- 
lished by scientists in the NEI, LMOD, Section 
on Cataract, to investigate the relationship 
between enzyme deficiency diseases and cata- 
ract. For example, a candidate gene study 
was initiated to determine whether a deficien- 
cy in sorbitol dehydrogenase in a family 
where several members have congenital cata- 
racts is due to changes in SDH gene structure 
or expression. This study is possible through 
the cooperation of the Unidad de Investiga- 
don Biomedica y Hospital de Pediatria, Insti- 
tuto Mexicano del Soguro Social, Guadalajara, 
Mexico. 

Sweden 

Many eye diseases, especially retinal degener- 
ations, could be successfully treated if human 
retinal transplantation were possible. In 
animal models, visual cells that have been 
transplanted do not develop and function 
normally. However, a new differentiating 
factor has been discovered and is being ex- 
pressed using molecular biology techniques at 
the NEI. This factor, which is a protein that 



causes neuronal-Uke differentiation, is being 
tested in vitro by NEI collaborators in Sweden 
at the University of Gothenburg and the 
University of Lund to determine if it wiU 
cause retinal cell differentiation. The ultimate 
purpose of these investigations is to develop 
cells that could be transplanted into the hu- 
man eye in vivo and function normally. 

In collaboration with protein biochemists 
at the Karolinska Institute in Stockholm, NEI 
cataract researchers are investigating the 
evolutionary relationships of ^-crystaUin, an 
enzyme /crystaUin of certain species, with 
other oxido-reductases. Establishing such 
relationships with enzymes of known function 
should help to identify the physiological roles 
of ^-crystalUn in the lens and in other tissues 
where it is present at low levels. A paper 
reporting these analyses is being prepared. 

The Early Marufest Glaucoma Trial is a 
randomized, controlled clinical trial to deter- 
mine whether and to what extent reduction of 
lOP influences the course of chronic open- 
angle glaucoma. Investigators at the Universi- 
ty of Lund are collaborating with investigators 
at the State University of New York at Stony 
Brook and will study an estimated 300 pa- 
tients with newly diagnosed disease. Partici- 
pants will be randomized either to pressure- 
lowering treatment or to observation without 
treatment. Both groups will be followed close- 
ly with computerized perimetry and fundus 
photography. Recruitment of patients began 
in 1993 and will continue for an estimated two 
years. FoUowup of patients will be conducted 
for four years. 

United Kingdom 

The UK Prospective Diabetes Study is a pro- 
spective randomized study of different thera- 
pies to determine whether improved blood 
glucose control or improved blood pressure 
control of noninsuUn-dependent diabetes will 
reduce morbidity and mortality. The study 
began in 1977 and has recruited more than 
5,100 newly diagnosed diabetic patients. 
Patients who fail to respond to diet therapy 
are randomized to diet therapy or active 



36 



International Program Activities 



therapy with sulfonylurea, insulin, or 
metformin. As part of the study, hypertensive 
diabetic patients have been randomized to 
tight blood pressure control (with either an 
angiotensin-converting enzyme inhibitor or p- 
blocker) or to less tight control. The develop- 
ment and progression of diabetic retinopathy 
in these patients are being assessed by retinal 
photography. The study is completing 11 
years of patient followup. 



Activities With International and 
Multinational Organizations 

During the past year, the NEI has supported 
investigations of bUnding eye diseases that 
have a worldwide effect. These studies are 
implemented through bilateral agreements 
between foreign countries and the United 
States; other types of country-to-country 
programs such as those supported by U.S. 
Agency for International Development; and 
collaborative activities with the WHO, the Pan 
American Health Organization, foundations, 
and private and voluntary organizations such 
as the Lions Clubs International. 

The NEI is continuing to provide technical 
advice to Lions Clubs International in the 
development of its $100 million SightFirst 
initiative, a global sight conservation program 
aimed at substantially reducing the prevalence 
and incidence of preventable and curable 
vision loss. 

In FY 1994, the ISTEI continued its activities 
as a WHO Collaborating Center for the Pre- 
vention of Blindness. The NEI director contin- 
ues to serve on the WHO's Special Advisory 
Panel in the Prevention of Blindness. Other 
NEI staff members have, on request, consulted 
to the WHO program. 



The NEI is working closely with nongovern- 
mental organizations in designing service and 
research programs to reduce the prevalence of 
bUndness, regardless of its etiology, through- 
out the world. A special emphasis last year 
and in the next few years will be an evalua- 
tion of program performance in selected coun- 
tries. 



Extramural Programs 

In FY 1994, NEI granted 14 awards to foreign 
institutions in six countries. Research and 
training projects were supported in lens and 
cataract, glaucoma, visual system develop- 
ment, photoreceptors, phototransduction, 
visual cortex, visual abnormahties, Leber's 
disease, nutrition of the eye, ocular compUca- 
tions of diabetes, and the prevention of blind- 
ness. Awards covered both basic and cUnical 
research projects. 



Intramural Programs and Activities 

The NEI continues to serve as an international 
center for research and training on eye dis- 
ease. In FY 1994, 25 visiting fellows, 21 visit- 
ing associates, 17 visiting scientists, 19 special 
volunteers, and three guest researchers from 
more than 20 countries conducted research at 
the NEI facilities in Bethesda, Maryland. 
Their work included basic laboratory investi- 
gations on the molecular structure and devel- 
opment of the visual system, sensory and 
motor disorders of vision, and the biochemical 
bases of retinal and corneal diseases and cata- 
ract development. In addition, visiting scien- 
tists collaborated with NEI investigators in 
clinical studies to define, treat, and prevent 
vision disorders, such as genetic and develop- 
mental defects, ocular inflammatory disease, 
and ocular compUcations due to systemic 
conditions such as diabetes. 



37 



Office of Science Policy and Legislation 



Report of the Associate Director for 
Science Policy and Legislation 



Michael P. Davis, M.S. 



The Office of Science Policy and Legis- 
lation is responsible for program plan- 
ning, analysis, and evaluation activi- 
ties; development and maintenance of a com- 
puterized management information system; 
and legislative and other program coordina- 
tion activities. In addition to the activities 
listed below, the office had one organizational 
change during the year. The Scientific Report- 
ing Branch was made an office within the 
Office of the Director and is now called the 
Office of Health, Education, and Communica- 
tion (OHEC). 



Policy, Legislation, Planning, and 
Evaluation Branch 

Carmen P. Moten, Ph.D., Chief 

During FY 1994, the Policy, Legislation, Plan- 
ning, and Evaluation Branch provided numer- 
ous reports concerning research-related activi- 
ties of the NEL Specific activities included the 
preparation of recurring and ad hoc program 
analyses in response to requests from the 
NIH, The U.S. PubUc Health Service (PHS), 
and the U.S. Department of Health and Hu- 
man Services (DHHS); serving as the focal 
point on program planning, analysis, evalua- 
tion, and legislation; and planning, coordinat- 
ing, carrying out, and monitoring NEI pro- 
gram evaluations. Principal activities for this 
branch are specified below: 



Preparation of NEI Scientific Advances- 
1995 Congressional Justification. 



-FY 



Preparation and submission for the Annual 
Report of Aging-Related Eye Disease Research. 
Eye diseases and disorders, such as AMD, 
cataract, glaucoma, and diabetic retinopa- 
thy, are causes of bHndness and visual 
impairment among older Americans. 

NEI submission for the Survey on Nursing 
Research and Related Activities Supported by 
NIH, FYs 1989 to 1992. 

Response to the Fogarty International 
Center (FIC) concerning the NIH report to 

the Senate on Biodiversity. 

NEI submission on Gene Therapy and He- 
reditary Rare Diseases. Much of the molec- 
ular genetics research that is conducted by 
NEI researchers is instrumental in devel- 
oping the basic understanding necessary 
to pursue gene therapy treatment strate- 
gies for rare ocular and visual system 
hereditary disorders. 

NEI submission for the top scientific ac- 
complishments in basic research, applied 
research, and clinical trials. 

Review of various PHS FY 1996 Legisla- 
tive Proposals from the Health Resources 
and Services Administration and the Food 
and Drug Administration. The NEI felt 
compelled to respond to the legislative 
proposal Regulation of Human Tissue for 
Therapeutic Use. The proposal intends to 
provide a regulatory program for banked 
human tissues that addresses the funda- 
mental differences between human tissues 
used for medical products. Fees wiU also 



41 



FY 1994 NEI Annual Report 



be charged for registration, operating 
permits, and inspections to support the 
cost of the program. The NEI believes 
that such fees could threaten human eye 
tissue transplantation. Almost all, if not 
all, eye banks of the Eye Bank Association 
of American are 501 (c)3 organizations. 
They depend heavily on philanthropic 
contributions in providing eye tissue for 
research or for patient care. Most rural 
and small eye banks would have serious 
difficulty with the imposition of fees, giv- 
en the difficulties of meeting current oper- 
ation budgets. 

NEI submission for supported projects 
that are evaluating cigarette smoking as a 
potential risk factor for eye dis- 
eases — Healthy People 2000 Tobacco Prior- 
ity Area. 

NEI submission for the 1994 NIH Disease 
Prevention Annual Report. The report 
highlighted basic research, applied re- 
search and cUnical investigation, interven- 
tion studies, and professional and public 
education. 

NEI submission report to the Office of the 
Director (OD), Office of Disease Preven- 
tion for the Fiscal Years 1992 and 1993 
Prevention Outlays and Full Time Equivalent 
Positions. 

Report to the OD, Office of Science PoUcy 
and Technology for the 2994 Minority 
Health Legislation. The NEI supports three 
demonstration projects aimed at develop- 
ing, implementing, and evaluating com- 
prehensive culture-specific and communi- 
ty-based education programs for the pre- 
vention of diabetic retinopathy. 

NEI Planning Activities Report, in response 
to the OD, Office of Strategic Planning 
and Evaluation request. 

NEI submission for the NIH Legislative 
Implementation Plan — NIH Revitalization Act 
of 1993. 



• NEI submission for the FY 1996 NIH Plan 
for HIV-Related Research. 

Analyses of draft materials from the PHS 
Office of Disease Prevention and Health 
Promotion for editorial review of the 
Guide to Clinical Preventive Services report 
of the U.S. Preventive Services Task Force. 

The branch also has been involved in 
researching, writing, and editing various 
reports requested by the NIH, PHS, DHHS, 
Congress, and nongovernmental organizations 
and individuals, including the following: 

NEI submission for the NIH Intramural 
Research Program to the Subcommittee on 
Appropriations Health and Education, 
Congressmen Louis Stokes. 

• Report to the Schepens Eye Research Insti- 
tute on the NEI extramural funding for 
various eye diseases and disorders. 

• NEI submission for the 1995 White House 
Conference on Aging. The mandate of such 
conferences is to produce recommenda- 
tions for aging policy to span the next 
decade. 

• Report of the NEI director for the 1993- 
1994 Biennial Report of the NIH. The report 
described research accomplishments, 
outlined future opportunities, and as- 
sessed important poUcy issues. 

• NEI submission for the Diabetes Mellitus 
Interagency Coordinating Committee 
(DMICC) Annual Report. The report in- 
cluded recent activities of the structure 
and function of polyol pathway enzjTnes, 
epidemiology of diabetic retinopathy, and 
advances in retinal cell biology. 

• NEI submission on Research Activities 
Related to Space. The report highlighted 
two areas of research, ultraviolet radiation 
on the deUcate tissues of the eye, and the 
structure and function of the vestibular- 
ocular reflex. 



42 



Science Policy and Legislation 



NEI submission for the Annual Legislative 
Weekend of the Congressional Black Caucus 
Health Braintrust. The report highlighted 
epidemiologic studies, clinical trials. Na- 
tional Eye Health Education Program, 
demonstration projects, and support for 
minority scientists, institutions, and stu- 
dents. 

The following information was submitted 
to the NEI Financial Management Branch: 

• NEI report of FY 1993 Actual Outlays for 
Trans-NIH Research Areas. The report 
areas included: accidents and injuries, 
aging and age-related diseases, 
Alzheimer's disease, arthritis and muscu- 
loskeletal disorders, breast cancer, cystic 
fibrosis, diabetes, diagnostic imaging /dia- 
gnostic radiology, drug development, 
funding for children (0 to 21), gene map- 
ping for both the Human Genome Project 
and in nonhumans, gene therapy research, 
health and behavior research, immunology 
research, infant mortality /low birth 
weight, kidney diseases, medical rehabil- 
itation research, minority aids, minority 
health and assistance, neurofibromatosis, 
neuroscience, nutrition, orphan drugs, 
prevention, sexually transmitted diseases, 
sickle cell diseases, smoking and health, 
space medicine, stroke, vaccine develop- 
ment, vaccine-related, and women's 
health. 

• Total NEI Basic /Applied/Development 
research by mechanism, in a tabular for- 
mat, for the Office of Financial Manage- 
ment and Budget Exhibit 44 A. 

• Written responses to questions submitted 
by Appropriations Subcommittee mem- 
bers. 

The branch contributed support for the 
Office of Health Education and Communi- 
cation (OHEC) concerrung the NEI grant port- 
folio. This portfolio included various types of 
eye disease-related research conducted by 
specific investigators such as: 



Retinal cell transplantation studies, RP 
and related diseases, cataract, dry eye, 
glaucoma, and tissue plasminogen activa- 
tor. 

The branch provided detailed information 
for various NIH offices, including: 

A description of NEI-ouflays for disease 
prevention research for the Office of Dis- 
ease Prevention. 

An analysis of all nutrition-related re- 
search supported by the NEI for inclusion 
in the Human Nutrition Research Information 
Management Re-port. 

NEI submission on Skin Diseases Activi- 
ties/Congressional Report. The report in- 
cluded a table of all skin disease-related 
research funded by NEI and research 
activites of the corneal angiogenesis, ge- 
netic studies of keratoconus, cloning of a 
human corneal desmosomal protein, and 
collaborative activities. 

• NEI table submission of all arthritis and 
musculoskeletal diseases-related research 
for the Arthritis and Musculoskeletal Diseases 
Interagency Coordinating Committee 
(AMDICC) Annual Report. 

The branch also provided editorial review 
of a variety of letters, reports, and other narra- 
tive materials for other offices within the NEI. 



Management Information Systems 
Branch 

David Scheim, Ph.D., Chief 

During the past fiscal year, the Manage- 
ment Information Systems Branch (MISB) has 
upgraded four of its network servers in Build- 
ing 31 and EPS to Windows NT advanced 
server, providing improved speed, rehability, 
and connectivity. MISB initiated and super- 
vised a contract for the extension of its local 
area network to a Windows NT server and 10 
workstations distributed among the NEI 



43 



FY 1994 NEI Annual Report 



intramural laboratories, allowing access to 
administrative data and computer support 
services. This has enabled MISB, for example, 
to grant intramural offices access to the Status 
of Funds program, which MISB also provided 
to additional users in the administrative and 
executive offices of Building 31. The integrat- 
ed architecture provided by the migration to 
the Windows NT network operating system 
allows all current and any additional users to 
run this program from one file server location. 

The MISB managed the installation of the 
TAIMS timekeeping system on approximately 
20 workstations within the NEI. Bringing this 
system to fuU production entailed assistance 
and problemsolving for users, particularly in 
the transmission of aggregated data. The 
MISB also provided similar assistance in the 
installation of the ATRAIN system for generat- 
ing training requests and installed an official 
airlines guide flight scheduling program for 
the administrative office. The NEI upgraded 
several users from Word Perfect 5.1 to 6.0 and 
arranged traiiung for NEI staff to make this 
transition. 

The MISB upgraded five printers to the 
Hewlett Packard LaserJet 4 series. Eight 
modems were upgraded from 2,400 to 14,400 
bits per second, allowing much faster commu- 
nications speeds for remote usage of the NEI 
LAN by NEI staff on tiavel or working after 
hours at home. MISB installed two CD ROM 
drives, primarily for use in software installa- 
tions and installed a magneto-optical drive for 
more reliable daily and weekly backups of all 
network data. The MISB, in addition, has con- 
tinued to provide support and maintenance 
for all 95 workstations in Building 31 and EPS, 
aU software used, and the network infrastruc- 
ture with no contractor support. 

The MISB planned and implemented a 
comprehensive documentation contiact for the 
NEI's microcomputer LAN, associated hard- 
ware and software, and aU custom-developed 
systems at a cost of $23,500. This project, 
when completed in FY 1995, wiU provide 
extensive textual documentation of NEI micro- 
computer systems; cabling diagrams; and 



online documentation of equipment, processes, 
and problem resolution procedures accessible 
to MISB and all NEI staff. Database tables 
and access systems for the online components 
of this effort were developed by MISB staff. 

The MISB began a joint effort with the 
NIAID for the development of a cUent-server 
personnel tracking system to maintain infor- 
mation on aU permanent and temporary em- 
ployees. Development will be performed by 
an NIAID contractor, but MISB input and 
design assistance will be provided in exchange 
for consideration of particular NEI require- 
ments. If successful, such collaborative efforts 
will be used for the subsequent development 
of a procurement tracking system using state- 
of-the-art software compatible with the NEI's 
current database platforms. 

The MISB evaluated automated grants 
awards systems in use at NIH and arranged a 
$14,000 contract for the customization of one 
such system for use by the NEI Extramural 
and Collaborative Program. This contract 
specifies capabilities provided to the NEI on 
par with those provided to another ICD 
through a contract costing about $1 million. 
Most of the work on this contact was complet- 
ed in FY 1994. 

MISB provided extensive enhancements to 
its grants information systems during FY 1994. 
Microsoft SQL Server, the database server for 
all systems, was upgraded to version 4.2 to 
allow enhanced functionality. JAM and 
JAM/DBI, the cUent tools for these systems, 
were also upgraded to allow additional func- 
tionality. The existing NEI snapshot, coundl 
letter, and grants coding systems were up- 
graded for this new environment. 

MISB reprogrammed the grants master on- 
line update and SCORE coding routines using 
the JAM cUent-server front end in which the 
NEI's other grant modules are developed. It 
also converted aU historical grants and SCORE 
data into cUent-server tables, using custom- 
developed data conversion and checking rou- 
tines. The NEI's council letter generation 
program was also modernized into a more 



44 



Science Policy and Legislation 



streamlined client-server process using R&R 
report writer, which, after evaluation of sever- 
al such tools, was selected as a future report- 
ing platform. These conversions have allowed 
the older and less reliable Paradox database 
applications to be completely phased out. 

Automated security, login, and usage 
tracking functions integrated with LAN login 
were developed by MISB staff. These func- 
tions provide automatic login to grants infor- 
mation systems for NEI staff and also auto- 
matic tracking of system usage. Between 
January and September of 1994, a total of 
4,198 logins to grants information systems 
were recorded, not including system testing 
by MISB staff. Also, daily check and backup 
procedures for aU active NEI databases, which 
run automatically each night, were implement- 
ed through custom programming by MISB. 

The NEI's weekly grants update batch 
procedures, comprising 24 procedures and 
100,000 lines of code previously written in 
Paradox 3.0, were completely reprogrammed 
by MISB staff in Microsoft SQL Server Trans- 
act SQL language, extensively tested, and 
implemented. The new batch update proce- 
dures run more quickly and reUably and 
contain extensive built-in system checks to 
automatically halt processing if an error is 
detected. The reprogramming was performed 
to consolidate the NEI's grants system by 
eliminating an obsolete platform, allowing for 
easier maintenance and future development in 
the more reliable, state-of-the art, cUent-server 
architecture. The new update procedures 
have run on weekends virtually error-free 
since mid-1994. 



The MISB has continued to provide cus- 
tom information reports to NEI staff for inter- 
nal use and pubUc distribution, with 70 new 
mainframe requests and an increasing volume 
of microcomputer-based production reports 
logged for FY 1994, with rapid turnaround 
achieved in every case. Weekly and monthly 
reports, as well, continue to be provided. In 
addition to its own programming efforts, the 
MISB has continued to support NEI staff in 
the use of information resources provided by 
DRG, the NLM, and other sources, including 
the DRG information system, CRISP, FOCUS, 
WYLBUR, MEDLINE, Grateful Med, Legislate, 
the electronic NIH library catalog. Gopher, 
and other specialized systems. 

The MISB has continued to handle a 
number of IRM functions for the NEI, includ- 
ing its environment and resources report, 
strategic plan, tactical plan, budget report, and 
security functions. MISB staff has continued 
to represent the NEI on a number of NIH- 
wide committees, including the Office Techni- 
cal Coordinators and its network subcommit- 
tee, the ADP Extramural Programs Coordi- 
nating Committee and its steering committee, 
the Database Technology Task Force, the NIH 
lead users group, the Campus Users Research 
Exchange, and the Technical LAN Coordina- 
tors Committee. 



45 



Office of Health, Education, and Communication 



Report of the Director of the Office of 
Health Education and Communication 



Judith A. Stein, M.A. 



In a reorganization, the NEI has estab- 
Ushed the OHEC within the NEI, OD. 
This new office replaces the Scientific 
Reporting Branch previously part of the NEI's 
Office of Science PoUcy and Legislation. The 
activities of this office include the National 
Eye Health Education Program (NEHEP); 
special activities such as the traveling science 
museum exhibit; dissemination of research 
results; publications; response to inquiries 
from pubUc, health professionals, and the 
media; and advising NEI staff on aU aspects of 
NEI and NTH scientific reporting, knowledge 
transfer, health education, and press relations. 



National Eye Health Education 
Program 

The NEHEP began the development of a 
diabetic eye disease education program for 
Hispanics/ Latinos with diabetes. Eight focus 
groups were conducted across the country to 
learn more about the knowledge, attitudes, 
and practices of this target audience as related 
to diabetes and eye health. Groups were con- 
ducted with Central Americans in Washing- 
ton, D.C.; Puerto Ricans in New York City; 
Mexican Americans in Los Angeles; and Cu- 
ban Americans in Miami. An ad hoc working 
group on Hispanic outreach met to provide 
recommendations to the NEHEP staff on the 
development of an education program. It is 
anticipated this program wiU be launched in 
spring 1995. 

A television pubUc service announcement 
(PSA) on glaucoma was produced and distrib- 



uted nationally in September 1994. The PSA 
stiesses the importance of eye examinations 
for people at risk for glaucoma especially 
African Americans older than age 40 and 
everyone older than age 60. In addition, radio 
and print PSA's on glaucoma and diabetic eye 
disease were distributed to media outlets 
reaching target audiences at risk for these 
diseases. 

The Third National Eye Health Education 
Conference was held in December 1993. The 
purpose of this conference was to provide an 
opportunity for the members of 51 organiza- 
tions in the NEHEP Partnership to share their 
program and interests and to develop collabo- 
rative eye health education programs at the 
community level. 

One idea that originated at the conference 
was to conduct an awareness campaign on 
diabetic eye disease during National Diabetes 
Month in November. In February, the Ameri- 
can Diabetes Association (ADA) and the NEI 
joined forces to coordinate this event. Nine 
other Partnership organizations offered sup- 
port to increase awareness among people with 
diabetes about the importance of an annual 
dilated eye examination. These organizations 
will coordinate local activities. A special bro- 
chure was developed for November, adapted 
from the existing NEHEP Don't Lose Sight of 
Diabetic Eye Disease brochure. The ADA wiU 
use its 1-800-DIABETES number as a place to 
call for a referral to an eye care professional 
and more information on diabetes and diabetic 
eye disease. The referral program is coordi- 
nated with the American Academy of Oph- 
thalmology and the American Optometric 



45 



FY 1994 NEI Annual Report 



Association. An extensive media campaign 
will also be conducted to complement local 
efforts. This includes a video news release, 
press release, print advertisement campaign, 
and a radio program targeted to African- 
American radio stations. 



25th Anniversary Program 

The NEI celebrated its 25th anniversary with 
a nationwide public education program to 
promote the benefits of vision research. The 
centerpiece of the celebration was the travel- 
ing exhibit VISION, which was developed 
to highlight the sight-saving results of vision 
research funded through American tax dollars. 



VISION premiered in San Francisco, 
CaHforiua at the Exploratorium in October 
1993. During 1994, it has been displayed at 
the Museum of Science and Industry in Chica- 
go, Illinois; the Museum of Discovery and 
Science in Fort Lauderdale, Florida; Union 
Station in Washington, D.C.; and at the Louisi- 
ana Nature & Science Center in New Orleans. 
An estimated 125,000 people have visited the 
exhibit to date. It will travel to approximately 
13 more cities, including Boston, Massachu- 
setts; Jacksonville, Florida; Houston, Texas; 
Los Angeles, California; Portland, Oregon; and 
Seattie, Washington, during the next few 
years. 

In each location where the exhibit is dis- 
played, NEI grantees and local chapters of the 
voluntary and/ or professional organizations 
plan a series of regional events designed to 
increase the public's awareness of vision re- 
search. These events include public lectures, 
vision screenings, press conferences, science 
writers seminars, and educational programs 
for school age children. 

The NEI also is developing a school curric- 
ulum program for children in grades four 
through eight. The program consists of three 
lesson plans, interactive classroom activities, 
and previsit and postvisit exercises. Topics 



covered include the anatomy and physiology 
of the eye and visual system, common eye 
diseases and disorders, and eye safety. The 
program is designed as a supplement to any 
science or health curriculum and can be used 
by either a guest speaker (vision researcher 
or eye care professional), or the classroom 
teacher. 

The curriculum will be pilot tested this fall 
in several communities throughout the coun- 
try and will be available in spring 1995. The 
Association for Research in Vision and Oph- 
thalmology will print and distribute the pro- 
gram to its 11,000 members this November 
and will highlight it during the association's 
annual meeting in May. A marketing and 
promotion plan wiU be developed to target 
science and health educators for children in 
grades four through eight nationwide. 



Public Inquiries Program 

The OHEC staff responded to more than 
16,000 inqviiries from the general public, pa- 
tients and their farrdlies, students, health pro- 
fessionals, legislators, and the media in FY 
1994, including 14 pieces of controlled corre- 
spondence representing five congressional 
inquiries and a Presidential proclamation. 
This reflects an approximate six percent in- 
crease in inquiries over last year. 

To handle the increase in pubUc inquiries 
more efficientiy, the OHEC staff developed 
standard information packets on commonly 
requested eye disorders and diseases, includ- 
ing sarcoidosis, blepharitis, RK, RP, and cata- 
ract surgery, and more. To assist health pro- 
fessionals find additional materials on specific 
eye diseases and disorders, staff members 
prepared listings of the materials found in the 
Eye Health Education subfile in the Combined 
Health Information Database. Two new bro- 
chures, written in an easy-to-read format, 
were developed for people at risk for cataract 
and AMD. 



50 



Office of Health, Education, and Communication 



The OHEC staff also handled 38 Freedom 
of Information requests. 

Scientific Reporting 

The NET publication Clinical Trials Supported by 
the National Eye Institute was developed and 
printed in faU 1993. This publication provides 
information on 22 extramural and intramural 
clinical trials supported by the NEl. Each 
dinical trial description includes the purpose 
and design of study, patient eligibility criteria, 
patient recruitment status, results to date, and 
participating cUnical centers. 



51 



Office of the Scientific Director 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00122-14 OSD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Anatomical Studies of the Primate Visual System 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Office of the Scientific Director 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

0.40 



PROFESSIONAL: 

0.40 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects □ (b) Human tissues [x] (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



This project involves the study of the anatomical properties and organization of cells in the visual system of 
primates, with emphasis on the retina and the visual cortex. The studies include the pattern distribution of 
selectively stained cones in the retina of macaques. The results have provided information on the probable 
retinal circuitry of the blue-sensitive cone pathway of primate retinal cells. 



55 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

To study the anatomical properties and neural 
organization of the primate visual system. 

Methods 

Retinal histological processing, intravitreal 
injection of dyes, computer modeling, and 
spatial statistical analyses of point and area 
patterns are used. 

Major Findings 

The anatomical studies of this project were 
affected once again by further underground 
construction work in a building just across the 
street, in front of the laboratory; in addition, 
the building housing the laboratory under- 
went structural renovation during most of this 
period. Such construction affected both mi- 
crotomy and microphotography, limiting work 
to the evening or night hours. 

The point pattern of blue-sensitive cones 
selectively stained with tissue-reactive 
nonfluorescent dyes (Procion black) was 
examined at various eccentricities of the 
macaque retina in distortion-free whole 
mounts. The point pattern was analyzed in 
terms of its angular structure and disorder 
using spatial statistical techniques described in 
earlier work. Comparison of the parafoveal, 
extrafoveal central retina, and peripheral 
retina point patterns indicates that the degree 
of disorder of the pattern increases slowly 



with eccentricity, from about 18 percent to 
about 30 percent. This is equivalent to dis- 
turbing each point of a lattice of unit area by 
0.18 to 0.30 units along a randomly selected 
azimuth. Despite this disorder, the pattern 
maintains its regularity with eccentricity, and 
side-by-side blue-sensitive cones are very 
rarely seen. 

These results indicate that the blue-cone 
pattern of macaque retina does not foUow a 
lattice distribution and support a previous 
model for the development of this point 
pattern, based on an exclusionary hard core 
surrounded by a probabilistic soft shell. 

Significance to Biomedical Research and the 
Program of the Institute 

Information on the anatomical properties of 
blue-sensitive cones is important not only to 
the functional properties of these cones inves- 
tigated in different basic disciplines but also to 
the clinical research and diagnosis of acquired 
retinal disease. The data obtained from the 
eye of diabetic human donors are particularly 
promising in this respect. 

Proposed Course 

These studies will be continued. 
NEI Research Program 

Retinal Diseases — Retinal Neuroscience 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00065-17 OSD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Physiological Studies of the Primate Visual System 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Office of the Scientific Director 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

0.60 



PROFESSIONAL: 

0.60 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects □ (b) Human tissues [x] (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project involves the study of the physiological organization of neurons of the visual system of nonhuman 
primates that may serve as a model for the human visual system. Emphasis is given to the spectral and spatial 
properties and central projections of retinal ganglion cells and cells from the lateral geniculate body and the 
visual cortex of macaques. Recordings from color-opponent retinal ganglion cells and parvocellular geniculate 
cells show essentially identical spectral response bandwidths under similar conditions of stimulation. On the 
basis of these bandwidth data, both types of cells can be grouped in subtypes which show a direct 
correspondence with specific color-opponent varieties. The bandwidth data essentially show no difference 
between geniculate and retinal color-opponent cells subserving similar areas of the visual field. 



57 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

To study the neural organization underlying 
the processing of visual information at differ- 
ent levels of the primate visual system. 

Methods 

Intracellular and extracellular recordings from 
single neurons, extracellular recordings of 
mass responses, computer video stimulation, 
tangent screen chromatic and spatial stimula- 
tion. 

Major Findings 

The single-ceU studies of this project were 
affected once again by further underground 
construction work in a building just across the 
stieet, in front of the laboratory; this work 
resulted in often violent shaking that defeated 
vibration-isolation measures. In addition, the 
buUding housing the laboratory underwent 
stiuctural renovations during most of this 
period. 

The response bandwidth of color-oppo- 
nent ganglion cells and parvocellular genicu- 
late cells was examined with spectral lights in 
conditions of neutral chromatic adaptation. 
The same stimulating conditions were used 
for both cells, which were typically recorded 
from the same anesthetized animals. As noted 
in previous work, spectral response band- 
widths have specific "signatures" when plot- 
ting bandwidth against the wavelength of the 
peak sensitivity. The average half-bandwidth 
at half-maximum sensitivity was 24 nano- 
meters (run) for retinal gangUon cells and 22 
nm for parvocellular geniculate cells. Aver- 
aged spectral bandwidths of these cells fall in 
various distinct signature subgroups matching 
subgroups based on color-opponent response 
varieties. 



No significant differences, either in aver- 
age half-bandwidth or spectral signature, were 
found between color-opponent, center-sur- 
round geniculate, and ganglion cells. In fact, 
for neurons subserving the same or similar 
areas of the central visual field, the geniculate 
and retinal bandwidth data could not be 
distinguished from one another. These results 
provide further support for an essentially one- 
to-one relationship between these neurons. 

Significance to Biomedical Researcli and the 
Program of the Institute 

Numerous behavioral, psychophysical, and 
electrophysiological studies show that the 
visual performance and characteristics of 
macaques and humans are extremely similar 
to one another, so that an understanding of 
nonhuman primate physiology provides a 
useful animal model for human visual func- 
tion. 

Proposed Course 

These studies will be continued. 

NEI Research Program 

Stiabismus, Amblyopia, and Visual Process- 
ing — Structure and Function of Central Visual 
Pathways. 

Publications 

de Monasterio FM: Operating system errors 
in DOS 5. J PC Tech 4:30-37, 1993. 

de Monasterio FM: Direct access to interrupt 
handlers in the system kernel. / PC Tech, in 
press. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00135-22 OSD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Biochemistry of Retina and Pig men ted Epithelium in Health and Disease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Helen H. Hess M.D. Medical Officer (Research) OSD, NEI 



COOPERATING UNITS (if any) 

Laboratory of Chemoprevention, National Cancer Institute (M. Anzano, Ph.D.) 



LAB/BRANCH 

Office of the Scientific Director 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

1.0 



PROFESSIONAL: 

1.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects Q (b) Human tissues [x] (c) Neither 

□ (a1) IVIinors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Effects of nutrition, oxidation, and other environmental factors (light intensity or darkness) on incidence and progress of posterior 
subcapsular opacities (PSO) associated with genetically influenced retinal degeneration are being studied in pink-eyed Royal College 
of Surgeons (RCS) rats, in which rod photoreceptor outer segment debris accumulates secondary to a phagocytic defect in retinal 
pigmented epithelium (RPE). Peroxidation in polyunsaturated fatty acids in debris lead to water-soluble toxic aldehydes, detectable 
in the vitreous and toxic to lens cells and membranes. Dystrophic rats fed a natural ingredient diet (NIH-07) were highly sensitive 
to retina light damage, beginning at an intensity of 10 to 40 lux, and 27 percent of the rats developed mature cataracts by 5 to 12 
mondis. Rhodopsin bleaching is essential for retina light damage and PSO. In vitro, free retinaldehyde has been shown to be a 
photosensitizer to generate singlet oxygen, an extremely damaging oxidant for both lipids and proteins, and this also may occur in 
vivo. 

In RCS rats reared at 10 to 40 lux, a purified diet (AIN-76A) fortified with antioxidants (0.4 percent p-carotene + 0.01 percent BHT) 
prevented PSO and mature cataracts. A diet containing additional antioxidants (1,000 mg/Kg diet of vitamin C and 150 mg/Kg vitamin 
E) retarded retinal degeneration during the time the cataracts would have had their onset (23 to 53 postnatal days) if NIH-07 had been 
fed. Higher concentrations of vitamin E did not show additional retardation of retinal degeneration. 

Effects of increasing environmental lighting in incidence of bilateral mature cataracts were studied in pink-eyed RCS rats fed the NIH- 
07. Incidence of bilateral mature cataracts (BMC) was 5 percent in rats reared in 10 to 40 lux of cyclic light; but was 25 percent in 
rats reared in 110 lux of constant light; 70 percent in 270 lux of constant light; and 100 percent in 65-day-old rats given 48 hours of 
high intensity light (7500 lux). After lengthy or intense illumination, occurrence of disturbed meridional rows of lens epithelial cells 
and posterior nucleated (Wedl) cells pointed to proliferation of germinative zone epithelial lens cells from deoxyribonucleic acid 
(DNA) damage. At low illumination, damage can be repaired (stationary cataracts and rare BMC). The results are consistent with 
the hypothesis of PSC causation by DNA damage to lens epithelial cells. Agents that can have this effect include products of 
peroxidation of polyunsaturated fatty acids, short wavelength radiation (UV, X-rays, P and y rays) and numerous chemical mutagens 
such as N-nitroso-N-methylurea (NMU). 

Normal albino rats, injected with NMU at a concentration and dosage sufficient to cause breast cancer, developed BMC by 5 months 
of age. These cataracts were PSC of a more severe nature than ever seen in RCS rats (exposed to excessive light), with abnormally 
large cells (some binucleate) not only at the posterior pole but encircling the lens. The retinal showed advanced degeneration, ^g 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 

J. Samuel Zigler, Ph.D. Chief, LMOD, NEI 

Jr. 

Joseph J. Knapka FhD. Nutrition Consultant, 
Veterinary Research 
Program (VRP), Nation- 
al Center for Research 
Resources (NCRR) 

Dennis Bernard M.S. Nutritionist, VRP, 

NCRR 

Maria Anzano Ph.D. Expert, Laboratory of 

Chemoprevention, NCI 

Objectives 

This project is designed to study the biochemi- 
cal and bionutritional relationships among 
lens, retinal photoreceptors, retina, retinal 
pigment epitheUum (RPE), and biological 
fluids in health and disease. It also involves 
exploring the possibilities for slowing the rate 
of retinal degeneration and preventing lens 
opacities and mature cataracts, which are 
often associated with retinal degeneration in 
rats and humans. 

The cataract that develops in the Royal 
College of Surgeons (RCS) rat is a posterior 
subcapsular cataract (PSC), a tj^e that makes 
up about 10 percent of cases of age-related 
cataract and occurs in 40 percent of cases of 
retinitis pigmentosa. PSC also is seen in other 
hereditary retinal diseases and in radiation 
damage to the lens. It is characterized by 
genetically damaged lens epitheUal cells, 
which fail to undergo normal differentiation 
into lens fibers and instead multiply and 
migrate to the posterior subcapsular region to 
form an opacity. 

PSC and retinal degeneration are pro- 
duced in rats by the chemical mutagen N- 
nitrosoN-methylurea (NMU). This agent has 
been used to produce breast and prostate 
tumors as well as gUal tumors of the brain in 
rats. Chemopreventive agents have been 
found to reduce the incidence of breast and 
prostate cancers in rats using dual agents, one 
to reduce or halt multiplication of cells, the 



other to cause differentiation of the cells. An 
objective will be to test whether these princi- 
ples can be used to reduce incidence of PSC 
and retinal degeneration in rats given NMU. 
The rat C-6 gUal cell hne, used for the past 30 
years, was derived from rat brain glial cells 
transformed by NMU in vivo; several clones 
were obtained, C-6 being one of the more 
differentiated. Transformation of lens epitheU- 
al cells in vivo by NMU potentially could yield 
a similarly useful ceU Une. 

Methods 

The RCS rat is being studied as an animal 
model of hereditary retinal degeneration that 
results from a defect in the RPE as well as a 
type of cataract that is secondary to retinal 
degeneration. Bionutrition has been used as a 
tool to combat Upid peroxidation in the RCS 
rat retina and to prevent water-soluble toxic 
aldehyde byproducts from reaching and 
damaging the lens. The RCS rat cataract is 
not genetic because the mutant gene is ex- 
pressed not in the lens but in the RPE; it is 
instead an outcome of environmental risk 
factors of both internal and external origin. 
Thus, the RCS rat is a living laboratory, and 
the cataracts are susceptible to orchestration 
by varjdng risk factors and preventive mea- 
sures. 

Defined diets were prepared and fed to 
congenic affected and unaffected RCS rats in 
controlled experiments. The diets were fed to 
young breeding pairs before they produced 
their first offspring and to their offspring after 
weaning so that the experimental animals 
received their diets from conception to date of 
observation. Clinical findings were recorded 
after indirect ophthalmoscopic and biomicro- 
scopic sHt-lamp examination. Postmortem 
examination of the eye included dissecting 
microscopy and light microscopy of stained 
specimens. At appropriate times, photogra- 
phy was used to record in vitro or in vivo data. 
Analytical methods included standard bio- 
chemical, fluorometric, and separation proce- 
dures. Special environmental lighting condi- 
tions were used to determine histopathological 
effects on the lens and retina. 



60 



Ojfice of the Scientific Director 



In collaborative studies with the National 
Cancer Institute (NCI), the direct acting de- 
oxyribonucleic acid (DNA) alkylating agent 
NMU was injected intiavenously in a dosage 
sufficient to produce cancer of the breast in 
female rats. Experimental rats were given 
chemopreventative agents. "Contiol" rats we 
studied were normals injected with NMU or 
saline; lenses and retinas were examined 
histopathologically. Eyes were fixed in 10 
percent neutral formalin, embedded in plastic, 
sectioned at 2 /xm, and stained with haema- 
toxyUn and eosin. 

Major Findings 

(1) Bilateral mature cataracts in RCS rats. 
Dystiophic RCS rats fed a natural ingredient 
diet and reared from birth at a low-hght 
intensity have a 27 percent incidence of ma- 
ture cataracts (MC) in one year, most of which 
are unilateral. In a study of incidence of 
bilateral MC (BMC) in the RCS rat as a func- 
tion of environmental light intensity, we 
found that at low-Ught intensity (10 to 40 lux) 
only 5 percent of rats had BMC in one year, 
while the remaining eyes had stationary 
cataracts (clear lens fibers between the opacity 
and capsule). In previous studies, when 
reared from birth in 100 lux of constant Ught, 
78 percent of rats has MC, 28 percent of which 
were BMC. Rats exposed to higher intensities 
of Ught starting at 22 to 28 postnatal days 
(when debris containing rhodopsin and poly- 
unsaturated fatty acids is maximal) has a 
higher incidence of BMC: at 270 lux of con- 
stant Ught, 70 percent of rats had MC, 70 
percent of which were BMC; and at 7500 lux 
for 48 hours, 100 percent of rats had MC, 100 
percent BMC. We concluded that rats reared 
in 10 to 40 lux cycUc Ught had DNA damage 
that was repaired in the majority of lenses, 
and BMC were rare. In constant Ught of 
higher intensity, repair did not occur and 
most or aU rats had BMC. The results support 
the hypothesis of cataractogenesis by toxic 
aldehydes from peroxidized retina Upids, 
damaged by singlet oxygen generated by the 
sensitizer retinaldhyde. Such aldehydes 
(detected in the vitreous) can damage pro- 
teins, Upids, and nucleic adds. Occurrence of 



disturbed meridional rows of lens epitheUal 
ceUs and posterior nucleated (Wedl) cells 
pointed to proliferation of germinative zone 
epitheUal lens ceUs, possibly from DNA dam- 
age. 

These results are consistent with a hypoth- 
esis of PSC causation by DNA damage to lens 
epitheUal ceUs in the germinative zone. 
Agents able to have this effect include prod- 
ucts or peroxidation of polyunsaturated fatty 
acids (abundant in retina rod outer segments 
and synapses), short wavelength radiation 
(ultraviolet, x-ray, P, and y rays), and numer- 
ous chemical mutagenic agents such as NMU. 

(2) BMC in NMU-injected normal albino 
rats. Examination of the "control" NMU-inject- 
ed normal rats, as compared with saline- 
injected rats, revealed BMC at five months of 
age in the NMU-injected rats. This histopath- 
ological appearance of the cataracts was that 
of posterior subcapsular cataracts of a severe 
nature with abnormaUy large ceUs of Wendl 
not only at the posterior pole but encircling 
the lens; some ceUs were binucleate. The 
retina showed advanced generate changes. 

(3) Antioxidant diets in RCS rats. Antioxi- 
dant diets that prevent the cataracts in pink- 
eyed RCS dystrophic rats have the effect of 
retarding retinal degeneration. None of the 
diets we have tried stops the degeneration, 
but, when a certain degree of retardation is 
achieved, the lens is protected. Last year we 
increased the concentiation of vitamin E in the 
AIN-76 purified diet that already had been 
supplemented with 0.4 percent p-carotene 
plus 0.01 percent of BHT (to prevent oxidation 
of carotene in the cage hopper) as well as 1000 
mg/Kg vitamin C and 150 mg/Kg vitamin E. 
We did not succeed in showing any additional 
retardation of the retinal degeneration (beyond 
55 days), perhaps because apoptosis occurs in 
this rat strain. 

Proposed Course 

As pink-eyed, tan-hooded dystrophic breeders 
become available from the National Institutes 
of Health (NIH) Foundation Colony, lens 



61 



FY 1994 NEI Annual Report 



epithelial cell whole mounts will be prepared 
to compare the numbers of cells in lenses with 
and without cataract. 

Collaborative studies of the mutagen 
NMU as a cataractogenic agent will continue. 
Principles learned from chemoprevention in 
rats with breast and prostate cancer would 
involve using dual agents that can (1) halt or 
slow multiplication of lens epitheUal cells and 
(2) cause differentiation into lens fibers. 

Collaboration to find the mutant autoso- 
mal recessive rdy gene on chromosome 3 of 
the RCS rat has been investigated. Until now, 
this study has not been feasible because the 
rat genome had not been well studied. How- 
ever, knowledge of the rat genome has been 
proceeding apace. A molecular biologist in 
NCRR, VRP, where "fingerprinting" of some of 
the strains of rats in the Foundation Colonies 
(including RCS rats) is being pursued, may be 
interested in this gene, which appears to be 
involved in RPE phagocytosis. 



NEI Research Program 

Lens and Cataract — Pathogenesis of Cataract 

Retinal Diseases — Retinitis Pigmentosa and 
Other Inherited Disorders 

Publications 

Hess HH: Incidence of bilateral mature cata- 
racts in Royal College of Surgeons (RCS) rats 
and environmental Ught stress. Invest Ophthal- 
mol Vis Sci 35(suppl):2137, 1994. 



62 



Laboratory of Immunology 



Report of the Chief 
Laboratory of Immunology 



Robert B. Nussenblatt, M.D. 



The Laboratory of Immunology (LI) has 
finished its eighth year. The sections 
of the laboratory include: the Immu- 
nology and Virology Section headed by Dr. 
John J. Hooks; the Section on Experimental 
Immunology headed by Dr. Igal Gery, who is 
also the deputy head of the Laboratory; the 
Section of Immunoregvdation headed by Dr. 
Rachel Caspi; the Section on Experimental 
Immunopathology headed by Dr. Chi-Chao 
Chan; the Section on Clinical Immunology 
whose acting head is Dr. Marc de Smet; and 
the Section on Molecular Biology headed by 
myself as acting head of an interdisciplinary 
group. 



Section on Clinical Immunology 

The Section on Clinical Immunology has 
continued to focus on major areas of clinical 
relevance, including new interventional stud- 
ies. The section has continued its study of 
patients with acquired immunodeficiency 
sjmdrome (AIDS) in collaboration with the 
National Institute of AUergy and Infectious 
Diseases (NIAID). A randomized study to 
evaluate a slow-release implant filled with 
ganciclovir was tested in AIDS patients with 
cytomegalovirus (CMV) retinitis. These im- 
plants are placed directly into the eye through 
the pars plana. They have been calibrated to 
release therapeutically effective doses of 
ganciclovir over an eight-month period. 
Recruitment for this study has come to an 
end, and the results wiU be tabulated and 



reported in the near future. This randomized 
study may yield important information about 
a new alternative to systemic anti-CMV thera- 
py for patients who cannot tolerate intrave- 
nous therapy or those who do not wish to be 
treated with systemic therapy. The potential 
for a marked improvement in quality of life in 
these patients is a serious consideration. 
Pediatric AIDS patients continue to be seen 
and evaluated for the incidence of ocular 
infection. This study is done in conjunction 
with Dr. Phihp Pizzo and the National Cancer 
Institute (NCI). 

The group has been extremely active in 
the development of new ways in which the 
ocular immune response can be interfered 
with. This has included the study of the effect 
of NPC- 15669, an inhibitor of neutrophil 
recruitment in uveitis. It was found that the 
injection of this compound into rats wiU 
significantly reduce the severity of endotoxin- 
induced uveitis (EIU), a disease that is mediat- 
ed mainly by pol5anorphonuclear cells as well 
as macrophages. Of great interest as well was 
that treatment of rats late in the process of 
experimental autoimmune uveitis (EAU), a 
disease thought to be mainly mediated by 
T-ceUs, could also have a marked inhibition of 
the evolution of their disease. Of great poten- 
tial interest as a therapeutic mode in the near 
future is the effectiveness of humanized 
anti-interleukin (IL)-2 receptor antibodies in 
the treatment of autoimmune uveoretinitis in 
monkeys. This work has been performed in 
collaboration with Dr. Thomas Waldmann of 
the NCI as well as Drs. James Raber and 



65 



FY 1994 NEI Annual Report 



Martin Kriete, from the Veterinary Research 
and Resources Section, National Eye Institute 
(NEI). The humanized antitact antibody is an 
anti-IL-2 receptor antibody originally pro- 
duced in mice but has been modified by 
replacing all but its binding region with hu- 
man immunoglobulin elements. Cynomolgus 
monkeys induced with EAU were treated with 
this antibody; the disease was markedly 
limited in the group treated with the antitact 
humanized antibody. These findings are in 
marked contradistinction to the inflammation 
that was seen in control groups. This work 
has great significance because of the implica- 
tions for the potential for human therapeutic 
studies to occur in the not-too-distant future. 
From a basic scientific point of view, these 
studies also implicate the potential role of IL- 
15 m this ongoing process. 



Section on Molecular Biology 

This new interdisciplinary group, the Section 
on Molecular Biology, has been in existence 
now for more than two years. The attempt is 
to better understand approaches to gene 
therapy, whether they be local or systemic in 
nature. The group has had a long-term inter- 
est in focusing on the regulation of the 
omithine-6-aminotransferase (OAT) gene. 
Work continued in the development of a 
knockout gene model for this disorder. Addi- 
tionally, interactions with a variety of individ- 
uals, including those from Johns Hopkins 
University and the State University of New 
Yf "k at Stony Brook, have begun to develop 
re ±ods by which therapy for gyrate atrophy 
could be introduced through skin cells. Work 
by this group has also emphasized studying 
direct ocular gene transfer using ElA deficient 
adenovirus constructs. These have been used 
to transfer genes into isolated retinal pigment 
epithelial cells (RPE) followed then by trans- 
plantation of these RPE cells. A mutant of 
transforming growth factor beta (TGF-P) has 
been placed into the adenovirus construct. It 
appears from early experiments that the RPE 
cells are capable of sustaining replication of 
ElA deficient adenovirus. 



Section on Immunoregulation 

The Section on Immunoregulation has main- 
tained its great interest in the development 
and study of animal diseases of experimental 
auto-ocular autoimmune disease. One aspect 
of the work has been to characterize further 
murine EAU because the mouse model offers 
quite important differences from other rodent 
models of uveitis. The section found a possi- 
ble correlation between the pathogenicity of 
autoimmune T-ceUs and their lymphokine 
production expression of functional adhesion 
molecules as well as the expression of some 
surface antigens in the examination of the 
Lewis rat model for uveitis. Antigen-specific 
Lewis rat T-ceU lines and sublines have been 
developed; one is specific for the major patho- 
genic epitope on the human retinal soluble 
S-antigen (S-Ag) and three are specific to the 
major pathogenic epitopes of bovine inter- 
photoreceptor retinoid-bin ding protein (IRBP). 
Though these lines have different degrees of 
uveitogenicity, the four T-ceU lines produce 
roughly equivalent amounts of interferon 
gamma (IFN-y) tumor necrosis factor, IL-3, IL- 
6, and TGF-p. 

Of interest, IL-4 cannot be detected. 
Similarly, there are essentially equal amounts 
of functional adhesion molecules being ex- 
pressed; however, the nonpathogenic subline 
was the poorest responder to antigen stimula- 
tion with respect to proliferation in IL-2 pro- 
duction. The nonpathogenic subline wiU 
show almost no expression of CD-4. These 
results would support the contention that class 
2 restricted recognition of autoantigens within 
the neuroretina by uveitogenic T-lymphocytes 
must occur as an initial step in the induction 
of experimental uveitis. Therefore, a defect in 
this step wiU preclude marked uveitogenicity 
of these cells. C5^okine genes within the eye 
in murine experimental uveitis have been 
studied. T-cells that have been specifically 
grown in the presence of the retinal protein 
IRBP or to its peptides can induce uveitis with 
adoptive transfer. These cell lines show an 
unrestricted cytokine profile in vitro. Looking 
at messenger ribonucleic add (mRNA) pro- 



66 



Laboratoiy of Immunolog]/ 



duction, a TH-1 type cytokine profile (IL-2, 
IFN-v, and tumor necrosis factor alpha 
[TNFa], was present in the eyes of mice that 
had EAU induced with the transfer of these 
cells Unes. These results suggest that these 
lymphokines are important for the induction 
of uveitis and that a lack of IL-4 mRNA in the 
eyes of mice that had experimental uveitis 
would argue that predominately TH-1 type 
cells were present. Additionally, the group 
has identified a major pathogenic epitope in 
the IRBP molecule that is recognized by mice 
of the H2R haplotype. 

Another approach of suppressing ocular 
autoimmunity was through the induction of 
oral tolerance. EAU susceptible B-IO.A mice 
were fed IRBP or a control solution. Results 
indicated that three feedings of 0.2 mL of 
IRBP every other day before immunization 
did not protect mice against this form of 
uveitis, whereas a similar regimen of five 
doses was in fact protective. However, of 
interest was supplementing the nonprotective 
three times regimen with one intraperitoneal 
administration of recombinant human (rHum) 
IL-2 resulted in disease suppression that was 
equal to that of the protective feeding regi- 
men. Analysis of the Peyer's patch cells of fed 
mice showed a large increase in the produc- 
tion of TGF-P, IL-4, and IL-10 in those animals 
that were fed IRBP and received IL-2, as 
compared with those animals that only re- 
ceived IRBP feedings. The group would 
propose that IL-2 treatment enhances the 
protection from experimental uveitis by stimu- 
lating regulatory cells that ultimately produce 
cytokines such as TGF-P, IL-4, and IL-10. This 
interest in immunosuppression oral tolerance 
has also resulted in an ongoing randomized 
masked study to look at the effectiveness of 
oral tolerization. This study continues to be in 
progress and will evaluate the usefulness of 
S-Ag oral adnvinistiation in the induction of 
tolerance in uveitis patients. It is hoped that 
this study will be completed in the next year. 



Section on Experimental 
Immunology 

The Section on Experimental Immunology has 
continued its long-term investigation of the 
pathogenesis of inflammatory eye diseases. In 
this ongoing interest, they reported the uveito- 
gerucity of recoverin this year. Recombinant 
recoverin was found to be highly uveitogenic 
in Lewis rats, inducing a severe experimental 
uveitis at doses as low as 10 /xg per rat. The 
clinical and histopathologic changes induced 
by recoverin were very similar to those that 
one sees with disease induced by the retinal 
S-Ag. Of great clinical interest is that recover- 
in has been suggested by many to be the 
antigen to which antibodies are developed in 
carcinoma-associated retinopathy syndrome. 
Work by the group has continued to look at 
the uveitogenicity and antigenicity of rHums- 
Ag in primates. Monkeys of three species 
have been immunized with recombinant S-Ag 
molecules. 

The ocular changes observed closely 
resemble those seen in previous studies of 
monkeys immunized with bovine S-Ag. 
Additionally, lymphocyte proliferative re- 
sponses were quite similar to those previously 
seen. Transgeruc mice were also evaluated by 
this group during the year. Transgenic mice 
that expressed foreign antigens in their lens 
were developed by collaborators at the Labo- 
ratory of Molecular and Developmental Biolo- 
gy (LMDB), NEI. These foreign antigens were 
expressed under control of the aA-crystalline 
promoter. Development of immunotolerance 
in transgenic mice was examined by measur- 
ing their capacity to mount specific immune 
responses following immunization with a 
corresponding antigen, which was emulsified 
in agevin. The main finding showed that the 
expression of chloramphenicol acetyltiansfer- 
ase (CAT) in the lens had littie effect on the 



67 



FY 1994 NEl Annual Report 



capacity of the transgenic nnice to respond 
against this antigen. Of interest was that in 
contrast to the findings with CAT, expression 
of human fibroblasts (HEL) in the lens pro- 
duced a state of complete tolerance to this 
antigen. HEL transgenic mice failed to devel- 
op any detectable antibody or ceUular immune 
response against this antigen. 

The group has also explored the phenome- 
non of oral tolerance by looking at the oral 
administration of the bacterial product 
N-acetyl-D-glucosaminyl-|3 (l-4)-N-acetyl-L- 
muranyl-L-alanyl-D-isoglutamine (GMDP) . 
When GMDP was given along with the uveit- 
ogenic peptide, it significantiy enhanced the 
level of tolerance induced by the peptide. The 
role of CD-8 positive lymphocytes in the 
process that produces oral tolerance was also 
examined by testing whether mice deficient of 
these cells were capable of developing oral 
tolerance. The CD-8 deficient animals used 
were p-2 m-mice. These mice then have very 
low expression of major histocompatibility 
complex (MHC) class 1 molecules and a 
severe deficiency of CD-8 positive cells. 
Feeding these mice with ovalbumin produced 
remarkable levels of immunotolerance that 
closely resembled those observed in the simi- 
larly treated control animals. This finding 
thus indicated that CD-8 positive cells are not 
essential for the induction of oral tolerance, at 
least when induced by the procedure used in 
this study. 

Further work by this group looking at 
transgenic models has evaluated the effects of 
IFN-Y on the physiology of the eye and the 
role of elevated MHC class 2 in the eye. Both 
transgenic rat and mice strains were generated 
by microinjection of deoxyribonucleic acid 
firagment containing a murine aA-crystalline 
promoter, which was then fused to the coding 
sequence of murine IFN-y gene. In both the 
rat and mouse models, ectopic expression of 
IFN-Y in the lens affecting the growth of the 
w^hole eye resulted in cataract thickening of 
the anterior lens capsule, rupture of posterior 
capsule, impairment of the lens fiber forma- 
tion as well as microphthalmia and micropha- 
kia. 



The group has an ongoing interest in the 
role of the T-ceU receptor and how it relates to 
autoimmune intraocular inflammatory disease. 
The goal is to develop anti-T-ceU receptor 
therapies for the treatment of uveitis. In 
doing this, athymic as well as euthymic rats 
were injected with cells from antigen-stimulat- 
ed T-ceU lines specific for a major pathogenic 
epitope for bovine IRBP. The findings suggest 
that the time of appearance of cytokine-pro- 
ducing T-cells in the retina is influenced by 
the immunologic status of the rat, the differ- 
ence in circulating cytokines in athymic rats 
might affect parameters such as vascular 
permeability and could facilitate penetration 
of T-ceUs into the eye. Additionally, the 
stiong TH-1 Uke cytokine profile was detected 
in R-16 rats, but this was not detected in 
AK-16 rats. This might suggest that the 
cytokine profile observed in the refina is 
influenced by the identity of the pathogenic 
epitope. 

The Clinical Branch, in collaboration with 
the Experimental Immunology Section, has 
looked at a variety of immunologic questions 
as they relate to ocular disease. A continuing 
interest in ceU adhesion molecules has 5delded 
new information. The effective treatment with 
the monoclonal antibody directed against 
lymphocyte function-associated antigen (LFA- 
1) and very late activation antigen (VLA)-4 
demonstrates that the anti-LFAl antibodies 
sigruficantiy inhibited the development of EIU. 
This was in contrast to the anti-VLA-4 anti- 
body that had no effect on the development of 
intraocular inflammation. They have also seen 
that systemic treatment with anti-IL-12 mono- 
clonal antibodies exacerbates the development 
of EIU. These findings were similar to previ- 
ous studies with anti-IFN-y antibody. They 
have also noted that topical heparin signifi- 
cantiy inhibited the development of allergic 
conjunctivitis in mice. 

This group has also been very active in 
the evaluation of uveitis in patients. The 
group has been involved in a number of 
studies designed to improve the treatment of 
uveitis. They have recentiy completed a 
prospective, double-masked randomized study 



68 



Laboratory of Immunology 



of diamox for uveitis cystoid macular edema. 
They have also completed a pUot study exam- 
ining the efficacy and toxicity of a chemother- 
apy regimen for therapy of patients with 
central nervous system (CNS) lymphoma 
involving the brain or eye. This has been 
done in collaboration with the Medicine 
Branch of NCI. A clinical trial is also under 
way evaluating heparin surface-modified 
intraocular lens in patients with uveitis. 



Section on Experimental 
immunopathology 

The Section on Experimental Immunopatholo- 
gy has continued to study the immunopathol- 
ogy of various inflammatory cells and ocular 
resident cells in a variety of experimental 
models of uveitis. This group has developed 
a particular expertise in in\munohistochemis- 
try and in situ hybridization techniques that 
have provided the whole laboratory with the 
ability to identify and topographically localize 
immunocompetent cells. 

Additionally, it analyzes the alteration of 
surface markers on ocular resident cells and 
the production of cytokines in experimental 
uveitic models as well as in tissue obtained 
from patients undergoing surgery. They have 
demonstrated that higher levels of S-Ag and 
its mRNA are expressed in nonretinal ocular 
cells such as the lens, the ciliary body, and the 
trabecular meshwork of EAU rats. This was 
particularly so in animals that received 
long-term steroid therapy. The group has also 
evaluated several new experimental models 
for uveitis. Experimental melanin induced 
uveitis (EMIU) can be induced with immuni- 
zation using bovine choroidal and RPE mela- 
run protein. EMIU is characterized by a 
bilateral recurrent iris, scleritis, and choroidi- 
tis. The main infiltrating cells in this model 
are T-cells of CD-4 origin seen in the early 
stage of the disease and CD-8 positive cells 
that infiltrate at later stages. 

There is an abundant expression of adhe- 
sion molecules and MHC class 2 antigens on 



the ocular resident cells one to two days 
before ocular inflammation is noted. Of 
interest in this disorder is the fact that recur- 
rences occur one month after the first attack 
becomes quiescent. The group has continued 
its interest in evaluating an animal model for 
acquired ocular toxoplasmosis. This is done 
by the infection of a virulent strain of Toxo- 
plasma gondii (ME49) into mice. Focal ocular 
inflammation as well as RPE involvement are 
seen about two weeks after the infection. 
About one month after infection, ocular in- 
flammation becomes stable and only occasion- 
al cysts can be seen. The group evaluated 12 
cases of intraocular lymphoma diagnosed at 
the NEI between 1984 and 1992. These were 
aU non-Hodgkin's large B-ceU lymphomas of 
the CNS. The prompt appropriate handling of 
specimens and the review by an experienced 
cytopathologist have been shown to be excep- 
tionally critical to the diagnosis of intraocular 
lymphoma. They have also evaluated three 
affected members of a Chinese-American 
family with Bietti's crystalline retinopathy. 
Crystalline lysosomal materials are observed 
in lymphoc5^es and skin fibroblasts of these 
patients. 



Section on Immunology and 
Virology 

The Section on Immunology and Virology has 
continued to emphasize its interest in the 
study of the RPE cells. This section has devel- 
oped a new method using RPE choroidal 
explants to initiate cell growth. By monitoring 
the clusters of cells growing around the ex- 
plants, they were able to select purely epitheU- 
al cells and discard the nonepitheUal cells at 
the primary culture stage. Using this tech- 
nique, they have established primary cell lines 
of human RPE from cells of elderly patients. 
The cell culture origin was confirmed by 
immunochemical staining for cytokeratin with 
monoclonal antibodies. Human RPE cultures 
secrete significant quantities of IL-6 and inter- 
cellular adhesion molecule 1 (ICAM-1) but no 
IL-1 in response to stimulation by inflammato- 
ry mediators. 



69 



FY 1994 NEI Annual Report 



These observations are important in un- 
derstanding posterior uveitis that may be 
caused by infections or an autoimmune pro- 
cess. Lymphocytes and macrophages infiltrate 
into the retina and secrete cytokines such as 
IL-1, TNFa, IFN-Y, and IL-2 that would initi- 
ate immune reactions. In response to these 
cytokines, the retinal resident cells could 
locally produce lL-6 as well as ICAM-1 to 
amplify the rmmunopathologic process. The 
group has continued its interest in the field of 
ocular toxoplasmosis. They were able to 
demonstrate 



in this model that 100 percent of mice develop 
cysts in the brain, but retinal cysts could be 
found late in the course of the disease. This 
section has also developed polymerase chain 
reaction techniques for the detection of a 
variety of viruses in the eye. This ability will 
be applied in the future for the evaluation of 
intraocular samples for the presence of a 
variety of viruses that are thought to be path- 
ogenic in the eye. 



70 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00280-03 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) 

Transgenic Rat and Mouse Models for the Study of Intraocular Effects of IFN-y and Autoimmunity 



PRINCIPAL INVESTIGATOR (List other professiortal personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 
PI: Charles E. Egwuagu Ph.D., M.P.H. Senior Scientist LI, NEI 

Otheis: Robert B. Nussenblatt 

Chi-Chao Chan 
Ana B. Chepelinsky 



Jorge Sztein 
Rashid Mahdi 



M.D. 


Scientific Director 




NEI 


M.D. 


Head, Section on Immi 


nology 


U, NEI 


Ph.D. 


Head, Section on 
Regulation of Gene 
Expression 




LMDB, NEI 


D.V.M., Ph.D. 


Visiting Associate 




U, NEI 


B.S. 


Biologist 




U, NEI 



COOPERATING UNITS (if any) 



Laboratory of Immunology 



SECTION 

Section on Experimental Immunology 



INSTITUTE AND LOCATION 

NEI, Nm, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL: 



1.2 



1.2 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues \x\ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

To Study the possible role of interferon (IF^^)-Y in ocular pathogenesis and, specifically, the linkage between its induction 
of aberrant major histocompatibility complex (MHC) class II expression and predisposition to ocular autoimmune 
diseases, we generated transgenic mice with constitutive expression of IFN-7 in the eye. Although the mouse has. up 
to this point proven to be a useful model, the rat is the preferred strain for the study of experimental autoimmune 
uveoretinitis (EAU). EAU is an animal disease that shares essential features with several human uveitic diseases such 
as sympathetic ophthalmia, birdshot retinochoriodopathy, Behget's disease, and Vogt-Koyanagi-Harada (VKH) syndrome. 
Consequently, we have generated a IFN-7 transgenic Sprague Dawley rat strain. In FY 1993-1994 we focused on 
characterizing both the rat and mouse IFN-7 transgenic models with the goal of establishing a comprehensive and 
complementary transgenic animal system that would be useful for studying the in vivo effects of IFN-y in the eye. 



71 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The objectives of this project include the study 
of the possible role of interferon gamma (IFN- 
Y) in the eye. Aberrant expression of the 
major histocompatibihty complex (MHC) class 
n molecules is an early event in a number of 
human autoimmune diseases, and IFN-y 
induces high levels of MHC class II protein 
biosynthesis. Therefore, we generated trans- 
genic animals with selective secretion of IFN-7 
in their eye tissues. These transgenic mice 
and rats are ideally suited for studying the 
effects of IFN-7 on the physiology of the eye 
and the role of elevated MHC class II predis- 
position to intraocular autoimmune diseases. 

Methods 

Transgenic rat and mouse strains were gener- 
ated by microinjection of a deoxyribonucleic 
add fragment containing the murine aA-crys- 
taUin promoter (aACry) fused to the coding 
sequence of murine IFN-y gene. Polymerase 
chain reaction (PCR) and reversed transcrip- 
tion-PCR (RT/PCR) were used to screen for 
the presence of the transgene and conduct 
messenger ribonucleic acid (mRNA) analyses, 
respectively. Methacrylate-embedded eye 
sections were analyzed for morphology and 
cryosections for immunoperoxidase antibody 
staining. 

Major Findings 

In both rat and mouse models, ectopic expres- 
sion of IFN-y in the lens affected the growth 
of the whole eye, resulting in cataract, thicken- 
ing of anterior lens capsule, rupture of posteri- 
or capsule, impairment of lens fiber formation, 
microphthalmia, and microphakia. Additional 
effects in the mouse include blepharophimosis, 
arrest of retinal differentiation, serous retinal 
detachment with presence of macrophages in 
the subretinal space, persistent hyperplastic 
primary vitreous, and corneal vascularization. 
Unlike the mouse, the rat anterior chamber is 
well formed, and the retina is intact with focal 



retinal serous detachment. MHC class 11 
mRNA levels were significantly increased in 
the transgenic mouse eyes, and MHC class 11 
proteins were expressed in their corneas, 
irises, cihary bodies, choroids, lens, and retinal 
pigment epthelia. At the molecular level, the 
pattern of lens gene expression was perturbed, 
and expression of gene coding for adhesion 
molecules and IFN-y-inducible transcription 
factor was up-regulated in transgenic eyes. 

Significance to Biomedical Researcti and ttie 
Program of ttie Institute 

The aACry /IFN-y transgeruc rat is the first 
transgenic rat strain generated for vision 
research. Constitutive expression of IFN-y, 
and its induction of MHC class II molecules in 
the eye, provides a useful model to address: 
(1) the linkage between aberrant MHC class 11 
expression and predisposition to autoimmuni- 
ty, (2) the role of IFN-y in the treatment of 
inflammatory eye diseases and in ACAID, and 
(3) understanding cytokine signaling during 
embryonic eye development. The rat and 
mouse models complement each other for 
elucidation of the in vivo effects of IFN-y in 
the eye. 

Proposed Course 

We intend to continue studying the molecular 
basis of IFN-y actions in the eye with particu- 
lar emphasis on the rat. A major focus will be 
to estabUsh primary and long-term cultures of 
IFN-y-expressing epithelial lens cells as these 
cell lines would be valuable in studies aimed 
at understanding the mechanism of transcrip- 
tional activation in the aACry-IFN-y animals. 

NEI Research Program 

Retinal Diseases — ^Inflammatory Diseases 

Publications 

Egwuagu CE, Sztein J, Reid W, Chan C-C, 
Mahdi R, Nussenblatt RB, Chepehnsky AB: 
Gamma interferon expression disrupts lens 



72 



Laboratory of Immunology 



and retinal differentiation in bransgenic mice. 
Dev Biol, in press. 

Egwuagu CE, Sztein J, Reid W, Chan C-C, 
Mahdi R, Nussenblatt RB, Chepelinsky AB: 
Transgenic rat and mouse models for stii dying 
the role of gamma interferon and MHC Class 
n in intiaocular diseases and autoimmunity, 
in Nussenblatt RB, Gery 1 (eds). Sixth Interna- 
tional Symposium of the Immunology and Immu- 
nopathology of the Eye. Amsterdam, Nether- 
lands, Elsevier Press, in press. 



Egwuagu CE, Sztein J, Reid W, Chan C-C, 
Mahdi R, Nussenblatt RB, Chepelinsky AB: 
Ectopic expression of gamma interferon in the 
eyes of transgenic mice induces ocular pathol- 
ogy and MHC class II gene expression. Invest 
Opthalmol Vis Sci 35:332-341, 1994. 

Egwuagu CE, Sztein J, Reid W, Chan C-C, 
Mahdi R, Nussenblatt RB, Chepehnsky AB: 
Transgenic rat and mouse models for the 
study of intraocular effects of IFN-y and 
autoimmunity. Invest Opthalmol Vis Sci 35/4 
(suppl):3391, 1994. 



73 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00262-05 LI 



1 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Analysis of T Lymphocytes and Cytokines Involved in Experimental Autoimmune Uveoretinitis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: Charles E. Egwuagu 

Others: Igal Gery 

Robert B. Nussenblatt 
Rachel Caspi 
Rashid Mahdi 
Alexanda T. Kozhich 
Phyllis B. Silver 



Ph.D., M.P.H. Senior Research Scientist 
Ph.D. Head, Section on 

Experimental Immunology 
M.D. Scientific Director 
Ph.D. Visiting Associate 
B.S. Biologist 
Ph.D. Visiting Fellow 
B.S. Biolog ist 



LI, NEI 
LI, NEI 

NEI 
LI, NEI 
LI, NEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Experimental Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.7 



PROFESSIONAL: 



0.7 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease that serves as a model 
of human intra-ocular inflammatory diseases (uveitis). It is initiated in susceptible animals by immunization 
with retinal antigens such as interphotoreceptor retinoid-binding protein (IRBP) or S-antigen (S-Ag) or by 
adoptive transfer of activated S-Ag- or IRBP-specific uveitogenic T lymphocytes. We had previously 
demonstrated that VpS-expressing T cells accumulate in the retina during EAU. In FY 1993-1994, we sought 
to define the T-cell subsets and cytokines present in the retina of athymic and euthymic Lewis rats after 
adoptive transfer of uveitogenic or nonuveitogenic T lymphocytes. Our results indicate that (1) the temporal 
appearance of cytokine-producing T cells in the retina is influenced by the immunological status of the rat and 
might affect parameters such as vascular permeability that influence the penetration of lymphocytes into the 
eye. (2) Cytokine messenger ribonucleic acid (mRNA) transcripts were detected in the retinas of animals 
immunized with uveitogenic T lymphocytes, as well as in the retinas of rats injected with Con A-specific T 
cells. However, rats injected with Con A-specific T cells neither developed EAU nor was there detection of 
VpS* T cells in their retinas. (3) Detection of interferon (IFN)-y transcripts was temporally correlated with 
the appearance of VpS"^ T cells in the retina and the onset of disease. Taken together, our data suggest that 
infiltration of the retina by activated T-cells is not sufficient for disease induction; ocular-antigen specific Thl- 
like VpS^ lymphocytes appear to be necessary for EAU induction. 



74 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



Project Description 

Objectives 

This project is aimed at determining the clo- 
nality of the T lymphocytes that mediate 
intraocular autoimmune diseases. Identifica- 
tion of the pathogenic T-cell subset in experi- 
mental autoimmune uveoretinitis (EAU) is 
relevant to our goal of developing anti-T-ceU 
receptor (TCR) therapies for the treatment of 
uveitis. Our effort during fiscal year 
1993-1994 focused on analyses of T cells pres- 
ent at the autoimmune site and the lympho- 
kines that they produce. 

Methods 

Athymic and euthymic rats were injected with 
cells from an antigen (Ag)-stimulated T-ceU 
line specific to the major pathogenic epitope of 
bovine interphotoreceptor retinoid-binding 
protein (IRBP) (peptide R16: amino acids 
1177-1191; R16 rats) or with concanavalin A 
(ConA)-stimulated splenocytes (ConA rats). 
In a separate experiment, euthymic rats were 
injected with an Ag-stimulated T-cell line 
specific to the pathogenic epitope of rat IRBP 
(peptide AK16: amino acids 273-283; AK16 
rats) or with purified proton derivative 
(PPD)-stimulated primed lymph node cells 
(PPD rats). Retinas were sampled every 12 
hours after uveitogervic challenge and reversed 
transcription polymerase chain reaction 
(RT/PCR) was used to analyze messenger 
ribonucleic acid (mRNA) levels for TCR VpS, 
interferon gamma (IFN-y), tumor necrosis 
factor alpha (TNF-a), interleukin (IL)-2, IL-6, 
CD-4, and CD-8. 

Major Findings 

(1) Analyses of cytokine mRNAs in retinas of 
athymic and euthymic ConA and R16 rats 
showed that in athymic rats IFN-y, TNF-a, IL- 
2, IL-6, CD-4, and CD-8 mRNAs were detected 
after 12 hours, whereas in the euthymic rats 
they were only detected after 24-84 hours. 
Retinas of ConA rats showed essentially the 
same cytokine profile as retinas of R16 rats. 



Cytokine mRNAs were not detected in retinas 
of animals that did not receive cells. 

(2) Analyses of cytokine mRNAs in 
euthymic AK16 and PPD rats showed that 
TNFa, IL-6, CD-4, and CD-8 mRNAs were 
detected after 24 hours, but IFN-y and IL-2 
transcripts were not detectable even after 120 
hours. In contrast to ConA rats, cytokine 
mRNA expression was not detected in retinas 
of PPD rats. (3) In aU experiments, the ap- 
pearance of Vp8* T cells in the retina coincid- 
ed with the temporal expression of IFN-y in 
the retinas of rats with experimental autoim- 
mune uveitis (EAU). A similar correlation 
was not observed for any of the other cyto- 
kines. 

Significance to Biomedical Research and the 
Program of the institute 

(1) The time of appearance of cytokine-pro- 
ducing T cells in the retina appears to be 
influenced by the immunological status of the 
rat. Differences in levels of circulating cyto- 
kines in athymic rats might affect parameters 
such as vascular permeability and could 
facilitate penetration of T cells into the eye. 

(2) A Th-1-like cytokine profile was 
detected in R16 rats, whereas it was not de- 
tected in AK16 rats. This might suggest that 
the cytokine profile observed in the retina is 
influenced by the identity of the pathogenic 
epitope. 

Proposed Course 

Analyses of uveitogervic T-ceU clonotypes and 
the lymphokines they produce during EAU 
will be continued to identify the relevant 
autoaggressive T cells involved. Our study 
will be expanded to include analyses of speci- 
mens obtained from patients with ocular 
sarcoidosis and anterior and posterior uveitis. 

NEI Research Program 

Retinal Diseases — ^Inflammatory Disorders 



75 



FY 1994 NEI Annual Report 



Publications 

Egwuagu CE, Bahmanyar S, Mahdi R, 
Nussenblatt RB, Gery I, Caspi R: Predominant 
usage of Vp8.3 T cell receptor in a T ceU line 
that induces experimental autoimmune uveo- 
retinitis. Clin Immunol & Immunopathol 65:152, 
1992. 

Egwuagu CE, Caspi R, Mahdi R, Gery I, 
Nussenblatt BR: Evidence for selective accu- 
mulation of Vp8+ T lymphocytes in experi- 
mental autoimmune uveoretinitis induced by 
two different retinal antigens / Immunol 
151:1627, 1993. 



Kozhich AT, Kawano Y, Egwuagu CE, Caspi 
RR, Maturi RK, Berzofsky JA, Gery I: A 
pathogenic autoimmune process targeted at a 
surrogate epitope. / Exp Med, in press 

Mahdi RM, Caspi RR, Kozhich AT, Kozhich 
OA, Silver PB, Nussenblatt RB, Egwuagu CE: 
C3^okine mRNA expression following adop- 
tive transfer of uveitogenic T cells into 
athymic and euthjonic Lewis rats. Invest 
Opthalmol Vis Sci 35/4 (suppl):1432, 1994. 



76 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00069-17 LI 



PERIOD COVERED 



October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Immune Responses to Ocular Antigens 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 



Igal Gery 

Alexander Kozhich 
Eddy Anglade 
Scott M. Whilcup 
Chi-Chao Chan 
Robert B. Nussenblatt 
Barbara Vistjca 
Nathan Felix 
James Lai 
Eric Wawrousek 

Christina M. Sax 



Ph.D. 
Ph.D. 
M.D. 
M.D. 
M.D. 
M.D. 
B.A. 
B.A. 
B.A. 
Ph.D. 

Ph.D. 



Head, Section on Experimental Immunology 

Visiting Fellow 

Senior Staff Fellow 

Associate Clinical Director 

Head, Section of Immunopathology 

Scientific Director 

Microbiologist 

Special Volunteer 

Guest Researcher 

Head, Section on Transgenic Animals and 

Genome Manipulation 

Senior Staff Fellow 



U, NEI 
LI, NEI 
U, NEI 
CB, NEI 
LI, NEI 
LI, NEI 
LI, NEI 
LI, NEI 
LI, NEI 
LMDS, NEI 

LMDB, NEI 



COOPERATING UNITS (if any) 

Biotechnology Unit, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and 
Digestive and Kidney Diseases (Joseph Shiloach, Ph.D.) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Experimental Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892-1858 



TOTAL STAFF YEARS: 



2.8 



PROFESSIONAL: 



2.4 



0.4 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ {a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Targeted at learning about inflammatory eye diseases grouped under the term "uveitis," this project continued to focus 
mainly on learning about ocular antigens capable of inducing experimental autoimmune uveoretinitis (EAU), an animal 
model for uveitis in humans, and procedures that modulate this disease. The major achievements of this project in FY 
1994 include: (1) We discovered that recoverin, a retinal calcium-binding protein, is highly uveitogenic in rats, producing 
severe inflammatory changes in eyes of rats of all four strains tested. In addition to identifying a new uveitogenic retinal 
molecule, this observation provides evidence to support the assumption that recoverin is the target for an autoimmune 
process that causes a condition termed cancer-associated retinopathy (CAR). (2) Recombinant human S-antigen, which 
has become available by recombinant deoxyribonucleic acid (DNA) technologies, was found to be uveitogenic in primates, 
inducing severe inflammatory ocular changes that closely resemble those induced by bovine S-antigen. Lymphocytes from 
the immunized monkeys were used to identify the peptides that are selected as the immunodominant determinants, i.e., 
the ones that are the target for the immune response in animals immunized with the whole protein. Monkeys of different 
species responded against different peptides, whereas four monkeys of the same species (cynomolgus) remarkably 
responded against the same selected peptides. (3) Blood lymphocytes collected at different time points from a human 
donor exhibited consistency in their responding strongly to the same selected peptides of human S-antigen. Yet, marked 
changes were observed in the capacity of individual dominant peptides to stimulate lymphocytes collected at different 
time points. (4) To examine the effect of sequestration on the immunogenicity of lens proteins, transgenic (TG) mice 
were developed in which foreign antigens are selectively expressed in the lens. Two different types of response were 
observed: mice expressing chloramphenicol aminotransferase (CAT) responded to this antigen similarly to their wildtype 
controls, while TG mice expressing hen egg lysozyme (HEL) failed to respond against this antigen, due to a state of 
complete immunotolerance. (5) Oral tolerance, a procedure used to inhibit pathogenic autoimmune processes, was found 
to be enhanced by treating the fed animals with certain bacterial products, with the best effect achieved with glucosaminyl 
muramyl dipeptide (GMDP). (6) The role of CDS lymphocytes in the process of oral tolerance induction was examined 
by testing the capacity of mice deficient in these cells to develop oral tolerance. CDS deficient mice resembled their 
controls in developing tolerance, thus showing that CDS cells are not always essential for induction of oral tolerance. 

77 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

Studies conducted during fiscal year (FY) 1994 
were aimed at the following: (1) to examine 
the uveitogenicity of recoverin, a calcium- 
binding protein specific to the retina, that was 
reported to be the target for autoantibodies 
detected in the majority of cases with cancer- 
associated retinopathy (CAR); (2) to test the 
uveitogenicity of recombinant human S-anti- 
gen (rHumS-Ag) in primates and to identify 
the peptide determinants that are selected as 
the immunodominant epitopes by the immune 
system of the immunized monkeys; (3) to 
monitor at different timepoints the changes 
that may occur among the subpopulations of 
human blood Ij^nphocytes that respond 
against different epitopes on HumS-Ag; (4) to 
investigate the level of sequestration of pro- 
teins within the lens by testing the develop- 
ment of immunotolerance toward foreign 
antigens expressed in the lens of transgenic 
mice (expression of foreign proteins in other 
organs usually produces tolerance); (5) to test 
the capacity of bacterial products to enhance 
oral tolerance, a procedure that is extensively 
used to inhibit pathogenic autoimmune pro- 
cesses, including uveitis in man. In the sys- 
tem used here, feeding with S-Ag is used to 
inhibit the development of experimental 
autoimmune uveoretinitis (EAU) in rats; and 
(6) to investigate the role of CD-8 lymphocytes 
in the process that brings about oral tolerance 
by testing the capacity of CD-8 deficient mice 
to develop oral tolerance. 

Methods 

Recombinant recoverin was kindly provided 
by Dr. L. Stryer, from Stanford University; 
rHumS-Ag was prepared by Dr. Joseph 
Shiloach, from the National Institute of Diabe- 
tes and Digestive and Kidney Diseases 
(NIDDK), as described in our Annual Report, 
FY 1993; peptides were sjnithesized and puri- 
fied by Applied Biosystem, Foster City, Cali- 
fornia. Monkeys of different species (rhesus, 
artcoides, and cjmomolgus) were provided by 



the National Institutes of Health (NIH) animal 
facility, and R2 m -/- mice (CD-8 deficient) 
were provided by the NCI Twinbrook Facility. 
Blood samples were collected at different 
timepoints from a single donor, and the 
mononuclear leukocytes were separated on 
Isolymph gradients. Published conventional 
methods were used to immunize animals and 
measure their immune response as weU as 
development of EAU. Adoptive fransfer of 
EAU was carried out as described by 
Mochizuki et al. {Invest Ophthalmol Vis Sci 26:1, 
1985). Levels of chloramphenicol acetyltrans- 
ferase (CAT) were measured by a biphase 
CAT assay, and those of human fibroblasts 
(HEL) were determined by the particle con- 
cenfration fluorescence immunoassay. 

Major Findings 

Uveitogenicity of recoverin. Recombinant re- 
coverin was found to be highly uveitogenic in 
Lewis rats, inducing severe EAU at doses as 
low as 10 fig per rat. The clinical and histo- 
pathological changes induced by recoverin 
closely resemble those elicited in rats by S-Ag, 
reaching in many cases the maximum grade of 
severity of 4+. Recoverin is also found in the 
pineal gland, and most rats with EAU also 
developed pineal inflammation. In addition to 
Lewis rats, recoverin was found to induce 
EAU in three other rat sfrains — ^BN, WF, and 
ACL Similarly to observations with other 
uveitogenic antigens, however, the severity of 
changes in these strains was lower than in 
Lewis rats. EAU induced by recoverin was 
found to be ceU-mediated; it is readily and 
adoptively fransferred to naive recipients by 
IjTnph node or spleen cells from immunized 
donors. Moreover, antibodies to recoverin 
seem to play a minor role, if any, in the patho- 
genic process because recipients developed 
disease even in the absence of antibodies, as 
seen in rats injected with recoverin-sensitized 
spleen cells that were stimulated in culture 
with Concanavalin A. 

Uveitogenicity and antigenicity ofrHum S-A8 
in primates. Monkeys of three different species 
(artcoides, rhesus, cynomolgus) developed 
uveitis when immunized with rHumS-Ag. 



78 



Laboratory of Immunology 



The ocular changes closely resembled those 
observed in previously published studies in 
eyes of monkeys immunized with bovine S-Ag 
(Nussenblatt RB, et al.. Arch Ophthalmol 
99:1090, 1981). Peripheral blood lymphocytes 
from the immunized monkeys responded well 
against the rHumS-Ag molecule as well as 
against bovine S-Ag. 

The monkey lymphocytes were also tested 
for their proliferative response against 40 
synthetic overlapping peptides that span the 
entire sequence of HumS-Ag. Lymphocytes 
from monkeys of the three tested species 
recognized and responded well against differ- 
ent peptides; a finding that indicates that 
different determinants of HumS-Ag serve as 
the "immunodominant epitopes" for the im- 
mune system of monkeys of different genetic 
makeups. On the other hand, all four cyno- 
molgus monkeys remarkably recognized the 
same immunodominant regions of HumS-Ag 
at sequences 21-61, 71-90, 121-140, 171-200, 
and 281-310. 

Response of human lymphocytes against 
HumS-AS peptide determinants: Specificity 
pattern is conserved. Peripheral blood lympho- 
cytes collected from a single donor at different 
timepoints were used to examine the possible 
changes in the repertoire of responsiveness 
toward HumS-Ag and its 40 overlapping 
peptides. Six blood samples, collected at 
timepoints spanning over six months, were 
tested in the present study. Significant re- 
sponses were found at all timepoints against 
four regions, localized at sequences 71-90, 
121-140, 171-200, and 341-360. Marked varia- 
tions were noted, however, among the six 
blood samples in the level of response to each 
of these four peptides, thus producing differ- 
ences in the hierarchy of their stimulating 
capacity. 

Immune responses in transgenic mice express- 
ing foreign antigens in their lens. Transgenic 
mice that express foreign antigens in their lens 
were developed by collaborators at the LMDB, 
NEl. Mice expressing CAT were developed 
by Dr. Chris Sax, but mouse Unes expressing 
HEL were established by Dr. Eric Wawrousek. 



Both foreign antigens were expressed under 
the control of the aA-crystaUin promoter. 
Sensitive immunological tests (see Methods 
section) showed high concentrations of CAT 
or HEL in the lens, but neither antigen could 
be detected in any other organ or in the blood 
of the transgenic mice. Development of im- 
munotolerance in the transgenic mice was 
examined by measuring their capacity to 
mount specific immune responses following 
immunization with the corresponding antigen, 
emulsified in complete Freund's adjuvant. 
The main findings include: (1) Expression of 
CAT in the lens had Uttie effect on the capaci- 
ty of the transgenic mice to respond against 
this antigen. Transgenic mice immunized 
with CAT produced antibodies to CAT with 
levels similar to those of their wild-type con- 
trols. The transgenic mice also developed 
cellular immune response to CAT, albeit with 
levels sUghtiy lower than those monitored in 
the wild-type controls. (2) In contrast to the 
findings with CAT, expression of HEL in the 
lens produced a state of complete tolerance to 
this antigen; HEL transgenic mice failed to 
develop any detectable antibody or cellular 
immunity against HEL following immuniza- 
tion with this antigen. 

Enhancement of oral tolerance by bacterial 
products. Oral administration of the bacterial 
product N-acetyl-D-glucosaminyl-P (l-4)-N- 
acetyl-L-muramyl-L-alanyl-D-isoglutamine 
(GMDP) along with a uveitogenic peptide 
significantly enhanced the level of tolerance 
induced by the peptide. The study was car- 
ried out in rats in which EAU is induced by 
peptide 1181-1191 of bovine interphotorecep- 
tor retinoid-binding protein (IRBP). Feeding 
with peptide 1181-1191 reduces the disease 
development, and the level of inhibition was 
further eivhanced by cofeeding with GMDP. 
A sUght enhancing effect was also induced by 
bacterial Upopolysaccharide (LPS), but Salmo- 
nella typhimurium mitogen (STM) and the des- 
(N-acetyl-D-glucosaminyl) analog of GMDP 
had no detectable effect in this system. The 
capacity of GMDP to enhance oral tolerance 
was further demonstrated by the finding that 
the cellular immunity to peptide 1181-1191 
was iivhibited remarkably more in rats fed 



79 



FY 1994 NEI Annual Report 



with the combination of this peptide and 
GMDP than in those fed with the peptide 
alone. 

Induction of oral tolerance in CD-8 cell-defi- 
cient mice. The role of OD-S lymphocytes in 
the process that produces oral tolerance was 
examined by testing whether mice deficient of 
these cells are capable of developing oral 
tolerance. The QD-S-deficient animals used 
here were p2m -/- mice, i.e., animals in which 
the disruption of the p2-microglobulin gene 
causes very low expression of MHC class I 
molecules and severe deficiency of CD-S"^ ceUs. 
Feeding these mice with ovalbumin (three or 
five times, 1 mg per mouse) produced remark- 
able levels of immunotolerance that closely 
resembled those observed in the similarly 
treated control animals. This finding indicates 
that the CD-8 cells are not essential for the 
induction of oral tolerance, at least when 
induced by the procedure used in this study. 

Significance to Biomedicai Research and the 
Program of the Institute 

(1) The finding that recoverin is highly 
uveitogenic underscores the unique character- 
istic of the retina, i.e., its content of mvdtiple 
molecules with the capacity of initiating path- 
ogenic autoimmune processes; previous stud- 
ies have identified four other uveitogenic 
proteins in the retina. In addition, the capaci- 
ty of recoverin to initiate a pathogenic autoim- 
mune process supports the notion that this 
protein is the target for the putative autoim- 
mune process that brings about the retinal 
damage in CAR. 

(2) The present study is the first to show 
that HumS-Ag is highly uveitogeruc in pri- 
mates. Moreover, this observation supports 
the notion that autologous S-Ag plays a major 
role in the immunopathogenic process of 
uveitis in man. This study also provides, for 
the first time, information concerning the 
peptide determinants of HumS-Ag that are 
immunodominant in monkeys in which im- 
munization with HumS-Ag produced uveitis. 
As expected, monkeys of different species 
varied in their selection of the dominant 



peptides, but the similarity among the four 
cynomolgus monkeys was quite surprising 
because these animals are outbred. The latter 
observation may suggest that uveitic patients 
with partial identity of histocompatibility anti- 
gens may also exhibit similarity in their selec- 
tion of immunodominant epitopes. (Such an 
observation was made with multiple sclerosis 
patients). 

(3) The study of responsiveness to HumS- 
Ag peptides of a human donor at different 
timepoints has provided information concern- 
ing the fluctuations among lymphocyte clones 
with specificity toward autologous peptides. 
The finding that responses to the same few 
epitopes were observed in aU blood samples 
thus indicates that only small and quantitative 
fluctuations occur among the clones of Ijmi- 
phocj^es that recognize the dominant epitope 
of an autologous antigen. 

(4) The experiments with the transgenic 
mice have yielded new information on the 
development of immunotolerance against 
antigens expressed inside the encapsulated 
lens. The findings so far, of two patterns of 
response against CAT or HEL suggest that 
lens proteins can be treated in different ways 
by the immune system. 

(5) The observation that the efficacy of 
oral tolerance can be enhanced by cotreating 
the animals with GMDP provides a new 
strategy for the inhibition of pathogenic im- 
munemediated processes such as uveitis. The 
potential to erihance oral tolerance is of great 
importance because the feeding procedure 
usually produces only partial inhibition of 
autoimmune diseases. 

(6) The finding that CD-8-deficient mice 
develop oral tolerance shows that this subpop- 
ulation of lymphocytes is not essential for the 
induction of oral tolerance in the system used 
in the present study. CD-8 ceUs were shown 
in other studies to play a major role in the 
induction of oral tolerance (Weiner H, et al., 
Annu Rev Immunol 12:809, 1994), and thus, our 
data provide direct evidence to the notion that 



80 



Labomtoiy of Immunology 



at least two different mechanisms participate 
in this process. 

Proposed Course 

Our future efforts will focus on the following 
issues: (1) Other retinal proteins, mainly those 
related to recoverin, will be tested for uveito- 
genicity; (2) the response to HumS-Ag of 
primates and human subjects will be further 
analyzed, mainly by attempts to establish and 
analyze cell Unes with specificity toward this 
molecule and its peptide detemunants; (3) the 
development of tolerance against foreign 
antigens expressed in the lens will be further 
analyzed, mainly with regard to the mecha- 
nisms that bring about tolerance and the 
difference between the responses to CAT and 
HEL; and (4) more effort wiU be focused on 
studies aimed at enhancing oral tolerance, the 
mechanisms involved in this phenomenon, 
and its usage for suppression of pathogenic 
autoimmune processes. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 



Publications 

Chan C-C, Hikita N, Dastgheib K, Whitcup 
SM, Gery I, Nussenblatt RB: Experimental 
melanin-protein induced uveitis in the Lewis 
rat: Immunopathological processes. 
Ophthalmology 101:1275-1280, 1994. 

Gery I, Chanaud NP III, Anglade E: 
Recoverin is highly uveitogenic in Lewis rats. 
Invest Ophthalmol Vis Sci 35:3342-3345, 1994. 

Gery I, StreUein JW: Autoimmunity in the eye 
and its regulation. Cuir Opinion Immunol, in 
press. 

Kasner L, Chan C-C, Whitcup SM, Gery I: 
The paradoxical effect of tumor necrosis factor 
alpha (TNF-a) in endotoxin-induced uveitis. 
Invest Ophthalmol Vis Sci 34:2911-2917, 1993. 

Kozhich AT, Kawano YI, Egwuagu GE, Gaspi 
RR, Maturi RK, Berzofsky JA, Gery I: A 
pathogenic autoimmune process targeted at a 
surrogate epitope. / Exp Med 180:133-140, 
1994. 

Sasamoto Y, Kawano YI, Wiggert B, Chader 
GJ, Gery I: Induction of unresponsiveness in 
adult rats by immunodominant and nondomi- 
nant peptides. Cell Immunol 152:286-292, 1993. 



81 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00298-01 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Inhibition of EAU in Monkey With Humanized Anti IL-2 Receptor 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: 



Francois G. Roberge 



Others: 



Yan Guex-Crosier 


M.D. 


Igal Gery 


Ph.D. 


Chi-Chao Chan 


M.D. 



Robert B. Nussenblatt 



M.D. Visiting Scientist LI, NEI 

Special Volunteer LI, NEI 

Deputy Laboratory Chief LI, NEI 

Head, Section on LI, NEI 
Immunopathology 

M.D. Scientific Director LI, NEI 



COOPERATING UNITS (if any) 

VRRS, NEI (James Raber, Ph.D.); VRRS, NEI (Martin Kriete, Ph.D.); NCI (Thomas Waldmann, M.D.); 
Hoffmaim LaRoche, Nutley, NJ (John Hakimi) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Clinical Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.7 



PROFESSIONAL: 



0.7 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Experimental autoimmune uveoretinitis (EAU) was induced in Cynomolgus monkey by immunization with 
recombinant human retinal S-antigen. At the onset of ocular disease, animals were treated for 28 days with 
intravenous injection of humanized anti-Tac, an anti-interleukin (IL)-2 receptor antibody originally produced 
in mouse but modified by replacing all but its binding region with human immunoglobulin elements. Controls 
were treated with vehicle alone. The animals were examined twice a week during the treatment period. The 
progression of the disease was markedly limited in the group treated with anti-Tac-H, while the severity of 
the inflammation continued to increase in the control group. The in vivo results were correlated with a 
significant inhibition of monkey lymphocyte proliferation stimulated by IL-2 when anti-Tac-H was added to 
the culture medium. 



82 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



Project Description 

Clinical Protocol Number 

93255 

Objectives 

General Goal of the Study. Evaluate the effec- 
tiveness of humanized anti-interleukin (IL)-2 
receptor antibodies for the therapy of autoim- 
mune diseases. 

Specific Objectives 

(1) Evaluate the effect of two humanized 
anti-IL-2 receptor antibodies, anti-Tac-H, and 
Mik-bl-H, respectively directed at the a and p 
chain of the IL-2R, in the treatment of active 
uveoretinitis in monkey. 

(2) Evaluate the possibUity of a beneficial 
therapeutic effect in using anti-Tac-H and 
Mik-bl-H in combination or in the form of 
recombinant hybrid molecules. 

(3) Correlate the therapeutic response 
with in vitro study of inhibition of prolifer- 
ation of monkey lymphocytes by anti-Tac-H 
and Mik-bl-H. 

(4) Measure the response of treated mon- 
keys against the antibodies used for treatment. 



Methods 

Animal Immunization and Examination. Cyno- 
molgus morJceys, between 2.0 and 3.0 kg, are 
immuriized with recombinant human retinal 
S-antigen at 40 /ig/kg emulsified in Hunter's 
TiterMax adjuvant by subcutaneous injection 
at the nape of the neck. 

Twice a week, starting 14 days after im- 
munization, the animals are sedated with 
ketamine, the pupils dilated with topical 
atropine, and the eyes examined by indirect 
ophthalmoscopy for signs of uveoretinal 
inflammation. 



Treatment Protocol. The animals are ran- 
domized in two groups of five individuals. 
Treatment consists of intravenous injection of 
anti-Tac-H or Mik-bl-H at 2.0 mg/kg, or 
control vehicle. The treatment is instituted on 
the day of presentation of the first sign of 
experimental autoimmune uveitis. Antibody 
injections are done following a schedule of 
alternating three- and four-day periods, for a 
total of eight injections, covering a total treat- 
ment period of 28 days. In addition, the 
pupils are kept dilated with the application of 
atiopine ophthalmic ointment 0.5 percent. 

Tests Performed 

Preimmunization 

• Blood drawn for complete blood count 
(CBC) and serum for baseline measure- 
ments. 

At initiation of therapy 

• Funduscopy and fundus photography. 

• Fundus fluorescein angiogram. 

• Measurement of intraocular pressure with 
a Tonopen tonometer. 

• Blood drawn for CBC, serum creatinine, 
slL-2R, and serum for baseline measure- 
ments. 

Once a week 

• Fundus photography. 

• Measurement of intraocular pressure with 
a Tonopen tonometer. 

• Blood collection for anti-idiotypic antibody 
and sIL-2R, anti-S-Ag Ab, and level of 
anti-Tac-H or Mik-bl-H. 

At termination of experiment 

• Funduscopy and fundus photography. 

• Fundus fluorescein angiogram. 

• Measurement of intraocular pressure with 
a Tonopen tonometer. 

• Blood drawn for CBC, serum creatinine, 
anti-idiotypic antibody, sIL-2R, anti-S-Ag 
Ab, level of anti-Tac-H or Mik-bl-H. 

• Eyes are collected for histopathological 
examination. 



83 



FY 1994 NEI Annual Report 



Major Findings 

On average, the ocular inflammation was 
stabilized in the animals treated with 
anti-Tac-H. There was a marked progression 
of the disease in the control group. Given the 
caveat of the small sample number, the statis- 
tical analysis of the present data by the 
Mann- Whitney test on the averaged variation 
per animal showed significance at a value of 
p < 0.01. We also observed a marked decrease 
of the intraocular pressure (-50 percent) in the 
inflamed eyes. There was no significant 
difference between the two treatment groups. 

We have also tested the activity of various 
anti-IL-2 receptor antibodies in the inhibition 
of IL-2 driven proliferation of monkey periph- 
eral blood lymphocytes. The results con- 
firmed the inhibitory effect of anti-Tac-H and 
a hybrid of anti-Tac-H and Mik-bl-H that had 
been observed with human lymphocytes. 
Mik-bl-H alone was not inhibitory but pro- 
duced an increased inhibition when used in 
combination with anti-Tac-H. We were also 
surprised to find that the antibody 7G7/B6, 
which does not block the IL-2 signal on hu- 
man lymphocytes, was a strong uihibitor of 
monkey lymphocyte proliferation. 

In conclusion, the results of this first 
experiment indicate that the humanized 
anti-Tac antibody could be useful in the thera- 
py of some autoimmune diseases. We are 
now conducting a similar experiment to evalu- 
ate Mik-bl-H. In light of the results of that 
experiment, we should decide in what direc- 
tion to develop the work. Already we feel 
that the next experiment should include a 
group to confirm the results obtained with 
anti-Tac-H. We are also trying to obtain IL-15 
to pursue the in vitro evaluation of the activity 
of the monoclonal antibodies on the monkey 
lymphocytes. 



Significance to Biomedical Research and the 
Program of the Institute 

The effectiveness of humanized anti-Tac in 
monkey suggests that this therapeutic ap- 
proach could be useful in the treatment of 
autoimmune diseases in human. Because the 
molecule is mostly of human origin, it appears 
that it would be better tolerated in patients, 
thereby avoiding the production of neutraliz- 
ing antibodies. 

Proposed Course 

We will conduct a similar experiment to 
evaluate Mik-bl-H. In light of the results of 
that experiment, we will decide in what direc- 
tion to develop the work, possibly using a 
combination of antiTac-H with Mik-bl-H. A 
forthcoming experiment shovdd include a 
group to confirm the results obtained with 
anti-Tac-H. We are also trying to obtain IL-15 
to pursue the in vitro evaluation of the activity 
of the monoclonal antibodies on the monkey 
lymphocytes. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 



84 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00300-01 LI 



PERIOD COVERED 



October 1, 1993 to September 30 , 1994 



TITLE OF PROJECT (80 characters or less. Title must fit or} orte line between the borders.) 

Study of the Effect of NPC 15669, an Inhibitor of Neutroph il Recruitment iiLUyeitis^ 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: Frangois G. Roberge M.D. Visiting Scientist LI, NEI 



Others: 



Margaret Cheung 
Kourosh Dastgheib 
Seiji Hayashi 
Chi-Chao Chan 



M.D. 
M.D. 
M.D. 
M.D. 



Ph.D. 



Senior Staff Fellow 
Visiting Fellow 
Volunteer Fellow 
Head, Section on 
Immunopathology 



LI, NEI 
LI, NEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 

SECTION 

Clinical Immunology 

INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.9 



PROFESSIONAL 



0.9 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Polymorphonuclear neutrophils (PMN) participate in the inflammatory infihrate of uveitis. At present, the 
importance of the role played by these cells is largely unknown. We have studied the influence that an 
inhibitor of PMN recruitment could have on two types of uveitis: (1) endotoxin-induced uveitis (EIU), a 
disease mediated mainly by PMN and macrophages, and (2) experimental autoimmune uveoretinitis (EAU), 
a T-cell-mediated disease in which the early infiltrate is composed mainly of PMN. We have found that 
injection of NPC 15669 in rats significantly reduced the severity of EIU. More surprisingly, treatment of rats 
late in the process of EAU induction also inhibited the evolution of this disease. 



85 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

(1) Evaluate the effect of NPC 15669 in the 
inhibition of protein exudation and cellular 
infiltration in the anterior chamber of the eye 
after subcutaneous injection of endotoxin. 

(2) Evaluate the effect that the inhibition 
of polymorphonuclear neutrophils recruitment 
could have on the evolution of experimental 
autoimmune uveitis (EAU) in the rat. 

Methods 

Uveitis was induced in Lewis rats with a 
subcutaneous injection of Upopolysaccharride 
(LPS). Treahnent with NPC 15669 started 
three hours after injection of endotoxin. NPC 
at doses of 12, six, or three mg/kg or vehicle 
alone were given by intraperitoneal (i.p.) 
injections repeated six times at two-hour 
intervals. After 24 hours, one eye was collect- 
ed for histology, and the aqueous humor was 
aspirated from the other eye for the measure- 
ment of protein and cells in the exudate. For 
EAU, Lewis rats were immuruzed with 
S-antigen (S-Ag) in Hunter's adjuvant. NPC 
was given three times a day at 30 mg/kg i.p. 
from day 10 to day 16 after immunization. 
EAU was evaluated by histopathology on the 
eye collected at the end of the treatment 
period. 

Major Findings 

Treatment with NPC produced a dose-depen- 
dent inhibition of endotoxin-induced uveitis 
(EIU). At the dosages of 12, six, and three 
mg/kg, there was a dose-dependent reduction 
in the leukocyte count in the aqueous humor, 
accompanied by a parallel decrease in the 
protein concentration. Histological examina- 
tion also showed a reduction in the inflamma- 
tory infiltrate of the iris and ciliary body. NPC 
was also effective in preventing the induction 
of EAU. In a representative experiment, four 
out of 14 NPC-treated rats developed mild 
EAU with an average severity grading of 1.37, 



whereas 10 out of 14 vehicle-treated rats 
developed EAU at a disease severity of 3.5. 

Significance to Biomedical Researcii and tlie 
Program of ttie Institute 

We conclude that the inhibition of neutrophil 
recruitment by NPC 15669 is effective in 
preventing intraocular inflammation. The 
treatment is effective even when instituted 
very late in a T-ceU mediated disease such as 
EAU. This finding suggests that the recruit- 
ment of PMN leukocytes plays a determining 
role in the dynamic of intraocular inflamma- 
tion. 

Proposed Course 

The mechanism of action of NPC 15669 in 
uveitis will be studied. The method wiU 
consist of measuring the expression of CD-18 
adhesion molecule on the surface of inflamma- 
tory cells, stimulated in vitro and in vivo in the 
presence or absence of the drug. 

NEI Research Program 

Retinal Diseases — ^Inflammatory Diseases 
Publications 

Cheung MK, Dastgheib K, Chan C-C, Roberge 
EG: Inhibition of PMN recruitment by NPC 
15669 prevents endotoxin induced uveitis. 
Invest Ophthalmol Vis Sci 35(4):1684, 1994. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00299-01 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one Ime between the borders.) 

Study of the Role of Nitric Oxide in Uveitis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: Frangois G. Roberge M.D. 

Others: Seiji Hayashi M.D. 

Chi-Chao Chan M.D. 

David Parks M.D. 
Margaret Cheung M.D., 
NamTram Pham M.D. 
Kourosh Dasthgieb 



Ph.D. 



Visiting Scientist 
Volunteer Fellow 
Head, Section on 
Immunopathology 
Senior Staff Fellow 
Senior Staff Fellow 
Volunteer Fellow 
Visiting Fellow 



LI, NEI 
LI, NEI 

LI, NEI 

LI, NEI 
LI, NEI 
LI, NEI 
LLNEI 



COOPERATING UNITS (if any) 



National Institute of Allergy and Infectious Diseases, Laboratory of Parasitic Diseases (Ricardo Grazzinelli, 
Ph.D.) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Clinical Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.9 



PROFESSIONAL: 



1.9 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

We have studied the role of nitric oxide (NO) in two types of uveitis: (1) Anterior uveitis represented by the 
model of endotoxin-induced uveitis (EIU) in the rat and (2) toxoplasma retinochoroiditis using a mouse model. 
In EIU, we have found NO levels that peak in the anterior chamber of the eye 2 hours before cellular 
infiltration and a sharp rise in protein exudation. Inhibition of NO production with a structural analog of 
L-arginine prevented the induction of EIU. In contrast, inhibiting NO synthesis during infection with 
Toxoplasma gondii caused an exacerbation of the disease. Spleen cell cultures from infected mice produced 
a large amount of NO. NO production was associated with a reduced viability and proliferation of the 
lymphocytes to toxoplasma antigen. The lymphocyte proliferative response was restored by blocking NO 
production. In conclusion, it appears that NO has diverse, even opposing roles in uveitis, depending on the 
type and mechanism of the inflammatory process involved. 



87 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

(1) Measure the production of nitric oxide 
(NO) in the eye in the course of uveitis in- 
duced by endotoxin. 

(2) Evaluate the effect of inhibiting the 
production of NO on the evolution of endo- 
toxin-induced uveitis (EIU). 

(3) Examine the effect of inhibiting NO 
production on the evolution of Toxoplasma 
gondii (T. gondii) infection in mice. 

(4) Evaluate the role of NO in the im- 
mune response against T. gondii. 

Methods 

EIU. EIU was induced in Lewis rats with a 
subcutaneous injection of Upopolysaccharide 
(LPS) at 300 fig/ kg. Aqueous humor was 
collected from groups of five rats every two 
hours for the first 12 hours, at 16 hours, and 
24 hours. NO levels in the aqueous humor 
were measured by a colorimetric assay based 
on the Griess reaction. Protein levels and the 
number of leukocytes were also determined. 
In some experiments, rats were treated by 
intraperitoneal injections with N'"-nitro-L- 
arginine methyl ester (L-NAME), an L-argin- 
ine analogue acting as a specific inhibitor of 
NO synthesis. The aqueous humor of one eye 
was collected after 24 hours, and the contralat- 
eral eye was examined by histopathology. 

Toxoplasmosis. Infection w^as induced in 
C57bl/B6 mice by i.p. injection of 10-20 cysts 
of T. gondii strain ME49. Aminoguanidine 
(AMG), an inhibitor of NO sjmthase, or vehi- 
cle control w^as administered i.p. every eight 
hours at 35 mg/kg. At the end of the second 
week, the eyes and brain were collected for 
histopathological examination. In other exper- 
iments, the spleens from infected and normal 
mice were collected tw^o, four, and six weeks 
after infection and the cells stimulated with 
toxoplasma antigen. The proliferative res- 



ponse was measured by ^H-thymidine incor- 
poration in the presence or absence AMG at 1 
mM. In parallel cultures, the level of NO 
produced was measured by a colorimetric 
diazotization assay of nitrite accumulated in 
the supernatant. Interferon gamma (IFN-y) 
and prostaglandin E2 (PGE2) were also mea- 
sured by specific enzyme-Unked immuno- 
sorbent assay. 

Major Findings 

In EIU, the analysis of the aqueous humor 
after LPS injection showed a sharp peak of 
NO at eight hours, followed two hours later 
by a rise in protein and cell entry into the eye. 
Treatment of rats with L-NAME markedly 
reduced the level of NO and inhibited the 
induction of ocular inflammation by LPS. The 
aqueous humor protein exudate decreased by 
73 to 82 percent, and the cellular infiltration 
was abrogated. The histopathological exami- 
nation of the eyes also showed a similar 
inhibition of iris and ciliary body tissue infil- 
tration in the treated rats. 

The inhibition of NO production with 
AMG in mice infected with T. gondii resulted 
in the exacerbation of disease. On histopatho- 
logical examination, the eyes of the infected 
mice treated with AMG showed increased 
inflammation in the retina, the choroid, and 
the vitreous compared with the infected con- 
trols. The brain showed an increased number 
of toxoplasma tachizoites infiltrating the tissue 
accompanied by a marked inflammation in 
AMG-treated animals. In addition, there was 
an accelerated evolution toward intraceUvdar 
cysts formation. When spleen ceUs from 
infected mice were cultured in the presence of 
toxoplasma antigen, there was a negative 
eftect on the survival and proliferation of the 
lymphocytes. This effect was correlated with 
high levels of IFN-y, NO, and PGE2 produc- 
tion. The presence of AMG in the culture 
medium reestablished the lymphocyte prolifer- 
ative response. Preventing PGE2 secretion 
with indomethacin also increased this re- 
sponse in proportion to an effect on NO 
production. 



Laboratoiy of Immunology 



Significance to Biomedical Research and the 
Program of the Institute 

In conclusion, it appears that NO has diverse 
opposing effect in uveitis depending on the 
type and mechanism of the inflammatory 
process involved. In anterior uveitis, the use 
of NO inhibitors could lead to improved 
therapy. In toxoplasmosis, however, it would 
be indicated to avoid drugs that inhibit NO 
synthesis. An important implication of these 
observations concerns the management of 
toxoplasma uveitis. It is a common practice to 
add corticosteroids to the antibiotic regimen in 
severe sight-threatening toxoplasmosis. In 
view of the negative regulation of NO produc- 
tion by corticosteroids, the use of antiinflam- 
matory drugs devoid of effect on the NO 
synthase should be considered. 

Proposed Course 

Research wiU be directed at identifying ocular 
cells that may produce NO. Finding such 
cells in the anterior segment of the eye could 
lead to a new therapeutic approach in the 
treatment of uveitis. 



the levels expressed in sensitive and resistant 
strains with toxoplasmosis. We will also 
study the possible effect of NO inhibition on 
triggering reactivation of chronic latent toxo- 
plasmosis. 

NEI Research Program 

Retinal Diseases— Inflammatory Diseases 

Publications 

Hayashi S, GazzineUi R, Chan C-C, Pham N, 
Roberge FG: In vivo inhibition of nitric oxide 
enhances ocular and CNS inflammation in 
murine toxoplasmosis. Invest Ophthalmol Vis 
Sci 35(4):1685, 1994. 

Parks DJ, Cheung MK, Chan C-C, Roberge FG: 
The role of nitric oxide in uveitis. Arch 
Ophthalmol 112:544, 1994. 

Roberge FG, Hayashi S: Nitric oxide is re- 
sponsible for the inhibition of lymphocyte 
proliferation in Toxoplasma gondii infection. 
Invest Ophthalmol Vis Sci 35(4):1685, 1994. 



We will also study the kinetics of NO pro- 
duction in vivo in mice infected with T. gondii. 
In particular, we will try to identify cells 
expressing NO S3rnthase in the eye and the 
brain during infection. Later we wiU compare 



89 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00279-03 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Study of Immunosup pressants for the Treatment of Uveitis in Animal Models 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 



PI: Frangois G. Roberge 

Others: Chi-Chao Chan 

Marc D. de Smet 
Robert B. Nussenblatt 
Dan Martin 
Margaret Cheung 
David Parks 



M.D. 


Visiting Scientist 


LI, NEI 


M.D. 


Head, Section on 
Immunopathology 


LI, NEI 


M.D. 


Visiting Scientist 


LI, ISfEI 


M.D. 


Scientific Director 


NEI 


M.D. 


Senior Staff Fellow 


LI, NEI 


M.D., Ph.D. 


Senior Staff Fellow 


LI, NEI 


M.D. 


Senior Staff Fellow 


LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Clinical Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 







PROFESSIONAL: 







0.0 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



THIS PROJECT HAS BEEN TERMINATED. 



90 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00301-01 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Ocular Gene Transfer 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Karl G. Csaky M.D., Ph.D. Medical Officer LI, NEI 

Others: Daniel Sullivan Ph.D. IRTA Fellow LI, NEI 

Eddy Anglade M.D. Staff Fellow LI, NEI 

Csaba Salaman M.D. Visiting Scientist LI, NEI 

Robert Interrante M.S. Special Volunteer LI, NEI 



COOPERATING UNITS (if any) 

S.U.N.Y. at Stony Brook, Stony Brook, ^fY (L. Taichman, M.D., Ph.D.); Department of Pediatrics, The Johns Hopkins 
University, BaUimore, MD (D. Valle, M.D.); Human Genome Center, NTH, Bethesda, MD (M. Blaese, M.D.); 
Massachusetts Institute of Technology, Department of Chemical Engineering, Boston, MA (D. Mooney, Ph.D.) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section of Ocular Gene Therapy 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

2.75 



PROFESSIONAL: 

2.25 



0.5 



CHECK APPROPRIATE BOX(ES) 

[xj (a) Human subjects [x] (b) Human tissues □ (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

An ocular gene therapy section is being developed that emphasizes the study of direct ocular gene transfer 
using ElA-deficient adenovirus constructs, the transfer of genes ex vivo into isolated retinal pigment epithelial 
cells prior to their transplantation, and the use of ornithine aminotransferase (OAT) transfected keratinocytes 
to treat patients with gyrate atrophy. 

A mutant of transforming growth factor beta-1 (TGF-P), which is secreted in an active form, has been placed 
into an adenovirus construct. Studies involving transduction of retinal pigment epithelial cells have shown 
that these cells can be efficiently transducted at a low multiplicity of infection and are capable of producing 
high levels of a biologically active TGF-(3. Adenovirus-TGF-P has also been injected into the anterior 
chamber of rats. Immunohistochemical analysis of these eyes has suggested the presence of secreted TGF-p. 

Human retinal pigment epithelial cells (RPE) have been transfected with plasmids containing beta-galactosidase 
(P-gal). Studies are underway to examine the optimal modes of transfection. Parallel studies are being done 
with isolated rat RPE cells. Both cell lines are capable of growth on thin synthetic basement membrane 
polymers. These polymers are transplanted, as a single cell layer, into the subretinal space of rats. 

OAT has been successfully transfected into OAT-deficient fibroblasts (CHO cells) and into keratinocytes, 
isolated from patients with gyrate atrophy. Studies are presently underway to examine ways to increase OAT 
levels and activity in these cells. 



91 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

An ocular gene therapy section is being estab- 
lished, the objective of which is to apply 
molecular biology techniques in the treatment 
of human ocular disease. Major areas of 
interest include direct ocular gene transfer 
using El A-deficient adenoviruses, transfection 
of retinal pigment epithelial cells ex vivo before 
their transplantation and the transfection of 
omithine-6-aminotransferase (OAT) into 
keratinocytes from patients with gyrate atro- 
phy (GA). Optimization of expression of OAT 
in cultured keratinocytes from patients with 
GA will allow us to determine if transfected 
keratinocytes can be used as a sufficient 
source of OAT to treat this disorder. 

Methods 

Using standard cloning techniques, adenovirus 
constructs are being synthesized. Using 
polymerase chain reaction. Northern, Western, 
Southern, and enzyme-linked immunosorbent 
assay (ELISA) methods, sequence and expres- 
sion data from various adenovirus constructs 
such as transforming growth factor (TGF)-p 
and OAT are being generated. 

Site-directed mutagenesis of the OAT gene 
and alteration in the promoters and their 
proximity to translational start sites are being 
performed. This will aUow us to identify OAT 
constructs that, when transfected into kera- 
tinocytes, will most efficiently degrade circu- 
lating ornithine. Standard tissue-culture 
techniques and the study of S5mthetic base- 
ment membrane polymers are used to study 
the transplantation of retinal pigment epitheli- 
um (RPE) into rodent eyes. This approach 
win allow the determination of the usefulness 
of these techniques in the treatment of animals 
with ocular neovascularization and diabetic 
macular edema. 



Major Findings 

Human RPE cells appear to be efficiently 
transduced by El A-deficient adenovirus. By 
using a lac-Z/ElA-adenovirus construct, 
human RPE were shown to express P-galacto- 
sidase activity in 100 percent of transduced 
cells. 

El A-deficient adenovirus constructs with 
TGF-P have been produced. As a result of 
site-directed mutagenesis, the TGF-|3 construct 
is secreted in an active form as a 24 kDa 
protein. This is unlike endogenous TGF-pi, 
which is secreted as a 90 to 110 kDa protein 
and requires activation by a latent factor to 
become biologically active. We have been able 
to show that the efficient transduction of 
human RPE can be readily achieved with the 
use of ElA-deficient adenovirus-TGF-p. Even 
at low multiplicity of infection, studies have 
shown that expression of active TFG-P can be 
achieved to pharmacological levels. TFG-P 
can be quantified by an ELISA assay, has been 
shown to be biologically active, and correlates 
with ribonucleic acid expression. The virus 
also has been injected into the anterior cham- 
ber of rats. At one to seven days following 
injection, immunohistochemical staining has 
demonstrated the presence of TGF in the 
corneal and iris stroma. Injection with nuU 
ElA-deficient adenovirus lacked TGF-p ex- 
pression, suggesting specificity of the expres- 



Human and transformed rat RPE have 
been isolated and grown in culture. Transfec- 
tion of cytomegalovirus (CMV)-lacZ constructs 
into these cell Unes, with Upofectant or calci- 
um phosphate, have jdelded transfection 
efficiencies of 20 to 30 percent. Transfection 
efficiency, into human RPE, was increased by 
contransfecting attenuated adenovirus with 
lipofectamine. This achieved a transfection 
efficiency of 90 percent. Both cell types have 
been grown, as a monolayer, on a 20 fim thick 
polylactic/polyglycoUc poljoner. Transplanta- 
tion of these sheets into the subretinal space of 
rats is now being performed. 



92 



Laboratory of Immunology 



Keratinocytes from patients with GA have 
been isolated and grown in culture. They 
exhibit low levels of endogenous OAT activi- 
ty. Various OAT constructs have been gener- 
ated. Expression of full-length OAT inserts, 
under a CMV or long terminal repeat (LTR) 
promoter, have been tested in CHO ceUs. 
Under LTR control, this construct yielded a 
twentyfold higher OAT activity than mock 
transfected ceUs. However, when transfected 
into the isolated keratinocytes, the increase 
was only threefold. Several OAT constructs 
were made with varying lengths of untranslat- 
ed regions and portions of the mitochondrial 
leader to the protein removed. Studies are 
now underway to determine which of these 
constructs is most efficient at expressing 
biologically active OAT. 

Significance to Biomedical Research and the 
Program of the Institute 

Direct ocular gene transfer has the potential 
for treatment in hereditary ocular diseases. In 
diseases such as GA where a deficiency of 
OAT is responsible for the clinical findings, 
gene therapy offers the possibility of cure. 
From a biological perspective, direct gene 
transfer into ocular tissues allows the study of 
the biology of overexpression of certain pro- 
teins into the eye. When animal models of 
disease are available, the use of direct gene 
transfer v^th adenovirus or transplantation of 
transfected RPE cells can identify critical 
proteins in the amelioration of these diseases. 
For example, TGF-P has been proposed as a 
downregulator of the immune response. By 
overexpressing TGF-P in animal models of 
uveitis, direct examination of this hypothesis 
can be obtained. 

In the event that keratinocytes from pa- 
tients with GA transfected with a construct of 
OAT exhibit sufficient OAT activity to de- 
grade significant serum ornithine, these cells 
wdll then be replaced onto the patient's skin. 
If serum ornithine levels fall, this will offer the 
first gene therapy for an ocular disease. 



Proposed Course 

The biology of overexpression of TGF-P in 
rodent models of uveitis will be further exam- 
ined. Various other inflammatory cytokines 
and inhibitors such as interleukin (IL)-IO and 
IL-1 receptor antagonist will also be investi- 
gated in this fashion. Further investigation of 
the effect of transduction by adenovirus on 
RPE cells will be performed. Subretinal injec- 
tions of adenovirus constructs will result in 
transduced RPE cells overexpressing selected 
proteins in vivo. The biologic significance of 
this overexpression in the subretinal space can 
then be examined. 

We will examine RPE ceUs, transplanted in 
a monolayer in the subretinal space, to deter- 
mine if these cells are able to function normal- 
ly. The kinetics of gene expression fiom these 
cells, transfected ex vivo, will also be deter- 
mined. 

The development of other mutants of OAT 
will continue. Once assayed for biological 
activity in degrading ornithine, these con- 
structs will be examined for expression and 
activity in cultured keratinocytes from GA 
patients. If sufficient OAT is produced that is 
capable of degrading adequate amounts of 
serum ornithine, further steps wdll be taken to 
replace these keratinocytes in the GA patients. 
Adenovirus constructs containing OAT will be 
created, and the study of their expression 
following ocular injections in mouse OAT 
knockout models of GA will be accomplished. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and 
Other Inherited Disorders 



93 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00268-04 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

The Diagnosis and Treatment of Human Uveitis and AIDS-Related Ocular Disease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Scott M. Whitcup M.D. Staff Medical Officer CB, NEI 

Others: Robert B. Nussenblatt 
Marc D. de Smet 
Chi-Chao Chan 



M.D. 


Scientific Director 


NEI 


M.D. 


Visiting Scientist 


LI, NEI 


M.D. 


Medical Officer 


LI, NEI 



COOPERATING UNITS (if any) 

Department of Medicine, The Johns Hopkins University, Baltimore, MD (David R. MoUer, M.D.) 



LAB/BRANCH 

Clinical Branch/Laboratory of Immunology 



SECTION 

Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



1.0 



PROFESSIONAL: 



1.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

fxl (a) Human subjects 
n (a1) Minors 
|~1 (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The title of this project has been changed from "The Diagnosis and Treatment of Human Uveitis" to "The Diagnosis and 
Treatment of Human Uveitis and AIDS-Related Ocular Disease," to reflect the expanded scope of the project to include 
the study of ocular disease in patients with AIDS. In addition, this project was previously listed in the Laboratory of 
Immunology but is now listed in the Clinical Branch. The goal of this project is to develop improved methods for 
diagnosing and treating human uveitis and AIDS-related ocular disease. This project encompasses clinical trials 
evaluating new diagnostic and therapeutic approaches and immunologic and histologic studies on blood and tissue 
specimens obtained from patients. 

A number of studies have focused on improving diagnostic tests for uveitis. Examination of lacrimal gland and 
conjunctival biopsies from patients with sarcoidosis has demonstrated lymphocytic infiltration and specific T-cell receptor 
repertoires despite a lack of granuloma formation, and these results should help improve the diagnostic yield of biopsies 
for the diagnosis of sarcoidosis. We have also shown that lumbar puncture can miss malignant involvement of the 
leptomeninges in patients with intraocular lymphoma and that ventricular taps are needed. 

The Clinical Branch is involved in a number of studies designed to improve the treatment of uveitis and ocular 
malignancy. A prospective, double-masked, randomized study of acetazolamide for uveitis-associated cystoid macular 
edema has just completed enrollment, and data will be analyzed this year. In addition, we completed a pilot study 
examining the efficacy and toxicity of a chemotherapy regimen for therapy of patients with central nervous system 
lymphoma involving the brain or eye. A complete response was obtained in seven of eight patients; the other patient 
had a partial response. A clinical trial evaluating a heparin surface-modified intraocular lens in patients with uveitis is 
under way, and a natural history study of patients with uveitis and good vision is in progress. 

Finally, the Clinical Branch is involved in studies with ocular complications related to AIDS. We are retrospectively 
reviewing the ophthalmologic examinations of 550 AIDS patients to improve diagnosis of ocular complications such as 
infection. In addition, we are participating in an open-label study of cidofovir for the treatment of resistant 
cytomegalovims (CMV) retinitis in patients with AIDS. 

94 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



Project Description 

Additional Personnel 



Emily Chew 


M.D. 


Visiting Scientist 

BEP, NEI 


Frederick Ferris, 


MD. 


Chief, Clinical Trials 


m 




Branch 
BEP, NEI 


George P. 


MX). 


Medical Officer 


Chrousos 




NICHD/DEB 


George 


M.D. 


Visiting Scientist 


Mastorakos 




NICHD/DEB 


Igal Gery 


PhD. 


Deputy Chief 
LI, NEI 


Susan Mellow 


R.N. 


Nurse Specialist 
CB, NEI 


R. Christopher 


MD. 


Senior Staff Fellow 


Walton 




LI, NEI 


Eddy Anglade 


M.D. 


Senior Staff Fellow 
LI, NEI 



Clinical Protocol Numbers 

87-EI-0104 
91-EI-0030 
91-EI-0139 
92-EI-0070 
92-EI-0108 
92-EI-0138 
92-EI-0157 
94-EI-0189 

Objectives 

The title of this project has been changed from 
"The Diagnosis and Treatment of Human 
Uveitis" to "The Diagnosis and Treatment of 
Human Uveitis and AIDS-Related Ocular 
Disease," to reflect the expanded scope of the 
project to include the study of ocular disease 
in patients with acquired immunodeficiency 
syndrome (AIDS). The goal of this study is to 
develop better methods for the diagnosis and 
treatment of human uveitis and ocular disease 
associated with AIDS. We are also interested 
in defining the pathophysiology of inflamma- 
tory eye diseases by performing immunologic 
and histologic studies on tissue specimens and 
blood samples obtained from patients. 



Methods 

Diagnosis of Uveitis and Malignancy 

(1) To improve the diagnostic yield of con- 
junctival and lacrimal gland biopsies for 
sarcoidosis, tissue specimens are examined 
using immunohistochemical staining and 
polymerase chain reaction (PCR). Conjuncti- 
val and lacrimal gland biopsies are performed 
and snap frozen in OCT from patients with 
known sarcoidosis. 

Immunohistochemical staining is per- 
formed using primary monoclonal antibodies 
against T-ceU markers, T-ceU receptors, Kveim 
antigen, and various interleukins. The ceU 
receptor repertoire is also studied using PCR 
techniques. Results will be compared with 
biopsies from patients with other uveitic 
conditions such as Behcet's disease to deter- 
mine the specificity of these results. 

(2) Intraocular lymphoma often masquer- 
ades as an idiopathic uveitis that delays the 
start of appropriate therapy. We continue to 
prospectively coUect data on patients with 
intraocular lymphoma to improve diagnosis in 
these patients. 

(3) Samples of aqueous humor, iris, and 
vitreous are obtained from patients undergo- 
ing ocular surgery for uveitis. Samples wiU be 
assayed for both cytokines and ceU-adhesion 
molecules and compared with samples ob- 
tained from patients undergoing routine 
cataract surgery in an attempt to identify 
immunologic markers for ocular inflammation. 

(4) Animals with experimental autoim- 
mune uveitis develop an associated pineaUtis. 
It is not known whether patients with human 
uveitis also have associated inflammation of 
the pineal gland. We are examining patients 
with uveitis for the presence of pinealitis 
using magnetic resonance imaging (MRI). 

Treatment of Uveitis of IVIalignancy 

(1) The efficacy of acetazolamide for the 
treatment of uveitis-associated macvdar edema 



95 



FY 1994 NEI Annual Report 



is being evaluated in a masked crossover 
study comparing acetazolamide with placebo. 
Visual acuity and the height of the macular 
edema measured on fluorescein angiography 
wiU be used as primary endpoints. 

(2) We are investigating the tieatment of 
patients with non-Hodgkin's lymphoma 
involving the brain or eye. The NEI is partici- 
pating in a pilot study to evaluate the efficacy 
and toxicity of a chemotherapy regimen for 
primary therapy of patients presenting with 
central nervous system lymphoma involving 
the brain or eye. High-dose methotrexate and 
leucovorin rescue is used in combination with 
thiotepa, vincristine, dexamethasone, and 
intrathecal cytarabine, and G-colony stimulat- 
ing factor (G-CFS) when needed. Radiation 
therapy is deferred until patients show evi- 
dence of disease progression. Patients are 
followed by cUnical ophthalmologic examina- 
tions as well as MRI and nuclear magnetic 
resonance spectroscopy. Laboratory correlates, 
including immunohistochemistry and viral 
sequence detection, are performed on available 
tissue. The data obtained from this study wUl 
be used to design a more inclusive study of 
chemotherapy in ocular and central nervous 
system lymphoma. 

(3) The purpose of this project is to evalu- 
ate the ability of a heparin surface-modified 
intraocular lens to reduce the incidence and 
severity of postoperative inflammation in 
patients with uveitis undergoing cataract 
surgery. Patients with a history of uveitis and 
catar ts, scheduled for cataract extraction 
with ^_^iacement of an intraocular lens, will be 
randomized to receive either a heparin sur- 
face-modified intraocular lens or a nonmodi- 
fied lens. The primary endpoint of the study 
is inflammatory cell deposits in the intraocular 
lens. Secondary endpoints will include other 
measures of ocular inflammation and visual 
acuity. Eighty patients will be recruited for 
the study. 

(4) The clinical course of patients with 
Behcet's disease who were treated wdth cyclo- 
sporine alone was compared retrospectively 



with patients who were treated with com- 
bined cyclosporine and prediusone. 

AIDS 

(1) We are retrospectively reviewing the 
ophthaLmologic examinations of 550 patients 
with AIDS in an attempt to improve the 
diagnosis of cytomegalovirus (CMV) retinitis. 
Information, including patient symptoms, 
visual acuity, and ocular examination data 
and CD-4 T-lymphocyte counts, wiU be ana- 
lyzed to establish criteria for screening pa- 
tients for ophthalmologic disease. We are also 
using laser interferometry to measure anterior 
chamber flare to determine whether this 
device can provide a sensitive and specific test 
for CMV retinitis in patients with AIDS. 

(2) The NEI is participating in an open- 
label, randomized, multicenter study of the 
safety and efficacy of cidofovir for the treat- 
ment of relapsing CMV retinitis in patients 
with AIDS. Patients with evidence of CMV 
retinitis progression while receiving ganci- 
clovir and /or foscamet therapy wiU be ran- 
domly assigned to one of two dose levels of 
cidofovir. Patients will undergo ophthalmo- 
logic examination at baseUne and at regular 
intervals thereafter. The primary endpoints 
are safety and time to progression of CMV 
retinitis. 

(3) Eyes obtained at autopsy are studied 
in an attempt to understand the pathophysiol- 
ogy of AIDS-related ocular disease. In addi- 
tion, the cUnical course of aU patients with 
AIDS and ocular disease are prospectively 
studied. 

Major Findings 

Diagnosis of Uveitis and IMalignancy 

(1) We have recruited nine patients with 
biopsy-proven sarcoidosis into the protocol 
examining conjunctival and lacrimal gland 
biopsies. None of the biopsies had gramiloma 
formation diagnostic for sarcoidosis. Never- 
theless, substantial areas of lymphocytic infil- 
tration were present on most biopsies, and we 



96 



Laboraton/ of Immunology 



are assessing the T-cell receptor repertoire in 
these specimens. 

(2) Evaluation of the diagnostic data from 
patients with intraocular lymphoma has 
shown that despite the absence of malignant 
cells in the cerebral spinal fluid obtained by 
lumbar puncture, malignant cells were demon- 
strated in a sample simultaneously obtained 
from an Omaya reservoir placed in the ventri- 
cles of five patients. The data suggest that 
malignant leptomeningeal involvement may 
be missed by lumbar puncture alone. 

(3) Studies on the levels of soluble inter- 
cellular adhesion molecule 1 in the serum, iris 
specimens, and aqueous of patients with 
uveitis are in progress. 

(4) We continue to recruit patients in the 
protocol using MRl to evaluate the pineal 
gland in patients with uveitis. 

Treatment of Uveitis and Malignancy 

(1) We have completed enrollment of patients 
in the crossover study of acetazolamide for the 
treatment of uveitic cystoid macular edema. 
The last patient is completing the study 
course; the randomization will be unmasked, 
and the data will be analyzed during the next 
year. 



Previous treatment with corticosteroids alone 
failed to control the uveitis in aU patients. 
Ten patients were given cyclosporine therapy 
alone (mean dosage, 8.6 mg/kg of body 
weight per day), and nine patients were given 
lower dosages of cyclosporine (mean dosage, 
6.2 mg/kg of body weight per day) in combi- 
nation with prednisone (mean dosage, 29.4 mg 
per day). The mean foUowup on therapy was 
51 months. After three months of therapy, a 
trend toward greater improvement in visual 
acuity was noted in patients treated with 
combined cyclosporine and prednisone com- 
pared with those receiving cyclosporine alone 
(17.8 letters vs. 10.2 letters, p = .24); after one 
year, little difference was observed in the 
improvement between the two groups (5.8 
letters vs. 3.3 letters, p = .80). However, a 
trend toward greater renal toxicity was seen in 
patients treated with cyclosporine alone after 
both three months and one year of therapy. 
Because of either a suboptimal therapeutic 
response or adverse effects, all patients treated 
with cyclosporine alone at baseline had pred- 
nisone added to their regimen after a mean 
time of 23.5 months. Overall, visual acuity 
remained stable or improved in 28 of 37 eyes 
(75.7 percent) over the course of therapy. The 
data suggest that combined cyclosporine and 
prednisone therapy is an effective treatment 
for Behget's uveitis and may be less toxic than 
therapy with cyclosporine alone. 



(2) Eight patients were eru-oUed in this 
pilot study and recruitment was completed in 
November 1993. Seven of eight patients had 
complete remission on this protocol; one 
patient had a partial response. Defiiute grade 
rV toxicity occurred in or\ly one patient. The 
phase 11 trial examining chemotherapy for the 
treatment of central nervous system lympho- 
ma has just started recruiting patients. 

(3) Seven patients have been enrolled in 
the protocol examining the heparin surface- 
modified intraocular lens in patients with 
uveitis. 

(4) We reviewed 19 patients with severe 
ocular Behget's disease treated with combined 
cyclosporine and corticosteroid therapy. 



AIDS 

(1) Laser interferometry has showm increased 
flare in the anterior chambers of patients with 
CMV retinitis and may be a useful screening 
test of ocular inflammatory disease in patients 
with AIDS. 

(2) Recruitment into the trial of ddofovir 
for CMV retinitis has started. 

(3) We have found an increased severity 
of CMV retinitis in children with AIDS as 
compared with adults. This increased severity 
appears to be related to a delay in diagnosis 
because chUdren are less apt to complain of 
visual symptoms and seek ophthalmologic 
evaluation than adults. Increased vigilance in 



FY 1994 NEI Annual Report 



screening children with ADDS for the develop- 
ment of ocular compUcations appears warrant- 
ed. We also described atypical findings of 
mild peripheral retinopathy in a patient with 
acute retinal necrosis following chicken pox, 
which may have been related to early acy- 
clovir therapy. 

Significance to Biomedical Research and the 
Program of the Institute 

Uveitis accounts for about 10 percent of the 
visual impairment in the United States. A 
major goal of the NEI is to improve the meth- 
ods for diagnosing and treating uveitis in an 
attempt to preserve useful vision in patients 
with inflammatory eye disease. In addition, 
many patients with AIDS develop severe, 
sight-threatening ocular disease. CMV retini- 
tis is the most common cause of impaired 
visual acuity in patients with AIDS, occurring 
in 10 to 30 percent of AIDS patients. An 
important goal of the NEI is to improve our 
methods of diagnosing AIDS-related eye 
disease and to develop and test new therapies 
for these disorders. 

Proposed Course 

We will continue to recruit patients with 
intraocular lymphoma, uveitis, and AIDS- 
related ocular disease into the clinical trials 
detailed earUer. A new protocol that looks at 
novel therapeutic approaches for CMV retini- 
tis will be started during the next year. We 
have completed the enrollment of patients into 
the crossover study of acetazolamide for 
uveitic cystoid macular edema and will start 
the analysis of the data. In addition, we have 
completed our collection of ophthalmologic 
data from the 550 patients with AIDS. Our 



plan is to analyze the compiled data and issue 
recommendations for the screerving of patients 
with AIDS for ocular disease. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases, 
Cancer 

Publications 

Callanan DG, Cheung MK, Martina DF, de 
Smet MD, Whitcup SM, Nussenblatt RB: 
Outcome of uveitis patients treated with long- 
term cyclosporine. Invest Ophthalmol Vis Sci 
35(suppl):2094, 1994. 

Friedman SM, Mames RN, Whitcup SM: 
Acute retinal necrosis after chickenpox in a 
patient with acquired immunodeficiency 
syndrome. Arch Ophthalmol 111:1607-1608, 
1993. 

Whitcup SM, Salvo EC Jr, Nussenblatt RB: 
Combined cyclosporine and corticosteroid 
therapy for sight-threatening uveitis in 
Behcet's disease. Am J Ophthalmol 118:39-45, 
1994. 

Whitcup SM, Nussenblatt RB: Treatment of 
autoimmune uveitis. Ann N Y Acad Sci 
696:307-318, 1993. 

Whitcup SM, de Smet MD, Rubin BI, Palestine 
AG, Martin DF, Bumier M Jr, Chan C-C, 
Nussenblatt RB: Intraocular lymphoma: 
Clinical and histopathologic diagnosis. Oph- 
thalmology 100:1399-1406, 1993. 



98 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00222-09 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) 

Immunopathology in Eyes With Experi m ental and C linical Ocular Diseases^ 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PL- Chi-Chao Chan M.D. Head, Section on LL NEI 

Immunopathology 

Others: Qian Li M.D. Visiting Associate LI, NEI 

Kourosh Dastgheib M.D. IRTA Fellow LI, NEI 

Bo Peng M.D. Visiting Fellow LI, NEI 

Deborah Luyo Technician LI, NEI 

Scott M. Whitcup M.D. Associate Director CB, NEI 

Charles E. Egwuagu Ph.D. Medical Officer LI, NEI 

Franjois G. Roberge M.D. Visiting Scientist LI, NEI 

Rachel R. Caspi Ph.D. Visiting Scientist LI, NEI 

Igal Gery Ph.D. Deputy Chief LI, NEI 

Robert B. Nussenblatt M.D. Scientific Director NEI 



COOPERATING UNITS (if any) 

Regulation of Gene Expression Section, LMDB, NEI (Ana B. Chepelinsky, Ph.D.); Ophthalmic Genetics and Services Branch, NEI 
(Muriel Kaiser- Kupfer, M.D.); Department of Ophthalmology, University of New South Wales, Sydney, Australia (Denis Wakefield, 
M.D.); Department of Ophthalmology, Kurume University, Kurume, Japan (Manabu Mochizuki, M.D.) 



Laboratory of Immunology 



SECTION 

Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



5.0 



PROFESSIONAL: 



4.0 



1.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects □ (b) Human tissues [x] (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The identity and topographic localization of immunocompetent cells and the alteration of surface markers on 
ocular resident cells in animals with various experimental uveitis were analyzed by immunohistochemical 
studies and in situ hybridization. Previously, we have demonstrated that T lymphocytes were the 
predominantly infiltrating cells in experimental autoimmune uveoretinitis (EAU), yet both macrophages and 
polymorphonuclear neutrophils were the predominantly infiltrating cells in endotoxin-induced uveitis (EIU). 
Cytokines (e.g., interferon gamma), inflammatory mediators (e.g., nitric oxide), and some retinal proteins (e.g., 
S-antigen (S-Ag)) play an important role in the immunopathogenesis of EAU and EIU. Agents that modulate 
them can alter the immunopathology of the experimental model. For example, prolonged corticosteroid 
therapies enhance S-Ag expression in nonretinal ocular tissues of rats with EAU. 

Several new animal models, including experimental melanin-protein-induced uveitis (EMIU), experimental 
blepharitis, murine allergic conjunctivitis, and murine toxoplasmosis, have been developed and studied. They 
represented different entities of uveitis in humans. EMIU closely resembles Vogt-Kayanagi-Harada syndrome 
and sympathetic ophthalmia; experimental blepharitis induced by immunization of monoclonal antibody of 
Id 16/6 resembles idiopathic blepharitis; murine allergic conjunctivitis induced by compound 48/80 resembles 
allergic conjunctivitis; murine toxoplasmosis infected with T. gondii resembles acquired ocular toxoplasmosis. 

Specimens from human ocular tissues with various diseases, including uveitis, retinal and corneal diseases, 
tumors, and metabolic genetic disorders, are studied using immunohistochemical and in situ hybridization 
techniques as well as light and electron microscopic examinations. In uveitis, immunocompetent cells and 
lymphokines besides the stimulators (e.g., infectious organisms) are valuable adjuncts to the clinical diagnosis 
and the understanding of pathogenesis of the diseases. In nonuveitic conditions, alteration of cellular 
membrane surface markers and intracytoplasmic organelles of the ocular resident cells (e.g., crystalline 
inclusions in Beitti's crystalline dystrophy; B-cell marker in intraocular lymphoma) may reflect cellular 
damage and abnormalities in these diseases. Elucidating the immunopathological role of the relationships 
between infiltrating inflammatory or malignant cells and other resident cells in the clinical behavior of various 
diseases will increase our understanding of human ocular disorders and provide better treatment. 

99 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

This program is designed to evaluate the 
clinical manifestation, histopathology, and 
immunopathology of the ocular tissue when 
experimental uveitis (experimental autoim- 
mune uveitis [EAU], endotoxin-induced uve- 
itis [EIU], experimental melanin-induced 
uveitis [EMIU], murine toxoplasmosis, etc.) are 
induced and /or modulated by different im- 
munosuppressive agents in various animal 
species. Ocular tissues obtained from patients 
with various diseases, including inflammatory 
and noninflammatory disorders, are all includ- 
ed. The infiltrating inflammatory cells, ocular 
resident cells and their products, cytokines, 
and metaboUtes are examined. These findings 
will help us understand ocular inflammation 
and the pathogenesis of each disease exam- 
ined in humans. 

Methods 

CUnical examinations include flashlight, sUt- 
lamp and fundus examination, under the 
dissecting microscopes of experimental ani- 
mals and patients. Pathological examinations 
include routine histologic techniques for Ught 
and electron microscopy, immunofluorescence 
and avidin-biotin-peroxidase complex method, 
and in situ hybridization techniques. Enzyme- 
linked immunosorbent assay technique is also 
performed for cytokines or protein analysis of 
the serum and intraocular fluids. 

Major Findings 

We have continued to study the immunopa- 
thology of various inflammatory cells and 
ocular resident cells in different experimental 
models of uveitis. We have demonstrated that 
higher levels of S-antigen (S-Ag) and its mes- 
senger ribonucleic acid (mRNA) are expressed 
in nonretinal ocular cells (the lens, the ciUary 
body, and the trabecular meshwork) of rats 
with EAU, in particular those that also re- 
ceived long-term corticosteroid treatment. 
This experimental result supports our previ- 



ous finding of S-Ag and its mRNA detected in 
irises of some uveitic patients who received 
long-term steroids. 

Several new experimental models for 
uveitis in humans have been investigated by 
us. EMIU is induced by immuruzation with 
bovine choroidal and retinal pigment epitheli- 
um (RPE) melanin protein and is characterized 
by bilateral, recurrent iridocycUtis and choroi- 
ditis. In EMIU, Uke EAU, the main infiltrating 
cells are T lymphocytes of QD-4 cells seen in 
the early stage and CD-8 cells seen in the late 
stage of the disease. Expressions of adhesion 
molecules and major histocompatibility (MHC) 
class II antigens are observed on ocular resi- 
dent cells one to two days before ocular in- 
flammation (eight to 10 days postimmuniza- 
tion). However, unlike EAU, recurrence 
occurs one month later. 

Acquired ocular toxoplasmosis is devel- 
oped by the infection of an avirulent strain of 
Toxoplasma gondii (T. gondii) (ME49) in 
C57BL / 6 mice. Focal ocular inflammation and 
RPE involvement are observed after 15 days of 
infection. Four weeks after infection, ocular 
inflammation becomes stable and rare cysts 
can be found. Treatment of mice with anti- 
bodies (CD-4 plus CD-8) or cj^okines (interfer- 
on gamma [IFN-7] or tumor necrosis factor 
alpha [TNF-a]) results in a marked increase of 
ocular inflanrmiation associated with the pres- 
ence of the parasite. 

In the study of transgenic mice v^th 
constitutive expression of IFN-y in the eye, we 
have reported that the growth of the eye is 
affected, resulting in microphthalmia, cata- 
racts, arrest of retinal differentiation, and 
enhancement of the expression of MHC class 
n. In a study of the role of nitric oxide, we 
have demonstrated that competitive blocking 
of NO formation with the L-arginine analogue 
is sufficient to inhibit the induction of EIU. 

Using immunopathological techniques, we 
examine ocular tissues obtained from patients 
with various octilar diseases to help visualize 
the pathology and the kinetics of the specific 
disease process. The findings provide useful 



100 



Laboratory of Immunology 



information for understanding the pathologi- 
cal mechanisms of the disease, determining 
the diagnosis, and guiding the subsequent 
management of the patient. Iris biopsies from 
patients with uveitis and cataracts were stud- 
ied. All except one specimen from uveitic 
patients had T lymphocyte and macrophage 
infiltration. The consistent spatial correlation 
between the presence of T cells and IFN-y as 
well as between the presence of macrophages 
and defensin are observed. 

Retinal necrosis, neovascularization, 
marked chorioretinal inflammation, particular- 
ly of T lymphocytic infiltration, and the ab- 
sences of bradyzoites are the characteristic 
findings in fetal eyes infected with T. gondii. 
Polymerase chain reaction (PCR), a sensitive 
and specific technique to identify infectious 
agents, and immunohistochemistry are helpful 
for the diagnosis of ocular toxoplasmosis. 

Twelve cases of intraocular lymphoma 
diagnosed at the NEI between 1984 and 1992 
were retrospectively reviewed. AU were non- 
Hodgkin's large B cell lymphoma of the cen- 
tral nervous system. The prompt, appropriate 
handling of specimens and review by an 
experienced cytopathologist are critical to the 
diagnosis of intraocular lymphoma. Malig- 
nant cells often are present in the vitreous 
before and /or in the cerebral spinal fluid. 
Multiple vitrectomies and lumbar punctures 
may be necessary before the correct diagnosis 
is made. 

Didanosine (DDI), used in the treatment of 
acquired immunodeficiency syndrome, is 
associated with toxic retinopathy. We have 
studied the first pathological report and dem- 
onstrated numerous membranous lamellar 
inclusions and cytoplasmic bodies in the 
affected RPE cells. These data show that the 
DDI retinopathy resiilts from the pathology of 
RPE cells. 

We evaluated three affected members of a 
Chinese-American family with Bietti's crystal- 
line retinopathy. Crystalline lysosomal mate- 
rials are observed in Ijmiphocytes and skin 
fibroblasts of these patients. The advanced 



panchorioretinal atrophy with crystals and 
complex Upid inclusions in the choroidal 
fibroblast was first documented in this study. 

Significance to Biomedical Research and the 
Program of the Institute 

Immunopathological findings on experimental 
uveitides have provided information on vari- 
ous inflammatory cells and ocular resident 
cells during the process of ocular inflamma- 
tion. This information helps us to better 
understand the mechanisms of uveitis and 
select or evaluate novel pharmacological 
agents as well as provide suitable therapeutic 
intervention of uveitis in humans. Studies of 
ocular tissues obtained from patients with 
various disorders have enabled us to gain 
information on the pathogenesis, diagnosis, 
and management of these ocular diseases. 

The finding that corticosteroids enhance S- 
Ag expression in nonretinal ocular tissues of 
rats with EAU may have clinical implication. 
The alteration of protein (such as S-Ag) ex- 
pression on the lens, the ciliary body, and the 
trabecular meshwork may contribute to the 
ocular side effects induced by prolonged 
corticosteroid therapy in patients. 

Melanin protein is capable of inducing 
autoimmune uveitis, resembHng noninfectious 
recurrent iridocyclitis and choroiditis in hu- 
mans. EMIU is another useful model for the 
study of uveitis such as Vogt-Kayanaki- 
Harada Sjmdrome and the evaluation of 
immunomodulating agents. 

Murine toxoplasmosis is a practical model 
for us to understand the respective roles of T. 
gondii proliferation and immune mechanisms 
involved in the pathogenesis of acquired 
ocular toxoplasmosis. T lymphocytes as well 
as TNF-a and IFN-y are crucial elements in 
controlling parasite growth. In immunocom- 
promised hosts such as AIDS patients, ocular 
lesions can be more severe and result from 
parasite proliferation rather than from an 
autoimmune process. 



101 



FY 1994 NEI Annual Report 



Early indicators of ocular toxoplasmosis in 
the fetus are infiltrating T cells and the ab- 
sence of tachyzoite antigens in the lesions. 
PCR for the diagnosis of ocular toxoplasmosis 
is helpful. 

The study demonstrates that T cells and 
macrophages are part of the inflammatory 
response in uveitis and that they secrete IFN-y 
and defensrn, respectively. These findings 
may have implications for the treatment of 
ocular uveitis. Antibody or specific immuno- 
modulatory therapy that abrogates the effects 
of cytokines such as IFN-y or defensins may 
decrease the extent and severity of ocular 
inflammation. 

Proposed Course 

AU experimental models, including EAU, EIU, 
EMIU, allergic conjunctivitis, experimental 
blepharitis, and murine toxoplasmosis will be 
studied clinically, histopathologicaUy, and 
immunopathologically in different strains and 
species. Various pharmacological agents and 
the role of cytokines, enzymes, metaboUtes, 
and cellular markers will be evaluated in these 
models. Also, we propose continuation of the 
analysis of human specimens to understand 
their immunopathogenesis. 

NEI Research Program 

Retinal Diseases — ^Inflammatory Diseases 

Publications 

Anderson W, Chan C-C, Nussenblatt KB, 
Whitcup SM: Topical heparin inhibits com- 
pound 48/80 induced allergic conjunctivitis. 
Invest Ophthalmol Vis Sci 35(4):1291, 1994. 

Brezin AP, Kasner L, Thulliez P, Li Q, Daffos 
F, Nussenblatt KB, Chan C-C: Ocular toxo- 
plasmosis in the fetus: Immunohistochemistry 
and DNA ampUcation. Retina 14:19-26, 1994. 

Caspi RR, Chan C-C, Grubbs B, Silver PB, 
Wiggert B, Parsa CF, Bahmanyar S, BiUiau A, 
Heremans H: Endogeneous systemic interfer- 
on-gamma has a protective role against ocular 



autoimmunity in mice. / Immunol 152:890-899, 
1994. 

Chan C-C, Gery 1, Nussenblatt RB, Mozes E, 
Singer DS: Periocular inflammation in mice 
with experimental systemic lupus erythemato- 
sus (SLE): A new experimental eye disease 
and its modulation. Invest Ophthalmol Vis Sci 
35(4):1988, 1994. 

Chan C-C, Hikita N, Dastgheib K, Whitcup 
SM, Gery 1, Nussenblatt RB: Experimental 
melariin-protein induced uveitis in the Lewis 
rat: Immuno-pathological processes. Ophthal- 
mology 101:1275-1280, 1994. 

Chan C-C, Palestine AG, Li Q, Nussenblatt 
RB: The diagnosis of ocular toxoplasmosis by 
the use of immunocytology and the polymer- 
ase chain reaction. Am J Ophthalmol 117:803- 
805, 1994. 

ChepeUnsky AB, Robinson ML, Overbeek PA, 
Parker DM, Chan C-C, Jamieson S, Dickson C: 
FGF-3 ectopic expression induces differentia- 
tion of central lens epitheUa and appearance of 
secretory epitheUa in the eyes of tiansgeruc 
mice. Invest Ophthalmol Vis Sci 35(4):1998, 
1994. 

Cheung MK, Martin DF, Chan C-C, Callanan 
DG, Cowan CL, Nussenblatt RB: Reactive 
lymphoid hyperplasia: Diagnosis by chorio- 
retinal biopsy. Am J Ophthalmol, in press. 

Cheung MK, Dastgheib K, Chan C-C, Roberge 
RG: Inhibition of PMN leukocyte recruitment 
by NPC 15669 prevents endotoxin-induced 
uveitis. Invest Ophthalmol Vis Sci 35(4):1684, 
1994. 

Dastgheib K, Hikita N, Sredni B, Albeck M, 
Sredni D, Nussenblatt RB, Chan C-C: Ocular 
inflammation stimulated by the immunomo- 
dulator AS 101 (ammonium trichloro (dioxy- 
ethelene-o-o') teUurate). Curr Eye Res, in 
press. 

Dastgheib K, Hikita N, Walton RC, Hayashi S, 
Chan C-C: Experimental melanin-protein 
induced uveitis (EMIU): Susceptibility and 



102 



Laboratory of Immunology 



recurrence. Invest 
35(4):1541, 1994. 



Ophthalmol Vis Sci 



DeBarge LR, Chan C-C, Greenberg SC, 
McLean IW, Yannuzzi LA, Nussenblatt RB: 
Chorioretinal, iris and ciliary body infiltration 
by juvenile xanthogranuloma masquerading as 
uveitis. Surv Ophthalmol 39:65-71, 1994. 

Egwuagu CE, Sztein J, Chan C-C, Mahdi R, 
Nussenblatt RB, Chepelinsky AB: Gamma 
interferon expression disrupts lens and retinal 
differentiation in transgenic mice. Dev Biol, in 
press. 

Egwuagu CE, Sztein J, Chan C-C, Reid W, 
Mahdi R, Nussenblatt RB, Chepelinsky AB: 
Ectopic expression of gamma interferon in the 
eyes of transgenic mice induces ocular pathol- 
ogy and MHC class 11 gene expression. Invest 
Ophthalmol Vis Sci 35(4):332-341, 1994. 

Egwuagu CE, Sztein J, Chan C-C, Mahdi R, 
Nussenblatt RB, ChepeUnsky AB: Transgenic 
rat and mouse models for the study of intraoc- 
iilar effects of gINF and autoimmunity. Invest 
Ophthalmol Vis Sci 35(4):1987, 1994. 

GazzineUi RT, Brezin A, Li Q, Nussenblatt RB, 
Chan C-C: Toxoplasma gondii: Acquired 
ocular toxoplasmosis in the murine model, 
protective role of TNF-alpha and IFN-gamma. 
Exp Parasitol 78:217-229, 1994. 

Hayashi S, GazzineUi R, Chan C-C, Pham N, 
Roberge FG: In vivo inhibition of nitric oxide 
enhances ocular and CNS inflammation in 
murine toxoplasmosis. Invest Ophthalmol Vis 
Sci 35(4):1685, 1994. 

Hikita N, Dastgheib K, Mochizuki M, 
Nussenblatt RB, Chan C-C: Effect of topical 
FK506 on experimental melanin-protein in- 
duced uveitis (EMIU) in rats. Invest Ophthal- 
mol Vis Sci 35(4):1540, 1994. 

HoUand EJ, Olsen TW, Chan C-C, Bergstrom 
L, Palestine AG, Nussenblatt RB: Kinetics of 
corneal transplant rejection in the rat penetrat- 
ing keratoplasty model. Cornea 13:317-323, 
1994. 



Jang S, Li Q, Whitcup SM, Peng B, 
Nussenblatt RB, Chan C-C: Susceptibility to 
endotoxin induced uveitis varies in different 
murine strains. Invest Ophthalmol Vis Sci 
35(4):1685, 1994. 

Kaiser-Kupfer MI, Chan C-C, MarkeUo TC, 
Crawford MA, Caruso RC, Csaky KG, Guo J, 
Gahl WA: Bietti's crystalline dystrophy: 
Natural history, biochemical and clinical 
pathologic correlations. Am J Ophthalmol, in 
press. 

Lai JC, Wawrousek EF, Sipe JD, Whitcup SM, 
Chan C-C, Igal G: Ocular and systemic im- 
munological profile of interleukin-lb (IL-1) 
transgenic mice. Invest Ophthalmol Vis Sci 
35(4):1988, 1994. 

Li Q, Dastgheib K, Hibita N, Egwaugu C, 
Nussenblatt RB, Chan C-C: TGF-pi mRNA 
expression in experimental melanin-protein 
induced uveitis (EMIU) and in experimental 
autoimmune uveitis (EAU). Invest Ophthalmol 
Vis Sci 35(4):1807, 1994. 

Li Q, Abe T, Kikuchi T, Nussenblatt RB, 
Shinohara T, Chan C-C: Corticosteroids 
enhance S-antigen in non-retinal ocular tissues 
of rats with experimental autoimmune uveitis. 
Exp Mol Pathol 60:27-38, 1994. 

MacCumber MW, Dastgheib K, Bressler NM, 
Chan C-C, Harris M, Fine S, Green WR: 
CHnicopathologic correlation of the multiple 
recurrent serosanguineous retinal pigment 
epithelial detachments syndrome. Retina 
14:143-152, 1994. 

MiUer-Rivero NE, Rizzo LV, Chan C-C, 
Wiggert B, Nussenblatt RB, Caspi RR: Sup- 
pression of IRBP-induced EAU in mice by 
feeding IRBP and its potentiation by 
interleukin-2. Invest Ophthalmol Vis Sci 
35(4):1865, 1994. 

Murali S, Hardten DR, DeMartelaere S, 
Olevsky OM, Mindrup EA, Hecht ML, Chan 
C-C, Holland EJ: Effect of topical adminis- 
tered platelet-derived growth factor on corneal 
wound strength. Curr Eye Res, in press. 



103 



FY 1994 NEI Annual Report 



Parks DJ, Cheung MK, Chan C-C, Roberge FG: 
The role of rutric oxide in endotoxin-induced 
uveitis. Invest Ophthalmol Vis Sci 35(4):1685, 
1994. 

Parks DJ, Cheung MK, Chan C-C, Roberge FG: 
The role of nitric oxide in uveitis. Arch 
Ophthalmol 112:544-546, 1994. 

Peng B, Li Q, Roberge F, Whitcup SM, Luyo 
D, Chan C-C: Topical rapamycin inhibits 
allergic conjunctivitis in a murine model. 

Invest Ophthalmol Vis Sci 35(4):1292, 1994. 

Rizzo LV, Silver PB, GazzineUi RT, Chan C-C, 
Wiggert B, Caspi RR: Expression of cytokine 
genes within the eye in murine EAU. Invest 
Ophthalmol Vis Sci 35(4):1862, 1994. 

Sartani G, Silver PB, Strassmann G, Chan C-C, 
Caspi RR: Effect of suramin treatment on 
induction of EAU. Invest Ophthalmol Vis Sci 
35(4):1862, 1994. 

Silver PB, Rizzo LV, Chan C-C, Donoso LA, 
Wiggert B, Caspi RR: Identification of a major 
pathogenic epitope in the IRBP molecule 
recognized by mice of the H-2' haplotype. 
Invest Ophthalmol Vis Sci 35(4):2061, 1994. 

Suh EDW, Vistica BP, Chan C-C, Raber JM, 
Gery I, Nussenblatt RB: Splenectomy abro- 
gates the induction of oral tolerance in experi- 
mental autoimmune uveoretinitis. Curr Eye 
Res 12:833-839, 1993. 



Walton RC, Lai JC, Chanaud NP HI, Chan C- 
C, Gery I, Whitcup SM: Inhibition of experi- 
mental autoimmune uveitis by MDL 28,842. 
Invest Ophthalmol Vis Sci 35(4):1865, 1994. 

Wawrousek EF, Chan C-C, Lai JC, Gery I: 
Progressive inflammatory disease and 
neovascularization in the eyes of interleukin- 
Ib transgenic mice. Invest Ophthalmol Vis Sci 
35(4):1988, 1994. 

Whitcup SM, Hayashi S, Rizzo L, Lai JC, 
GazzineUi R, Nussenblatt RB, Chan C-C: 
Systemic anti-IL-12 antibody exacerbates 
endotoxin-induced uveitis. Invest Ophthalmol 
Vis Sci 35(4):1481, 1994. 

Whitcup SM, Dastgheib K, Nussenblatt RB, 
Walton RC, Pizzo PA, Chan C-C: A clinico- 
pathologic report of the retinal lesions associ- 
ated with Didanosine. Arch Ophthalmol, in 
press. 

Whitcup SM, Hikita N, Shirao M, Miyasaka 
M, Mochizuki M, Nussenblatt RB, Chan C-C: 
Monoclonal antibodies against CD54 and 
CDlla prevent and inhibit endotoxin-induced 
uveitis. Exp Eye Res, in press. 

Zierhut M, Chan C-C, Duijvestijn A, 
Nussenblatt RB, Whitcup SM: High endotheli- 
al venules in IRBP-induced experimental 
autoimmune uveitis. Invest Ophthalmol Vis Sci 
35(4):1807, 1994. 



Wakefield D, Li Q, McCluskey P, Nussenblatt 
RB, Chan C-C: Immunohistochemical localiza- 
tion of T lymphocytes and macrophages and 
expression of interferon gamma and defensin 
in uveitis. Ocul Immunol Inflam, in press. 



104 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00270-04 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Immunologic Mechanisms of Ocular Disease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Scott M. Whitcup M.D. Staff Medical Officer CB, NEI 



Others: Chi-Chao Chan M.D. 

Robert B. Nussenblatt M.D. 

Sei-Ichi Ishimoto M.D. 



Head, Section on 
Immunopathology 
Scientific Director 
Visiting Associate 



LI, NEI 

NEI 

U NH (CRADA) 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Clinical Branch/Laboratory of Immunology 



SECTION 

Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.4 



PROFESSIONAL: 



0.4 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The title of this project has been changed from "cell adhesion molecules in ocular inflammation" to "immunologic 
mechanisms of ocular disease" to reflect the broadening scope of experiments to encompass the role of cell adhesion 
molecules and other immunologic factors, including cytokines, in the pathogenesis of ocular diseases, particularly uveitis, 
ocular malignancy, and ocular disease related to AIDS. In addition, this project was previously listed in the Laboratory 
of Immunology but is now listed in the Clinical Branch. The goal of this project is to study the immunologic 
mechanisms involved in the pathogenesis of ocular inflammation and ocular malignancy and to develop and test therapies 
based on these data. Recently, we have concentrated on the role of cell adhesion molecules and cytokines in the 
development of ocular inflammatory disease. Cell adhesion molecules (CAMs) are surface proteins important for antigen 
sensitization and the migration of leukocytes to sites of inflammation. We are currently investigating compounds that 
block CAMs as a treatment for uveitis and other ocular inflammatory diseases. During the past year, we showed that 
a single injection with a monoclonal antibody against the CAMs, lymphocyte function-associated antigen-1 (LFA-1) and 
Mac-1, can inhibh the development of uveitis by more than 66 percent. In contrast, treatment with a monoclonal antibody 
against a third CAM, vascular adhesion molecule-1 (VCAM-1) failed to inhibit in vivo disease. Antibodies against these 
cell adhesion molecules also inhibited lymphocyte proliferation in vitro by up to 70 percent. 

We also studied the role of interleukin-12 on the development of acute intraocular inflammation and showed that 
systemically administered antibody against IL-12 exacerbates the endotoxin-induced uveitis. Similarly, intraocular IL-12 
inhibited disease. These data suggest that endotoxin-induced uveitis may be a Th-2-dependent disease. Finally, we 
investigated the effect of a number of therapeutic agents in animal models of ocular inflammation. We demonstrated 
that MDL 28,842, an inhibitor of S-adenosyl-L-homocysteine hydrolase, decreases the ocular inflammation in animals 
with experimental autoimmune uveitis (EAU). In other experiments, we showed that topically administered heparin 
inhibits the development of allergic conjunctivitis induced by mast cell degranulation. 



105 



PHS 6040 (Rev. 5/92) 



FY 1994 NEl Annual Report 



Project Description 

Additional Personnel 

Rachel Caspi Ph.D. Visiting Associate 

LI, NEI 

Igal Gery Ph.D. Deputy Chief 

LI, NEI 
Qian Li MD. Visiting FeUow 

LI, NEI 
R. Christopher MX). Senior Staff Fellow 

Walton LI, NEI 

Objectives 

The goal of this project is to study the immu- 
nological mechanisms involved in the patho- 
genesis of ocular inflammation and malignan- 
cy and to develop and test new therapies for 
these disorders. The project has focused on 
investigating the role of ceU-adhesion mole- 
cules and cytokines in ocular inflammation 
and testing new therapeutic agents using in 
vitro and in vivo models of ocular inflammato- 
ry disease. 

Methods 

Animal Models of Ocular Inflammation. Endo- 
toxin-induced uveitis (EIU) is induced by 
injecting 100 jig of Salmonella typhimurium 
endotoxin into one footpad of Lewis rats or 
200 /Ag into one footpad of C3H-Hen mice. 
Experimental autoimmune uveitis (EAU) in 
mice is induced by immunizing BIO.A mice 
with 50 /Lig of interphotoreceptor retinoid 
binding protein in complete Freund's adju- 
vant, with pertussis toxin injected 
intraperitoneaUy. Finally, allergic conjunctivi- 
tis was induced in mice using topical adminis- 
tration of compound 48/80, a mast cell 
degranulating agent. 

Histology and Immunohistockemistry of 
Ocular Inflammation. Enucleated arumal eyes 
and human ocular tissue are immediately 
snap frozen and embedded in OCT. The 
expression of ceU-adhesion molecules and 
presence of cytokines is then assessed with 
immunohistochemical staining using an avi- 



din-biotin-peroxidase complex method on 
frozen sections of ocular tissue. Eyes are also 
embedded in methyl methacrylate, and four 
micron sections are examined for histologic 
evidence of inflammation. In the model of 
compound 48/80 induced allergic conjunctivi- 
tis, tarsal and bulbar conjunctiva were re- 
moved and processed for routine histology or 
frozen for immunohistochemical staining as 
described earUer. 

In vitro lymphocyte proliferation assays are 
performed as previously detailed {Cell 
Immunol 122:251, 1989). 

Major Findings 

(1) In a study examining the effect of treat- 
ment with a monoclonal antibody against 
lymphocyte function-associated antigen (LFA)- 
1 and very late activation antigen (VLA)-4, we 
demonstrated that anti-LFA-1 antibody signifi- 
cantly inhibited the development of EIU. In 
contrast, anti-VLA-4 antibody had no effect on 
the development of intraocular inflammation. 
The data suggest that compounds blocking 
LFA-1 but not VLA-4 should be effective for 
the treatment of acute ocular inflammation. 
Studies in vitro showed that antibodies against 
LFA-1 and Mac-1 inhibited lymphocyte prolif- 
eration but that anti-VLA-4 antibody had less 
effect. These data suggest that anti-LFA-1 and 
anti-Mac-1 antibody may be useful in the 
treatment of lymphocyte-mediated ocular 
inflammatory disease. Finally, increased 
expression of ceU-adhesion molecules was 
noted in corneal specimens with allograft 
failure, suggesting that antibodies against 
adhesion molectiles may prevent rejection. 

(2) We found that systemic treatment 
with anti-interleukin (IL)-12 monoclonal anti- 
body exacerbates the development of EIU. The 
mean number of inflammatory cells ± S.E.M. 
was 41.6 ± 9.3 for mice treated with anti-IL-12 
antibody, 17.6 ± 3.5 for mice treated with IFN- 
Y IL-12, and 17.7 ± 21 for control mice. Addi- 
tional studies showed that intraocular admin- 
istration of IL-12 inhibits the development of 
EIU. Similar to previous studies with anti- 
interferon gamma (IFN-y) antibody, systemic 



106 



Laboratory of Immunology 



anti-IFN-y antibody exacerbates intraocular 
inflammation. This may be due to decreased 
generation of Th-1 cells and increased genera- 
tion of Th-2 ceUs. 

(3) MDL 28,842, a potent irreversible 
inhibitor of S-adenosyl-L-homocysteine hydro- 
lase was shown to inhibit the development of 
EAU in mice. Animals treated with MDL 
28,842 at 2.5 and 5.0 mg/kg/day had signifi- 
cantiy less disease when compared with 
contiols (p < 0.009). Importantiy, there was 
no significant weight loss in the treated ani- 
mals. These data suggest that MDL 28,842 
may be a useful therapeutic agent in the 
treatment of uveitis in humans. 

(4) Topical heparin significantiy inhibited 
the development of allergic conjunctivitis in 
mice. Preliminary studies revealed no ocular 
toxicity. These findings suggest that topical 
heparin may provide a well-tolerated and 
effective treatment for allergic conjunctivitis. 

Significance to Biomedical Research and ttie 
Program of the Institute 

One major mission of the NEl is to under- 
stand the mechanisms of sight- threatening 
eye disease so that new and effective therapies 
can be developed. The expression of cell- 
adhesion molecules appears to be a funda- 
mental mechanism in the development of 
intraocular inflammation. Cytokines are also 
important in the pathogenesis of ocular in- 
flammation, and certain cytokines such as IL- 
12 and IL-IG appear to have a regulatory role 
on uveitis. With this understanding, we hope 
to develop new anti-inflammatory therapy for 
ocular inflammation. The testing of these 
therapeutic agents in models of ocular inflam- 
mation allows the development of new thera- 
pies for patients with ocular inflammatory 
disease, which accounts for approximately 10 
percent of the visual impairment in the United 
States. 

Proposed Course 

We plan to continue our experiments investi- 
gating the role of ceU-adhesion molecules and 



cytokines in uveitis. In addition to using 
monoclonal antibodies against adhesion mole- 
cules, we are testing small molecules to block 
the cell adhesion molecules because these 
agents may be administered topically. We 
have started experiments studying the role of 
CAMs and cytokines in other inflammatory 
diseases, including secondary glaucoma, 
corneal allograft rejection, and intraocular 
malignancies. We are also continuing our 
experiments with MDL 28,842. 

NEl Research Program 

Retinal Diseases — Inflammatory Diseases 

Publications 

Anderson W, Chan C-C, Nussenblatt RB, 
Whitcup SM: Topical heparin inhibits com- 
pound 48/80 induced allergic conjunctivitis. 
Invest Ophthalmol Vis Sci 35(suppl):1291, 1994. 

Jang S, Li Q, Whitcup SM, Peng B, 
Nussenblatt RB, Chan C-C: Susceptibility to 
endotoxin induced uveitis varies in different 
murine strains. Invest Ophthalmol Vis Sci 
35(suppl):1685, 1994. 

Walton RC, Lai JC, Chanaud NP, Chan C-C, 
Gery I, Whitcup SM: Inhibition of experimen- 
tal autoimmune uveitis by MDL 28,842. Invest 
Ophthalmol Vis Sci 35(suppl):1865, 1994. 

Whitcup SM, Hayashi S, Rizzo, Lai JC, 
Gazzinelli R, Nussenblatt RB, Chan C-C: 
Systemic anti-IL-12 antibody exacerbates 
endotoxin-induced uveitis. Invest Ophthalmol 
Vis Sci 35(suppl):1481, 1994. 

Whitcup SM, Nussenblatt RB, Price FW Jr, 
Chan C-C: Expression of cell adhesion mole- 
cules in corneal graft failure. Cornea 12:475-80, 
1993. 

Whitcup SM: The role of cell adhesion mole- 
cules in endotoxin-induced uveitis. Regional 
Immunol, in press. 

Whitcup SM, Hikita N, Shirao M, Miyasaka 
M, Tamatani T, Mochizuki M, Nussenblatt RB, 



107 



FY 2994 NEI Annual Report 



Chan C-C: Monoclonal antibodies against Patents 

CD54 (ICAM-1) and CDlla (LFA-1) prevent 

and inhibit endotoxin-induced uveitis. Exp U.S. patent (applied for) 

Eye Res, in press. 

Contract/CRADA Reports 

Zierhut M, Chan C-C, Duijvestijn A, 

Nussenblatt RB, Whitcup SM: High endotheU- Allergan CRADA 

al venules in IRBP-induced experimental 

autoimmune uveitis. Invest Ophthalmol Vis Sci 

35(suppl):1807, 1994. 



108 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00269-04 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title mus( fit on one line between the borders.) 

Ocular Toxicity of 2',3'-Dideoxvinosine (ddl) 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Scott M. Whitcup M.D. Staff Medical Officer CB, NEI 

Others: Robert B. Nussenblatt M.D. Scientific Director NEI 

Rafael Caruso M.D. Visiting Scientist OGCS, NEI 



COOPERATING UNITS (if any) 

Pediatric Branch, National Cancer Institute (Philip A. Pizzo, M.D.); Laboratory of Immunoregulation, National 
Institute of Allergy and Infectious Diseases (Clifford H. Lane, M.D.); Clinical Oncology Program, National 
Cancer Institute (Robert Yarchoan, M.D.) 



Clinical Branch/Laboratory of Immunology 



Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

0.4 



PROFESSIONAL: 



0.4 



0.0 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects □ (b) Human tissues □ (c) Neither 

|~1 (a1) Minors 
□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project, "Ocular toxicity of 2',3'-Dideoxyinosine (ddl)", was previously listed in the Laboratory of 
Immunology but is now a project in the Clinical Branch, ddl is a purine analog with antiretroviral activity 
currently used to treat patients with the acquired immunodeficiency syndrome (AIDS), both adults and 
children, in clinical protocols at the NIH. The purpose of this study is to prospectively follow patients treated 
with ddl for the development of ocular complications secondary to drug toxicity. Ninety-five children with 
symptomatic (CDC class P-2) HIV infection were enrolled in a Phase I-II study to assess the safety and 
antiretroviral activity of ddl. More than 100 children treated with ddl have been examined, and 6 children 
have developed peripheral atrophy of the retinal pigment epithelium during ddl therapy. Eyes with ddl- 
associated retinal lesions have now been examined histologically. Microscopic examination of these lesions 
revealed multiple areas of retinal pigment epithelial (RPE) loss, some surrounded by areas of hypertrophy or 
hypopigmentation of the RPE. Partial loss of the choriocapillaris and neurosensory retina were also noted in 
areas of diseased RPE. Transmission electron microsocopy showed numerous membranous lamellar inclusions 
and cytoplasmic bodies in the RPE cells. These data show that didanosine primarily affects the RPE and that 
the choriocapillaris and overlying neurosensory retina are also dystrophic in areas of RPE loss. We also 
continue to follow a group of 75 adults treated with ddl with periodic fundus examinations and electro- 
oculograms. One aduh previously developed retinal lesions while treated with ddl, but no additional adult 
patients have developed retinal lesions. 



209 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 

R. Christopher MD. Senior Staff Fellow 

Walton LI, NEI 

Objectives 

The goal of this study is to monitor patients 
treated with dideoxyinosine (ddl) for the 
development of ocular complications. 

Methods 

(1) Patients treated with ddl are given com- 
plete eye examinations every three to four 
months, including dilated ophthalmoscopy 
and fundus photography of any abnormal 
retinal findings. Patients treated with the 
higher dosages of ddl also receive periodic 
electro-oculograms to assess the electrophy- 
siologic function of the retinal pigment epithe- 
Hum (RPE). 

(2) Eyes from one child with ddl-associat- 
ed retinal lesions were studied histologically. 
Eyes were fixed in 10 percent buffered forma- 
lin for routine histology and electron micros- 
copy. Sections were stained with hematoxylin 
and eosin, periodic acid Schiff, and Gomori's 
methenamine silver. Small fragments of 
chorioretinal tissue in the area of the lesions 
were embedded in an epoxy resin, and ultra- 
thin sections were stained with uranyl acetate 
and lead citrate for transmission electron 
microscopy. 

Major Findings 

(1) Six children have now developed periph- 
eral atrophy of the RPE during ddl therapy. 
The lesions are scalloped areas of RPE atrophy 
with hyperpigmented borders and occur 
predominantly in the midperiphery of the 
fundus in both eyes. These retinal lesions 
slowly progress if ddl therapy is continued, 
but central visual acuity remains unaffected. 
During the past year, one child developed a 
few retinal lesions while on ddl. 



(2) No additional adults developed retinal 
lesions. 

(3) Gross examination of the eyes re- 
vealed multiple 1 to 2 mm round lesions of 
RPE loss, many with areas of RPE hyperpig- 
mentation at their periphery or centrally 
located. The lesions spanned from 3 mm 
posterior of the ora serrata to the midperi- 
phery and extended circumferentially in each 
eye. Microscopic examination revealed a 
normal cornea, iris, cihary body, lens, and 
optic nerve in both eyes. There were multiple 
areas of RPE loss, and some of these areas of 
RPE hypertrophy and/ or RPE hypopigmenta- 
tion were located at their margins. Chorioreti- 
nal adhesion and partial loss of the choriocapi- 
llaris and outer retina to the level of the inner 
nuclear layer were also noted in areas of RPE 
loss. Transmission electron microscopy re- 
vealed enlarged RPE cells with large, spherical 
melanin granules (RPE hypertrophy) and 
other RPE cells with a reduced number of 
pigment granules (RPE hypopigmentation). 
Degenerating RPE and neural cells were also 
seen. Bruch's membrane was intact, except in 
the areas of chorioretinal adhesion. A large 
number of membranous lamellar inclusions up 
to 20 nm in thickness and concentric membra- 
nous cytoplasmic bodies measuring up to 2 
/xm in diameter were clustered between the 
melanin granules of some RPE cells. 

Significance to Biomedical Research and the 
Program of the Institute 

It has been determined that ddl is a drug with 
in vitro and in vivo activity against human 
immunodeficiency virus infection. One of the 
missions of the NEI is to monitor patients for 
the development of ocular toxicity and assess 
the effect such toxicity has on vision. 

Proposed Course 

The detailed study of the retinal lesions associ- 
ated with ddl has been completed. Although 
aU HIV patients followed on NIH protocols 
will continue to be followed for the develop- 
ment of ocular complications, this specific 
project was completed on September 30, 1994. 



110 



Lahoratory of Immunology 



NEI Research Program 

Retinal Diseases — Photoreceptors and Pigment 
Epithelium 

Publications 

Nguyen B-Y, Shay LE, Wyvill KM, Pluda JM, 
Brawley O, Cohen RB, Whitcup SM, Venzon 
DJ, Broder S, Yarchoan R: A pUot study of 
sequential therapy with Zidovudine plus 
acyclovir, didanosine, and dideoxycytidine in 
patients with severe human immunodeficiency 
virus infection. / Infect Dis 168:810-817, 1993. 



Whitcup SM, Dastgheib K, Nussenblatt RB, 
Walton RC, Pizzo PA, Chan C-C: A clinico- 
pathologic report of the retinal lesions associ- 
ated with didanosine. Arch Ophthalmol, in 
press. 



Ill 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00264-04 LI 



PERIOD COVERED 

October 1, 1992 to Septemb e r 30, 1993 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Cytokines and Ocular Antig e ns in the Eye 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Chi-Chao Chan M.D. Head, Section on LI, NEI 

Immunopathology 



Others: Robert B. Nussenblatt 
Igal Gery 

Qian Li 
Louis Kasner 



M.D. Scientific Director LI, NEI 

Ph.D. Head, Section on LI, NEI 

Experimental Immunology 
M.D. Visiting Fellow LI, NEI 

M.D. Fellow LLNEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.0 



PROFESSIONAL: 



0.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x| (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project has been terminated and combined with project number ZOl EY 00222-08 LI. 



112 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00115-16 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 



Cvclosporine Therapy in Uveitis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Robert B. Nussenblatt M.D. Scientific Director NEI 



Others: 



Marc D. de Smet 


M.D. 


Scott Whitcup 


M.D. 


Chi-Chao Chan 


M.D. 



Visiting Scientist 
Senior Staff Fellow 
Medical Officer 



LI, NEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS; 



0.5 



PROFESSIONAL: 



0.5 



0.0 



CHECK APPROPRIATE BOX(ES) 

fxl (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Cyclosporine (Cs), an endecapeptide fungal product with specific anti-T-cell characteristics, is being 
administered to patients with sight-threatening ocular inflammatory disease of noninfectious origin who have 
failed on either corticosteroid or cytotoxic agent therapy to test its efficacy in the treatment of uveitis. Within 
the context of these ongoing studies, the combined use of CsA and ketaconazole has been tested in a 
randomized masked study of a small group of patients whose uveitis was well controlled with Cs. The 
combination allowed a significant reduction in the dose of Cs needed to control the disease. In some 
instances, the dose could be reduced by as much as 90 percent. No significant increase in side effects was 
noted. A phase I/II randomized trial using CsA and CsG has ended. There is a definite trend showing that 
combined use of a Cs and low to moderate steroid doses is efficacious in preventing the progression of uveitis. 
An effective dose of Cs appears to be around 5 mg/kg. At this dosage, toxicity has been reduced for up to 
12 months of followup. CsG was more effective than CsA in treating cystoid macular edema. Patients who 
remain on Cs long term continue to be followed to gain further information about renal (or other) toxicity. 



113 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Biologist 
LI, NEI 



Project Description 

Additional Personnei 

Barry Grubbs 

Clinical Protocol Number 

81-EI-33 



Objectives 

Cyclosporine (Cs), an endecapeptide obtained 
from fungi, has been shown to have specific 
anti-T-cell activity {Transplant Proc 12:234, 
1980). We have reported cyclosporine' s excep- 
tional effectiveness in preventing the induction 
of S-antigen autoimmune uveitis in rats as 
w^eU as inhibiting the disease once immuniza- 
tion has occurred (J Clin Invest 67:1228, 1981). 
The goal of this study is to test CsA versus 
CsG to test their efficacy in treating patients 
with bilateral sight-threatening posterior 
uveitis of an autoimmune nature. 

Methods 

Patients 18 years of age or older, of either sex 
(females not pregnant), who have not done 
weU on more conventional medical therapy 
were admitted to this study. All patients 
must have a bilateral sight-threaterung uveitis 
of noninfectious etiology that was not satisfac- 
torily controlled by either corticosteroid or 
cytotoxic agent therapy. Lymphocyte cultures 
are prepared, and the immune cells are tested 
against various crude ocular extracts as well 
as purified human S-antigen to assess evi- 
dence of cellular immune memory, which is 
considered to be the in vitro equivalent of the 
anamnestic response in vivo. Patients chosen 
are treated with CsA or a new analog called 
CsG in a phase I/II trial to evaluate the safety 
and activity of CsG versus CsA. During this 
period, the patients' cHnical and immunologic 
courses are closely monitored. Specific atten- 
tion is given to renal function changes, a 
frequent side effect. Patients who need to 
continue CsA because of their ocular disease 



for more than one year may be asked to 
undergo renal biopsy to evaluate the revers- 
ible and irreversible components to CsA renal 
toxicity. Some patients who have been en- 
tered on previous CsA studies and have 
continued to be followed in the eye clinic wiU 
continue to be monitored for their renal func- 
tion and to determine how and when cyclo- 
sporine can safely be tapered. 

Major Findings 

CsA has been effective in the treatment of 
some cases of posterior uveitis. An improve- 
ment in the inflammatory activity and visual 
acuity was seen in most patients treated to 
date. The particular responsiveness of pa- 
tients with the ocular manifestations of 
Behcet's disease to this agent has been corrob- 
orated by a masked, randomized trial per- 
formed in Japan. The improvement in the 
clinical condition was supported by a concom- 
itant improvement in electrophysiologic test- 
ing, particularly contrast sensitivity. 

Patients treated with CsA had no abnor- 
malities of natural killer cell activity before the 
initiation of therapy, nor was any noted after- 
ward. CsA significantiy decreased skin test 
responsiveness but did not alter lymphocj^e 
proliferation or antibody production in pa- 
tients. Renal toxicity has been noted in some 
patients on long-term therapy, necessitating 
the addition of systemic corticosteroids and a 
decrease in CsA dosage. At three months, 
approximately 78 percent of the patients 
entering this open study were considered 
therapeutic successes, but 62 percent were 
considered successes at one year. 

Seventeen patients treated long term with 
CsA underwent renal biopsy. These biopsy 
specimens were read in a masked fashion by 
a group of renal disease specialists who com- 
pared these biopsies with those from age-mat- 
ched controls. An irreversible component of 
CsA toxicity covdd be identified in the main 
being renal tubular afrophy accompanied by 
interstitial fibrosis. The majority of the indivi- 
duals' biopsies had normal serum creatinine 
values, but a correlation could be made 



114 



Laboratory of Immunology 



between the alterations noted and previous 
serum creatinine elevations for some period of 
time. The Cs A/G trial has shown that CsG 
and CsA have overall equal value in treating 
uveitis. However, CsG was more effective in 
reducing cystoid macular edema than was 
CsA, particularly at lower dosages. 

Significance to Biomedical Research and tiie 
Program of ttie Institute 

Uveitis is one of the most frustrating problems 
in all of ophthalmology. Present modes of 
therapy for patients with severe ocular inflam- 
matory disease are inadequate and nonspecif- 
ic. CsA appears effective in treating posterior 
uveitis of noninfectious etiology. This is the 
first new agent in decades to be found useful 
in treating the severe form of this condition; 
therefore, it is important that the optimum 
therapeutic schedule be developed. Newer 
therapeutic strategies have already begun. 



Proposed Course 

Newer studies to look at various Cs combina- 
tions will continue. 

NEI Research Program 

Retinal and Diseases — Inflammatory Diseases 

Publications 

de Smet MD, Nussenblatt, RB: Clinical use of 
cyclosporine in ocular disease. Int Ophthalmol 
Clin 33(4):31-45, 1993. 

Nussenblatt RB, deSmet MD, Rubin B, Freidlin 
V, Whitcup SM, Davis J, et al: A masked 
randomized, dose response study between 
cyclosporine A and G in the treatment of 
sight-threatening uveitis of noivinfectious 
etiology. Am J Ophthalmol 115:583-591, 1993. 



215 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00278-03 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Oral Administration of Antigen and the Ocular Immune Response 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and instittJte affiliation) 

PI: Robert B. Nussenblatt M.D. Scientific Director NEI 



Others: Igal Gery 



Susan Whitcher 
Marc D. de Smet 



Ph.D. 

M.S. 
M.D. 



Head, Section on LI, NEI 
Experimental Immunology 

Clinical Protocol Assistant LI, NEI 

Visiting Scientist LI, NEI 



COOPERATING UNITS (if any) 



Laboratory of Immunology 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.8 



PROFESSIONAL 



0.3 



0.5 



CHECK APPROPRIATE BOX(ES) 

[xj (a) Human subjects 
|~| (a1) Minors 
|~| (a2) Interviews 



□ (b) Human tissues □ (c) Neitiier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The effect of the oral administration of various antigens on the ocular immune response has been tested in the 
animal model for severe intraocular inflammatory disease, experimental autoimmune uveioretinitis, which is 
induced by both retinal S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). Oral 
tolerance could be induced by repeatedly feeding rats with S-Ag. A putative suppresser cell that was CDS 
positive could be isolated from the spleen of such animals and transferred to other animals to induce a similar 
toleragenic effect. In addition, the role of the spleen was confirmed in ongoing animal experiments. A 
randomized masked trial to evaluate the usefulness of S-Ag feeding in patients with intraocular inflammatory 
diseases continues. A pilot study was performed in two patients that showed the induction of such tolerance, 
and these patients continue to be followed. 



116 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



Project Description 

Objectives 

Exploring means at imniunon^odulation has 
been a major role of this laboratory. Although 
extensive experimentation has used various 
immunosuppressive agents, there has also 
been a major thrust in an attempt to use other 
modes of immunosuppression. The goal of 
this series of experiments, in animals as well 
as in humans, is to test the efficacy of oral 
tolerance with uveitogenic antigens in the 
treatment of animals induced with experimen- 
tal autoimmune uveitis and in patients with 
bilateral sight-threatening posterior and inter- 
mediate uveitis of an autoimmune nature. 

Methods 

Six- to 10- week-old Lewis rats of either sex are 
used in these experiments. Animals are fed 
various antigens before and after the induction 
of experimental uveioretinitis. The feeding 
regimen includes whole molecules such as the 
retinal S-antigen (S-Ag) and interphotorecep- 
tor retinoid-binding proteins as well as their 
fragments. In a subset of experiments, some 
animals will also undergo splenectomy before 
the initiation of these experiments, but others 
will receive sham procedures. We are at- 
tempting to evaluate the clinical course of 
their disease and corroborate the cUnical 
observations with histopathology at various 
points after the initiation of the experiments. 
The goal is to evaluate the role of the spleen 
as well as the role of various fragments in the 
ability to induce this tolerogenic state. 

In the studies to be performed with pa- 
tients, those individuals who have bilateral 
uveitis of a noninfectious cause and who are 
18 years or older of either sex wiU be consid- 
ered for the study. Additionally, their lym- 
phocytes must demonstrate an in vitro preUf- 
orative response to the retinal S-Ag. The 
patients also need to be on systemic immuno- 
suppressive therapy, whether it be corticos- 
teroids, cytotoxic agents, or cyclosporine. The 
goal of this study will be to assess, in individ- 



uals who need high amounts of immunosup- 
pressive therapy to control their disease, 
whether the addition of oral feeding of retinal 
antigens wiU induce a toleragenic state. 

This study will be performed in a random- 
ized, double-masked fashion in which some 
patients wiU receive S-Ag, other patients wiU 
receive a retinal mixture containing several 
antigens, and still others wiU receive placebo. 
The intent is to reduce the amount of immu- 
nosuppressive therapy they are taking with 
the hope that a toleragenic state can be in- 
duced by the feeding of these antigens. 

Major Findings 

In the animal work, the spleen appears to play 
an important role in the induction of oral 
tolerance of S-Ag. In addition, the spleen is 
essential for adoptive transfer of tolerance by 
splenocytes from S-Ag fed donors. Thus, it 
would be logical to assume that the spleen 
acts as a sight for induction and /or ampUfica- 
tion of ceUs with suppressive activity. 

The pilot study has demonstrated that, at 
least in two patients, a toleragenic state could 
be induced with the feeding of antigen at the 
dosages that are planned for this study. One 
patient with par planitis and the other v^dth 
Behcet's disease have been able to discontinue 
their medication completely or reduce it to 
exceptionally low dosages. 

Significance to Biomedical Researcf) and the 
Program of the Institute 

Uveitis is one of the most frustrating problems 
in all of ophthalmology. The present modes 
of therapy for patients with severe ocular 
inflammatory disease all have limitations, 
particularly because of their secondary side 
effects. In identifying patients with an im- 
mune response to the retinal S-Ag, we will 
have been able to induce an immunosuppres- 
sive state without the use of pharmacologic 
agents. Furthermore, the induction of this 
tolerance would be antigen specific. 



ii: 



FY 1994 NEI Annual Report 



Proposed Course Publications 

The randomized study will begin shortly. Nussenblatt RB, de Smet MD, Weiner HL, 

Gery I: The treatment of the ocular complica- 

NEI Research Program tions of Behcet's disease with oral tolerization, 

in Wechsler B, Godeau P (eds): Sixth Interna- 

Retinal Diseases — Inflammatory Diseases tional Conference on Behget's Disease. New 

York, Excerpta Medica, 1993. 



118 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00184-12 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit or) one line between the borders.) 

Cellular and Immunogenetic Mechanisms in Uveitis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Rachel R. Caspi Ph.D. Visiting Scientist LI, NEI 

Others: Phyllis Silver B.S. Biologist LI, NEI 

Luiz Rizzo M.D. Visiting Associate LI, NEI 

Gil Sartani M.D. Visiting Fellow LI, NEI 

Xu Hui Ph.D. Visiting Fellow LI, NEI 

Sun Bing Ph.D. Visiting Fellow LI, NEI 

Chi-Chao Chan M.D. Medical Officer LI, NEI 



COOPERATING UNITS (if any) 

National Institute of Child Health and Development (Lawrence M. Nelson, M.D.); Arthritis and Rheumatism Branch, National Institute 
of Arthritis and Musculoskeletal and Skin Diseases (Ronald L. Wilder, M.D., Ph.D.); Bone Marrow Transplantation Unit, National Cancer 
Institute (Frances Hakim, Ph.D.); Research and Development, Wills Eye Hospital, Philadelphia, PA (Larry A. Donoso, M.D., Ph.D.) 



LAB/BRANCH 

Laboratory of Immunology 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 

5.1 



PROFESSIONAL: 

5.1 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects □ (b) Human tissues [x] (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Cellular mechanisms in ocular immunologically mediated disease are being studied in animal models of 
experimental autoimmune uveoretinitis (EAU). Rats and mice are immunized with retina-derived antigens or 
synthetic peptides, representing fragments of these antigens, to induce EAU. Susceptibility to disease induction 
is being evaluated in various strains of known genetic makeup, in the hope of delineating the hereditary 
mechanisms that predispose to uveitis. In vivo functional long-term T-cell lines and clones are developed from 
lymphoid organs of rats and mice immunized with uveitogenic ocular proteins. The functional properties and 
antigen receptors of these cells are being studied to develop strategies for in vivo targeting of the autoiiimiune 
cells. EAU in rats and mice serves as a template for the evaluation of new drugs and compounds as well as 
for the study and characterization of the participating cells and their factors. The goals of these studies are 
to identify the immunogenetic factors predisposing to uveitic disease, learn about the pathogenic mechanisms 
involved, characterize the immunoreactive cells and their mediators, and finally to utilize this knowledge for 
designing rational approaches to immunotherapy. 



119 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 



Robert B. 


M.D. 


Scientific Director, NFEI 


Nussenblatt 




Chief, LI, NEI 


Charles E. 


PhD. 


Staff FeUow, NEI 


Egwuagu 






Rashid Mahdi 




Biologist, LI, NEI 


Igal Gery 


Ph.D. 


Head, Section on 
Experimental 
Immunology, NEI 



Objectives 

The development and study of animal models 
of experimental ocular autoimmune disease 
permits the study of cellular and genetic 
factors that may be generally involved in 
ocular autoimmunity. Experimental autoim- 
mune uveitis (EAU) in rats and mice serves as 
a template for the evaluation of new drugs 
and compounds as well as for the study and 
characterization of the participating cells and 
their factors. Long-term maintenance of T 
cells in vitro permits the investigators to sepa- 
rate and selectively grow various T-cell sub- 
sets. The goals are to use the EAU model in 
rats and mice for the study of cellular mecha- 
nisms in ocular autoimmunity. Specifically, 
we are trying to delineate discrete stages in 
the immunopathogenic process and to devise 
strategies to specifically disrupt them at criti- 
cal points. 

One means to this end is to establish, 
characterize, and use retinal antigen-specific T- 
cell Unes and clones. These lines and clones 
permit us to learn about cells capable of 
ocular immunomodulation in the positive and 
negative sense, to earn about migration and 
localization of autcmmune lymphocytes, and 
to study their interactions with other lym- 
phoid and nonlymphoid cells in eliciting 
effector mechanisms. Finally, we use the EAU 
model for the study of hereditary mechanisms 
controlling genetic susceptibility and resis- 
tance to ocular autoimmune disease. The 
study and understanding of these parameters 
will help not only in the development of new 



therapies but also possibly in the prevention 
of ocular disease. 

Methods 

Rats and mice of various strains are immu- 
nized with purified S-antigen (S-Ag) or with 
interphotoreceptor retinoid-binding protein 
(IRBP) in complete Freund's adjuvant or with 
various pathogenic peptides derived from 
these proteins that are synthesized in the 
laboratory. After the development of disease, 
eyes are processed for histopathology and 
examined for disease, and lymphoid cells from 
the blood, lymph nodes, or eyes are taken. 
Cells thus obtained are placed in culture either 
with mitogen or with the retinal antigen with 
which the donor aiumal was immunized. 
Responses of the immune cells are studied. 

Cells are also expanded in culture and 
used to transfer EAU to nonimmune aiumals 
to study the cell population responsible for 
disease induction. Similar methods are used 
to study cells responsible for disease suppres- 
sion. Long-term ceU lines are developed and 
in some cases are cloned. These cell Unes and 
clones are then tested for functional character- 
istics such as the ability to induce or suppress 
ocular disease, production of soluble media- 
tors, expression of various cell-surface mole- 
cules, response to therapeutic agents, and 
interactions with other cells in culture. 

Major Findings 

The possible correlation between the pathoge- 
nicity of autoimmune T cells and their lym- 
phokine production, expression of functional 
adhesion molecules and expression of some 
surface antigens, was examined in the Lewis 
rat EAU model. We used four retinal antigen- 
specific Lewis rat T-cell lines and sublines: one 
specific to the major pathogenic epitope of the 
human retinal soluble antigen (S-Ag; residues 
337-356), and three specific to the major patho- 
genic epitope of the bovine IRBP (residues 
1177-1191). The lines have different degrees 
of uveitogenicity, from highly pathogenic to 
nonpathogenic. AU four T-cell Unes produced 
roughly equivalent amounts of interferon 



120 



Laboratory of Immunology 



gamma (IFN-y), l5nTiphotoxin/ tumor necrosis 
factor (TNFa/p), interleukin (IL)-3, IL-6, and 
transforming growth factor beta (TGF-P). IL-4 
activity could not be detected. The lines also 
expressed similar levels of functional adhesion 
molecules, as measured by binding to cultured 
rat aorta endothelial cells. The nonpathogenic 
subline, however, was the lowest responder to 
antigenic stimulation with respect to proUfera- 
tion and IL-2 production. Examination of ceU- 
surface antigens showed that in contiast with 
the other lines, the majority of cells in the 
nonpathogenic subline lacked detectable 
expression of QD-4. No difference was found 
in the level of expression of the IL-2 receptor 
and T-ceU antigen receptor between the four 
lines. Because CD-4 is the restricting element 
in these lines, reduced QD-4 expression in the 
nonpathogenic subUne may at least partially 
explain its poor response in vitro to antigenic 
stimulation. 

All three attributes could be coimected to 
lack of pathogenicity of this line in vivo. 
These results support the contention that class 
H-restricted recognition of autoantigen within 
the neuroretina by uveitogenic T lymphocytes 
must occur as an initial step in the pathogene- 
sis of EAU. A defect in this step wiU preclude 
pathogenesis, regardless of some other func- 
tional attributes possessed by effector T cells 
such as production of inflammatory lympho- 
krnes and expression of adhesion molecules. 

We have also studied the expression of 
cytokine genes within the eye in murine EAU. 
We have previously described several T-ceU 
Unes specific to the retinal protein IRBP or to 
its peptides that can induce EAU on adoptive 
transfer into naive mice. We have also shown 
that such cell Unes elaborate an unrestricted 
cytokine profile in vitro. 

The purpose of this study was to investi- 
gate what set of cytokines was being ex- 
pressed by these cells in vivo within the target 
organ. C57BL/6 athymic mice and BIO. A 
euthymic mice were injected intravenously 
with histocompatible uveitogenic T cells. 
Control animals were immunized with an 
established uveitogenic regimen of IRBP. The 



eyes were harvested 11 to 21 days later after 
perfusing the animals with 10 mL of 
PBS /heparin (to ensure that the cytokines 
detected did not originate from passenger 
lymphocytes passing through the retinal 
vessels). One eye was sent for histological 
examination, and the other eye was processed 
for ribonucleic acid (RNA) extiaction. The 
RNA was reverse-transcribed, and the result- 
ing complementary deoxyribonucleic acid was 
ampUfied using primers for several cytokines 
of interest. The results showed that although 
the uveitogenic T-ceU Unes and clones had an 
unrestricted cytokine profile in vitro, predomi- 
nantiy Th-1-type cytokine messenger ribonu- 
cleic acid (mRNA) (IL-2, IFN-y, and TNF-a) 
were present in eyes of mice that had EAU 
induced with those cells. Eyes of actively 
immuruzed animals showed a less restricted 
lymphokine profile, with most eyes having 
detectable mRNA for IL-2, IFN-y, and IL-4. 
Eyes of control animals that had neither been 
immunized nor adoptively transferred with T 
cells were negative for all cytokines tested. 
These results suggest that IL-2, IFN-y, and 
TNF-a are involved in murine EAU. Taken 
together with a lack of IL-4 mRNA in eyes of 
mice that had EAU induced by adoptive 
transfer, the data argue that predominantiy 
Th-1 tjrpe ceUs were present in the uveitic 
eyes. Because adoptive EAU was induced 
with T-ceU lines having an unrestricted cyto- 
kine profile (representing a Th-0 or a mixed 
Th-l+Th-2 population), this might imply that 
either a differentiation or a selection event 
occurred during development of EAU. 

In the mouse model of EAU, we have 
identified a major pathogenic epitope in the 
IRBP molecule that is recognized by mice of 
the H-2r haplotype. Overlapping 20-amino 
add peptides, spanning the entire human 
IRBP molecule, were synthesized and used to 
immunize C57BL/10 (H-2b), BIO.BR (H-2k), 
and BlO.Rlll (H-2r) mice. Bovine IRBP was 
used as a positive control. EAU was 
examined by histopathology 28 days after 
immunization. In vivo and in vitro 

immunological responses were assessed by 
delayed type hypersensitivity (DTH) and 
lymphocyte proliferation, respectively. 



121 



FY 1994 NEI Annual Report 



Peptide 161-180, spanning the sequence 
SGIPYVISYLHPGSTVSHVD, was found to be 
highly pathogenic for BIO.RIII mice but not for 
the other strains. EAU occurred in 20/20 
BIO.RIII mice after immunization with 50 yitg 
of peptide. The average disease score was 2.7 
as compared with 3.2 for IRBP-immunized 
mice. A dose-response curve showed that 
peptide 161-180 remained maximally patho- 
genic at 25 /ig, but incidence and scores were 
reduced at 10 fxg. The truncated 14-mer 165- 
178 was also highly pathogenic (100-200 
/ig/ mouse), suggesting that it contained the 
pathogenic epitope. Mice immunized with the 
peptide, or with whole IRBP, had positive 
DTH and lymphocj^e responses in vitro to the 
immunizing as weU as to the reciprocal anti- 
gen. These results indicate that peptide 161- 
180 appears to contain an epitope that is 
pathogenic to mice of the H-2r, but not H-2b 
or the H-2k haplotjq^es. High incidence and 
high severity scores as well as immunological 
crossrecognition between the peptide and 
IRBP in vivo and in vitro suggest that this 
peptide contains a major pathogenic epitope. 

In another study, the compound suramin 
was evaluated for its efficacy to prevent EAU. 
Suramin has been in clirucal use for treatment 
of parasitic diseases and some types of cancer 
and is known to have immunosuppressive 
properties. EAU was induced in BIO.A mice 
by immunization with the whole IRBP protein 
and in Lewis rats by immunization with 
peptide 35 of S-Ag or by adoptive transfer of 
a T-ceU Une specific to this peptide (SP-35 
Une). 

Actively immunized animals were treated 
with suramin (30-100 mg/kg, l.P.) to cover 
either the afferent or the efferent stage of EAU 
(days and seven or days seven and 14, 
respectively). Adoptively transferred animals 
(considered to represent efferent-stage disease) 
were treated on day minus one. Control 
animals were injected with phosphate-buffered 
saline at the corresponding times. EAU was 
assessed by cHrucal evaluation and by histopa- 
thology performed approximately one week 
after onset. Immunological responses were 
assessed by DTH and lymphocyte prolifera- 



tion to the immunizing antigen. The results 
revealed that treatment of BIO.A mice with 
100 mg/kg of suramin completely prevented 
disease when given during the afferent stage 
and ameliorated disease when given during 
the efferent stage of EAU. The same dose and 
regimen were somewhat less effective in 
preventing EAU in Lewis rats: Afferent 
treatment lowered the incidence and ameUo- 
rated disease scores by approximately 50 
percent, whereas efferent treatment (of either 
immunized or adoptively transferred rats) had 
Uttie or no effect on disease. Effect on DTH 
responses and lymphocyte proliferation rough- 
ly paralleled the effect on EAU. Thus, afferent 
treatment with suramin suppressed EAU and 
immunological responses, whereas treatment 
during the efferent stage was less effective, 
suggesting interference with antigen priming. 
The response to treatment may in part be 
dependent on the species in that mice re- 
sponded to the same treatment better than 
rats. To our knowledge, this is the first report 
of the successful use of suramin for treatment 
of autoimmunity. 

Another approach to suppressing ocular 
autoimmunity was through induction of oral 
tolerance. It was previously shown that EAU 
in rats can be suppressed by feeding of S-Ag. 
We wished to: (1) test whether a similar 
phenomenon exists in mice and (2) evaluate 
the feasibility of potentiating it by tmmuno- 
manipulation. For this purpose, EAU-suscep- 
tible BIO.A mice were fed IRBP or control 
solution using various regimens and were 
subsequently challenged with a uveitogenic 
regimen of IRBP. EAU was assessed by 
histopathology 21 days after immuruzation. 
Immunological responses measured included 
DTH, l5miphocyte proliferation, and cytokine 
production. 

The results indicated that three feedings of 
0.2 mg IRBP every other day before immuni- 
zation did not protect mice against EAU, 
whereas a similar regimen of five feedings of 
0.2 mg IRBP every other day was protective. 
However, supplementing the nonprotective 3x 
regimen with one intraperitoneal administra- 
tion of 400 units of recombinant human IL-2 



122 



Laboratory of Immunology 



on the day of immunization resulted in dis- 
ease suppression that was equal to that of the 
protective 5x regimen (p <. 0.02 compared with 
unfed controls). Analysis of cytokines pro- 
duced by Peyer's Patch ceUs of fed mice 
showed a large increase in production of 
TGFb, IL-4, and IL-10 in the 3x-fed and IL-2- 
treated animals compared with animals given 
the nonprotective 3x (no IL-2) feeding regimen 
and animals given the protective 5x feeding 
regimen. We propose that the IL-2 treatment 
enhances protection from EAU by stimulating 
regulatory cells that produce TGF-p, IL-4, and 
IL-10. Furthermore, the differences in lym- 
phokine production patterns among the exper- 
imental groups suggest that the mechanism of 
protection induced by the 3x + IL-2 regimen 
may differ from that induced by the 5x regi- 
men. It is conceivable that in the former case, 
protection from EAU was achieved by active 
suppression of the uveitogenic effector cells, 
whereas a mechanism of deletion or anergy 
might predominate in the latter. 

Significance to Biomedical Research and the 
Program of the Institute 

It has become increasingly clear that the 
cellular mechanisms and possibly the genetic 
mechanisms observed in animal models of 
uveitis reflect the mechanisms that operate in 
ocular immune-mediated disease in humans. 
The identification and characterization of the 
ceUs involved in ocular autoimmunity, and of 
their functions, will provide new understand- 
ing of inflammatory ocular diseases. Success- 
ful immunomodulation of EAU in animal 
models has thus far usually served as a good 
predictor of the clinical success of a given 
therapeutic modality. The continued study of 
basic mechanisms involved in the immuno- 
pathogenesis of uveitis in animal models will 
aid in the development of novel immunother- 
apeutic approaches for the control of uveitis in 
humans. 

Proposed Course 

This project will continue so that more infor- 
mation about the basic mechanisms in experi- 
mental uveitis may be obtained. 



NEI Research Program 

Retinal Diseases — Inflammatory Diseases 
Publications 

Caspi RR, Chan C-C, Grubbs BG, Silver PB, 
Wiggert B, Heremans H: Interferon-gamma at 
the systemic level protects mice against experi- 
mental autoimmune uveoretinitis. Reg Im- 
munol, in press. 

Caspi RR, Chan C-C, Fujino Y, Najafian F, 
Grover S, Silver PB, Hansen CT, WUder RL: 
Recruitment of naive T cells plays a pivotal 
role in experimental autoimmune uveoretinitis 
(EAU). Reg Immunol, in press. 

Caspi RR, Nussenblatt RB: Natural and thera- 
peutic control of ocular autoimmunity — rodent 
and man, in Goutinho A, Kazatchkine M 
(eds): Autoimmunity: Physiology and Disease. 
Wiley-Liss, Inc., New York, 1994, pp 377-405. 

Caspi RR, Parsa C, Chan C-C, Grubbs BG, 
Bahmanyar S, Heremans H, BUliau A, Wiggert 
B: Endogenous systemic interferon-gamma 
has a protective role against ocular autoimmu- 
nity in mice. / Immunol 152:890-899, 1994. 

Caspi RR, Silver PB, Chan C-C, Wiggert B, 
Redmond TM, Donoso LA: Immunogenetics 
of experimental autoimmune uveoretinitis 
(EAU). Reg Immunol, in press. 

Caspi RR: Experimental autoimmune 

uveoretinitis in rats and mice, in Cohen I, 
Miller A (eds.): Guidebook to Animal Models for 
Autoimmune Diseases. Academic Press, in 
press. 

Caspi RR: Thl and Th2 lymphocytes in exper- 
imental autoimmune uveoretinitis, in: Advanc- 
es in Ocular Immunology (Proceedings of the 
Sixth International Symposium on the Immu- 
nology and Immunopathology of the Eye, 
Bethesda, MD, 1994). Amsterdam, Elsevier 
Science Publishers B.V., International Congress 
Series, 1994, pp 55-58. 



123 



FY 1994 NEI Annual Report 



Kozhich AT, Kawano Y, Egwuagu CE, Caspi 
RR, Berzofsky JA, Gery I: A pathogenic 
autoimmune process targeted at a surrogate 
epitope. ; Exp Med 180:133-140, 1994. 

Mahdi RM, Caspi RR, Kozhich AT, Kozhich 
OA, Silver PB, Nussenblatt RB, Egwuagu CE: 
C5^okine mRNA expression following adop- 
tive transfer of uveitogenic T cells into 
athymic and euthymic Lewis rats. Invest 
Ophthalmol Vis Sci 35(4):1561, 1994. 

Malty R, Caspi RR, Nelson LM: Neonatal 
th5miectomy causes persistence into adulthood 
of the neonatal capacity for increased IL-4 
production. FASEB } 8:A483, 1994. 

Miller-Rivero NE, Rizzo LV, Chan C-C, 
Wiggert B, Nussenblatt: RB, Caspi RR: Sup- 
pression of IRBP-induced EAU in mice by 
feeding IRBP, and its potentiation by 
interleukin-2. Invest Ophthalmol Vis Sci 
35(4):1865, 1994. 

Prendergast RA, Coskuncan NM, Lutty GA, 
McLeod DS, Caspi RR: Induction of adoptive 
T ceU-mediated EAU: Temporal appearance 
of specific and control activated cells in retinal 
tissue. Invest Ophthalmol Vis Sci 35(4):1561, 
1994. 

Prendergast RA, Coskuncan NM, MacLeod 
DS, Lutty GA, Caspi RR: T ceU tiraffic and the 
pathogenesis of experimental autoimmune 
uveoretinitis, in: Advances in Ocular Immunolo- 
gy (Proceedings of the Sixth International 
S5miposium on the Immunology and Immuno- 
pathology of the Eye, Bethesda, MD, 1994). 
Amsterdam, Elsevier Science Publishers B.V., 
International Congress Series, 1994, pp 59-62. 

Rizzo LV, Miller-Rivero NE, Chan C-C, 
Wiggert B, Nussenblatt RB, Caspi RR: Inter- 
leukin-2 treatment potentiates induction of 
oral tolerance. FASEB J 8:A476, 1994. 



Rizzo LV, MiUer-Rivero NE, Chan C-C, 
Wiggert B, Nussenblatt RB, Caspi RR: Effect 
of interleukin-2 on the induction of oral toler- 
ance in experimental autoimmune uveoreti- 
nitis, in: Advances in Ocular Immunology (Pro- 
ceedings of the Sixth International Sjonposium 
on the Immunology and Immunopathology of 
the Eye, Bethesda, MD, 1994). Amsterdam, 
Elsevier Science Publishers B.V., International 
Congress Series, 1994, pp 221-224. 

Rizzo LV, Miller-Rivero NE, Chan C-C, 
Wiggert B, Nussenblatt RB, Caspi RR: 
lnterleukin-2 treatment potentiates induction 
of oral tolerance in a murine model of autoim- 
munity. / Clin Invest 94:1668-1672, 1994. 

Rizzo LV, Silver PB, Gazzinelli RT, Chan C-C, 
Wiggert B, Caspi RR: Expression of cytokine 
genes wdthin the eye in murine EAU. Invest 
Ophthalmol Vis Sci 35(4):1862, 1994. 

Sartani G, Silver PB, Sh-assmann G, Chan C-C, 
Caspi RR: Effect of suramin treatment on 
induction of EAU. Invest Ophthalmol Vis Sci 
35(4):1862, 1994. 

Savion S, Oddo S, Grover S, Caspi RR: 
Uveitogenic T lymphocytes in the rat: Patho- 
gerucity vs. lymphokine production, adhesion 
molecules and surface antigen expression. / 
Neuroimmunol, in press. 

Silver PB, Rizzo LV, Chan C-C, Donoso LA, 
Wiggert B, Caspi RR: Identification of a major 
pathogenic epitope in the IRBP molecule 
recognized by mice of the H-2r haplotype. 
Invest Ophthalmol Vis Sci 35(4):2061, 1994. 



124 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00266-05 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on arte line between the borders.) 

Characterization of Immune Responses to Retinal Specific Antigens 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI 



Others: Igal Gery 



Robert B. Nussenblatt 
Frangois Roberge 



Ph.D. Head, Section on LI, NEI 

Experimental Immunology 

M.D. Scientific Director NEI 

M.D. Visiting Scientist LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.6 



PROFESSIONAL: 



0.6 



0.0 



CHECK APPROPRIATE BOX{ES) 

fxl (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

One of the characteristics of S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP), which 
are retinal-specific antigens, is the ability to induce an intense autoimmune inflammation in the eyes of 
experimental animals when injected in the presence of an adjuvant. This disease, called experimental 
autoimmune uveitis (EAU), is critically dependent on T cells and antigen processing by appropriate antigen- 
presenting cells (APCs). Antigen processing, which occurs within the endocytic vesicles of the APCs, results 
in the production of small polypeptide subunits. These small polypeptides must then be protected from further 
degradation and transported to the cell surface where the interaction with the T cell takes place. 

In FT 1994 we further characterized the intracellular protein first identified in FY 1993. We confirmed that 
the protein does belong to the heat shock family of proteins by partial amino acid sequencing of the 70kD 
peak. We also identified the peak at 40kD as being actin. Testing with different peptide moieties has shown 
that binding of immunogenic peptides by hsp70 is a selective process. Certain peptides bind well to hsp70, 
although other peptides have no affinity for hspVO. The process is also selective in that it requires ATP in 
order to release the bound peptide. We have also found increased levels of hsp antibody in the serum of 
patients with Behget's disease. The increase serum levels corresponded to periods of ocular inflammatory 
activity in the absence of any evidence of active systemic disease. 



125 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 



Sumeet Mainigi 




Biologist, LI, NEI 


Sakpana 


PhD. 


Biolgist, LRCMB, NEI 


Rengerajan 






Gerald J. Chader 


Ph.D. 


Chief, LRCMB, NEI 


Barbara Wiggert 


PhD. 


Head, Section on 
Biochemistry, LRCMB, 

NEI 



Clinical Protocol Numbers 

84-EI-214 
79-EI-49 

Objectives 

In fiscal year (FY) 1994, we further character- 
ized the intracellular protein first identified in 
FY 1993. We confirmed that the protein does 
belong to the heat shock family of proteins by 
partial amino add sequencing of the 70kD 
peak. We also identified the peak at 40kD as 
being actin. Testing with different peptide 
moieties has shown that binding of immuno- 
genic peptides by heat shock proteins (HSP) 
70 is a selective process. Certain peptides 
bind weU to HSP 70, but other peptides have 
no affinity for HSP 70. The process is also 
selective in that it requires adenosine triphos- 
phate to release the bound peptide. We have 
also found increased levels of HSP antibody in 
the serum of patients with Behcet's disease. 
The increased serum levels corresponded to 
periods of ocular inflammatory activity in the 
absence of any evidence of active systemic 
disease. 

Methods 

Using B-cell lysates from Epstein-Barr virus 
(EBV)-fransformed cells and from B-ceU iso- 
lates of rat spleens, the HSP 70 capable of 
binding to interphotoreceptor retinoid-binding 
progression (IRBP) fragment 1169-1191 was 
isolated and further characterized by amino 
acid sequencing. Isolation was carried out on 
an activated Sepharose 4b column to which an 
appropriate peptide was Hnked. Several 



experiments were carried out to determine the 
affinity of various peptide substitutes to 
infraceUular HSP 70. Human serum was 
tested in a standard enzyme-Unked immuno- 
sorbent assay using a commercial HSP 70 
antigen. 

Major Findings 

We have determined that the 70 kD binding 
protein belongs to the heat shock family of 
proteins and that it is a new member of the 
HSP 70 family of proteins because part of its 
sequence has only 40 percent homology with 
other HSP members. The 40 kD protein is 
actin. The exact role of actin in antigen pre- 
sentation is still unknown. One simple expla- 
nation is that actin binds through a simple 
bystander phenomenon because the protein is 
isolated from a ceU lysate. However, mono- 
meric actin may weU play a more active role 
in antigen presentation, particularly of cyto- 
soUc proteins. Further studies will be neces- 
sary to further elucidate its role. 

Using cell lysates of EVB-fransformed 
cells, we were able to show that the binding of 
HSP 70 to various peptide fragments is depen- 
dent on the peptide sequence. Substitution of 
certain nonpolar amino acids by charged 
molecules changes the binding characteristics. 
Differences were also noted between patients 
with Behcet's disease and normal individuals 
in terms of binding to various residues or 
analogs of IRBP. Of particular interest was 
the finding that serum levels of HSP 70 anti- 
bodies varied with the level of ocular inflam- 
mation. Patients had levels of HSP 708 anti- 
bodies that were above two standard devia- 
tions of confrols, the level of HSP 70 antibod- 
ies rose significantiy in patients with ocular 
inflammatory episodes. This is a unique 
finding in ocular inflammatory disease, where 
it is rare to find a systemic marker of inflam- 
matory activity in the eye. 

Proposed Course 

In the coming year, the main emphasis will be 
on further elucidating the role of hsps in 
anfigen presentation by studying the effect of 



226 



Laboratoiy of Immunology 



cellular stress on antigen presentation in cell 
lines and clones. In addition, we will attempt 
to produce a specific polyclonal and mono- 
clonal antibody to the HSP 708 that was 
isolated in the course of these studies. Once 
the antibody has been generated, we will look 
at several more patient populations to fiarther 
define the role of hsp in uveitis. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 
Publications 

de Smet MD, Bitar G, Roberge FG, Gery I, 
Nussenblatt RB: Human S-antigen: Presence 
of multiple immunogenic and immuno- 
pathogenic sites in the Lewis rat. J Autoimmun 
6:587-599, 1993. 

de Smet MD, Rengerajan K, Chader GJ, 
Wiggert B: Characterization of B ceU proteins 
binding specifically to uveitopathogenic pep- 
tide 1169-1191 of bovine IRBP. Invest Ophthal- 
mol Vis Sci 35(suppl):1865, 1994. 



Mainigi S, Rengarajan K, Wiggert B, Chader 
GJ, Nussenblati RB, de Smet MD: Elevation of 
serum antibody levels to heat shock protein 70 
during ocular inflammatory episodes in 
Behcet's patients. Invest Ophthalmol Vis Sci 
35(suppl):2098, 1994. 

Rengarajan K, de Smet MD, Chader GJ, 
Wiggert B: Affinity of HSP70 fiom EBV 
transformed human B cells for bovine and 
human IRBP peptides. Invest Ophthalmol Vis 
Sci 35(suppl):1864, 1994. 

Rengarajan K, de Smet MD, Chader GJ, 
Wiggert B: Identification of heat shock pro- 
tein binding to an immunodominant 
uveitopathogenic peptide of IRBP. Curr Eye 
Res 13:298-296, 1994. 



127 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00276-03 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Surgical Management of Uveitis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI 

Others: 



Frangois Roberge 


M.D. 


Visiting Scientist 


LI, NEI 


Margaret Cheung 


M.D. 


Senior Staff Fellow 


LI, NEI 


David Parks 


M.D. 


Senior Staff Fellow 


LI, NEI 



COOPERATING UNITS (if any) 

Clinical Oncology Program, Medicine Branch, National Cancer Institute (Robert Wittes, M.D.) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 







PROFESSIONAL: 











CHECK APPROPRIATE BOX(ES) 

fx| (a) Human subjects 
r~l (a1) Minors 
□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



THIS PROJECT IS INACTIVE. 



128 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00218-09 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Ocular Manifestations of the Acquired Immune Deficiency Syndrome 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: Marc D. de Smet M.D. Visiting Scientist LI, ^fEI 



Others: 



Robert B. Nussenblatt 
Scott Whitcup 
Margaret Cheung 
David Parks 
Frangois Roberge 
Chi-Chao Chan 



M.D. 
M.D. 
M.D. 
M.D. 
M.D. 
M.D. 



Scientific Director 
Assistant Clinical Director 
Senior Staff Fellow 
Senior Staff Fellow 
Visiting Scientist 
Head, Section on 
Immunopathology 



NEI 

CB, NEI 
LI, NEI 
LI, NEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 

Department of Critical Care Medicine, Clinical Center (Henry Masur, M.D.); Laboratory of Immunoregulation, 
National Institute of Allergy and Infectious Diseases (H. Clifford Lane, M.D.); Pediatric Branch, National 
Cancer Institute (Phil A. Pizzo, M.D.) ^^^^^^^^^ 



LAB/BRANCH 



Laboratory of Immunology 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



2.0 



PROFESSIONAL 



2.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

fxl (a) Human subjects 
[x] (a1) Minors 
□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Patients suffering from AIDS (acquired immunodeficiency syndrome) are at risk of developing significant 
ocular problems, either as a result of HIV (human immune deficiency virus) itself or as a result of 
opportunistic infection. Some of these problems can lead to blindness if left untreated. Among the many 
pathogens that can lead to blindness, cytomegalovirus (CMV) is by far the most common. 

In FY 1994, we have evaluated the effectiveness of an intraocular delivery device in preventing the spread 
of CMV retinitis. We have now nearly completed recruitment for this study. Although no analysis has yet 
been done of the data, no significant adverse reaction has been noted, and in all cases progression of CMV 
retinitis has been prevented. 



129 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Nurse Spedalist CB, 
NEI 



Project Description 

Additional Personnel 

Susan Mellow RN 

Clinical Protocol Number 

90-EI-208 



Objectives 

Patients suffering from AIDS (acquired immu- 
nodeficiency s5Tidrome) are at risk of develop- 
ing significant ocular problems, either as a 
result of human immunodeficiency virus itself 
or as a result of opportunitistic infection. 
Some of these problems can lead to blindness 
if left untreated. Among the many pathogens 
that can lead to blindness, cytomegalovirus 
(CMV) is by far the most common. 

In fiscal year (FY) 1994, we have evaluated 
the effectiveness of an intraocular delivery 
device in preventing the spread of CMV 
retinitis. We have nearly completed recruit- 
ment for this study. Although no data analy- 
sis has yet been done, no significant adverse 
reaction has been noted, and in all cases 
progression of CMV retinitis has been pre- 
vented. 

Methods 

This project entails the clinical evaluation, 
diagnosis, and treatment of retinitis in AIDS 
patients. It also involves the development of 
novel methods of therapy for the various 
forms of retirutis observed. Study of patho- 
logic tissue also is used to better understand 
the nature of the infectious processes. 

Major Findings 

In the past year, our major effort has centered 
on the treatment of CMV retinitis by using a 
slow release intraocular device. CMV retinitis 
is a major vision-threatening infection found 
in patients with advanced stages of AIDS. 



Current therapeutic modalities commit pa- 
tients to life-long intravenous (IV) therapy 
with anti-CMV drugs. These drugs require 
close monitoring because of their systemic 
toxicity. They also require the placement of a 
permanent IV access and hence place the 
patient at risk for both local and systemic 
infection. Recurrence of disease also tends to 
occur in the majority of patients. An intraocu- 
lar, slow-release device avoids these side 
effects by providing continuous antiviral 
therapy above the minimum inhibitory con- 
centration for up to eight months. Recruit- 
ment of patients for the study that was started 
in FY 1993 is now completed. This study 
compares patients in whom an intraocular 
device is placed immediately with patients in 
whom placement is differed until the CMV 
retinitis has progressed by 750 /im. Although 
recniitment has been completed, some patients 
are still in the active phase of the study. No 
serious side effects have been encountered, 
and good control of the CMV retinitis has 
been achieved. Full analysis of the study 
parameters wiU be undertaken shortly. Partic- 
ular attention wiU be given to the rate of 
bilateraHzation and to the survival of patients 
treated with the intraocular device. 

Despite treatment, CMV retinitis has a 
tendency to recur. With larger areas of retinal 
involvement, the risk of CMV detachment 
increases considerably, particularly in patients 
with peripheral involvement where vitro- 
retinal traction is greatest. Up to 30 percent of 
patients with CMV retinitis wiU develop a 
retinal detachment. Repair of detachments 
reqviires the use of a variety of vitro-retinal 
techniques from classical retinopexy to the use 
of more advanced vitreal-retinal approaches, 
including the use of silicone oU. The best time 
for intervention still remains to be determined. 
In FY 1994, we have begun to explore the 
various surgical approaches that are available 
for the repair of retinal detachments in pa- 
tients with CMV retinitis. For patients with 
detachments involving noninfected retina, 
standard scleral buckling procedures appear to 
be adequate. If a retinal detachment develops 
in involved retina, scleral buckling with sili- 
cone oil appears to be the most effective 



130 



Laboratory of Immunology 



approach. Many patients, however, do not 
recover their fuU central visual acuity. The 
cause of this decrease is not known and is 
being investigated. 

Significance to Biomedicai Research and the 
Program of the Institute 

The AIDS epidemic is a major pubUc health 
concern. CMV retinitis remains the number 
one cause of blindness among patients infect- 
ed with the AIDS virus. Early diagnosis is 
important because all drugs currently avail- 
able are only virostatic and not viroddal; thus, 
some progression of the lesion is seen in more 
than 50 percent of patients, despite anti-CMV 
therapy. No therapeutic modaUties that are 
cost effective and that reduce the incidence of 
progression are necessary. 

Management of the compUcations of CMV 
retinitis, particularly after recurrence and pro- 
gression of the disease will be an ever-increas- 
ing challenge as patients survive longer. Of 
these, the threat of retinal detachment is a 
major concern. The means of prophylaxis and 
therapy need to be developed. 



Proposed Course 

In the coming fiscal year, we are planning to 
evaluate the slow-release device further as 
well as the means of preventing second eye 
involvement in patients who are treated with 
the device. We also will study further means 
of treating patients with viral-related retinal 
detachments and means of preventing visual 
loss at the time of surgery. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 

Publications 

de Smet MD, Nussenblatt RB: Ocular mani- 
festations of HIV in the pediatric population, 
in Pizzo PA, Wilert CM (eds): Pediatric AIDS: 
The Challenge of HIV Infection in Infants, Chil- 
dren, and Adolescents. Baltimore, Williams & 
Wilkins, 1994 pp. 457-466. 

Maturi RK, Nussenblatt RB, de Smet MD: 
Prevalence of tear hyposecretion and vitamin 
A deficiency in patients with AIDS. Invest 
Ophthalmol Vis Sci 35(4)(suppl):1308, 1994. 



131 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00232-09 LI 



PERIOD COVERED 

October 1. 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on arte Ime between the borders.) 

Interferon System in Cellular Function and Disease 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: John J. Hooks Ph.D. Head, Section on LI, NEI 

Immunology and Virology 



Others: Caroline Percopo M.S. Biologist 

Chandrasekharam Nagineni Ph.D. Visiting Scientist 



LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 

Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) 



LAB/BRANCH 

Laboratory of Immunology 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.5 



PROFESSIONAL: 



0.2 



0.3 



CHECK APPROPRIATE BOX(ES) 

l~l (a) Human subjects 
r~l (al) Minors 
□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Cytokines, such as interferon gamma (IFN-y) and interleukin (IL)-2, are a group of specialized hormone-like 
proteins that exert profound influences on cellular development and on a variety of cellular functions. This 
project has concentrated on studying the ways in which cytokines interact with cells of the immune system 
and with cells in the ocular microenvironment. We have shown that IFN-y and IL-2 are found within the 
inflamed eye in association with T-cell infiltration and major histocompatibility complex (MHC) class II 
antigen expression on infiltrating cells and on retinal pigment epithelial (RPE) cells. Furthermore, IFN-y- 
activated RPE cells can process and present antigens to helper T lymphocytes. 

Experimentally we demonstrated that isolated human RPE cells can be induced to produce another 
lymphokine, IL-6, and soluble intercellular adhesion molecule- 1 (ICAM-1) following incubation with IFN-y. 
IL-6 is a potent inflammatory cytokine capable of enhancing antibody production and cytotoxic T-cell 
activities. ICAM-1 is an adhesion molecule that mediates cell adhesion, an essential component for several 
immunologic functions. These studies indicate that cytokine-mediated activation of RPE cells may be a basic 
component of ocular immunity and an important aspect of RPE cell transplantation. 

These observations indicate that IFN-y induces MHC class I, class II antigen and ICAM-1 expression and IL-6 
secretion by RPE cells. All of these factors may serve as a local amplification system in autoimmune and 
inflammatory eye disease. A better understanding of the role of cytokines in the mechanisms involved in the 
development of autoimmunity and inflammation may be beneficial in developing treatments for these diseases. 



132 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



Project Description 

Objectives 

This project is designed to determine the 
bioregulatory actions of interferon (IFN) and 
other cytokines and to evaluate their regulato- 
ry actions in the pathogenesis of disease. 

Methods 

We assayed human IFN using inhibition of 
vesicular stomatitis virus plaque formation in 
human amnion or WISH cells. IFNs were 
characterized by neutralization of antiviral 
activity with monoclonal anti-IFN immuno- 
globulin. Interleukin (IL)-2 biological activity 
was assayed by induction of proliferation of 
CTL cells. Soluble intercellular adhesion 
molecule 1 (ICAM-1) and IL-6 activity was 
assayed by an enzyme-Unked immunosorbent 
assay (ELISA) and immunoblot assays. 
ICAM-1 and IL-6 messenger ribonucleic acid 
(mRNA) were evaluated by Northern blot 
assays. Analytical flow cytometry was used to 
quantitate retinal proteins. Gene transcription 
techiuques such as Northern blot analysis and 
nuclear runoff transcription assays are being 
used to evaluate interferon gamma (IFN-y) 
modulation of retinal proteins. 

Major Findings 

Numerous studies indicate that a variety of 
autoimmune diseases are associated with the 
IFN-y-induced tissue-specific expression of 
major histocompatibility complex (MHC) class 
n molecules. During the past five years, we 
have identified various steps that may be 
involved in ocular immunopathologic mecha- 
nisms. In these studies of retinal degenera- 
tions and autoimmune diseases, we showed 
that a critical regulatory ceU in the retina, the 
retinal pigment epitheUal (RPE) cell, is capable 
of expressing MHC class n determinants. We 
can also detect IFN-7, in situ, in retinas from 
patients with inflammatory eye diseases as 
well as MHC class n positive RPE ceUs. In 
addition, freshly isolated human RPE ceUs can 
express these determinants following treat- 



ment with IFN-y. In animal model systems, 
we found that inoculation of recombinant 
IFN-y induces la expression of ocular cells, 
and treatment with anti-la antibodies can 
eliminate or inhibit experimental autoimmune 
uveitis. Recently, we showed that the RPE 
cell may be playing an important role in 
ocular immunity, acting as a resident antigen 
presenting cell in the retina. 

Also, we found that the RPE cell is capa- 
ble of producing the cytokine, rL-6. During 
the past year, we have evaluated lCAM-1 
production by cytokine-activated RPE cells. 
RPE cell cultures were estabUshed from hu- 
man donor eyes. Stimulation of RPE cells 
with IL-la, IL-P, tumor necrosis factor alpha 
(TNF-a), and IFN-7 resulted in the production 
and release of ICAM-1. In addition, there was 
a concomitant increase in the RPE ceU surface 
expression of ICAM-1 and the production of 
ICAM-1 mRNA. In contrast, IL-6 and hpo- 
polysaccharide were not capable of inducing 
ICAM-1 secretion by RPE ceUs. Taken togeth- 
er, these studies indicate that the proinflam- 
matory cytokine such as IL-1, TNF, and IFN-y 
can activate RPE cells to release both IL-6 and 
soluble ICAM-1. These studies further sub- 
stantiate the concept that cjiiokine-mediated 
activation of RPE ceUs may be a basic compo- 
nent of ocular immunity and may have major 
immunological consequences for RPE ceU 
transplantation studies. 

Significance to Biomedical Research and the 
Program of the Institute 

The studies described herein highlighted the 
fact that the release of cytokines such as 
IFN-y, within the ocular microenvironment 
and the subsequent induction of cytokines and 
MHC class I and 11 antigen expression on 
resident and infiltrating cells may be critical 
elements in a cascading effect that leads to 
ocular cell destruction. The cell within the 
retina that may play a critical role in autoim- 
mune uveitis is the RPE cell. IFN-y induced 
activation of RPE cells may participate in 
autoimmune disease in the ocular microenvir- 
onment. 



133 



FY 1994 NEI Annual Report 



Cytokines produced and localized in the 
eye may play a critical role in normal physiol- 
ogy, pathogenic mechanisms, and therapeutic 
approaches. Because the RPE cell is a pivotal 
regulatory cell in the retina, an understanding 
of how cytokines interact with this cell will 
shed light on avenues for therapeutic interven- 
tion in pathogenic states and transplantation. 

Proposed Course 

We plan to continue our evaluation of the role 
of cytokines in autoimmunity and inflamma- 
tion. We are now developing systems in rat 
models to monitor directly the effects of 
altering c)^okine production on inflammatory 
eye diseases. Moreover, we will continue to 
characterize the antigen-presenting ability of 
the RPE cell to a variety of antigens and 
viruses. 

NEI Research Program 

Retinal Diseases — ^Inflammatory Diseases 



Publications 

Detrick B, Hooks JJ: Cytokines and effector 
molecules in human immunology, in Leffell 
MS, Bias WB, Donnenberg AD, (eds): CRC 
Handbook of Human Immunology. Boca Raton, 
Florida, CRC Press Inc., in press. 

Nagineni CN, Detrick B, Hooks JJ: Synergistic 
effects of gamma interferon on inflammatory 
mediators that induce interleukin-6 gene 
expression and secretion by human retinal 
pigment epithelial cells. Clin Diag Lab Im- 
munol 1:569-577, 1994. 

Nagineni CN, Detrick B, Hooks JJ: Human 
RPE cells secrete cytokines in response to 
inflammatory mediators, in Proceeding of the 
Sixth International Symposium on the Immunolo- 
gy and Immunopathology of the Eye. Amster- 
dam, Elsevier Science Publishers, 1994. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00233-09 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title rrtust fit art one line between the borders.) 

Studies on the Bioregulato r y Aspects of the R etinal Pigment Epithelial Cell 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: John J. Hooks Ph.D. Head, Section on LI, NEI 

Immunology and Virology 

Others: Chandrasekharam Nagineni Ph.D. Visiting Scientist LI, NEI 

Caroline Percopo M.S. Biologist LI, NEI 

T. Michael Redmond Ph.D. Research Biologist LRCMB, NEI 

R. Krishnan Kutty Ph.D. Senior Staff Fellow LRCMB, NEI 

David Parks M.D. Senior Staff Fellow LI, NEI 

Marc D. de Smet M.D. Visiting Scientist LI, NEI 

Robert B. Nussenblatt M.D. Scientific Director LI, NEI 



COOPERATING UNITS (if any) 

Hopital St. Louis, Paris, France (Lawrence Boumsell, M.D.); University of Nice, France (Alain Bernard, M.D.); National Institute of 
Dental Research (Reuben Siraganian, M.D.); The Johns Hopkins University (Stanley A. Vinores, Ph.D.; Peter Campochiaro, M.D.); 
Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI, NIH, Betbesda, MP 20892 



TOTAL STAFF YEARS: PROFESSIONAL: 

0.9 0.5 



0.4 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects x (b) Human tissues (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The retinal pigment epithelial (RPE) cell plays a basic role in maintaining the structural and physiological 
integrity of the neural retina. We have isolated and propagated RPE cells in vitro and have developed 
monoclonal antibodies directed against human RPE cells. The RPE epitope is a 67 kDa protein that is closely 
associated with the microsomal membrane. A complementary deoxyribonucleic acid (cDNA) clone has been 
isolated that codes for a protein that does not match any other sequences in the databases. We are using these 
techniques and reagents to evaluate molecular, biochemical, and biological properties of the RPE cells. 

We have propagated human RPE cells in vitro and evaluated their ability to respond to cytokine activation. 
Transforming grov^^th factor - p (TGF-P) is a potent cytokine that modifies a variety of cellular functions. 
This cytokine has been identified within the retina. We found that TGF-p induces gene expression and 
production of Heme oxygenase (HO-1). Since HO-1 is a protective agent against oxidative damage in an O, 
rich environment, RPE production of this protein may protect the retina against oxidative damage. 

Studies are also in progress to propagate and transplant RPE cells in various animals. We have established 
a graft rejection model by transplanting human RPE cells into the rat retina. These studies demonstrate that 
both cellular and humoral aspects of the immune response are activated to reject RPE cell transplants. These 
studies provide the framework to evaluate cytokines and immune reactivity in RPE cell transplantation. 



135 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The aim of this project is to evaluate the 
molecular, biochemical, and varied biologic 
properties of retinal pigment epitheUal (RPE) 
cells in normal and disease states. Moreover, 
we are evaluating RPE cell transplantation. 

Methods 

RPE cells are isolated and propagated in vitro. 
Monoclonal antibodies were generated in mice 
by fusing mouse spleen cells with myeloma 
cells. Antibodies to RPE cells are evaluated by 
immunoperoxidase assays and Western blot 
assays. The effect of drugs and cytokines are 
evaluated by cell viability and proliferation 
assays and nuclear transcription runoff assays. 

Major Findings 

(1) Cytokine Activation of RPE Cells. Trans- 
forming growth factor beta (TGF-P) is a potent 
cytokine that modifies a variety of cellular 
functions. This cjdiokine has been identified 
within the retina. We found that TGF-p 
induces messenger ribonucleic acid (mRNA) 
for heme oxygenase-1 (HO-1) and induces 
HO-1 protein expression in human RPE cells. 
Because HO-1 is a protective agent against 
oxidative damage in an oxygen rich environ- 
ment, RPE production of this protein may 
provide dowrvregulation of oxidative damage. 

(2) RPE cell transplantation. Recent studies 
indicate that RPE cell transplantation may be 
beneficial in restoration of retinal architecture 
in selected retinal degenerations. It is essen- 
tial to develop methods for large-scale prepa- 
rations of RPE ceU ciiltures for somatic cell 
genetic engineering manipulations. We are in 
the process of evaluating various parameters 
for human and rat RPE cell culture and trans- 
plantation. Preliminary studies show that w^e 
can successfully transplant human RPE cells 
into the rat retina. We have used this xenoge- 
neic RPE transplant in the rat as a graft rejec- 
tion model. We demonstrated that the trans- 



fer of cultured adult human RPE cells to the 
Lewis rat subretinal space eUcits a graft rejec- 
tion peaking at seven days posttransplanta- 
tion. Infiltrating cells consisted of phagocytic 
CD-llc/CD-18 cells as well as CD-4 and CD-8 
positive cells. Systemic antibodies to the 
human RPE cells developed in the rats by 
seven to 21 days. These data indicate that 
both cellular and humoral aspects of the 
immune response are activated in this graft 
rejection model. 

Significance to Biomedical Research and the 
Program of the Institute 

The monoclonal antibodies developed in this 
study are the first directed solely at the RPE 
cell. These antibodies are potentially useful in 
identification of RPE cells in situ and in vitro. 
These antibodies, which can be used to moni- 
tor RPE cellular functions, may be used in 
providing a better understanding of the role of 
RPE cells in retinal degenerative disorders. 
Identification of the complementary deoxyri- 
bonucleic acid and proteins detected by the 
monoclonal antibodies may provide the frame- 
work to evaluate specific RPE cell functions. 
RPE cell transplantation to correct retinal 
degenerative processes is being actively inves- 
tigated in a number of laboratories. The 
studies reported here provide the framework 
to evaluate RPE ceU transplantation. 

Proposed Course 

(1) We will continue to characterize these 
antibodies as well as the effect of these anti- 
bodies on ceU function in vivo and in vitro. 

(2) We will isolate, propagate, and charac- 
terize RPE cells for transplantation studies in 
arumals and man. We will design effective 
ways to maintain the cell in culture and de- 
sign ways to measure and monitor ceU func- 
tion. 

NEI Research Program 

Retinal Diseases — Photoreceptors and Pigment 
Epithelium 



Laboratoiy of Immunology 



Publications Kutty RK, Kutty G, Nagineni CN, Hooks J], 

Chader GJ, Wiggert B: RT-PCR assay for 

Kutty RK, Nagineni CN, Kutty G, Hooks J], heme oxygenase-1 and henne oxygenase-2: A 

Chader GJ, Wiggert B: Increased expression sensitive method to estimate cellular oxidative 

of heme oxygenase-1 in human retinal pig- damage. Ann NY Acad of Sci, in press, 
ment epithelial cells by transforming growth 
factor-beta. / Cell Physiol 159:371-378, 1994. 



137 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00240-08 LI 



PERIOD COVERED 

October 1, 1993 to September 30. 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Virus Infections in the Eye 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: John J. Hooks Ph.D. Head, Section on LI, NEI 

Immunology and Virology 



Others: 



Caroline Percopo 
Yun Wang 
Miguel Bumier 
Ingeborg Kirch 
Yusuke Komuraski 



M.S. 
M.D. 
M.D. 
M.D. 
M.D. 



Biologist 
Guest Worker 
Visiting Scientist 
Guest Worker 
Guest Worker 



LI, NEI 
LI, NEI 
LI, NEI 
LI, NEI 

LI, NEI 



COOPERATING UNITS (if any) 

Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.); Department of Pathology, Uniformed Services 

University of the Health Sciences (Katherine Holmes, Ph.D.); Department of Ophthalmology, Ruprecht-Karl's University, Heidelberg, Germany (Ellen 

Kraus-Mackiw, M.D.); Laboratory of Biology, NCI, NIH (Charles H. Evans, M.D., Ph.D.); Department of Medicine, The Johns Hopkins Medical School, 

Baltimore, MD (William Burns, M.D.); Laboratory of Clinical Investigations, NIAID, NIH, (Jeffrey 1. Cohen, M.D., and Steven Strauss. M.D.) 

LAB/BRANCH 



Laboratory of Immunology 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 



NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



1.0 



PROFESSIONAL: 



0.8 



0.2 



CHECK APPROPRIATE BOX(ES) 

i~| (a) Human subjects 
□ (a1) Minors 
r~| (a2) Interviews 



[x| (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Our studies of various virologic and immunopathologic processes that occur when viruses replicate in the ocular 
microenvironment comprise four areas: (1) coronavirus infection in ocular and optic nerve cells; (2) the possible roles 
of viruses in human diseases; (3) molecular diagnosis and pathogenesis of cytomegalovims (CMV) infections in humans, 
and (4) Varicella - zoster vims infections of the retina. 

We have established that murine coronavims can induce ocular disease and may be used as a model system for studying 
retinal degenerative diseases. This model has many unique features. The virus is capable of inducing an acute infection 
in the presence of mild retinal vascular inflammation. Initial retinal damage is followed by clearance of infectious vims 
and progressive retinal degeneration. In situ hybridization techniques identified that the viral RNA persists within the 
Muller cells and RPE cells throughout the course of the disease. Recent studies show that there are genetic and 
immunologic components to this disease. The retinal degenerative pathologic manifestations of the disease can be 
influenced by the genetics of the host. That is, some strains of mice are resistant to vims-induced retinal degenerative 
changes. The pathologic changes are also closely related to the development of anti-retinal and anti-RPE antibodies. 
These findings suggest a role for autoimmunity in the pathogenesis. This disease may be considered a model for 
degenerative diseases of the pigment epithelium and photoreceptors in humans. 

Human CMV is a herpesvims that is a major cause of blindness in children bom with congenital infections and in 
immunocompromised individuals. It is difficult to study CMV latency in man. Therefore cell culture models of CMV 
replication and latency may provide insight into a rationale for alternative treatment modalities. We identified that CMV 
replicates in human RPE cells. Virus replication is extremely slow and is associated with a low expression of IE viral 
proteins. These may be critical variables in viral persistence and viral activation within the retina. 



138 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



Project Description 

Objectives 

This project was designed to determine the 
various effects of virus infections on the ocular 
microenvironment and to study modes of 
antiviral therapy. 

Methods 

This study involves the propagation and 
quantitation of viruses such as herpes simplex 
virus type 1, coronaviruses, and cytomegalovi- 
rus (CMV) in vitro and in vivo as weU as 
immunocytochemical analysis of infected cells 
and tissues. Techniques used in the character- 
ization of virus infection include flow cyto- 
metric analysis. Western blot analysis. North- 
em blot analysis, in situ hybridization, and 
amplification of viral genes by polymerase 
chain reaction (PCR). Techniques used in 
characterization of antivirus antibodies include 
enzyme-Unked immunosorbent assay and 
neutralization assays. 

Major Findings 

(1) Coronavirus Infection in the Eye. The mu- 
rine coronavirus, mouse hepatitis virus 
(MHV), JHM strain, induces a retinal degener- 
ative disease in adult Balb/c mice. In the 
early, acute phase one to seven days after 
inoculation, a niild retinal vasculitis is ob- 
served. The second stage is seen by day 10 
and progresses for several months. This stage 
is characterized by a retinal degeneration in 
the absence of vasciiUtis or inflammation. This 
degenerative process is associated with a 
reduction of the photoreceptor layer, loss of 
interphotoreceptor-binding protein, abnormali- 
ties in the retinal pigment epitheUum (RPE) 
and retinal detachments. The development of 
the degenerative phase of the disease is con- 
trolled by a genetic predisposition of the host 
and is associated with the development of 
antiretinal and anti-RPE cell autoantibodies. 
During the past year, we have evaluated three 
aspects of this disease process: (a) virus 
persistence within the retina, (b) immunologic 



aspects of the disease, and (c) genetic predis- 
position to the disease. 

One of the intriguing aspects of this dis- 
ease process is the nature of viral clearance. 
The acute phase of the disease is associated 
with the presence of viral proteins and the 
detection of infectious virus within the retina. 
However, after day eight, infectious virus and 
viral proteins are not detected. Nevertheless, 
the retinal tissue damage characterized as 
retinal degeneration becomes apparent at day 
10 and continues for months. The purpose of 
this study was to determine if the virus per- 
sists within the retina and other tissues during 
the course of this disease process. In situ 
hybridization was selected as a way to deter- 
mine if the virus persists and to identify the 
cellular location of this persistent infection. 

The presence of viral ribonucleic add 
(RNA) was detected by in situ hybridization 
with a viral complementary deoxyribonucleic 
add (cDNA) probe, and viral proteins were 
identified by immunocytochemical staining. 
cDN A probe representing MHV-A59 genes 4-6 
was prepared by digesting the plasmid DNA 
(clone 2-2) with Pst 1. cDNA was labeled 
v^dth digoxigenin-11-dUTP and hybridized 
with tissue sections. The resultant hybridized 
probe was identified with antidigoxigenin 
antibody conjugated to alkaline phosphatase. 
When uninfected (normal) mouse eye sections 
were incubated with the viral cDNA probe, no 
reactivity was observed. 

In contrast, when JHM virus-infected 
mouse eye sections were incubated with the 
viral cDNA probe; positive reactivity was 
noted within the retina from day one to day 
60 postinoculation. During the acute phase of 
the infection, viral RNA was found in the 
retina, RPE, ciliary body epitheUum, and the 
iris epitheUum. During the late phase of the 
infection, viral RNA was almost exclusively 
found within the retina and RPE and not in 
the anterior segment of the eye. Within the 
retina, viral RNA was detected in the ganghon 
ceU layer, the inner retina, the outer retina, 
and the RPE ceU. Pretreatment of infected 
eyes with RNase inhibited the reactivity and 



FY 1994 NEI Annual Report 



incubation of infected eyes with plasmid 
cDNA resulted in no reactivity. Immuno- 
cytochemical staining identified viral protein 
within the retina only from day one to day 
eight. This ocular disease is also associated 
with a persistent systemic infection. Both 
viral RNA and viral proteins were identified 
within the liver during the first eight days. 
However, only viral RNA is detected in the 
liver from day eight to 60. These studies 
show that MHV establishes an acute infection 
(days one to eight) where infectious virus and 
viral proteins are identified. This is followed 
by a persistent infection within the retina and 
Uver where only viral RNA can be detected by 
in situ hybridization. 

The coronavirus-induced retinopathy 
model allows us to explore some of the cellu- 
lar and molecular mechanisms involved in the 
autoimmune aspects of retinal tissue damage. 
During the past year, we have characterized 
the autoantibodies associated with the retinal 
degenerative process. BALB/c mice were 
inoculated by the intra vitreal route with 10^^ 
TCID50 / 5 ^tl of virus or media. At varying 
times after inoculation, sera and eyes were 
removed. The presence of antiretinal and 
anti-RPE cell antibodies were identified by 
immunocytochemical staining on normal rat 
eye sections and by immunoblot analysis. 
Sera collected from BALB/c mice from 12 to 
70 days after JHM virus infection contained 
antiretinal autoantibodies. 

These antibodies are not found in the sera 
from normal or mock-injected mice. Antibod- 
ies to retinal tissue were identified as two 
distinct patterns of autoantibodies — retinal 
and RPE autoantibodies. Absorption studies 
were performed to characterize the autoanti- 
bodies. Absorption of sera with 10 percent 
retina, brain, or kidney tissue did not alter 
antiretinal or anti-RPE cell autoantibody 
reactivity. However, absorption of sera with 
lyophilized soluble fractions of rat or cow 
retina did inhibit both antiretinal and anti-RPE 
autoantibodies. Incubation of sera with lyoph- 
ilized soluble fractions of rat kidney or Uver 
did not inhibit autoantibody reactivity. The 
studies identify that the autoantibodies in- 



duced by the virus infection react with soluble 
proteins identified in the rat and cow retinas. 
This animal model of postvirus retinopathy is 
associated with the production of retinal- 
specific autoantibodies and may provide 
insight into the study of humoral autoimmune 
responses in human retinal degenerations. 

Because the genetic composition of the 
host and the virus can determine the response 
to infection and the resulting pathology, the 
third phase of our studies evaluated the effect 
of MHV infections on different strains of mice. 
We reported in the past year that the patho- 
logic manifestations of a virus infection in the 
retina can be influenced by the genetics of the 
host. BALB/c mice developed both the retinal 
vasculitis and the retinal degenerative phases 
of the disease, whereas CD-I mice developed 
only the early retinal vasculitis and were 
spared the degenerative disease. During the 
past year, we have found that the A59 virus 
strain as well as the JHM virus stiain both 
induce a biphasic retinal disease. 

In contrast, the inoculation of MHV-3 
strain by the intravitreal route did not resvdt 
in a retinal degenerative disease. Within three 
to five days, aU of the animals died. Evalua- 
tion of the brain did not reveal pathologic 
damage. However, the liver contained patho- 
logic changes consistent with fvdminant acute 
hepatitis. These remarkably different diseases 
are induced by different strains of the same 
virus. The ability of different MHV strains to 
cause fatal acute hepatitis or persistent retinal 
disease may help to decipher the mechanisms 
of viral tissue tropism in this strain-specific 
pathogenesis. These studies demonstrate that 
the genetics of the virus can profoundly affect 
the pathology generated by a virus using the 
eye as a portal of entry. 

In summary, this model is characterized 
by the replication of JHM virus in the retina; 
producing an acute necrotizing disease of the 
sensory retina, resulting in only a mild inflam- 
matory response and a long-lasting disease 
(longer than 14 weeks). These studies identify 
that a progressive degenerative disease in the 
retina may be initiated by an acute virus 



Laboratory of Immunology 



infection in the absence of major inflammatory 
response. These studies during the past year 
clearly indicate that this retinal degenerative 
process has a persistent viral component, an 
immune component, and a genetic component. 
How these genetic and immunologic factors 
interact to influence the development of reti- 
nal degenerations are the intriguing aspects of 
this model. 

(2) Possible Role of Viruses in Human Eye 
Diseases. We have initiated studies to evaluate 
the possible involvement of viruses in the 
pathogenic processes of a variety of human 
eye diseases. We are now collecting serum 
samples and ocular tissue to use seroepi- 
demiologic approaches to detect virus and 
viral antigens via immunocytochemical stain- 
ing, in situ hybridization, and PCR assays. 

(3) Cytomegalovirus replication within the 
retina. CMV infections are frequent complica- 
tions in kidney and bone marrow transplant 
patients and human immunodeficiency virus 
patients. The mechanisms by which CMV is 
activated and repUcates within the retina is 
not known. We evaluated the ability of hu- 
man CMV to initiate replication in human 
RPE cells and compared this with studies in 
human fibroblasts (HEL) and human amnion 
epitheHal (WISH) cells. Human RPE ceUs 
were obtained from donor eyes and propagat- 
ed in vitro. CeUs were infected with CMV 
(AD169 strain) at an input multiplicity of 1. 
CMV replication was evaluated by immuno- 
fluorescence, flow cytometry. Western blot 
analysis, and infectivity assays. Cellular 
protein expression was evaluated by immuno- 
fluorescence and flow cytometry with mono- 
clonal antibodies. CMV induces a cytopathic 
effect, infectious virus in RPE cells, and hu- 
man fibroblast cells but fails to replicate in 
another epitheUal ceU— WISH ceUs. CMV 
replication in RPE cells is characterized by a 
prolonged period of low-virus protein expres- 
sion. Less than one percent of the cells con- 
tain viral proteins (IE, E, L) during the first 10 
days. These viral proteins are not detected by 
flow cytometry or Western blot analysis until 
15 days after inoculation. In contrast, CMV 
proteins are found in HEL ceUs within 24 



hours. Untieated human RPE cells in vitro, 
express MHC class I molecules, P-2 microglob- 
ulin, and intracellular adhesion molecule 1 
(CD-54). The CMV infection of RPE cells 
results in a downregulation in the expression 
of MHC class I molecules on the RPE cells. 
Cytotoxic T cells recognize the virus-infected 
cell only in association with MHC class I 
molecules on the cell surface. This study 
demonstrates that CMV can repUcate slowly 
within the RPE cell and that this repUcation 
can be monitored by flow cytometry. CMV 
replication in RPE cells is associated with the 
modulation of cellular protein expression, and 
this alteration may contribute to viral persis- 
tence within the retina. 

(4) Varicella-zoster virus (VZV) infections of 
the retina. VZV is a herpes virus that is fre- 
quenfly associated with acute retinal necrosis 
and other forms of retinal tissue damage. 
Nevertheless, there are no good in vivo or in 
vitro models to evaluate virus replication and 
latency. During the past year, we have initiat- 
ed studies to develop models of VZV infection 
of the retina and retinal ceUs. PreUminary 
studies indicate that human VZV can repUcate 
in the guinea pig retina, and this replication is 
associated with a chronic uveitis. In vitro 
studies indicate that VZV can replicate within 
human RPE cells. These preliminary findings 
suggest that we now have two approaches to 
evaluate VZV replication in retinal tissues. 

Significance to Biomedical Research and the 
Program of the Institute 

Elucidating the factors involved in viral 
spread and pathogenesis will yield a better 
understanding of diseases of viral etiology. 
We have established a new virus model for 
retinal degenerative processes in adult ani- 
mals. This model has many unique features. 
The virus is capable of inducing an acute 
infection in the presence of mild retinal vascu- 
lar inflammation. The initial retinal damage is 
followed by persistence of viral RNA and 
progressive retinal destruction, even months 
after infectious virus is gone. Moreover, the 
development of retinal degenerative process is 
determined by the genetics of the host and 



Ml 



FY 1994 NEI Annual Report 



involves the development of antiretinal auto- 
antibodies. This model should assist us in 
understanding the pathogenesis of selected 
human diseases of unknown etiology. 

We have developed new systems to evalu- 
ate two herpes virus infections of the retina, 
CMV, and VZV. We have shown that CMV 
repUcates within RPE cells in a slow, limited 
manner. The evaluation of the molecular 
aspects of this defect may provide critical 
clues in terms of the virus' ability to estabUsh 
persistent infections and the factors initiating 
viral activation within the retina. We have 
developed two new approaches to evaluate 
VZV replication within the retina. 

Proposed Course 

(1) We will continue to evaluate coronavirus 
infections of the eye. The role of genetic fac- 
tors and autoantibodies in the pathogenesis of 
retinal degenerations will be evaluated. The 
data obtained will be correlated with what is 
known about human retinal degenerative 
disorders. 

(2) We wiU initiate studies to determine 
whether certain viruses can replicate in retinal 
tissues and cells. Infected cells wall be evalu- 
ated for the release or expression of uveito- 
genic proteins. 

(3) We will continue to collect samples 
and initiate studies to detect the involvement 
of viruses in human eye diseases. 

(4) We will evaluate the molecular diag- 
nosis and pathogenesis of CMV and VZV 
infections in the eye. 



NEI Research Program 

Retinal Diseases — Inflammatory Diseases 
Publications 

Bumier M, Wang Y, Detrick B, Hooks JJ: 
Retinal manifestations of a murine coronavirus 
infection: A histopathological and ultrastruc- 
tural study. Exp Pathol, in press. 

Hooks JJ: Ocular Virology, in Tabarra K (ed): 
Infections of the Eye. Boston, Little, Brown & 
Co. PubUshers, 1993. 

Hooks JJ, Wang Y, Komurasald Y, Percopo C, 
Nagineni CN, Detrick B: Molecular and 
immunologic mechanisms involved in corona- 
virus induced retinopathy, in Proceedings of the 
Sixth International Symposium on the Immunolo- 
gy and Immunopathology. Amsterdam, Excerpta 
Medica, Elsevier Science Publishers, 1994. 

Wang Y, Percopo CM, Bumier MN, Detrick B, 
Hooks JJ: Genetics of the virus determines 
retinal tissue damage induced by corona- 
viruses, in Proceedings of the Sixth International 
Symposium on the Immunology and Immunopa- 
thology. Amsterdam, Excerpta Medica, 
Elsevier Science Publishers, 1994. 



PROJECT NUMBER 
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE ! 

NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00277-03 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between (he borders.) 

Role of Retinal Pigment Ep ith elium in Re tina l Dis orders 



PRINCIPAL INVESTIGATOR (List ottier professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Chandrasekharam N. Nagineni Ph.D. Visiting Scientist LI, NEI 

Others: John J. Hooks Ph.D. Head, Section on Immunology LI, NEI 

and Virology 



COOPERATING UNITS (if any) 

Department of Pathology, George Washington University, Washington, DC (Barbara Detrick, Ph.D.) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 

1.0 



PROFESSIONAL: 

1.0 



OTHER: 

0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects [x] (b) Human tissues □ (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The retinal pigment epithelium (RPE) plays a critical role in the regulation of retinal and choroidal function in normal 
and disease states. Due to limited availability of human tissues, an in vitro cell culture system is desired. Therefore, 
we have developed and characterized the primary cell lines of human RPE from donor eyes obtained from eye banks. 
Using human RPE cell cultures as a model, we conducted investigations to examine the various roles of RPE in the 
pathophysiology of retinal disorders. 

Human RPE cultures secreted significant quantities of interleukin-6 (IL-6) and intercellular adhesion molecule- 1 
(ICAM-1) but not interleukin-1 (IL-1) in response to the stimulation by inflammatory mediators. Interferon gamma 
(IFN-7) exhibited synergistic effects in the secretion of IL-6 and ICAM-1 in the presence of suboptimal levels of tumor 
necrosis factor a (TNF-a) or IL-1. Cellular expression of ICAM-1, mostly localized to intercellular junctions, was 
observed in RPE treated with TNF-a and IFN-y. There is a close correlation between IL-6 and ICAM-1 secretion and 
IL-6 and ICAM-1 mRNA levels, respectively, suggesting the regulation at the gene transcription. The responses of RPE 
to inflammatory mediators in IL-6 and ICAM-1 secretion was rapid and sustained in the presence of stimulants but 
reversed to control levels quickly upon withdrawal of the stimulants, indicating the reversibility of the responses of RPE 
to inflammatory signals. These results show that RPE responds to inflammatory stimuli by increased cellular expression 
and secretion of IL-6 and ICAM-1, which may in turn perpetuate immune reactions in the pathogenesis and/or prevention 
of retinal and choroidal diseases. 



143 



PHS 6040 (Rev. 5/92) 



FY 1994 NEl Annual Report 



Project Description 

Additional Personnel 

Krishnan R. Kutty Ph.D. LRCMB, NEI 
Barbara Wiggert PhD. LRCMB, NEI 



Objectives 

The primary objectives of this research project 
are to: (1) establish primary cell lines of 
human retinal pigment epithelial (HRPE) cells 
from donor eyes, (2) develop serum-free 
media and other factors that can induce differ- 
entiation and pigmentation of HRPE, (3) 
investigate the role of inflammatory mediators 
and growth factors on cellular and molecular 
aspects of HRPE structure and function, and 
(4) evaluate the usefulness of HRPE and rat 
retinal pigment epithelial (RPE) cultures for 
transplantation studies to restore retinal func- 
tions in hereditary, autoimmune, and 
age-related disorders. 

Methods 

Primary cell cultures of HRPE are prepared by 
initial seeding of either freshly isolated RPE 
cells or RPE-choroid explants. Cells are 
grown in minimum essential medium supple- 
mented with 10 percent fetal calf serum and 
nonessential amino adds and antibiotics. We 
are developing serum-free, hormonally de- 
fined media and extracellular matrix factors 
that would induce differentiated morphology 
for the RPE cells. 

Techniques required for cell culture, im- 
munofluorescence, C5d:okine, and intercellular 
adhesion molecular 1 (ICAM-1) assays by 
enzyme-hnked immunosorbent assay, gel 
electrophoresis. Western and Northern blot- 
ting for proteins and ribonucleic acid (RNA), 
reverse tianscription polymerase chain reac- 
tion (RT/PCR) are developed and standard- 
ized in our laboratory to carry out these 
studies. 



Major Findings 

In the past, age of the donor was considered 
very critical in preparing human RPE cultures 
because eyes from donors older than 50 years 
of age did not yield fruitful cell Unes, proba- 
bly due to senescence-associated loss of viabil- 
ity. In these experiments, RPE cells were first 
disassociated from the eye cups by digestion 
with proteolytic enzymes, a treatment that 
might have caused initial contamination with 
nonepitheUal cells from which it is impossible 
to purify epithelial cells. Therefore, we have 
developed a new method using RPE-choroid 
explants to initiate ceU growth. By careful 
monitoring of clusters of cells growing around 
the explants, we were able (on the basis of 
morphology combined with experience) to 
select pure epithelial cells and discard nonepi- 
theUal cells at the primary culture stage. 

Using this technique, we established 
primary cell Unes of human RPE from eyes 
obtained from 81- and 87-year-old donors. 
The epitheUal nature of these ceU Unes was 
confirmed by immunochemical staining for 
cyiokeratin with monoclonal antibodies. All 
of the ceUs expressed cytokeratin at different 
passages. Immunoblotting analysis of cellular 
proteins indicated cytokeratin-18 was the 
predominant cytokeratin in these ceUs. Be- 
cause RPE is the only epitheUal ceU in the 
posterior segment (choroid-RPE-retina), these 
results estabUsh without doubt that the ceU 
Unes developed are, in fact, RPE. 

Human RPE cultures secrete significant 
quantities of interleukin (IL)-6 and ICAM-1, 
but not IL-1, in response to the stimulation by 
inflammatory mediators. IL-1 is the most 
potent stimulant of IL-6 secretion followed by 
tumor necrosis factor alpha (TNF-a) and 
Upopolysaccaride (LPS). Interferon gamma 
(IFN-y) had only minimal effects on IL-6 
secretion. In confrast to IL-6 secretion, ICAM-1 
secretion was not influenced by LPS. TNF-a, 
IFNy, and IL-1 had almost similar effects on 
ICAM-1 secretion by HRPE. The effect of 
IFN-Y is striking in that it can act synergjs- 
tically in the presence of suboptimal levels of 



144 



Laboratory of Immunology 



TNF-a or IL-1 to augment secretion of both 
IL-6 and ICAM-1. More than 98 percent of 
IL-6 produced by HRPE cells was secreted 
promptly into the medium. Western blot 
analysis of secreted IL-6 revealed multiple 
molecular forms suggesting that HRPE are 
capable of carrying out posttranslational 
glycosylation processes that may be important 
for functional activities. Cellular expression of 
IL-6 was not detected by immunofluorescence 
staining of the cells. In contrast, intense 
staining for ICAM-1 — that was mostly local- 
ized to intercellular junctions of the monolayer 
of epithelial cells — was observed in HRPE 
treated with TNF-a and /or IFN-y. Analysis 
of IL-6 and ICAM-1 messenger ribonucleic 
add (mRNA) expression by Northern blotting 
indicated rapid and sustained responses of 
HRPE to inflammatory cytokines that can be 
reversed quickly on withdrawal of the stimu- 
lus. There is a close correlation between IL-6 
and ICAM-1 secretion as weU as IL-6 and 
ICAM-1 mRNA levels, respectively. 

Our results indicate that HRPE respond to 
LPS and inflammatory cytokines (TNF-a, IL-1, 
and IFN-y); and enhance IL-6 and ICAM-1 
gene expression and secretion of proteins. 
During posterior uveitis of the eye caused by 
infections or autoimmune diseases, macro- 
phages and lymphocytes infiltrate into the 
retina and secrete cytokines such as TNF-a, 
IL-1, IFN-Y, 3rid IL-2 that would initiate im- 
mune reactions. These cytokines in their turn 
act on retinal resident cells to locally produce 
IL-6 and ICAM-1 to ampUfy the immunopath- 
ological processes. IL-6, a multipotent cyto- 
kine, plays a major role in the autoimmune 
and inflammatory disorders by its abUity to 
induce proliferation and differentiation of 
lymphocytes and production of antibodies. 
ICAM-1, an adhesive glycoprotein, participates 
in inflammatory reactions by recruiting leuko- 
cytes to the sites of inflammation, lymphocyte 
proliferation, cytotoxic T-ceU function, and T- 
cell mediated B-ceU activation. 

Several Hnes of evidence support that 
bacterial endotoxins and cytokines TNF-a, 
IL-1, IFN-Y, IL-2, and IL-6 play a critical role 
in uveitis and other inflammatory diseases of 



the eye. Intravitreal injection of IL-1, TNF-a, 
or IL-6 has been shown to cause uveitis in 
experimental animal models. Moreover, 
elevated levels of IL-6, IFN-y, IL-1, and TNF-a 
have been found in aqueous humor and 
vitreous aspirates of patients with uveitis, 
proliferative vitreoretinopathy, and other 
noncomplicated retinal detachments. Upregu- 
lation of the expression of ICAM-1 on retinal 
ceUs and epiretinal membranes and an in- 
crease in soluble ICAM-1 in vitreous of pa- 
tients with inflammatory retinal diseases 
suggest a vital role for ICAM-1 in various 
diseases. Our studies suggest that RPE reacts 
to inflammatory stimuU and secrete IL-6 and 
ICAM-1, thereby elevating these proteins in 
the local environment for the perpetuation of 
immunopathological processes. The synergis- 
tic actions of IFN-y on IL-6 and ICAM-1 secre- 
tion by HRPE cells in the presence of other 
cytokines would result in an effective amplifi- 
cation mechanism because several cytokines 
are produced simultaneously during iitflam- 
mation. 

The roles of growth factors, basic fibro- 
blast growth factor, transforming growth 
factor beta (TGF-P) and platelet-derived 
growth factors in RPE functions, and the 
regulation of secretion of these growth factors 
by RPE are being investigated. We found that 
these growth factors had no effect on the 
expression and secretion of IL-6, ICAM-1, and 
IL-1. The expression of heme oxygenase-1 
(HO-1) was increased by TGF-P in HRPE ceUs 
by fourfold to fivefold within hours of stimu- 
lation. However, LPS, inflammatory cyto- 
kines, and other growth factors had no effect 
on HO-1 levels. Among ocular tissues, RPE 
has the highest activity of HO-1. HO-1 cata- 
lyzes the oxidation of heme into biliverdin 
and carbon monoxide. Biliverdin is converted 
by nonlimiting enzymatic reaction into biliru- 
bin, an antioxidant that offers protection of 
cells against heat and oxidative stress. These 
results suggest that TGF-P upregulates de- 
fense mechanisms of RPE, a phagocytic cell 
that is constantly subjected to chemical stress, 
by engulfing the shed outer segments of 
retinal rods and cones for protection against 
oxidative damage. 



145 



FY 1994 NEI Annual Report 



Significance to Biomedical Research and the 
Program of the Institute 

Primary cell lines of human RPE are an ideal 
in vitro model for evaluation of several func- 
tions of RPE and for further elucidation of the 
mechanisms of RPE involvement in the patho- 
genesis of retinal and choroidal diseases. 
These cells are potentially useful in cellular 
transplant therapy to correct hereditary and 
age-related macular degeneration defects in 
humans. 

Proposed Course 

Two of the major problems associated with 
human RPE cell cultures are: (1) progressive 
loss of pigmentation on serial passaging of 
cells and (2) lack of clear intercellular junc- 
tions and in vivo-]ike morphological appear- 
ance. These changes could probably be due to 
cytoskeletal reorganization and partial dedif- 
ferentiation. Development of such fully differ- 
entiated RPE cell lines is crucial not only to 
understanding cellular functions but also for 
cellular transplant therapy. Our goal is to 
examine the mechanisms by which RPE cul- 
tures can be induced to resume in vivo charac- 
teristics. Preliminary studies show that HRPE 
cells assume differentiated morphology on 
incubation in serum-free medium containing 
insuhn, transferrin, selenium, hydrocortisone, 
prostaglandins, and tri-iodothyronine. Further 
studies will be conducted in defining and 
selecting specific media composition and /or 
culturing on suitable extracellular matrix. 

The effects of inflammatory cytokines and 
bacterial endotoxins on HRPE wUl be evaluat- 
ed: (1) on the secretion and expression of IL-8 
and granulocyte-macrophage colony-stimulat- 
ing factors; (2) on the role of anti-inflammato- 
ry cytokines TGF-p, IL-4, and IL-10 on ihe 
influences of inflammatory mediators; (3) on 
cellular cytoskeletal organization, intercellular 
junctions, and adhesion properties; and (4) 
characterization of growth factors and proteo- 
lytic enzymes secreted by RPE in response to 
various stimuli. These studies are likely to 
shed light on the role of RPE in the patho- 
physiology of retina and choroid, the tissues 



that are in close vicinity to and have direct 
influence on RPE. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases, 
Macular Degeneration, Photoreceptors and 
Pigment Epithelium 

Publications 

Kutty RK, Nagineni CN, Kutty G, Hooks JJ, 
Chader GJ, Wiggert B: Increased expression 
of heme oxygenase-1 in human retinal pig- 
ment epithelial cells by transforming growth 
factor-b. / Cell Physiol 159:371-378, 1994. 

Nagineni CN, Detrick B, Hooks JJ: Synergistic 
effects of gamma interferon on inflammatory 
mediators that induce interleukin-6 gene 
expression and secretion by human retinal 
pigment epithelial cells. Clin Diagnos Lab 
Immunol, (in press). 

Nagineni CN, Detrick B, Rhame J, Hooks JJ: 
Inflammatory cytokines IFN-y, TNF-a and 
IL-1 induce ICAM-1 secretion /shedding by 
human retinal pigment epithelial cells. Invest. 
Ophthalmol. Vis Sci 35 (4):2040, 1994. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00287-02 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Toxoplasmosis Infections in the Eye 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: John J. Hooks Ph.D. Head, Section on LI, NEI 

Immunology and Virology 

Others: M. Cristina Martins 

Chandrasekharam Nagineni 
Miguel Bumier 
Robert B. Nussenblatt 



M.D. 


Guest Worker 


LI, NEI 


Ph.D. 


Visiting Scientist 


LI, NEI 


M.D. 


Visiting Scientist 


LI, NEI 


M.D. 


Scientific Director 


LI, NEI 



COOPERATING UNITS (if any) 

National Institute of Allergy and Infectious Diseases (R. Gazzinelli, M.D.) 



Laboratory of Immunology 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS; 

0.5 



PROFESSIONAL: 

0.5 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects [x] (b) Human tissues □ (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Toxoplasma gondii infections are a major source of visual loss and blindness. Ocular toxoplasmosis may 
occur as a result of congenital infections, acquired infections, and as a manifestation of immunosuppression, 
particularly as a result of transplantation or acquired immunodeficiency syndrome (AIDS). Due to the recent 
resurgence of acquired ocular toxoplasmosis in Brazil and the worldwide complications of toxoplasmosis in 
HIV infections, we initiated studies to develop a model of acquired toxoplasmosis to evaluate the molecular 
mechanisms of pathogenesis and therapeutic strategies. 

We have developed a murine model of ocular toxoplasmosis that is characterized by retinal inflammation, 
chorioretinal scaring, retinal disorganization, and cyst formation. Retinal disease occurs in three different 
strains of mice following inoculation with toxoplasmosis by the subcutaneous or intraperitoneal routes. This 
model of acquired ocular toxoplasmosis is being used to evaluate the efficacy of new antiparasitic agents in 
controlling the development of retinal cyst formation and retinal inflammation. 



147 



PHS 6040 (Rev. 5/92) 



FY 1994 NEl Annual Report 



Project Description 

Objectives 

This project was designed to develop an 
animal model of acquired ocular toxoplasmo- 
sis and in vitro models of toxoplasmosis repli- 
cation within the retina. These models will be 
used to evaluate molecular mechanisms of 
ocular pathogenesis and to evaluate new 
antiparasitic drugs and cytokines. 

Methods 

This study involves the propagation and 
quantitation of Toxoplasmosis gondii (T. gondii) 
strains in vitro and in vivo, as weU as immuno- 
cytochemical analysis of infected ceUs and 
tissues. Techniques used in the characteriza- 
tion of T. gondii infections include histopathol- 
ogy, immunocytochemistry, in situ hybridiza- 
tion, and Western blot analysis. Techniques 
used in characterization of anti-T. gondii anti- 
bodies include enz)Tne-linked immunosorbent 
assays. 

Major Findings 

Adult Swiss Webster, C57BL6 and BALB/C 
mice were inoculated by the subcutaneous 
route or intraperitoneal route with 10 T. gondii 
cysts (S2C9 or ME49 strains) in a 1 mL vol- 
ume. At various times after inoculation (day 
seven, 14, 21, 28, and 42) the mice were sacri- 
ficed, and eyes and brains were removed and 
fixed in 10 percent buffered formalin. Fifteen 
hematoxylin and eosin sections of brain and 
eye were evaluated for the presence of T. 
gondii cysts. 

By day 14, 100 percent of the mice devel- 
oped cysts in the brain. Retinal inflammation 
was also noted in 100 percent of the animals 
by day 14. Chorioretinal scars were also ob- 
served in mice inoculated with both strains of 
T. gondii. Retinal cysts were found in mice 28 
and 42 days after inoculation with ME49 
strain and 14 and 42 days after inoculation 
with S2C9 strain in Swiss Webster mice. T. 
gondii cysts in the retina were also detected in 



C57BL6 mice at 14, 21, 28, and 42 days after 
inoculation with the S2C9 strain. This study 
identities an animal model of ocular toxoplas- 
mosis characterized by retinal inflammation, 
chorioretinal scaring, retinal disorganization, 
and cyst formation. 

Preliminary studies on an in vitro cell 
culture model for T. gondii replication in 
retinal tissues has revealed that T. gondii can 
infect and replicate in human retinal pigment 
epithelial cells. Initial studies also indicate 
that this replication can be inhibited by the 
addition of recombinant human interferon 
gamma. 

Significance to Biomedical Research and the 
Program of the Institute 

This is the first animal model of acquired 
toxoplasmosis that consists of retinal iriflam- 
mation, degeneration, and parasitic cyst for- 
mation. This model wUl allow us to evaluate 
the efficacy of new antiparasitic drugs in 
controlling the development of retinal cyst 
formation and retinal inflammation and scar- 
ring. 

Proposed Course 

We will evaluate drugs and cytokines in 
controlling the ocular manifestations of ac- 
quired T. gondii infections. 

NEl Research Program 

Retinal Diseases — ^Inflammatory Diseases 



248 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00293-01 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Gene Targeting of Invariant Chain Gene: A Tool To Study Immunoregulation in Autoimmune Diseases 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 



Moncef Jendoubi 



Others: Noriko Esumi 

Daniel H. Lacorazza 
Luis J. Rivero 



Ph.D. 

M.D., Ph.D. 

Ph.D. 

Ph.D. 



Visiting Scientist 

Visiting Associate 
Visiting Fellow 
Visiting Fellow 



LI, NEI 

LI, NEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 

Section of Genetics and Molecular Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.9 



PROFESSIONAL: 



3.9 







CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Class II antigens of the major histocompatibility complex (MHC) are essential in the immune response because 
they bind processed polypeptides for presentation to T lymphocytes. The interactions of class II MHC 
molecules on the antigen-presenting cell with a responding T lymphocyte are complex because they involve 
molecular contacts with an antigen peptide, an antigen-specific T-cell receptor, and the CD4 molecule 
expressed on the T lymphocytes. Most antigens must be processed in order to bind to MHC molecules and 
to be recognized by T lymphocytes. 

In this context, the majority of peptides bound by MHC class II molecules during antigen loading are not 
derived from ingested and processed foreign proteins but instead are primarily derived from a finite number 
of self-proteins. During progression outward through the exocytic pathway in the process of antigen 
presentation, class Il-invariant chain complexes play a critical role. 

Experimental autoimmune uveoretinitis (EAU) in rodents is a T-cell-mediated autoimmune response, 
particularly against the photoreceptors of the neural retinal cells, and can serve as a model for human uveitis. 
The roles of MHC and non-MHC genes have been strongly associated with EAU in rats and mice. To further 
study the MHC class II participation in mice animal model, we decided to generate deficient mice for the 
invariant chain gene (li). This glycoprotein combines with MHC class II heterodimers from the beginning of 
their synthesis in the endoplasmic reticulum, travels through the Golgi apparatus, and ends up in endosomal 
compartments where it is either proteolytically cleaved or degraded. The absence of li has been shown to 
affect the transport of class II molecules, resulting in a poor antigen presentation. 



149 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

Targeting Vector. A replacement vector has 
been designed to introduce a mutation in li 
murine gene, where the neomycin phospho- 
transferase gene was inserted in the middle of 
the second exon. Two fragments of 0.3 Bgl II- 
Sac I and 9.2 Kb Sac I-EcoR I were added 
upstream and downstream of the neomycin 
gene, respectively. Furthermore, two copies of 
herpes simplex virus thymidine kinase (HSV- 
tk) that allow the negative selection were 
cloned at the end of the targeting vector. The 
targeting fragment used for the electropora- 
tion was excised from the backbone vector by 
digestion with Not I, dialyzed against TE 
(Tris/HCl 10 mM, pH 7.5, EDTA 1 mM), 
precipitated with ethanol, resuspended in an 
appropriate buffer at a concentration of 1 
mg/ml, and analysed on agarose gel. 

Methods 

Electroporation. Embryonic stem cells (D3, 
from 129 /5v stiain) were cultured in standard 
condition on an inactivated layer of embryonal 
fibroblasts and fed two and one-half hours 
before electroporation. Cells were trypsinized, 
washed with DMEM, and resuspended in HBS 
(Hepes 25 mM, NaCl 134 mM, 5 mM KCl, 
Na2P04 0.7 mM, pH 7.1) at 2x10' ceUs/ml. 
The same volume of HBS containing 100 
/ig/ml of targeting vector was added to the 
cell suspension and incubated 10 minutes on 
ice. The electroporation was carried out using 
a 600 BTX electroporator with the following 
conditions: Volts: 250; /tParadays: 300; Resis- 
tance: R8. 

Transfected cells were left at room temper- 
ature for 10 minutes and seeded at a concen- 
tration of 5x10* cells/ 10 cm Petri dish. Twen- 
ty-four hours later, the ceUs were selected 
using G418 (200 /ig active substance/ml) and 
ganciclovir (2 fiM). 

Blastocyst Injection. Blastocysts, three and 
one-half days old, were collected by flushing 



the uterus of super ovulated C57BL/6 females. 
Mutated cells in the U gene were injected into 
blastocysts before their transfer to pseudo- 
pregnant B6D2 FI foster mothers. 

Major Findings 

A total of 8x10' D3 cells were electroporated 
with the targeting vector and selected with 
G418 and ganciclovir for 10 days. Double- 
resistant clones were picked up individually 
and expanded in a 24-weIl plate. Deoxyribo- 
nucleic acid (DNA) was isolated from each 
clone and analyzed by polymerase chain 
reaction (PCR) for homologous recombination 
events, using a combination of primers that 
allow the discrimination of random integra- 
tions. In addition, genonuc DNA was isolated 
from aU double-resistant clones and analyzed 
by Southern blot, using both inside and out- 
side probes for the invariant chain gene. For 
further confirmation, the same studies were 
repeated, using various restriction enzymes. 
Taken together, the results of PCR and South- 
em blot analysis confirmed that three out of 
130 double-resistant clones scored positive. 

These three targeted clones were further 
expanded and injected into blastocysts, and 
the embryos were later transferred into the 
foster mothers. Offspring carrying the mutat- 
ed li gene were identified by their chimeric 
coat color. Again, DNA was purified from all 
chimeric mice and analyzed by PCR as well as 
by Southern blot. The obtained results 
showed that the targeted mutation has been 
transmitted to the offspring. Heterozygous 
mice were set up for mating to generate 
homozygous mice mutated on both alleles of 
the invariant chain gene. Presentiy, we were 
able to create homozygous mice and to estab- 
lish a colony of these animals. Homozygous 
mice bom in the colony are normal and grow 
up without showing any obvious abnormality. 

Significance to Biomedical Researcti and ttie 
Program of the Institute 

The creation of a deficient mouse for the 
invariant chain gene represents a previously 
unavailable tool to study several important 



150 



Laboratory of Immunology 



phenomenons in the immune system, both in 
normal and pathological conditions. 

Degenerative and inflammatory diseases 
of the posterior pole of the eye are common 
causes of impaired vision and blindness 
throughout the world. Between 500,000 and 
1,000,000 Americans suffer severe visual 
impairment from retinal and choroidal dis- 
eases. Retinal degenerative disorders consist 
of a diverse group of diseases frequently 
associated with a genetic predisposition such 
as major histocompatibUity complex (MHC) 
class II. However, in many ocular diseases the 
causes are unknown. In this respect, invariant 
chain-deficient mice wiU be very useful to 
study some of these ocular disorders. Thus, 
these mice will be used to study the implica- 
tion of MHC class n genes in ocular immuno- 
pathological diseases. 

Proposed Course 

In the future, we will focus our work on the 
following: 

(1) We will use the invariant chain gene- 
deficient mice to study endotoxln-induced 
uveitis. 

(2) We will study the effect of viral infec- 
tion such as murine coronavirus that induces 
an acute, long-lasting disease of the retina to 
clarify the degenerative and inflammatory 
diseases that affect the retina and the choroid 
in many ocular disorders (this study will be 
conducted in collaboration with Dr. John 
Hooks in the LI). 

(3) We wiU use these deficient mice as 
models to study allergic conjunctivitis, in 
collaboration with Dr. Chi-Chao Chan in the 
LI. 

(4) We will use these deficient mice as 
models to study experimental autoimmune 
uveitis, in collaboration with Dr. Rachel Caspi 
in the LI. 



NEI Research Program 

Retinal Diseases — Inflammatory Diseases 
Publications 

Lacorazza DH, Rivero LJ, Jendoubi M: Ex- 
pression of the human OAT gene after retro- 
viral transfer into CHO deficient cell line. 
NIH Research Festival E05:35, 1993. 

Rivero LJ, Jendoubi M: Creation of invariant 
chain gene deficient mice as a tool to study 
autoimmune disease and uveitis. Sixth Inter- 
national Symposium on Immunology and Ophthal- 
mology, NIH, June 22, 1994. 

Rivero LJ, Jendoubi M: Gene targeting of 
invariant chain gene by homologous recombi- 
nation as a tool to study immunoregulation in 
autoimmune diseases. Advances in Ocular 
Immunology, p. 163, Amsterdam, Netherlands, 
Elsevier Press, 1994. 

Rivero LJ, Jendoubi M: Embryonic stem cell 
lines carrying insertional mutation. Cold 
Spring Harbor M Mol Gen, p 172, 1992. 

Rivero LJ, Jendoubi M: Mutagenesis of ESC 
and generation of chimeric mice. NIH Research 
Festival 4:23, 1992. 

Rivero LJ, Kozhich A, Nussenblatt RB, 
Jendoubi M: Retrovirus expression of orni- 
thine 6aminotransferase in vitro toward gene 
therapy. Invest Ophthalmol 34(4):807, 1993. 

Rivero LJ, Kozhich A, Nussenblatt RB, 
Jendoubi M: Retrovirus-mediated gene trans- 
fer and expression of human ornithine 6- 
aminotransferase into embryonic fibroblast. / 
Cell Biol 17E:251, 1993. 

Rivero LJ, Lacorazza DH, Kozhich A, 
Nussenblatt RB, Jendoubi M: Retrovirus- 
mediated gene transfer and expression of 
human ornithine delta transferase into embry- 
onic fibroblasts: An alternative approach to 
somatic gene therapy. Hum Gen Ther 5:701, 
1994. 



151 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00292-01 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Retinal Survival in Transgenic Mice Expressing Human Ornithine 3-Aminotransferase 

PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI 



Others: Noriko Esumi 

Daniel H. Lacorazza 
Luis J. Rivero 
Chi-Chao Chan 



M.D., Ph.D. Visiting Associate LI, NEI 

Ph.D. Visiting Fellow LI, NEI 

Ph.D. Visiting Fellow LI, NEI 

M.D. Head, Section on Immunology LI, NEI 



COOPERATING UNITS (if any) 



Laboratory of Immunology 



Section of Genetics and Molecular Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



3.9 



PROFESSIONAL: 



3.9 







CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues Jx] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Gyrate atrophy (GA) is a severe human recessive eye disease resulting in progressive loss of vision due to 
chorioretinal degeneration. The disorder is associated with a deficiency of the mitochondrial matrix enzyme, 
ornithine 5-aminotransferase (OAT), which catalyzes the interconversion of ornithine and a-ketoglutarate to 
A-pyrroline-5'carboxylate and glutamate. GA patients with activity have 10- to 20-fold higher levels of plasma 
ornithine as compared with controls. Dietery and specific hormonal administration in rats and mice have been 
shown to modulate the regulation of this enzyme in different tissues, suggesting OAT's important 
physiological role in vivo. To test this hypothesis and to find out whether ornithine 6-aminotransferase could 
be a common mediator for retinal cell growth, we produced two transgenic lines, expressing the human OAT 
gene in strains of mice normally exhibiting a progressive retinal degeneration. Here we show that transgenic 
mice expressing human ornithine 8-aminotransferase exhibit less severe retinal degeneration than control mice 
littermates. 



152 



PHS 6040 (Rev, 5/92) 



Laboratory of Immunologi/ 



Project Description 

Objectives 

Certain inbred strains of mice, such as FVB/N, 
present a progressive retinal degeneration 
beginning a few days after birth and becoming 
complete a few months later. Retinal degener- 
ation in these mice has been associated at least 
in part with a deficiency in a phosphodiester- 
ase activity, which leads to the accumulation 
of cyclic GMP in affected retina degenerative 
photoreceptors. We postulated that other 
gene products such as ornithine-6-aminotrans- 
ferase (OAT), which is associated with retinal 
degeneration in humans may be involved. 
Thus, persistent expression of human OAT 
(hOAT) in retinal degenerated mice could 
have a role in the development of retinal cell 
layers. To study the physiological relevance 
of OAT in vivo and to determine whether its 
expression could rescue the retinal cell layers 
from degeneration, we produced two 
transgenic lines (OATtg) expressing the hOAT 
gene in strains of mice normally exhibiting a 
progressive retinal degeneration. Therefore, it 
can be seen that transgenic mice expressing 
hOAT exhibit less severe retinal degeneration 
than control mice litter mates. 

Methods 

Production of Transgenic Mice. hOAT comple- 
mentary deoxjrribonucleic acid was cloned 
under the transcriptional element of Moloney 
murine leukemia retrovirus long terminal 
repeat (LTR-MoMuLV), and the construct was 
injected into zygotes of FVB/N mice strain 
genetic background. 

Biochemical Analysis of Transgenic Mice. 
Cells were prepared from different tissues 
from both transgenic mice and control litter 
mates; the cells were lysed in 500 mM NaCl, 
50 mM Tris pH 7.5, one percent NP40. 30 /ig 
of protein extracts from each sample was 
subjected to sodium dodecyl-sulfate polyacryl- 
amide gel electrophoresis (SDS/PAGE) and 
stained with coomassie blue or transferred to 
nylon filters for immunoblot analysis. 



Histopathology. Enucleated eyes were 
prefixed in 4 percent phosphate-buffered 
glutaraldyde for one hour after being fixed in 
10 percent formaldehyde overnight, dehydrat- 
ed, and embedded in methacrylate. Four /xm- 
thick sections were cut horizontally along the 
pupiUary-optic nerve plane of the eye and 
were stained with hematoxyhn-eosin. Sections 
were evaluated, and microphotographs were 
taken at a magnification of 400 X. 

Major Findings 

Biochemical Analysis. OATtg mice were bom 
normal. The expression of the transgene was 
assessed primarily by Northern blot analysis 
and then in several tissues using immunoblot 
analysis and specific antibodies raised against 
hOAT. When the protein extracts were sepa- 
rated on SDS/PAGE and stained with 
coomassie blue, we saw the presence of new 
polypeptides, and /or an enhancement of 
several others, at all range of molecular 
weights only in the OATtg tissue protein 
extracts. 

OATtg and control litter mates were 
analyzed for their expression of hOAT and its 
consequences on cell growth, particularly in 
the eye. The expression of the transgene was 
assessed primarily by Northern blot analysis 
and then in several tissues using immunoblot 
analysis. The correct size of hOAT protein in 
transgenic mice, as detected with specific 
antibodies raised against hOAT, indicated that 
our transgene encoded the entire protein. 

Histopathology. To further determine 
whether the expression of hOAT would have 
any consequence on the retinal cell layer 
development, we examined histopathologically 
OATtg and the control litter mates derived 
from both founders at different timepoints. At 
birth, no significant differences of the retinal 
structure, mainly the inner and outer neuro- 
blast layers, between wild-type and OATtg 
mice were observed. At one week, wild-type 
retina showed, as expected, an early degenera- 
tive development of the outer segments of the 
photoreceptors with a sUght reduction of the 
outer and inner nuclear layers (ONL and 



153 



FY 1994 NEI Annual Report 



INL). On the contrary, OATtg retina showed 
better preserved inner and outer segments of 
the photoreceptors (IS /OS) with larger num- 
bers of intact nuclei both in the ONL and ESTL. 

At two weeks, the retinal thickness be- 
tween the wild-type and OATtg showed 
remarkable differences. The ONL of wild-type 
retina showed no more than one row of pho- 
toreceptor nuclei, and the IS /OS had become 
a debris of degenerating membranes, owing to 
the defect in FVB/N with congenital retinal 
degeneration. In contrast, at least three rows 
of cells with relatively fewer pyknotic nuclei 
remained in the ONL of OATtg retina. In the 
transgenic mice, the thickness of the IS /OS 
was ahnost double that of the wild-type 
retina. The INL and the outer plexiform layer 
(OPL) showed better differentiation and more 
thickness in OATtg than in wild type. 

After two months, retina in the control 
litter mates showed complete degeneration 
and atrophy with total loss of all photorecep- 
tor cells, including the nuclei, the IS /OS, and 
OPL. The ONL was in direct contact with the 
retinal pigment epithelium (RPE). The residu- 
al single row of degenerated photoreceptor 
nuclei and the remains of IS /OS and OPL 
were identified in some regions. During the 
retinal development, the most striking differ- 
ence between OATtg and wild type was 
observed at two weeks. All three wild types 
showed a retinal degeneration, but the OATtg 
mice exhibited a retardation of the degenera- 
tive process. 



Significance to Biomedical Researcii and the 
Program of ttie Institute 

Although the physiological role of OAT and 
how it contributes to retinal cell growth, 
whether directly or indirectly, remains to be 
understood. The present study provides 
strong evidence that OAT plays a critical role 
in rescuing neural cell lines in the retina. In 
the human, many ocular diseases affect both 
retina and choroid and lead to blindness. This 
is the first time that we show a physiological 
role of OAT and demonstrate that it partici- 
pates in some extent to the survival of retinal 
cells. Thus, by virtue of its role as a growth- 
factor, OAT represents a valuable model that 
may aid studies of the pathogenesis and 
treatment of human retinal degeneration. 

Proposed Course 

In the future we will focus on the following: 

(1) We will breed these OATtg+ mice 
with OAT-deficient mice to restore the missing 
enzymatic activity. 

(2) We will study the consequences of the 
overexpression of the exogenous OAT on 
other tissues. 

(3) We will try to find out how the over- 
expression of OAT leads to the survival of 
retinal cells. 

NEI Research Program 

Retinal Diseases— Retinitis Pigmentosa and 
Other Inherited Disorders 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00294-01 LI 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Enzymatic Correction of OAT Deficiency: Progress Toward Gene Therapy to Ocular Genetic Disease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI 



Others: Noriko Esumi 

Daniel H. Lacorazza 
Luis J. Rivero 



M.D., Ph.D. 

Ph.D. 

Ph.D. 



Visiting Associate 
Visiting Fellow 
Visiting Fellow 



LLNEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 



Laboratory of Immunology 



Section of Genetics and Molecular Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.9 



PROFESSIONAL: 







CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Gyrate atrophy (GA) of the choroid and retina is an autosomal recessive chorioretinal degeneration, caused 
by deficiency of the mitochondrial matrix enzyme omithine-6-aminotransferase (OAT). This deficiency results 
in the accumulation of ornithine in the body fluids and leads to hyperomithinemia. Although the clinical 
phenotype is largely confined to the eye, OAT deficiency is a systemic disorder. 

With the final goal of applying gene therapy to this human genetic disease, we have established an in vitro 
model to test the correction of OAT enzymatic deficiency in mammalian cells, using OAT-recombinant 
retroviruses. 

Herein, we report the construction of several Moloney murine leukemia virus (MoMLV)-based recombinant 
retrovirus vectors, in which the human OAT cDNA was placed under the transcriptional control of the mouse 
phosphoglycerate kinase (PGK) promoter or under the enhancer-promoter regulatory element derived from 
MoMLV long terminal repeat (LTR). The retrovirus constructs were packaged in the PG13-GALV cell line 
and used to transduce C9, an OAT-deficient cell line derived from Chinese hamster ovary cells (CHO-Kl). 
We showed that the recombinant retrovirus transfers the hOAT gene into C9. Expression of the hOAT gene 
in the transduced C9-deficient cell line exceeded the level of endogenous human fibroblasts, OAT mRNA, and 
enzymatic activity. 



155 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

Omithine-6-aminotransferase (OAT) (L-omi- 
thine:2-oxo-acid aminotransferase, 2.6.1.13) is 
a nuclear-encoded mitochondrial matrix en- 
zyme that catalyzes the reversible intercon- 
version of ornithine and a-ketoglutarate to 
glutamate semialdehyde and glutamate. OAT 
monomers are synthesized as 49-kDa precur- 
sors and processed to 45-kDa forms upon 
entry into the mitochondrial matrix where 
they assemble into the active homohexameric 
form of the enzyme. The OAT gene in hu- 
mans has been characterized and mapped to 
the site 10q26. Both rat and human comple- 
mentary deoxyribonucleic acid (cDNA) have 
been cloned. This has greatly facilitated the 
study of OAT's physiological role. In previ- 
ous studies, it has been show^n that gyrate 
atrophy (GA) patients have a high concen- 
tration of ornithine in their body fluids, up to 
20 times the normal level. This hyperomithi- 
nemia was associated with the absence of 
OAT enzymatic activity. More recently, se- 
quencing of OAT cDNA from GA patients 
revealed the presence of mutations— mostly 
point mutations — that cause frameshift, non- 
sense, and missense mutations and lead to the 
inactivation of the OAT gene and ultimately to 
the absence of enzymatic activity typical of 
GA disease 

The hyperomithinemia associated with GA 
of the choroid and retina can be lowered to 
normal levels with a low-protein and low- 
arginine diet in all cases studied so far, and 
with pyridoxine hydrochloride (vitamin B6) in 
some cases. Because an arginine-free diet 
cannot be easily followed for a long period of 
time, this treatment is temporary and pallia- 
tive rather than curative. With the view of 
apphang a genetic tiierapy to GA patients and 
correcting the OAT enzymatic deficiency by 
supplying a functional gene, we established an 
in vitro model system in which we attempted 
to correct the enzjmnatic deficiency in an OAT- 
defident cell line as a first step toward a 
somatic gene therapy. 



Methods 

We constructed several OAT-recombinant 
retroviruses bearing human OAT (hOAT) 
cDNA under different regulatory elements, 
transduced them into cells — an OAT-deficient 
cell line — and finally studied the efficiency of 
OAT gene expression following retrovirus- 
mediated transfer. Using Southern, Northern, 
and Western blot analyses as well as specific 
OAT enzymatic assays we show that OAT 
recombinant retroviruses efficientiy transfer 
the gene to recipient cells leading to high 
expression of an active OAT enzyme. 

Major Findings 

Establishment and Characterization of a Recombi- 
nant Retrovirus Producer Cell Line. Four retro- 
viral vector constructs bearing the hOAT 
cDNA as well as the neomycin phosphotrans- 
ferase (neo^) gene as selectable marker were 
packaged into virions using the PG13-GALV 
cell Une. The PG13-GALV packaging ceU line 
was used to produce virions with Gibbon 
leukemia virus host range and to infect ham- 
ster cell Hnes, including C9. 

The retrovirus producer cells were 
screened for the presence of an unrearranged 
OAT recombinant retrovirus, the production 
of high-titer virus and the functional expres- 
sion of the inserted hOAT cDNA. The pattern 
of the Southern blot indicates that the retro- 
virus constructs were integrated into the 
packaging cell genome without rearrange- 
ment. Moreover, the hOAT expression was 
confirmed by Northern blot analysis, and the 
results showed the presence of the cor- 
responding OAT transcripts in the four trans- 
fected packaging cell Hnes. 

To determine whether the inserted hOAT 
recombinant retrovirus is able to produce an 
active enzyme, we performed an enzymatic 
assay on cell lysates from PG13-GALV retro- 
virus producer cell lines previously transfected 
with the constructs described above. The four 
different lysates showed a high expression of 
OAT enzyme, however, the comparison of the 
enzymatic activity between them demonstrate 



56 



Laboratory of Immunology 



that the PG13-GALV retrovirus producer cell 
line transfected by LPOSN expresses the 
highest OAT activity. 

Correction of OAT Deficiency in C9-Trans- 
duced Cell Line. To further assess the expres- 
sion of integrated hOAT sequences, we ana- 
lyzed the production of the OAT messenger 
ribonucleic acid (mRNA). We purified total 
cellular ribonucleic acid (RNA) from wild-type 
C9 cells and transduced ones with LPOSN 
retrovirus as well as from normal human 
fibroblasts. Total RNA from these cells was 
processed for Northern blot analysis. The 
results showed strong hybridization on a 5.0- 
kbp and a weaker hybridization on a 3.6-kbp 
transcript, which might suggest that the major 
transcript could be derived from the viral long 
terminal repeat and the minor transcript from 
the phosphoglycerate kinase (PGK) promoter. 

This result may suggest an interference be- 
tween viral and PGK promoter. The native 
hOAT transcript from human fibroblasts 
showed the corresponding size or 2.1-kbp. 
The hOAT transcripts in the transduced C9 
cell line are in greater abundance than the 
endogenous hOAT mRNA in the normal 
human fibroblasts taking into account that the 
lanes contain the same amount of total RNA 
as confirmed by hybridization with a P-actin 
probe. To test if the mRNA transcript from 
the proviral insert was being appropriately 
translated and processed into a mature pro- 
tein, we performed a Western blot analysis 
using protein extracts from transduced and 
nontransduced C9 as well as from human 
fibroblasts, and an anti-hOAT antibody direct- 
ed against a 19-mer peptide located in the N- 
terminal domain of the OAT protein. No 
sigruficant immunological reaction was ob- 
served in C9 wild-type extract, while in trans- 
duced cells a strong reaction was seen on one 
polypeptide, which comigrates with an appar- 
ent molecular weight of 45-kDa with that 
detected in human fibroblasts. From these 
results, we infer that the OAT produced by 
the transduced cells has been well processed 
into a mature protein. 



To further assess whether the expressed 
OAT protein in the transduced cells is enzy- 
matically active, cell lysates were prepared 
from transduced and nontransduced C9 and 
from human fibroblasts and were analyzed for 
the presence of an active OAT. The deficient 
cell line has almost no OAT enzymatic activi- 
ty, but the human native fibroblast cells 
showed a normal activity (specific activity 
[SA] = 24 ± 2 nmol A^-pyrrohne-5-carboxylate 
[A'P5C] formed /mg of protein /h), whereas 
the transduced C9 cells produced at least a 
threefold increase of OAT activity as com- 
pared with human fibroblasts (SA = 65 ± 9 
nmol A^P5C formed/mg of protein/h). These 
results show that the LPOSN provirus is 
capable of expressing high levels of functional 
hOAT enzyme that represents at least 10-fold 
more than the OAT residual activity in defi- 
cient cells. 

Our results demonstrate that the retro- 
virus-mediated transfer of hOAT results in a 
stable integration of the transferred gene 
without rearrangement into the transduced 
cells genome. In addition, we have shown 
that the gene was transcribed, translated, and 
processed into a protein recognized by a 
specific hOAT antibody and able to metabo- 
lize the ornithine in its natural substrate. 

Significance to Biomedicai Research and ttie 
Program of tlie Institute 

OAT deficiency is associated with hyperomi- 
thinemia and degeneration of the choroid and 
retina, suggesting that the accumulation of 
ornithine may produce a toxic effect on eye 
tissue. 

The introduction of a normal OAT gene 
via retrovirus transfer into somatic cells of GA 
patients consequentiy may turn the hyperomi- 
thinemia to normal level and lead to an im- 
provement in visual function. Retiovirus gene 
delivery has been extensively used in in vitro 
studies and in animal models as well as for 
somatic gene therapy in humans. Following 
this idea, we designed and made OAT retro- 
virus vectors that would provide optimal gene 
expression in deficient himian somatic cells 



FY 1994 NEI Annual Report 



and used this gene delivery system to evalu- 
ate OAT expression in vitro in an OAT-defi- 
dent cell line. 

In our previous studies, we were able to 
express the hOAT gene in murine embryonal 
fibroblasts. With the aim of achieving a 
higher expression of hOAT, we analyzed 
several recombinant retroviral vectors. Al- 
though all OAT retrovirus constructs present 
equivalent levels of functional OAT protein, 
the enzymatic activity was particularly higher 
with retrovirus, which was used to transduce 
an OAT-deficient cell line. Expression of the 
provirus was studied in the transduced C9 
cells, both the 5' LTR and the internal PGK 
promoters were active, giving rise to two RNA 
transcripts. Enzymatic activity in this trans- 
duced deficient cell Une was at least three 
times higher over that of hOAT in normal 
human fibroblasts. However, in GA patients 
the OAT residual activity is about 5 percent 
the normal level (5 nmol A^P5C 
formed /mg/h); therefore, the obtained result 
of enzymatic activity in the transduced cells 
(65 nmol A^P5C formed/mg/h) would corre- 
spond to more than a 10-fold increase as 
compared with residual activity in GA pa- 
tients. Therefore, the level of OAT expression 
by the LPOSN retrovirus in the in vitro system 
is in the physiologic range needed for the 
correction of congenital OAT deficiency. 

In GA patients, OAT deficiency results in 
retinal and choroidal degeneration, indicating 
that OAT function is mostly needed in the 
eye. This raises the question about the target 
tissue for somatic gene delivery for gene 
therapy of this ocular genetic disease. Obvi- 
ously, the best tissue would be the retinal 
pigmented epithilium; however, these cells are 
not surgically accessible for removal, manipu- 
lation, and transplantation into the eye of GA 
patients. In addition, when these cells were 
isolated from rat or human eyes after biopsy, 
they were very difficult to maintain in culture. 
OAT is also highly expressed in liver, where 
it plays an important role in ornithine metabo- 
lism; therefore, hepatic cells could be consid- 
ered for OAT gene delivery to GA patients. 
These cells have been previously used success- 



fully by others to correct inborn genetic defi- 
ciency in animal models and in humans using 
both adenovirus and retrovirus. Although 
both systems have their limitations, the adeno- 
virus gene transfer remains an additional 
alternative worth pursuing for OAT gene 
therapy. 

There are currently more than 40 ap- 
proved human gene therapy protocols; never- 
theless, to date none of these trials involves 
ocular diseases. Our data provide the basic 
knowledge necessary to focus on the appropri- 
ate human cells as a target tissue for a future 
gene therapy trial in this ocular genetic dis- 
ease. 

Proposed Course 

Our future efforts will focus on the following 
issues: 

(1) We are correcting the enzymatic 
activity in cell Unes from GA patients. 

(2) We will assess the enzymatic activity 
in vivo in animal models after tissue engraft. 

(3) We will use the deficient animal that 
we are creating to assess the feasibility of gene 
therapy in ocular genetic disease. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and 
Other Inherited Disorders 

Publications 

Lacorazza DH, Rivero JL, Jendoubi M: Ex- 
pression of the human OAT gene after retro- 
viral transfer into CHO deficient cell Une. 
NIH, Research Festival, E05:35, 1993. 

Lacorazza DH, Jendoubi M: Correction of 
OAT deficiency in chinesehamster ovary cell 
line mediated by retrovirus gene transfer. 
Sixth International Symposium on Immunology 
and Opthalmology, NIH, June 22, 1994:25. 



Laboratory of Immunology 



Lacorazza DH, Jendoubi M: Correction of Rivero LJ, Laccorazza DH, Kozhigh A, 
genetic and enzymatic activity of ornithine 6- Nussenblatt RB, Jendoubi M: Retrovirus- 
aminotiansferase into mammalian deficient mediated gene transfer and expression of 
cell Unes using retrovirus mediated gene human ornithine delta tiansferase into embry- 
transfer. ARVO Annual Meeting, Sarasota, onic fibroblasts: An alternative approach to 
Florida, May 1-6, 1994: 1705. somatic gene therapy. Hum Gen flier 5:701, 

1994. 
Lacorazza DH, Rivero LJ, Jendoubi M: Genet- 
ic and enzymatic correction of ornithine 6- 
aminotransferase into CHO deficient cell line. 
Gene Therapy, in press. 



159 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



PROJECT NUMBER 



ZOl EY 00295-01 LI 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Isolation and Characterization of the Mouse OAT Gene for Gene Targeting 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI 

Others: Noriko Esumi M.D., Ph.D. Visiting Associate LI, NEI 

Daniel H. Lacorazza Ph.D. Visiting Fellow LI, NEI 

Luis J. Rivero Ph.D. Visiting Fellow LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



Section of Genetics and Molecular Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



PROFESSIONAL: 

3.9 



OTHER: 



TOTAL STAFF YEARS: 

3.9 

CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects □ (b) Human tissues (xl (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Gyrate atrophy (GA) of the choroid and retina is an autosomal recessive eye disorder involving a progressive 
loss of vision due to chorioretinal degeneration. A variety of omithine-6-aminotransferase (OAT) gene 
mutations have been reported in GA patients and suspected to associate with this ocular disease. However, 
the precise mechanism by which the OAT deficiency and hyperomithinemia lead to the chorioretinai 
degeneration remains unknown. To elucidate the pathophysiological role of OAT, we are attempting to create 
OAT-deficient mice by gene targeting via embryonic stem (ES) cells. Toward this ultimate goal, we isolated 
murine OAT gene to construct OAT targeting replacement vector. 



260 



PHS 6040 (Rev. 5/93) 



Labor at oiy of Immunologic 



Project Description 

Objectives 

Isolation of Mouse OAT Functional Gene. The 
129 mouse genomic library, OLA 129-yGEM- 
12, was kindly provided by Dr. Anton Berns 
from the Cancer Institute, Netherlands. This 
library was screened with a nearly full-length 
rat omithine-6-aminotransferase (OAT) com- 
plementary deoxyribonucleic acid (cDNA) 
probe [4] labeled with ^^P-dCTP by random 
oUgonucleotide priming. Screening proce- 
dures were performed using standard proto- 
cols. 

Methods 

Polymerase Chain Reaction (PCR) Analysis. To 
determine the structure of positive genomic 
clones, PCR was performed with primers from 
each exon based on mouse cDNA sequence 
(Genebank, X64837) to ampHfy each exon and 
intron. Standard reaction conditions were 
used. 

Sequencing. The promising Clone 11 was 
subcloned into BlueScript vector (Stratagene, 
La Jolla, California) and designated 
pBSmOAT6. This plasmid was sequenced by 
dideoxy nucleotide chain termination method 
of Sanger using ^^S-dATP and the CircumVent 
Thermal Cycle Dideoxy DNA Sequencing Kit 
(New England Biolabs, Beverly, Massachu- 
setts). 

Construction of Targeting Vector. The 
mouse genomic fragment in pBSmOAT6 was 
16 kb in length and used almost entirely to 
make a targeting vector. Neomycin resistance 
gene (Neo") was purified from pMClneo 
PolyA kindly provided by Dr. Mario Capecchi 
and inserted at Sea I site of exon 4 to disrupt 
the OAT gene. Herpes simplex virus thymi- 
dine kinase (HSV-TK) gene was purified from 
TGV-TK2 (Moncef Jendoubi, unpublished) 
originated from pIC19R/MCl-TK provided by 
Dr. Capecchi and added at the 3' end of the 
OAT fragment in targeting construct. Standard 



procedures were used in all subcloning and 
constructing processes. 

Major Findings 

Isolation and Clmracterization of Mouse OAT 
Gene. By genomic library screening, 16 clones 
were isolated from 6 x 10" phage plaques. AU 
clones were analyzed by PCR, and the results 
were compared with the PCR amplification 
pattern with mouse genomic DNA as a tem- 
plate. Among 16 clones. Clone 11 seemed to 
encode the functional OAT gene containing 
the 5' flanking region and the coding region 
up to exon 5. 

Because the OAT gene has been reported 
to have at least several pseudogenes and 
related sequences in human and rat genome 
[11-14], it is highly suspected that mouse 
genome also has at least several related se- 
quences. Therefore, sequencing of Clone 11 
was performed to confirm that this clone 
encoded the functional OAT gene. Sequences 
of a total of 444 bases from exons 3, 4, and 5 
were comparable to the sequence of the mouse 
OAT cDNA. 

Construction of Targeting Vector. Targeting 
vector was constructed according to a posi- 
tive-negative selection strategy. Using 16 kb 
fragment in pBSmOATG, exon 4 was disrupt- 
ed by insertion of Neo' gene at Sea I site for 
positive selection by G4I8. Two targeting 
vectors were made in which Neo' gene was 
infroduced in a forward and an opposite 
direction as referred to the coding sfrand of 
OAT gene. HSV-TK gene was added at the 3' 
end of the genomic fragment for negative 
selection by ganciclovir. The length of homolo- 
gous region was 14 kb upsfream from Neo"^ 
and 2.2 kb downstream from Neo*". This con- 
sfruct is designed for three favorable features: 
that genomic fragment used is isogenic to ES 
cell (129 sfrains of mice), that a positive-nega- 
tive selection can be used for identifying 
clones after elecfroporation into ES cells, and 
that a long homologous region is expected to 
yield a high frequency of homologous recom- 
bination events. 



FY 1994 NEI Annual Report 



We made several electroporations using 
different ES cells for their capacity to contrib- 
ute to germ-Une transmission. Cells were 
electroporated with OAT targeting construct 
replacement vector and selected with both 
G418 and ganciclovir, to enrich the homolo- 
gous recombination events. Two weeks later, 
resistant clones were picked up individually 
and grown for further analysis. Genomic 
DNA was purified from all clones and ana- 
lyzed by Southern blot using different restric- 
tion digests and differents probes. The results 
showed that several clones were recombinants 
on one allele. Four of them already have been 
injected to generate deficient mice for OAT 
gene. Presently we are collecting the first 
litters of chimeric mice for the OAT. 

Significance to Biomedical Research and the 
Program of the Institute 

The identification, characterization, mapping, 
and sequencing of the OAT gene is a very 
significant achievement in itself because we 
were able to study the functional OAT gene as 
well as several pseudogenes. 

We used 16 kb to construct two targeting 
vectors to mutate the functional OAT gene in 
ES cells. We were able to obtain 17 targeted 
recombinant clones, four of them have been 
already injected into embryos to generate 
deficient animals for the OAT gene. These 
deficient animals will be invaluable in many 
respects: 

(1) This v^ be the first animal model for 
ocular genetic diseases. 

(2) We will be able to study the physio- 
logical relevance of the OAT gene in vivo. 

(3) We wdll also find out if there is any 
relationship between retina degeneration and 
the absence of functional OAT enzyme. 



Proposed Course 

Our work in the future will focus on the 
following: 

(1) We will study the ocular physiology 
in the OAT deficient mice. 

(2) We will assess the importance of the 
absence of a functional OAT enzyme in vivo. 

(3) We wall use this animal model to 
assess the feasibility of gene therapy for gyrate 
atrophy disease. 

(4) We will study how to correct the 
enzymatic deficiency in vivo by transferring 
cells from normal donors to affected animals. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and 
Other Inherited Disorders 

Publications 

Esumi N, Jendoubi M: Isolation and charac- 
terization of the mouse ornithine 6-amino- 
transferase gene for gene targeting by homolo- 
gous recombination. Sixth International Sympo- 
sium of the Immunology and Immunopathology of 
the Eye, 1994, p 25. 

Esumi N, Jendoubi M: Advances in Ocular 
Immunology. Amsterdam, Netherlands, Elsevier 
Press, p. 151, 1994. 

Esumi N, Jendoubi M: Isolation, characteriza- 
tion and sequencing of the mouse ornithine 6- 
aminotransferase gene for gene, in prepara- 
tion. 



162 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00288-02 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 199 3 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Gene Therapy for Oc ul ar Genetic Disease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI 



Others: Noriko Esumi 

Daniel H. Lacorazza 
Luis J. Rivero 
Robert B. Nussenblatt 



M.D., 
Ph.D. 
Ph.D. 
M.D. 



Ph.D. 



Visiting Associate 
Visiting Fellow 
Visiting Fellow 
Scientific Director 



LI, NEI 
LI, NEI 
LI, NEI 
NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section of Genetics and Molecular Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 







PROFESSIONAL 







CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



THIS PROJECT HAS BEEN TERMINATED. 



163 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00241-07 LI 



PERIOD COVERED 



October 1, 1 992 to Septembe r 30. 1 993 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Immuno pathology of Ocular Diseases in Humans 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 
Others: 



Chi-Chao Chan 

Robert B. Nussenblatt 
Qian Li 

Marc D. de Smet 
Raymond DeBarge 
Scon M. Whitcup 
Juan Lopez 
Miguel Bumier 
Richard Fenton 
Dev Shah 



M.D. 

M.D. 
M.D. 
M.D. 
M.D. 
M.D. 
M.D. 
M.D. 
M.D. 
M.D. 



Chief, Section on 
Immunopathology 
Scientific Director 
Visiting Fellow 
Visiting Scientist 
Senior Staff Fellow 
Staff Medical Officer 
Visiting Associate 
Visiting Scientist 
Staff Fellow 
Visiting Associate 



LI, NEI 

NEI 
U, NEI 
U, ^fEI 
LI, NEI 
U, NEI 
LI, NEI 
LI, NEI 
LLNEI 
LI, NEI 



COOPERATING UNITS (if any) 



Department of Ophthalmology, Armed Forces Institute of Pathology (Ian W. McLean, M.D.); University of 
Minnesota, Department of Ophthalmology (Edward J. Holland, M.D.); L'Hopital de la Pitie, Paris, France 
(Pbuc LeHoang, M.D.) 



LAB/BRANCH 

Laboratory of Immunology 



Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.0 



PROFESSIONAL: 



0.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project has been terminated and combined with Project No. ZOl EY 00222-08 LI. 



164 



PHS 6040 (Rev. 5/92) 



Laboratory of Mechanisms of Ocular Diseases 



Report of the Chief 
Laboratory of Mechanisms of Ocular Diseases 



J. Samuel Zigler, Jr., Ph.D. 



Investigators in the Laboratory of 
Mechanisms of Ocular Diseases 
(LMOD) are engaged in a broad range 
of studies relating to the biology of various 
tissues in the normal eye and the molecular 
mechanisms responsible for certain ocular 
diseases. Major emphasis has been on cataract 
and the various ocular complications of 
diabetes. 

The addition of Dr. Fred Bettelheim to the 
group as a part-time Special Volunteer and the 
initiation of new collaborative studies with Dr. 
Joseph Horwitz, from the Jules Stein Eye 
Institute, has broadened and strengthened our 
expertise in the biophysical aspects of lens 
opacification and in the use of human cataract 
samples in the laboratory. 



Section on Cataracts 

Dr. Donita Garland has developed new meth- 
ods for dissecting the human lens into distinct 
zones representing tissue produced during 
different stages of Ufe. Two-dimensional 
analysis of the proteins present in each of 
these regions provides a greatiy improved 
picture of the pattern of protein changes that 
occur as a function of aging and development 
in the lens. Marked differences are found 
between the nuclear and cortical protein 
patterns in both normal lenses and cataracts. 
Careful analysis of the protein content of 
dissected zones from intracapsular cataract 
lenses may provide the most definitive insight 



to date on the significance, with respect to 
cataractogenesis, of the changes that occur in 
both the lens crystallins and the metabolic 
proteins of the lens. 

Dr. Paul RusseU and his group are using 
a variety of model systems, e.g., whole animal, 
lens organ culture, and lens epitheUal ceU 
culture, to investigate the mechanisms that the 
lens uses to resist environmental and systemic 
stresses, which can lead to cataract. A great 
improvement in the efficiency of lens organ 
culture has resulted fiom the development of 
a simple and very effective means to identify 
lenses damaged during dissection. This al- 
lows elimination of bad lenses before experi- 
ments begin, thereby greatiy reducing the 
level of variability within experimental 
groups. 

Two new projects have been started dur- 
ing the past year. In one, transgenic mice that 
develop cataracts as the result of insertion of 
the human immunodeficiency virus protease 
gene wiU be studied to determine how expres- 
sion of this foreign protease in the lens leads 
to cataract. In the second new project, serum 
samples collected from patients with cataract 
and from normal volunteers wiU be analyzed 
for presence of autoantibodies in human lens 
proteins. By using two-dimensional elecfro- 
phoresis, the specific protein(s) to which 
antibodies have been produced can be identi- 
fied and correlated with cataract type and 
progression rate. 



167 



FY 1994 NEI Annual Report 



Dr. Fielding Hejtmancik and his group 
have had outstanding success in gene linkage 
studies on several ocular diseases. The locus 
for Usher's syndrome type I, which they 
previously Unked to chromosome llq, contin- 
ues to be refined using fine linkage mapping 
with the ultimate goal of identifying and 
characterizing the gene responsible for this 
disease. Linkage has also been obtained for 
two different cataract families during the past 
year. With the establishment of collaborative 
arrangements with clinical researchers in a 
variety of locations, including India, Italy, and 
Barbados, this group wiU have access to 
excellent material for the study of inherited 
cataract, retinal diseases, and glaucoma. 

Dr. Deborah Carper's laboratory has 
continued to study the role of the polyol 
pathway in the generation of diabetic compli- 
cations. Site-directed mutagenesis studies on 
aldose reductase (AR) have identified certain 
critical residues, information that wUl be 
invaluable in the effort to design more effec- 
tive inhibitors of this enzyme. Studies have 
also continued on sorbitol dehydrogenase, the 
second enzyme of the pathway, with determi- 
nation of the complete structure of the human 
gene as well as its chromosomal localization. 
Work is under way to identify the defect in 
this gene in a family that has cataracts associ- 
ated with sorbitol dehydrogenase deficiency. 



In Dr. J. Samuel Zigler's laboratory, work 
has concentrated on the testing of potential 
anticataract agents and on the analysis of the 
functions of lens crystallins and the role(s) 
they play in the normal lens and in the pro- 
cess of cataractogenesis. Studies done in 
collaboration with Dr. Joseph Horwitz are 
aimed at elucidating the physiological signifi- 
cance of the chaperone-Uke activity of a-crys- 
taUin. The possibility that the "enzyme/ 
crystallins" may be part of the lens' defenses 
against oxidation is being actively investigat- 
ed. Studies on the effect of smoke on the lens 
have provided further support for the view 
that it represents an important risk factor for 
cataract development. 



Section of Pathophysiology 

Dr. W. Gerald Robison and his colleagues are 
studying the process of diabetic retinopathy. 
By using advanced morphological and mor- 
phometric techiuques, they have analyzed the 
appearance of a variety of specific lesions in a 
rat model of this disease that are characteristic 
of changes seen in the human disease. They 
have also demonstrated that in the rat, these 
changes can be prevented by AR inhibitors. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00105-15 LMOD 



PERIOD COVERED 

October 1, 1 9 93 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Structure and Composition of L e ns Crysta llin s Wit h Respect to Cataractog enesi s 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J. Samuel Zigler, Jr. Ph.D. Research Biologist LMOD, NEI 



Others: Vasantha Rao Ph.D. Visiting Associate 

Pedro Gonzalez Ph.D. Visiting Associate 

Chuan Qin M.D. Visiting Fellow 

Frederick A. Bettelheim Ph.D. Special Volunteer 



LMOD, NEI 
LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (if any) 



Jules Stein Eye Institute, UCLA (J. Horwitz, Ph.D. and B. Bateman, M.D.); University of Tennessee (H.M. 
Jemigan, Jr., Ph.D.) National Cancer Institute (M. Krishna, Ph.D.); Centre for Cellular and Molecular Biology, 
Hyderabad, India (D. Balasubramanian, Ph.D. and M. Rao, Ph.D.) 



Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL 



4.0 



4.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project, directed toward elucidation of the molecular mechanisms responsible for cataractogenesis and 
the development of means of prevention of this disease, places special emphasis on the structure and function 
of the lens crystallins and the role these proteins play in lens opacification. Until recently, crystallins were 
thought to be simply structural elements of the lens matrix without specific quantifiable biological functions. 
Two recent discoveries have provided new insights and approaches to the physiological roles of the crystallins: 
(1) the crystallins are either functionally active enzymes or are at least related to proteins with specific 
biological activities, and (2) a-crystallin is a molecular chaperone that can prevent the aggregation of 
denaturing proteins. 

We believe that crystallins have specific biological functions in addition to their structural role in forming the 
transparent lens tissue. The strongest example to date is the chaperone-like function of a-crystallin. This 
major lens protein prevents the aggregation of other proteins that are undergoing modification and 
denaturation. In the lens, where protein turnover is extremely limited and where the long-lived crystallins are 
known to undergo extensive structural modification, a-crystallin may be essential in preventing protein 
aggregation and precipitation. Such precipitation would destroy the optical transparency of the lens by 
creating light scattering centers. We have also developed evidence that the enzyme/crystallins are contributing 
to the antioxidative capacity of the lens, primarily by markedly increasing the pool of reduced pyridine 
nucleotides in the lens. We can demonstrate the utilization of the nucleotide's reducing capacity in eliminating 
activated species of oxygen generated by Fenton chemistry or by other mechanisms. Further support for the 
view that enzyme/crystallins have specific functions was obtained from studies on the expression of C- 
crystallin in the lenses of guinea pigs and llamas. The data clearly demonstrate that the £,-crystallin gene was 
recruited by the lens independently in each of the two species. This finding strongly supports a selective basis 
for the recruitment rather than a neutral evolution mechanism and indicates that the protein must provide 
significant benefit to the lens of these species. 

169 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The primary objectives of this project are to: 
(1) elucidate processes responsible for cataract 
development at the molecular level, (2) inves- 
tigate the structures and functions of the lens 
crystallins, and (3) develop and use model 
systems for screening potential anticataract 
agents and test them in appropriate in vitro 
and animal model systems. 

Methods 

Conventional protein chemical techniques 
used are chromatography, electrophoresis, and 
isoelectrofocusing. Immunological studies of 
lens proteins use specific antisera. Physico- 
chemical analyses on the proteins are per- 
formed using high-pressure liquid chromatog- 
raphy, fluorescence, and circular dichroism 
techniques. Lens organ culture experiments 
use rat or monkey lenses and use active trans- 
port and membrane permeability parameters 
to monitor the effects of various stresses on 
the cultured lenses. 

Techniques used in analysis of nucleic 
adds include ribonucleic acid and deoxyribo- 
nucleic acid (DNA) isolation, complementary 
DNA and gene cloning, DNA sequencing, 
various electrophoretic methods, and the 
pol5anerase chain reaction. 

Major Findings 

(1) In collaboration with Dr. Joseph Horwitz, 
from the Jules Stein Eye Institute, we have 
further characterized the chaperone-like activi- 
ty of a-crystaUin and analyzed the specificity 
of the reaction with different target proteins. 
With some denaturing proteins, the presence 
of obligate cofactors is critical if a-crystaUin is 
to effectively prevent aggregation. Circular 
dichroism spectroscopy suggests that subtle 
conformational changes associated with the 
binding of the cofactor may be essential for 
interaction of a-crystallin with these proteins. 
Our findings suggest that a-crystaUin is spe- 



cifically adapted to function as a chaperone 
within the lens. 

(2) The putative promoter region previ- 
ously identified in the Uama ^-crystaUin gene 
has proved to be active by functional analysis. 
The studies in transfected cells also indicate 
that the promoter is highly lens specific, i.e., 
does not drive gene expression in other tis- 
sues. 

(3) The human gene for ^-crystallin has 
been characterized and shown to have essen- 
tially the same structure as found in the 
guinea pig and Uama. However, unlike the 
guinea pig and Uama genes, it lacks a second 
lens-specific promoter and consequentiy is not 
expressed at a high level in the lens. A pro- 
cessed pseudogene for ^-crystaUin was also 
found to be present in the human genome. 

(4) A catalytic activity that uses the re- 
ducing equivalents of pyridine nucleotides to 
eliminate free radical oxidants has been identi- 
fied in the lens. We beUeve that this activity, 
which appears to be associated with a protein, 
may be an important part of the lens' antioxi- 
dant capacity. Because NAD(P)H is renew- 
able via redox cycling, it is conceivable that its 
reducing equivalents may function in a man- 
ner analogous to the glutathione redox cycle. 

(5) Smoke has been identified as a risk 
factor for cataract in several epidemiological 
studies. In coUaboration with Dr. Ch. Mohan 
Rao, from the Center for CeUular and Molecu- 
lar Biology, Hyderabad, India, we have ex- 
posed organ-cultured rat lenses to a conden- 
sate of wood smoke. The studies indicate that 
components of the condensate accumulate 
within the lens, are metabolized, and cause 
damage to the ceU membranes. Histological 
analysis demonstrated early and severe injury 
to the lens epithelial ceUs. 

(6) Studies with Dr. Fred Bettelheim, also 
using rat lenses in organ culture, have estab- 
Ushed a calcium-induced cataract model as a 
system for studying the role of optical aniso- 
tropy fluctuations in lens opacification. Early 
results suggest a major role for the intermedi- 



170 



Laboratory of Mechanisms of Ocular Diseases 



ate filament protein, vimentin, in establishing 
and maintaining proper order within the 
cytoplasm of lens fibers. Loss of vimentin via 
calcium-induced proteolysis leads to optical 
anisotropy fluctuations that cause lens turbid- 
ity. 

(7) The testing of potential agents for the 
prevention or retardation of cataract develop- 
ment is being performed in the lens organ 
culture system on compounds with antioxida- 
tive capacity. Compounds that provide prom- 
ising results will be prepared for testing in 



Significance to Biomedical Research and the 
Program of the Institute 

Cataract is a major pubUc health problem 
worldwide. Better understanding of the 
biochemistry of the normal lens and of the 
molecular changes that occur during aging 
and cataract development are essential if this 
disease is to be controlled. Our studies are 
aimed primarily at elucidating the role of the 
lens crystallins, the primary structural ele- 
ments of the normally transparent lens matrix, 
in the processes leading to opacification. 
Such knowledge should contribute to the 
development of means of intervention that can 
prevent or delay the process of cataract devel- 
opment. 

Proposed Course 

We wiU: (1) work to establish viable model 
systems for testing anticataract agents and use 
these systems to assess the efficacy of various 
types of compounds, including antioxidants; 
(2) clarify the mechanism and the significance 
of the pjoidine nucleotide-dependent antioxi- 
dant system in the lens and investigate the 
possible role of enzyme /crystallins in this 
system; (3) continue to investigate the chaper- 
one-like function of a-crystallin and determine 
its physiological significance in the normal 
lens and in cataract; and (4) expand the two- 
dimensional analysis of proteins from human 
normal lenses and cataracts through the use of 
preliminary fractionation by affinity chroma- 
tography. Studies under way using blue 



Sepharose affinity columns suggest that this 
will be a very useful way to reduce the com- 
plexity of the protein mixture as well as to 
focus on specific classes of proteins. 

NEI Research Program 

Lens and Cataract— Pathogenesis of Cataract 

Publications 

Bettelheim FA, Qin C, Zigler JS Jr: Calcium 
cataract: A model for optical anisotropy 
fluctuations. Exp Eye Res, in press. 

Cui X-L, Qin C, Zigler JS Jr: Residual EDTA 
bound by lens crystallins accounts for their 
reported resistance to copper-catalyzed oxida- 
tive damage. Arch Biochem Biophys 308:207- 
213, 1994. 

Gonzalez P, Hemandez-CalzadiUa D, Rao PV, 
Rodriquez IR, Zigler JS Jr, Borras T: Compar- 
ative analysis of the ^-crystaUin/quinone 
reductase gene in guinea pig and mouse. 
Molec Biol Evolution, 11:305-315, 1994. 

Gonzalez P, Rao PV, Zigler JS Jr: Organiza- 
tion of the human I;-crystallin/qviinone reduc- 
tase gene (CRYZ). Genomics 21:317-324, 1994. 

Heinzmaim C, Kojis TL, Gonzalez P, Rao PV, 
Zigler JS Jr, Polymeropoulos MH, Klisak I, 
Sparkes RS, Mohandas T, Bateman JB: As- 
signment of the ^-crystallin gene (CRYZ) to 
human chromosome Ip22-lp31 and identifica- 
tion of restriction fragment length polymorph- 
isms. Genomics, in press. 

Persson B, Zigler JS Jr, JomvaU H: A super- 
fanuly (MOR) of medium chain dehydrogen- 
ases/reductases: Sublines including t-crystal- 
lin, alcohol and polyol dehydrogenases, qui- 
none oxidoreductases, enoyl reductases, VAT- 
1 and further proteins. Eur J Biochem, in 
press. 

Rao PV, Horwitz J, Zigler JS Jr: Chaperone- 
like activity of a-crystaUin. J Biol Chem 
269:13266-13272, 1994. 



FY 1994 NEI Annual Report 



Tumminia SJ, Qin C, Zigler JS Jr, Russell P: Zigler JS Jr: Lens proteins, in Albert DM, 
The integrity of mammaUan lenses in organ Jakobiec F (eds): Principles and Practice of 
culture. Exp Eye Res 58:367-374, 1994. Ophthalmology. Basic Sciences, Philadelphia, JB 

Saunders Co., 1994, pp 97-113. 
Tumminia SJ, Rao Pv, Zigler JS Jr, Russell P: 
Xenobiotic induction of quinone oxidoreduc- 
tase activity in lens epitheUal cells. Biochim 
Biophys Acta, 203:251-259, 1993. 



172 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00189-11 LMOD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Oxidation of Proteins in Cataractogenesis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI 



Others: 



Jose Jimenez 
Lx)renzo Merola 
Kenichi Matsuno 



Ph.D. 
M.S. 
Ph.D. 



Visiting Fellow 

Chemist 

IRTA 



LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.3 



PROFESSIONAL: 



3.3 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Oxidative processes are a major contributing factor in senile cataracts. We have demonstrated that metal 
catalyzed oxidation of the crystallins induces protein modifications that mimic those seen in aging, senile 
cataracts, and brunescent lenses. The importance of the role of metal in oxidative processes related to cataract 
is further supported by studies in other labs. The lens contains high levels of thiols such as glutathione that 
can participate in these metal-catalyzed oxidation reactions. Our lab has continued our studies on both the 
interaction of metals with crystallins and the mechanisms that protect the lens against deleterious oxidation 
reactions. 

A protein that protects enzymes specifically against inactivation by thiol-dependent metal-catalyzed reactions 
has been reported in yeast and rat tissues. (This activity is not related to catalase, glutathione peroxidase, or 
superoxide dismutase.) Our lab demonstrated the presence of a similar activity in lenses of bovine, guinea 
pig, human, pig, monkey, and rat. The antioxidant activity consistently copurifies with a subpopulation of 
glutathione S-transferase ju. 

Copper, zinc, and iron, but not calcium, induced aggregate formation in bovine lens extracts and solutions of 
the crystallins. Aggregation, measured by light scattering, was time dependent, occurring at metal-to-protein 
ratios greater than one and varying, depending on the metal and protein. Zinc induced the aggregation of P- 
and a- but not y-crystallin. The affinity of copper and zinc for these proteins is relatively low. The addition 
of EDTA, DETAPAC, L-histidine, or L-cysteine prevented zinc- and copper-induced protein aggregation and 
caused complete disaggregation. The treatment of a- and P-crystallin and trypsin inhibitor with 
diethylpyrocarbonate prevented aggregation induced by zinc but not by copper. The presence of salt decreased 
the metal-induced aggregation of a- and (3-crystallin. No metal-induced changes in secondary and tertiary 
structures of these proteins were observed by fluorescence and circular dichroism spectroscopy. 



173 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The long-term goal of this project is to under- 
stand the role of oxidation in cataract forma- 
tion. The immediate objectives are to: (1) 
study the effect of oxidation on structure and 
function of lens crystallins, (2) study the 
interaction between crystallins and those 
metals that are involved in oxida- 
tion/reduction reactions and the effects of 
these interactions on crystallin solubihty and 
aggregate formation, and (3) characterize the 
enzymes that protect lens proteins against 
thiol-dependent metal-catalyzed oxidation. 

Methods 

Bovine, rat, and guinea pig tissues were used 
for these studies. We used classical methods 
to purify proteins. Other methods used were 
standard procedures for studying proteins, 
polyacrylamide gel electrophoresis, high- 
pressure Uquid chromatography, ultraviolet 
spectroscopy, fluorescence, circular dichroism, 
electron spin resonance, amino acid analysis, 
immunotechniques, and capillary gas Uquid 
chromatography. 

Major Findings 

Oxidative processes are considered to be a 
major contributing factor in senile cataracts. 
We have demonstiated that metal-catalyzed 
oxidation of the crystallins induces protein 
modifications that mimic those seen in aging, 
senile cataracts, and brunescent lenses. The 
importance of the role of metal in oxidative 
processes related to cataract is further sup- 
ported by studies in other laboratories. The 
lens contains high levels of thiols such as 
glutathione that can participate in these metal- 
catalyzed oxidation reactions. Our laboratory 
has continued studies on both the interaction 
of metals with crystallins and the mechanisms 
that protect the lens against deleterious oxida- 
tion reactions. 



(1) A protein that protects enzymes spe- 
cifically against inactivation by thiol-depen- 
dent, metal-catalyzed reactions has been 
reported in yeast and rat tissues. (This activity 
is not related to catalase, glutathione perox- 
idase, or superoxide dismutase.) Our labora- 
tory demonstrated the presence of a similar 
activity in the lens of bovine, guinea pig, 
human, pig, monkey, and rat. Following the 
antioxidant activity, a protein was purified 
from bovine lenses. Seventy percent of the 
protein sequence was obtained. The protein 
was identified as glutathione S-transferase /i, 
a detoxification enzyme found in most cells. 
This enzyme is not known to possess an 
antioxidant activity. The antioxidant activity 
consistentiy purifies with a subpopulation of 
glutathione S-transferase through many differ- 
ent purification schemes, yet we have not 
proved that the antioxidant activity is part of 
the glutathione S-transferase molecule. It is 
possible that this subuiut may form a hetero- 
dimer with glutathione S-transferase. Studies 
are in progress to try to resolve this issue. 

Other Unes of evidence indicate the pres- 
ence in the lens of a protein(s) more closely 
related to the antioxidant protein characterized 
from yeast and rat brain. Antibodies made 
against the whole protein or to an internal 
peptide crossreact with proteins from lens but 
not glutathione S-transferase. Further North- 
em-blot analysis of total ribonucleic acid 
(RNA) from cow, human, monkey, and rat 
lens was performed using a complementary 
deoxyribonucleic acid (cDNA) probe made to 
the rat antioxidant protein. The results clearly 
demonstiated the presence of specific messen- 
ger RNA for this particular gene. 

(2) Copper, zinc, and iron, but not calci- 
um, induced aggregate formation in bovine 
lens extiacts and solutions of the crystallins. 
Aggregation, measured by light scattering, 
was time dependent, occurring at metal-to- 
protein ratios greater than one and varying 
depending on the metal and protein. Zinc 
induced the aggregation of p- and a- but not 
Y-crystaUin. Copper and zinc induced the 
aggregation of a number of other proteins, but 
they had no effect on lysozyme and papain. 



Laboratory of Mechanisms of Ocular Diseases 



One explanation for the lack of effect on these 
two proteins is that they are basic proteins. 
However, copper induced the aggregation of 
Y-crystallin, also a basic protein. 

The affinity of copper and zinc for these 
proteins is relatively low. The addition of 
EDTA, DETAPAC, L-histidine, or L-cysteine 
prevented zinc- and copper-induced protein 
aggregation and caused complete disaggrega- 
tion. The treatment of a- and |3-crystaUin and 
trypsin inhibitor with diethylpyrocarbonate 
prevented aggregation induced by zinc but 
not by copper. The presence of salt decreased 
the metal-induced aggregation of a- and p- 
crystallin. No metal-induced changes in 
secondary and tertiary structures of these 
proteins were observed by fluorescence and 
circxilar dichroism spectroscopy. 

In the presence of zinc, lens crystallins 
were least soluble at 2.0 - 2.5 pH units higher 
than their isoelectric points. The effects of 
varying pH were fully reversible. Solubility 
decreased further with increasing temperature. 
Lowering the temperature did not induce any 
disaggregation, indicating that the tempera- 
ture effect on zinc-induced aggregation was 
not reversible. 

The mechanism of metal-induced aggrega- 
tion is not clear. Effects of the metals on 
conformation could not be demonstrated. The 
data obtained to date support a neutralization 
mechanism of aggregation more than a cross- 
linking mechanism, but these studies are still 
in progress. 

(3) Capillary gas chromatography (GLC) 
is used in this laboratory to study sugar 
metabolism in the lens of two animal models, 
rat and monkey. Standard methodologies 
have been used to quantitate the common 
sugars. Methods have been devised in this 
laboratory that allow the separation of sorbitol 
and dulcitol, the alcohol derivatives of glucose 
and galactose. Sugar analyses have been done 
on serum of animals fed various sugar diets 
and rat lenses incubated in vitro with a variety 
of sugars. 



The chromatogram obtained from the 
capillary GLC analysis of animal blood serums 
and lens material contains, in addition to the 
expected sugar derivatives, a number of 
unidentified metabolites present in low quan- 
tities. Our laboratory is in the process of 
identifying these. One of those metabolites 
present in both rat and monkey lenses, has 
been tentatively identified as scylloinositol. 

Significance to Biomedical Research and tlie 
Program of the Institute 

Oxidative processes are known to contribute 
to cataractogensis. Metal-catalyzed oxidation 
of the crystallins leads to protein modification 
that mimic those seen in aging, senile cata- 
racts, and brunescent lenses. Studies on the 
interaction of metals with the crystallins are 
highly relevant to understanding the role of 
these oxidation reactions in lens. Understand- 
ing those mechanisms present in lens for 
protecting against oxidative damage is impor- 
tant for developing interventions. Studies on 
glutathione S-transferase are highly relevant to 
cataract. Recent reports correlate deletions on 
glutathione S-transferase /x with certain cata- 
racts. 

Proposed Course 

We wiU focus our studies for fiscal year 1995 
on the following: (1) pursuing the activity in 
lens that protects against thiol-dependent 
oxidation reactions, (2) assaying for glutathi- 
one S-transferases in epithelial layers from 
cataract surgeries, and (3) continuing the 
studies on the interaction of metals with the 
crystaUins. 

NEI Research Program 

Lens and Cataract — Pathogenesis of Cataract 

Publications 

Bettelheim FA, Reid MB, Garland D: Hydra- 
tion of gamma crystallins. Exp Eye Res 58:219- 
224, 1994. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00296-01 LMOD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994^ 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Studies on Human Lens Proteins 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI 



Others: J. Samuel Zigler, Jr. Ph.D. Senior Scientist 

Yvonne Duglas-Tabor B.A./B.S. Biologist 



LMOD, NEI 
OGCS, NEI 



COOPERATING UNITS (if any) 

Ophthalmic Genetics and Clinical Services Branch, NEI, NIH (Manuel B. Datiles, M.D.); Jules Stein Eye 
Institute, UCLA (J. Horwitz, Ph.D.) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL: 



0.7 



0.7 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This laboratory has in progress a study to characterize the proteins of the human lens. The lens consists of 
a few very high abundance proteins called the crystallins and several hundred lesser abundance proteins. 
During the life of the lens, the proteins become extensively modified. Methodologies have been developed 
to yield good separation of the proteins using two-dimensional polyacrylamide gel electrophoresis. Due to 
the extensive modifications, identification of the proteins is based on immunotechniques and sequencing. 
Normal donor lenses varying in age from fetal to 70 years and cataracts of different etiologies have been 
analyzed. The regions of the normal lenses are identified by structure patterns. The cortex has been separated 
into three different layers, and each of the developmentally defined nuclear regions has been separated. The 
protein patterns in each of the cortical regions are distinguishable as cortex with the characteristic large protein 
spots corresponding to each of the major crystallins. The protein patterns of the nuclear regions are all similar 
to each other but clearly unique from those of the cortical regions. Protein spots corresponding to the major 
crystallins are not readily visible. Many new spots are present, including numerous low molecular weight 
spots that are fragments of crystallins. These results have significance in understanding the potential role of 
protein modification in cataractogenesis. The proteins throughout the lens are being identified, yielding a 
database of information on the normal human lens. The protein patterns of numerous cataracts have been 
determined. These data are now being analyzed with respect to the cataract etiology. 



176 



PHS 6040 (Rev. 5/92) 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The long-term goal of this project is to under- 
stand those mechanisms involved in human 
cataractogenesis. 

The immediate objectives of this project are to: 

(1) identify the proteins of the human lens and 
characterize the changes in the protein compo- 
sition that occur with development and aging, 

(2) identify the covalent modifications that the 
human lens proteins undergo with develop- 
ment and aging, and (3) characterize the 
proteins of human cataracts of various 
etiologies. 

Methods 

Human lens material was obtained from 
donors' eyes and from cataract surgery. 
Immobilized pH gradients and the Isodalt 
two-dimensional electrophoresis system were 
used to separate the lens proteins. Other 
techniques used include high-pressure liquid 
chromatography, ultraviolet /visible spectros- 
copy, and immunotechniques. 

Major Findings 

Normal donor lenses of ages varying from 
seven months to 60 years were dissected, and 
the lens regions were identified by suture 
patterns. Adult lenses were easily and clearly 
separated into regions corresponding to elon- 
gating fiber cells, outer cortex, inner cortex, 
adult nucleus, fetal nucleus, and embryonic 
nucleus. In some lenses, another layer was 
observed that may correspond to the juvenile 
nucleus. These separations are consistent with 
the zones of discontinuity seen microscopically 



The proteins of each region were separat- 
ed by two-dimensional electrophoresis. For 
the three cortex layers, the two-dimensional 
protein patterns are distinguishable as cortex. 
These patterns are characterized by large 
protein spots corresponding to aA-, aB-, pBl- 



VS-, and (3B2-crystallins as well as 100 to 200 
lesser abundant spots. Comparisons of the 
three cortex regions yield some differences 
among the protein patterns. The inner cortex 
of the mature lens has an increase in the 
acidic forms of a crystallin, bBl-crystaUin 
spots are greatly diminished, and many lesser 
abundant spots are either increased or 
decreased. 

In contrast, the protein patterns of the 
nuclear regions of adult lenses are unique and 
distinguishable from those of the cortical 
regions. The crystaUins have undergone 
extensive modification. Protein spots corre- 
sponding to the major crystaUins are not 
visible. Many new spots are present, and 
there is a significant increase in the number of 
low molecular spots that are fragments of 
crystaUins. The protein patterns, including the 
low molecular weight species, are simUar in 
aU adult lenses analyzed. The clear distinction 
between the protein patterns of the cortex and 
those of the nucleus suggests that certain 
modifications of the crystaUins occur in the 
developmentaUy defined nuclear regions but 
not in the cortical regions. It is not clear if 
there are signals that initiate the modifications 
of crystaUins in the nucleus or if there are 
signals that prevention the modifications of 
proteins in the cortex. 

The protein patterns of the adult, fetal, 
and embryonic nuclear regions are simUar in 
the adiilt lens. The protein patterns of the 
nuclear regions of lenses fiom birth to about 
30 years change with increasing age from a 
pattern closer to a cortical pattern to that of a 
nuclear protein pattern. For lenses older than 
30 years, the protein patterns of the nuclear 
regions appear to remain stable. 

Predictable protein patterns are found in 
aU normal lenses in comparable regions of 
lenses of comparable ages. Thus, these results 
are consistent with the interpretation that the 
extensive modifications seen in the two-di- 
mensional protein patterns do not represent 
deleterious, random modifications that result 
from decreased lens viability or form exposure 
to noxious environmental agents. These 



177 



FY 1994 NEI Annual Report 



modifications are likely not related to cataract 
formation. Rather at appropriate developmen- 
tal stages, a series of reactions occur leading to 
the covalent modification of the crystallins. 
These modified forms of the crystallins may 
be better suited for function in the center of 
the lens in maintaining transparency. 

Numerous studies have demonstrated that 
with time many of the lens proteins undergo 
acidification. Our studies demonstrate that 
the enzymes such as glyceraldehyde 3-phos- 
phate dehydrogenase are present as multiple- 
charge species even in the outermost layers of 
the cortex. This suggests that the conversion 
of these enzymes to more acidic forms is not 
likely to be just the result of aging as has been 
suggested. 

The proteins of the cataractous regions of 
posterior subcapsvdar cataracts with diagnoses 
of serule, presenile, radiation, retinitis pigmen- 
tosa, and gyrate atrophy have been analyzed 
by two-dimensional electrophoresis. Some 
differences in the protein patterns exist, but, 
so far, no protein changes can be correlated 
with any disease etiology. 

The proteins of a large number of cortical 
and nuclear cataracts have been analyzed. 
Correlations between protein changes and 
cataract types have not yet been found using 
the present methods of classifying cataracts. 
The data obtained to date indicate that there 
are no obvious, unique crystallin modifications 
responsible for each type of cataract. 



Many of the proteins in the human lens 
have been identified using immunoblotting 
techniques. Degraded forms of aB-crystallin 
have been identified in both normal and 
cataractous lenses. 

Blots of human lens proteins separated by 
two-dimensional electrophoresis are being 
used to identify the targets of autoantibodies 
found in normal and cataract patients. 

Significance to Biomedical Researcti and ttie 
Program of tfie Institute 

These studies are directiy relevant to the NEI 
program. Characterization of the proteins of 
the normal human lens will provide informa- 
tion necessary to understand cataractogenesis. 
In addition, it wiU provide information on 
normal metabolic and developmental process- 
es in this unique tissue in contrast to those 
processes that occur as a function of aging. 

Proposed Course 

In fiscal year 1995, we will continue the char- 
acterization of human lens proteins. Our 
studies wiU include continued efforts to identi- 
fy the modified proteins and the modifications 
involved, the purification of modified crystal- 
lins for sequencing and mass spectroscopy, 
and the continued analysis of cataracts. 

NEI Research Program 

Lens and Cataract — Lens Development and 
Aging 



178 



PROJECT NUMBER 
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00201-10 LMOD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Structure and Exp re ssion of Polyo l Pathway Enzym es 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Deborah Carper Ph.D. Biologist LMOD, NEI 

Others: Susan Old Ph.D. Staff Fellow LMOD, NEI 

Takeshi Iwata Ph.D. Visiting Associate LMOD, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS; 

3.0 



PROFESSIONAL 

3.0 



0.0 



CHECK APPROPRIATE BOX{ES) 

□ (a) Human subjects [x] (b) Human tissues □ (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

In tissues that do not require insulin for glucose uptake, the high systemic level of glucose that develops 
during diabetic hyperglycemia readily translates into high tissue levels of glucose. Some of this excess glucose 
is metabolized by the polyol pathway. Aldose reductase (AR), the first enzyme of this pathway, reduces 
glucose to the organic osmolyte sorbitol while sorbitol dehydrogenase (SDH), the second enzyme of the 
pathway, oxidizes sorbitol to fructose. The diabetes-enhanced flux of glucose through the polyol pathway has 
been implicated in the etiology of diabetic complications, including cataract, retinopathy, and neuropathy. Our 
studies have been aimed at defining the structure and regulation of AR and SDH so that new approaches to 
the control of this pathway may be made available in diabetic tissues. 

Many aldose reductase inhibitors (ARIs) have been shown to have broad substrate specificity and undesirable 
side effects. Emphasis on the structure/function properties of the AR enzyme will help in the refinement and 
design of future inhibitors. To this end, catalysis and inhibition of aldose reductase was examined following 
site-directed mutagenesis. In the rat, our mutagenesis studies indicated that tyrosine 48, histidine 110, and 
cysteine 298 are important residues in the active site and that tyrosine 48 is most likely the proton donor 
during substrate reduction. In addition, mutation of histidine 110, an active-site residue, decreased inhibitor 
effectiveness by up to 2000-fold, indicating that this residue is important in inhibitor binding. Mutations of 
human AR indicated that although phenylalanine 122 putatively binds ARIs, substitution of this amino acid 
has little affect on the inhibitor-binding constant, K,. 

Sorbitol dehydrogenase has been reported to be linked to cataract formation in nondiabetics. We have 
determined the gene structure, tissue distribution, transcriptional initiation, and chromosomal localization of 
human SDH. In addition we have used SSCP analysis followed by sequence comparison to analyze congenital 
cataract patients with SDH deficiency. Through these studies, we hope to identify the gene defect in this 
family. 

179 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The objective of this project is to study the 
structure, function, and regulation of the two 
enzymes of the polyol pathway — aldose re- 
ductase (AR) and sorbitol dehydrogenase 
(SDH). 

Methods 

The methods used include molecular biology, 
protein chemistry, cell biology, and molecular 
genetics. 

Major Findings 

Structure-function studies of AR. X-ray crystal- 
lographic studies previously indicated that 
either Y48, HI 10, or C298 could potentiaUy 
function as the proton donor in the catalytic 
mechanism of AR. The mechanism involves 
binding of the substrate to the enzyme/ 
NADPH complex with subsequent hydride 
transfer from the rucotinamide ring to the 
carbonyl group. A proton is then donated to 
the carbonyl oxygen. Our studies showed that 
mutagenesis of rat lens tyrosine 48 to phenyl- 
alanine (Y48F) virtually abolished rat lens 
enzyme activity, even though the physical 
structure of Y48F appeared to be normal as 
evidenced by circular dichroism spectra and 
NADPH-binding affini*^v constants, Kd. 
Changes of histidine 110 to glutamine 
(HllOQ) and cysteine 298 to serine (C298S) 
resulted in mutants that were still active. Our 
findings would indicate that Y48 is the proton 
donor in the reduction reaction of rat lens AR. 

The potential role of the amino adds C298, 
HI 10, H187, and H200 in the inhibition of rat 
lens AR was examined by measuring the IC50 
values of five different AR inhibitors. No 
differences between the wild tjrpe and the 
mutants were observed, except for HllOQ. 
This AR mutant was less sensitive to inhibi- 
tion by both the carboxylic and hydantoin 
classes of compounds. The greatest increase 
in IC50 was seen with the hydantoins. There 



was a three hundredfold increase for sorbinil 
and a two thousandfold increase for tmeristat. 
The carboxylic acids such as tolrestat and 
statiJ gave increases of approximately fivefold. 
These inhibitor studies indicate that HI 10 is 
important in inhibitor binding. The mecha- 
nism involved may include perturbation of the 
positively charged anion weU formed by Y48, 
HI 10, and the nicotiniamide ring with variable 
effects on AR inhibitors due to their inherently 
different structural properties. The efficacy of 
AR inhibitors are evaluated by their ability to 
lower polyol levels and prevent or retard 
cataract formation, retinal microangiopathies, 
microalbuminuria, decreased motor nerve 
conduction velocity, and /or deterioration of 
nerve ultrastructure. Because the rat is the 
most widely used model of diabetic complica- 
tions, comparative information on the catalysis 
and inhibition of rat and human AR has 
provided a baseline for designing specific 
inhibitors. 

Ovu: studies on the mutagenesis of human 
AR have been focused on elucidating the 
residues involved in inhibitor binding. X-ray 
crystallographic studies indicated that a num- 
ber of hydrophobic interactions occur between 
the enzyme and the inhibitor. The inhibitor 
was proposed to be sequestered by a hydro- 
phobic bridge comprising phenylalanine 122 
and leucine 300. We have begun to evaluate 
this location by mutating phenylalanine 122 to 
t)n"osine and cysteine. Small changes in the 
affinity to various substrates. Km, and in the 
turnover number, Kcat, were observed. How- 
ever, contrary to the predicted importance of 
this residue, there was no significant change 
in the inhibitor-binding constant, Ki. 

Gene structure of sorbitol dehydrogenase. The 
complete complementary deoxyribonucleic 
acid (cDNA) sequence coding for human SDH 
and the genomic organization of this enz5nne 
were determined. SDH is arranged into nine 
exons and eight introns. The first exon con- 
tains 89 bp of 5' untranslated sequence, and 
exon nine contains 1,263 bp of 3' untranslated 
sequence. This considerably long stretch of 3' 
untranslated sequence comprises more than 60 
percent of the total cDNA sequence, the im- 



180 



Laboratoii/ of Meclmnisms of Ocular Diseases 



portance of which is unknown, although it is 
likely to include messenger ribonucleic acid 
stability and translational regulation. Homol- 
ogous human alcohol dehydrogenase genes 
and the human ^-crystaUin gene are also 
arranged into nine exons and eight introns, 
but none of the spUcing points coincide with 
the splice points of the SDH gene. At the 
promoter region of human SDH, no obvious 
TATAA or CCAAT box was found. 

Interestingly, different transcription initia- 
tion sites were observed in Uver and lens. 
These different tianscription initiation sites do 
not affect the tianslation initiation site 
(ATG-codon). At the 5' flanking region of this 
gene, which is reported to bind Spl tianscrip- 
tional factor, three Spl and a CACCC box 
were observed. The sequential motif of the 
promoter region resembles that of duck lactate 
dehydrogenase p/e-crystaUin that is highly 
expressed in heart as an enzyme and in lens 
as a crystaUin. That SDH may also be an 
enzyme /crystaUin is an interesting possibUity. 
Fluorescence in situ hybridization localized the 
SDH gene to human chromosome 15q21.1. 

Northern blot analysis demonstrated that 
the highest expression of SDH was in lens. 
Other tissues with high expression were 
kidney, heart, brain, testes, retina, and the 
retinoblastoma ceU hne Y79. The high expres- 
sion of SDH in human lens is of great interest 
in view of previous reports showing abnormal 
SDH activity in red blood cells of patients 
who develop cataracts. 

A congenital cataract patient from the 
largest known family with red blood cell SDH 
enzyme deficiency was examined for muta- 
tions by single standard conformation poly- 
morphisms and DNA sequencing. In this 
family, four out of five brothers and their 
father had bilateral cataracts. Although SDH 
activity is reduced 18 to 78 percent of normal 
in all members of this family, the occurence of 
cataract does not appear to be correlated with 
the severity of the red blood cell deficiency. 
However, this does not rule out a correlation 
between cataract and lens SDH activity. In 
this study, a polymorphic one bp mismatch in 



exon four and a two bp deletion followed by 
a one bp mismatch in exon nine were found, 
but no mutation within the coding sequence 
was detected. Thus, the enzyme deficiency is 
probably not due to abnormal protein struc- 
ture, and other possibilities such as reduced 
promoter activity are now being examined. 
We have demonstrated high expression of 
SDH in human lens compared with other 
tissues. A previous study reported higher 
enzyme activity in human lens compared with 
other species. These data suggest that SDH 
may play an important role in the human lens, 
and dysfunction of this enzyme may lead to 
alterations in the polyol pathway. 

Significance to Biomedical Research and ttie 
Program of tlie Institute 

The polyol pathway has been implicated in 
ocular and other compUcations of diabetes. 
Regulating the enzymes of this pathway — AR 
and SDH — in a manner that will decrease the 
flux of glucose through the polyol pathway 
should ameliorate these compUcations. Using 
structure /function studies, the localization of 
the inhibitor site of AR wiU provide a rational 
basis for inhibitor drug design. By character- 
izing SDH, we can begin to evaluate its role 
in the lens and its interrelationship with AR. 

Proposed Course 

Site-directed mutagenesis studies will continue 
to be used to determine the inhibitor-binding 
site of human AR. For SDH, the functional 
promoter wiU be defined, and the other mem- 
bers of the family with lowered SDH activity 
and cataracts wiU be examined. 

NEI Research Program 

Lens and Cataract — Molecular Genetics 

Publications 

Bateman JB, Kojis T, Heinzmann C, Klisak I, 
Diep A, Carper D, Nishimura C, Mohandas T, 
Sparkes R: Mapping of AR gene sequences to 



FY 1994 NEI Annual Report 



human chromosomes 1, 3, 7, 9, 11, and 13. Sato S, Old S, Carper D, Kador PF: Purifica- 
Genomics 17:560-565, 1993. tion and Characterization of Recombinant Human 

Placental and Rat Lens ARs Expressed in Esche- 
Iwata T, Carper D: Human Sorbitol Dehydroge- richia coU. New York, Plenum Publishing 



nase Gene. New York, Plenum Publishing Corp., in press. 
Corp., in press. 

Old SE, Carper DA, Hohman, TC: 
Na,K-ATPase response to osmotic stress in 
primary dog lens epithelial cells. Invest Ophth- 
almol Vis Sci, in press. 



182 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00237-09 LMOD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on or)e line between the borders.) 

Characterization of the Lens 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Paul Russell Ph.D. Research Chemist LMOD, NEI 



Others: 



Carolyn Chambers 
Geoffrey Kidd 
Santa Tumminia 



Ph.D. 
Ph.D. 
Ph.D. 



Senior Staff Fellow 
Senior Staff Fellow 
Senior Staff Fellow 



LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (if any) 

Oakland University (John Reddan, Ph.D.); Purdue University (Jean Smith, Ph.D.); University of East Anglia 
(George Duncan, Ph.D.); National Institute of Child Health and Human Development (Jose Pichel, Ph.D. and 
Heiner Westphal, Ph.D.) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.6 



PROFESSIONAL: 



3.6 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

We are continuing our efforts in characterizing the lens and processes that may occur in cataract development. There 
are three aims in our research efforts: determination of the seqeuences of human crystallins, development of the in vitro 
lens incubation system, and examination of stress on lenses and lens crystallins. Each of these areas complements the 
Other and builds a base on which to study how the lens resists cataract development and the stresses that will eventually 
lead of opacification. 

The first area of work has been the determination of the sequence of human p B2-crystallin and human yS-crystallin. 
The human p B2-crystallin is the principal p crystallin in the lens. Two human lens complementary deoxyribonucleic 
acid (cDNA) libraries were made. One of the libraries was to adult lens and one was to fetal lens. The p B2-crystallin 
cDNA was cloned and sequenced from these libraries and the deduced amino acid sequence was determined. Work also 
continued on the promoter region of this crystallin in order to develop a construct that would be developmentally 
regulated in transgenic animals. yS-crystailin is a protein that increases in content in the lens with age. The amount 
of this crystallin is very often decreased with the advent of human cataract formation. The yS-crystallin sequence was 
determined by a combination of cDNA sequencing, electrospray ionization mass spectrometry, and fast atom 
bombardment mass spectrometry. 

In vitro lens incubation is currently one of the only ways to study the effect of environmental stresses on whole lens 
metabolism. Work has progressed to establish criteria for determining which of the in vitro incubated lenses has 
metabolic integrity and has not been injured in the dissection process. A simple test has been developed to determine 
the integrity of the lens in vitro in as few as 30 minutes after the start of the incubation procedure. Studies have been 
done in vitro to determine the response of the whole lens to oxidative insult using the content and mRNA levels of 
catalase, an enzyme responsible for protection against H2O2. Glutathione levels of stressed lenses have also been 
measured to determine the reason for the loss of this vital constituent. 

The effects of environmental stresses have also been studied using cell culture systems. The cell line used in these 
studies constitutively expresses aB-crystallin. Effects of oxidative stress and heat shock have been investigated on this 
crystallin that is thought to serve as a molecular chaperon. 

183 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The purposes of this project are to: (1) deter- 
mine the components in the human lens, (2) 
develop model systems to examine how 
stresses such as oxidation can alter the tissues 
in the anterior segment of the eye, (3) define 
these model systems for use with agents that 
might delay cataract formation, and (4) ex- 
plore basic questions about the metabolism of 
the lens using cell and molecular biological 
methods. 

Methods 

Among numerous biochemical and molecular 
biological methods used in this research are 
Northern, Southern, and Western blotting of 
messenger ribonucleic acid, deoxyribonucleic 
add (DNA), and proteins. In addition, vari- 
ous methods for quantitation of these compo- 
nents such as slot-blotting are done. The 
polymerase chain reaction is used as is nucleic 
acid sequencing. 

Major Findings 

(1) Two libraries of the complementary 
cDNAs from human lenses have been made. 
One of the libraries is from adult lens, and 
one is from fetal lens. 

(2) The sequence of the human pB2-crys- 
tallin has been detemiined from cloned se- 
quences from the human lens libraries. The 
sequence of the human |3B2-crystallin is simi- 
lar to the sequence of the bovine and rat 
crystallins and shows the high level of homol- 
ogy in this crystallin. The deduced sequence 
of the pB2-crystallin matches exactiy the 
protein sequence that was published concur- 
rently by others. 

(3) The sequence of human vS-crystallin 
has been determined using a combination of 
cDNA sequencing, electrospray ionization 
mass spectrometry, and fast atom bombard- 
ment mass spectrometry. The human se- 



quence differs from the bovine sequence in 
several areas, including one peptide fragment 
that appears to be associated with cataract 
development. 

(4) A method for the determination of 
protein content in the incubation medium has 
been tested as a means to determine lens 
integrity in vitro. The method, which is rapid, 
can accurately predict which of the lenses in 
vitro has been metabolically compromised 
during the dissection procedure. 

(5) Studies on rat and monkey lenses 
incubated in vitro have suggested that the 
rapid loss of glutathione in the young rat 
lenses in vitro may be due to the rapid growth 
rate of these lenses. Older lenses and lenses 
from adolescent monkeys do not exhibit the 
rapid loss of this constituent. The loss of 
glutathione in the rat lens has long been an 
enigma. 

(6) aB-crystallin, a molecular chaperone, 
has been studied using a cultured cell line that 
constitutively expresses this protein. In the 
U373MG astroglioma ceU Hne, the accumula- 
tion of this crystallin has been shown to be 
stress dependent and phosphorylation inde- 
pendent. Heat shock will cause a rapid rise in 
the level of this protein, but within four to six 
hours the levels of aB-crystallin return to 
baseline values. Incubation of the cell with 
cobalt wiU also cause a rise but at a later time, 
and the level does not return to baseline 
within 72 hours. The aB-crystaUin also ap- 
pears to switch from water soluble to w^ater 
insoluble after the insult suggesting an alter- 
ation in the compartmentalization of this 
chaperone. The level of phosphorylation of 
the aB-crystaUin was not changed during the 
stress experiments, indicating phosphorylation 
was not an important event for the function of 
this protein in response to stress. 

Significance to Biomedical Research and the 
Program of the Institute 

The human lens needs to be characterized to 
determine which components are involved in 
the protection of this tissue from envirorunen- 



184 



1 



Laboratory of Mechanisms of Ocular Diseases 



tal pressures such as oxidative stress and 
which components are the more susceptible to 
these stresses. Once the constituents in the 
human lens are known, the development of 
systems to study the whole lens is vital to 
understanding the process of cataract forma- 
tion. Obtaining a reproducible in vitro system 
is important to the development of anticata- 
ract agents. Working under definable condi- 
tions will allow us to formulate mechanisms 
and agents to ameliorate cataract develop- 
ment. 

NEI Research Program 

Lens and Cataract — Lens Biochemistry and 
Biophysics 

Publications 

Chambers C, RusseU P: Sequence of the 
human lens pB2 crystallin encoding cDNA. 
Gene 133:295-299, 1993. 



Kidd GL, Reddan JR, RusseU P: Differentia- 
tion and angiogenic growth factor message in 
two mammalian lens epithelial cells lines. 
Differentiation 6:67-74, 1994. 

RusseU P, Tumminia SJ, Pichel JMC: Compari- 
son of lens epithelial ceU Unes from transgenic 
animals. Invest Ophthalmol Vis Sci 35:2204, 
1994. 

Tumminia SJ, Qin C, Zigler JS Jr, RusseU P: 
The integrity of mammaUan lenses in organ 
culture. Exp Eye Res 58:367-374, 1994. 

Tumminia SJ, RusseU P: aB-crystallLn expres- 
sion and chaperone function in human astro- 
gUoma ceU Une U373. Invest Ophthalmol Vis 
Sci 35:2212, 1994. 



185 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00289-01 LMOD 



PERIOD COVERED 

Oct obe r 1, 1993 to S eptember 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Lentic ular Expressi on of the HIV Protease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) 

PI: Paul Russell Ph.D. Research Chemist LMOD, NEI 



Others: Santa Tumminia 



Ph.D. 



Senior Staff Fellow 



LMOD, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.35 



PROFESSIONAL: 



0.35 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues Q (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Two transgenic mouse strains have recently been obtained. These strains contain the HIV protease coding sequence 
linked to the oA-crystallin promoter. One strain gets cataract in utero, while the other strain develops cataract at 
approximately 25 days. The strains are currently hemizygous. Efforts in the past several months have been directed to 
getting homozygous populations of these strains to determine the cause of the cataract formation and how this is related 
to the expression of the HIV protease in the lens. 



186 



PHS 6040 (Rev. 5/92) 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The purpose of this project is to determine 
how the expression of the human immunode- 
ficiency virus protease in the lens causes 
cataract formation. 

Methods 

We will use biochemical and molecular biolog- 
ical methods as well as Ught and electron 
microscopy to investigate cataract formation. 

Major Findings 

The homozygous mouse strains appear to get 
cataract formation a few days before the 
hemizygous animals; therefore, we are using 
homozygous selection. 



Significance to Biomedical Research and the 
Program of the Institute 

Cataract formation in the presence of a small 
amount of HIV protease in the lens could sug- 
gest that the protease is cleaving a specific 
protein that will start the process of cataract 
formation. Alternatively, the protease could 
be interfering v^th the process of differentia- 
tion in the lens. Either of these two hypothe- 
ses would have important imphcations either 
for cataract formation or for the action of the 
HIV virus in general. 

Proposed Course 

After developing the homozygous strains, we 
will investigate the mechanism involved in 
cataract formation in these animals. 

NEi Research Program 

Lens and Cataract — Pathogenesis of Cataract 



187 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00290-01 LMOD 



PERIOD COVERED 

October 1, 1993 to Septe mb er 3 0, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Autoantibo die s to Lens Crys tallins 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Paul Russell Ph.D. Research Chemist LMOD, NEI 



Others: J. Samuel Zigler, Jr. 
Donita Garland 
Yvonne Duglas-Tabor 



Ph.D. 


Senior Scientist 


Ph.D. 


Research Biologist 


B.A./B.S. 


Biologist 



LMOD, NEI 
LMOD, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.05 



PROFESSIONAL: 



0.05 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



Jx] (b) Human tissues □ (c) Neitlier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Experiments are under way to investigate the level of autoantibodies to lens crystallins in the serum of normal volunteers 
and of individuals with cataracts. The specific autoantibodies will be determined and measured using chemiluminescent 
Western blots of two-dimensional gels of human lens proteins. Initial efforts on some serum samples have indicated that 
autoantibodies are present, and these appear to react most commonly with the p-crystaUin components in the lens. 



188 



PHS 6040 (Rev. 5/92) 



Laboratoiy of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The purpose of this project is to determine if 
autoantibodies to lens are present in the 
serum and if the specific autoantibodies corre- 
late with cataract type or the rate of progres- 
sion of cataract. We will use chemilumines- 
cent Western blot analysis to investigate the 
increase in autoantibodies. 

Methods 

Autoantibodies will be determined using 
Western blots to lens proteins. The lens 
proteins will be separated by two-dimensional 
gel electrophoresis. 

Major Findings 

This project has just recently been undertaken; 
however, it has been determined that autoanti- 
bodies are present in the serum. The autoanti- 
bodies that have been observed are generally 
to proteins that are part of the p-crystallin 
family. 



Significance to Biomedical Research and the 
Program of the Institute 

This project sets out to determine if specific 
cataract type or the progression of cataract 
formation can be related to specific autoanti- 
bodies to lens crystallins that are present in 
the serum. If a correlation between these 
autoantibodies and progression of lens opacifi- 
cation can be made, it might be possible to 
develop a cUnical test to determine which 
individuals are most at risk for rapid cataract 
formation. 

Proposed Course 

Efforts will continue to determine the level of 
autoantibodies in the serum of normal indi- 
viduals, and then these levels will be com- 
pared with the ones found in the serum of 
patients who have cataracts. 

NEI Research Program 

Lens and Cataract — Pathogenesis of Cataract 



1S9 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00272-04 LMOD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Inherited Ocular Diseases 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: James Fielding Hejtmancik M.D., Ph.D. Medical Officer LMOD, NEI 



Others: 



John Hope 
Radha Ayyagari 
Ling Lee 
Masami Oguni 
Rita Mitra 
T. Padma 



Ph.D. 

Ph.D. 

M.S. 

M.D. 

Ph.D. 

Ph.D. 



Senior Fellow 
Visiting Associate 
Chemist 
Fogarty Fellow 
Special Volunteer 
Visiting Scientist 



LMOD, NEI 
LMOD, NEI 
LMOD, NEI 
LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (if any) 

Baylor College of Medicine (J. Towbin, M.D.); Univ. of Iowa (R. Smith, M.D.); Univ. of Texas-Houston (S. Daiger, Ph.D.); Ocular 
Genetics and Clinical Services Branch, NEI, NIH (M. Kaiser, M.D., R.Caruso, M.D., R. Sperduto, M.D.); Massachusetts Institute of 
Technology (G. Benedek, Ph.D., J. Pande, Ph.D.); Osmania Univ., Hyderabad, India (J.S. Murty, Ph.D., T. Padma, Ph.D.); L.V. Prasad 
F.yp Inst , Hyderabad, India (G.N. Ran, MP., S . Rasti, Ph.D.); Universita Degli Studi di Parma (G. Maraini. M.D., G. Alherti, M.D.) 



LAB/BRANCH 



Laboratory of Mechanisms of Ocular Diseases 



Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



5.67 



PROFESSIONAL: 



5.67 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 
\x\ (a1) Minors 
□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Study of inherited visual diseases provides a means by which both normal and aberrant visual processes might 
be understood. In addition to directly elucidating the pathophysiology of the inherited disease under study, 
these studies can provide insights into the structure-function relationships of the molecular components of the 
visual system and their normal physiology. This laboratory is using a number of approaches to study inherited 
visual diseases affecting the lens and retina. 

Lens crystallins make up more than 90 percent of the soluble protein of the lens and are heavily modified in 
most cataracts. The effects that specific modifications of P- and y-crystallin structure produce on crystaUin 
functions such as stability and formation of macromolecular aggregates are being expressed using SF9 cells 
transformed with bacculovirus vector containing coding sequences for normal and modified p A3/A1- and p 
A2-crystallin genes. Regions of the p-crystallin molecule of special interest include the amino and carboxy 
terminal arms, the connecting peptide, and the Greek key motifs of the core domains. In addition, the 
interactions of acidic and basic P-crystallins are being studied. 

A second approach to understanding inherited visual diseases uses principles of positional cloning to identify 
genes important in human inherited diseases. Human diseases currently undergoing linkage analysis, gene 
isolation, or characterization of mutations include Usher syndrome, long QT syndrome, cataracts, and a variety 
of X-linked syndromes. We are currently collecting families with autosomal recessive retinitis pigmentosa 
and Bietti syndrome in preparation for study of this important group of diseases. Finally, the effects of 
specific genetic aUerations, including red pigment gene polymorphisms and glutathione S-transferase Ml 
deletions on the visual process, are being studied. 



190 



PHS 6040 (Rev. 5/92) 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The long-range objectives of this project in- 
clude increasing the understanding of inherit- 
ed visual diseases, with the eventual aims of 
increasing the diagnostic ability for these 
diseases and providing a foundation for 
developing rational therapies based on a 
thorough knowledge of their molecular patho- 
physiology. These long-range objectives will 
be approached by pursuing the specific aims 
of identifying genes involved in inherited 
visual diseases and elucidating the mecha- 
nisms by which mutations in these genes 
cause disease. 

Methods 

Conventional cloning technology is used in 
preparing sequences for gene expression 
studies. These include Ugation with T4 deoxy- 
ribonucleic acid (DNA) Ugase, screerung by 
NaOH miniprep methodology, and ^^P-labeled 
DNA probes as well as aUele-specific oligonu- 
cleotide hybridization to screen for specific 
single-base settings. Sequence changes are 
introduced by site-specific mutagenesis using 
standard methodology. Gene expression is 
carried out in insect cells (Sf9) using the 
baculovirus expression system. Protein ex- 
pression is monitored by standard two-dimen- 
sional gel electrophoresis followed by immu- 
noblotting. Association behavior is assessed 
by elution volume on sieve FPLC. 

Crystallin and other complementary DN As 
and genomic fragments are isolated by library 
screening with cloned genes or oligonucleo- 
tides using routine methods. Sequencing is 
carried out by cycUng or using automated 
florescent technology. 

Until recentiy, HrJcage analysis has been 
carried out by conventional Southern blotting. 
Cell lines from patients and other family 
members are immortalized by Epstein Ban- 
virus transformation. DNA is isolated by 
standard methodology and digested by restric- 



tion endonucleases. After agarose gel electro- 
phoresis. Southern transfer is performed and 
the resulting blot probed with isolated DNA 
fragments labeled with [^^P] by oligonucleotide 
labeling. Recentiy, short tandem repeat (STR, 
microsateUite) markers have been analyzed by 
polymerase chain reaction performed in the 
presence of labeled oligonucleotides and 
analyzed on sequencing gels. Linkage data 
are recorded on a computerized spreadsheet 
and analyzed using both two-point and n^ulti- 
point analysis with the LINKAGE program 
package. 

Major Findings 

(1) The p-crystaUins, their structure, and the 
mechanisms by which heterogeneity arises 
among this family of proteins are being inves- 
tigated. The pA3-crystaUin is identical to pAl 
except for an additional 17 amino acid N- 
terminal extension. The same gene is believed 
to encode and express both polypeptides. In 
addition, the PA3/A1 coding sequences were 
inserted into the Bluebac expression vector 
(Stratagene) and expressed in SF9 cells. In SF9 
ceUs, a protein with the same amino terminal 
sequence as the pAl -crystallin is produced 
when the baculovirus-infected cells are grown 
past their prime. This is temporally correlated 
with the disappearance of the (3A3-crystaUin 
band, suggesting that the smaller band is 
created by processing or by degradation of the 
larger in this system. In addition, clones for 
the mouse pA2-, pBl-, pB2-, and pB3-crystal- 
Uns have been isolated and sequenced in 
preparation for characterization of their roles 
in p-crystallin aggregation. 

(2) An additional crystallin has been 
constructed in which the amino terminal arm 
is deleted and replaced by a glycine residue, 
so that this extension is identical to that found 
in Y2-crystaUin. This has been expressed in 
SF9 ceUs (Blubac vector) and has an appropri- 
ate migration on Laerrdi gels, CD-spectrum, 
and amino acid sequence. The activity of this 
P-crystaUin in association into the typical 200- 
250 kDa aggregates has been tested using 
FPLC on superdex 75 and superose columns. 
The normal pA3 polypeptide readily associ- 



191 



FY 1994 NEI Annual Report 



ates into homodimers, but the truncated (3A3 
associates minimally if at all. SF9 cells ex- 
pressing the recombinant crystallins were 
grown in '^^'S containing medium purified and 
reassociated with an excess of lens extract 
containing normal crystallins (unlabeled) 
using limited urea denaturation followed by 
dialysis. Association into p-crystallin aggre- 
gates was assessed by FPLC on sizing col- 
umns. The recombinant full-length P-crystal- 
lin peptide associates into both dimers and 
tetramers, with the dimer peak migrating 
slightly before the p-Ught peak. The truncated 
PA3-crystallin, however, migrates slightly 
behind the p-light peak and does not form 
obvious tetramers. These data strongly sug- 
gest that the amino terminal arm of P-crystal- 
Uns assists in the association of p-crystallins 
into higher order aggregates. 

(3) A pA3-crystallin has been constructed 
in which the entire connecting peptide from 
the first to the second domain has been re- 
placed with the corresponding sequence from 
Y2-crystallin. This tests the hj^othesi: that 
the connecting peptide, which crystallographic 
data show is extended in the p-crystallins and 
curved back on itself in the v-crystallins, is 
responsible in this fashion for the p-crystal- 
lins' tendency to dimerize. The p-crystallin 
with the modified connecting peptide was 
subjected to the same tests of association as 
described in number 2 and behaved essential- 
ly as the normal (unmodified) pA3-crystallin. 
The secondary structure of the modified P- 
crystallin is currentiy being confirmed with 
CD analysis. 

(4) The mouse pB2-crystallin has been 
subcloned into a baculovirus vector and ex- 
pressed in the SF9 cell culture system. The 
expressed protein is the appropriate molecular 
mass for an intact pB2-crystallin and forms 
dimers appropriately. Modifications, includ- 
ing deletion of the amino and carboxy termi- 
nal arms, are being engineered into the pB2- 
crystallin protein; the effects of these modifica- 
tions on its formation of homodimers and 
heterodimers with the normal and modified 
PA3-crystaIlin protein will be examined. 



(5) Studies of phase transition properties 
of the y-crystallin gene family have begun in 
collaboration with Dr. George Benedek, from 
the Massachusetts Institute of Technology, 
Boston. The bovine vB-crystallin has been 
modified at two of the four residues proposed 
to be critical for phase transition behavior. 
Phase transition analysis of the expressed, 
unmodified 72-crystallin has begun in Boston. 

(6) Ophthalmological diseases have been 
studied in humans by linkage analysis of 
restriction fragment length polymorphism 
markers. Diseases that we have mapped 
within the past year include Long QT syn- 
drome, two types of autosomal dominant 
congenital cataracts, and Usher's syndrome 
type I. In addition, family collection has 
begun on Bieti syndrome in anticipation of 
carrying out linkage analysis. Clinical and 
genetic heterogeneity of Usher syndrome 
within the Acadian population has been 
explored in detail. Genetic analysis confirms 
the cUnical impression that both type I and U 
Usher S5mdrome are found in the Acadian 
population and even within a single extended 
pedigree. The heterogeneity analysis des- 
cribed above implies that this must be due to 
segregation of two different and unlinked 
genes within this population. 

Two genes causing Usher sjmdrome type 
I have been mapped. In the Acadian popula- 
tion of Louisiana, the genetic locus is on 
chromosome lip, although in our British 
families, the gene is on chromosome llq. 
These findings have been subjected to hetero- 
geneity analysis using both the HOMOG2 
program and the M-test and are sigrvificant at 
p < .01 under the most stringent analyses. 
This surprising finding implies that multiple 
genes can cause the rather specific cUnical 
findings present in Usher syndrome. The 
location of the Usher syndrome gene on 
chromosome lip has been studied in detail 
using fine linkage mapping and haplotype 
analysis. The Usher syndrome gene has been 
localized to a 5 cM interval between the mark- 
ers D11S861 and D11S899. A YAC contig is 
being established over this region, and four 
YAC clones extending from D11S861 to 



192 



Laboratory of Mechanisms of Ocular Diseases 



D11S902 form the minimal tiling path over the 
assembled region, with about 12 additional 
YAC clones incorporated into this contig. 

(7) Several large families with autosomal 
dominant and recessive cataracts have been 
ascertained and studied clinically and samples 
collected. Genotyping of microsateUite mark- 
ers has begun in four of these families with 
attention initially concentrated in regions 
around candidate genes. Two families have 
5delded significant lod scores, one on chromo- 
some 2 and one on chromosome 17. 

(8) The possible association of various 
genetic mutations and polymorphisms v^th 
visual function is being carried out. The effect 
of various polymorphisms in the red pigment 
genes with visual function in heterozygous 
females is being studied in collaboration with 
Dr. Raphael Caruso (Ophthalmic Genetics and 
Clinical Services Branch), and the effect of 
deletion of the glutathione S-transferase Ml 
gene on the frequency of cataracts is being 
investigated in collaboration vsdth Dr. G. 
Maraini, from the Universita Degli Studi di 
Parma. 

Significance to Biomedical Research and the 
Program of the Institute 

Elucidation of the genetic defects causing 
visual disability will have ttnpUcations far 
beyond the patient population suffering from 
the specific syndrome under study. Inherited 
diseases provide a means by which the molec- 
ular physiology and pathophysiology of the 
visual system may be understood, and this 
knowledge can then be appUed to a broad 
spectrum of diseases as well as to the normal 
eye. This rationale also applies to the study of 
inherited diseases of which visual defects are 
only a small part. Thus, although our studies 
of myotonic dystrophy have already resulted 
in improved diagnostic abUities, the mecha- 
nism by which cataracts occur in this disease 
will provide insight into cataractogenesis in 
other hereditary syndromes as well as age- 
related and nonspecific cataracts. 



Proposed Course 

(1) Studies wiU continue on the structure- 
function relationships of lens crystallins, 
concentrating on the effects that modifications 
of the terminal arms, interconnecting peptide 
between the two domains, and surface charge 
and hydrophobic contact sites have on aggre- 
gation of both acidic and basic p-crystaUins. 
We wiU also continue to explore the effects 
that modification of the Greek key motifs have 
on crystaUin stability and, where applicable, 
lens transparency. In addition, the effects that 
modifications of y-crystaUin sequences have 
on the protein-phase transitions and their rela- 
tionship to cold cataract will continue to be 
explored. 

(2) Sample collection and linkage analysis 
of a variety of human diseases vdll continue. 
The main emphasis will be on inherited visual 
diseases, especially Usher syndrome type 1 
and cataracts. Fine mapping and physical 
mapping of the Usher s3mdrome type IC gene 
on Chromosome lip is being pursued, and 
candidate gene analysis will be initiated as 
soon as it would be meaningful. We are 
initiating fine mapping and candidate gene 
analysis of the autosomal dominant cataracts 
that we have mapped in families ascertained 
in collaboration with Dr. Muriel Kaiser-Kupfer 
and Dr. G.N. Rao and linkage analysis of the 
of autosomal recessive cataracts ascertained in 
collaboration wdth Dr. J.S. Murty in India. 
This will be coordinated with a new project 
categorizing and mapping expressed se- 
quences of the human lens and the ongoing 
mechanistic studies on lens crystallins de- 
scribed above. Together, these projects should 
provide a coordinated effort to elucidate the 
mechanisms of cataractogenesis in the human 
lens. 

(3) The studies of possible associations be- 
tween mutations and polymorphisms of genes 
functioning in the vision process and visual 
function wiU continue. Sample collections 
from both Italy and the National Institutes of 
Health (NIH) wiU continue for the cataract 
epidemiological study. Patients will be re- 



193 



FY 1994 NEI Annual Report 



cruited for these studies through the NIH eye 
dinic. 

(4) Families will be studied, and samples 
wUl be collected for a collaborative study of 
open angle glaucoma in Barbados. This will 
be carried out in collaboration with Dr. 
Christina Leske, from the State Uruversity of 
New York at Stony Brook, and wiU build on 
ongoing epidemiological studies. 

NEI Research Program 

Lens and Cataract — Molecular Genetics 
Publications 

Hejtmandk JF: Neurology of the visual sys- 
tem, in Conn PM (ed): Neurology. Philadel- 
phia, J.B. Lippincott Co., in press. 

Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky 
J: Inherited disorders of the eye lens, in 
Scriver CR, Beaudet AL, Sly WS, Valle D 
(eds): The Metabolic Basis of Inherited Disease. 
New York, McGraw HiU, in press. 

Hejtmancik JF, Ostrer H: DNA diagnosis of 
endocrinological diseases, in Becker KL (ed): 
Principles and Practice of Endocrinology and 
Metabolism. Philadelphia, Lippincott Co., in 
press. 

Hejtmancik JF, Piatigorsky J: Molecular and 
cell biology of the transparent and cataractous 
eye lens, in Bittar EE (ed): Advances in Molecu- 
lar and Cell Biology. Greenwich, JAI Press Inc., 
1994 



Hope JN, Chen H-C, Hejtmancik JF: Aggrega- 
tion of pA3-crystallin is independent of the 
specific sequence of the domain connecting 
peptide. / Biol Chem, in press. 

Hope JN, Chen H-C, Hejhnancik JF: 
pA3/Al-crystallin association: Role of the 
amino terminal arm. Protein Eng 7:445-451, 
1994. 

Nickerson JM, Hejtmancik JF: Molecular 
biology and genetics of the retina, in Tasman 
W, Jaeger E (eds): Duane's Foundations of 
Clinical Ophthalmology. Philadelphia, J.B. 
Lippincott Co., 1993, pp 1-49. 

Scott MH, Hejtmancik JF, Wozencraft LA, 
Reuter LM, Parks MM, Kaiser-Kupfer MI: 
Autosomal dominant congenital cataract: 
Interocular phenotypic heterogeneity. Ophthal- 
mology 101:866-871, 1994. 

Smith RJ, Berlin CI, Hejtmancik JF, Keats BJ, 
Kimberling WJ, Lewis RA, MoUer CG, Pelias 
MZ, Tranebjaerg L: Clinical diagnosis of the 
Usher syndromes. Usher Syndrome Consor- 
tium. Am J Med Genet 50:32-38, 1994. 

Towbin JA, Li H, Taggart RT, Lehmarm MH, 
Schwartz PJ, Satler CA, Ayyagari R, Robinson 
JL, Moss A, Hejtmancik JF: Evidence of genet- 
ic heterogeneity in Romano-Ward long-QT 
Syndrome (LQTS): Analysis of 23 famihes. 
Circulation, in press. 



Hejtmancik JF, Piatigorsky J: Molecular biolo- 
gy of the eye lens, in Alpert DM, Jakobiec FA, 
DowUng JE, Ra viola E (eds): Principles and 
Practice of Ophthalmology: Basic Sciences. 
Philadelphia, W.B. Saunders Co., 1994, pp 168- 
181. 



194 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00149-21 LMOD 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Ultrastructure and Func tio n of the Cells and T is sue s of the Eye^ 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: W. Gerald Robison, Jr. Ph.D. 

Others: Nora Laver M.D. 

Jorge Jacot Ph.D. 

Anne Groome B.S. 

Joe Hackett B.S. 

Evita Bynum B.S. 

Joel Glover B.S. 



Head, Section on Pathophysiology LMOD, NEI 

Special Volunteer LMOD, NEI 

IRTA Fellow LMOD, NEI 

Histology Technician LMOD, NEI 

Biologist LMOD, NEI 

Microbiologist LMOD, NEI 

Biologist LMOD, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 

SECTION 

Section on Pathophysiology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



6.0 



PROFESSIONAL: 



2.0 



4.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Diabetic retinopathy is the major cause of blindness in adults (20 to 74 years old) in the industrialized 
countries. Whereas systemic diabetes mellitus results from lowered availabihty and/or cellular recognition of 
insulin, the complications of diabetes, such as diabetic retinopathy, are caused by the chronic hyperglycemia 
itself. Exaggerating the hyperglycemic effect by feeding rats a galactose diet, we produced the first rat model 
for diabetic retinopathy. Intervention studies designed to simulate clinical trials were used to test the 
possibility of delaying, halting, or reversing retinopathy soon after the earliest capillary lesions could be 
documented. Weanling male Sprague-Dawley rats were divided into 10 groups, 4 of which received either 
normal lab chow or a 50 percent galactose diet with or without one of two aldose reductase inhibitors (ARIs: 
AL-3152 or WAY-121,509), and other groups that received 50 percent galactose for four, six, or eight months 
and then intervention either by addition of inhibitor or removal of galactose. From rats killed at four, six, 
eight, 16, 18, and 24 months, one retina was prepared for obtaining electron micrographs of capillary 
transections; the other was used for whole mounts of isolated retinal vessels. Images of whole and transected 
capillaries were captured and analyzed using computer hardware and programs specially designed for 1024-x 
1024-x 8-bit resolution. Based on several quantitative assessments, including basement membrane thickness, 
PAS stain intensity, acellularity, dilation, tortuosity, length, and microaneurysms, the retinopathy was graded 
on a scale of to 10. At 6 months, when intervention was begun, the untreated galactose-fed rats exhibited 
a 30 percent, statistically significant (p<0.01) increase in capillary basement membrane thickness and grade 
1 retinopathy overall. By 18 months, the same group had grade 7 retinopathy, whereas the rats receiving 
intervention with an ARI-enriched or galactose-free diet exhibited only grade 2 retinopathy, and the rats fed 
control diet or galactose plus AL-3152 throughout the 18 months showed no retinopathy. At 24 months, the 
untreated rats exhibited grade 10 retinopathy, and both intervention groups had a grade 8.5 retinopathy. In 
conclusion, intervention at 6 months delays but does not halt or reverse the progression of galactose-induced 
retinopathy. We plan to attempt, by dietary manipulation, to produce rat models that develop the diabetic-like 
retinal angiopathies sooner. Also, using cell and organ culture, we will investigate the possible mechanisms. 

195 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The objective of this project is to use special 
diets in vivo and controlled media in cell 
cultures of ocular tissues to mimic the diabetic 
state to determine if diabetic-like tissue chang- 
es can be prevented by inhibitors of aldose 
reductase (AR). 

Methods 

Weanling male Sprague-Dawley rats were 
divided into groups, some of which received 
either normal laboratory chow or a 50 percent 
galactose diet with or without an AR inhibitor 
(AL-3152 or WAY-121,509 at 11 mg/kg/day), 
and other groups that received 50 percent 
galactose for four, six, or eight months and 
then intervention either by addition of inhibi- 
tor or removal of galactose. Rats were killed 
at four, six, eight, 16, 18, and 24 months. A 
new enzyme digestion procedure (elastase 
method), which was developed in this labora- 
tory, was used on the retina of one of the eyes 
of each rat to remove aU the retinal tissues 
except the vessels. 

This provided a whole mount of the 
retinal vasculature and thus permitted the 
recognition of degenerated pericytes ("ghosts") 
and all the more advanced angiopathies by 
Ught microscopy. The retina of the other eye 
was sectioned and examined by electron 
microscopy. Images of whole and transected 
capillaries were captured and analyzed using 
computer hardware and programs specially 
designed for 1024 x 1024 x 8 bit resolution. 
Based on several quantitative assessments, 
including basement membrane thickness, 
periodic acid Schiff stain intensity, acellularity, 
dilation, tortuosity, length, and micro- 
aneurysms, the retinopathy was graded on a 
scale of one to 10. Tissue cultures of human, 
bovine, and canine retinal capUlary pericytes 
and lens epithelial cells were used to investi- 
gate the mechanism of underlying diabetic 
angiopathies. 



Major Findings 

Vascular whole mounts prepared by our new 
enzyme digestion procedure exhibited multi- 
ple retinal angiopathies identical to those 
typical of human background diabetic retinop- 
athy in the capillaries of rats fed galactose for 
24 months. These did not occur in the retinas 
of rats fed a galactose diet with an AR inhibi- 
tor. AR was shown to be present in cultured 
retinal pericytes: (1) by immunohistochemistry 
using antibody against human placental AR, 
(2) by its activity using measurements of 
xylitol production in cells grown in a medium 
supplemented with xylose, and (3) by the 
detection of messenger ribonucleic acid for 
AR. There was a compromised proliferation 
rate in pericytes compared with endotheUal 
cells when incubated in high (30 mM) sugar 
concentrations, suggesting toxicity of polyol at 
the cellular level. AR appears to be involved 
in all the retinal complications of diabetes, 
from pericyte degeneration to micro- 
aneurysms. 

Significance to Biomedical Research and the 
Program of the Institute 

Diabetic retinopathy is mainly a disease of 
retinal capillaries. Recently, potentially benefi- 
cial treatments and animal models have be- 
come available. However, demonstration of 
the earliest vessel lesions has relied on the 30- 
year-old trypsin digestion method for the 
isolation of retinal vessels. Until now, basic 
experimental studies and drug testing on 
diabetic retinopathy have been limited by the 
lack of reliable and convenient animal models. 
Now, besides the alloxan diabetic dog ai\d the 
galactosemic dog, there is a galactosemic rat 
model. All this has been possible because AR 
is involved in diabetic retinopathy. AR, w^hich 
has been implicated in sugar cataracts, certain 
corneal healing defects, and peripheral neu- 
ropathy of diabetic and galactosemic animals, 
now appears to be involved in all lesions of 
background diabetic retinopathy. Although 



196 



Laboratory of Mechanisms of Ocular Diseases 



the normal physiological role of this enzyme 
in most tissues remains unknown, under the 
conditions of high plasma sugar concentra- 
tions encountered in diabetes and galactose- 
mia, AR converts these sugars to their respec- 
tive sugar alcohols (polyols). These polyols 
are not readily metabolized nor do they pene- 
trate ceU membranes easily. Thus, once 
formed at significant rates, they may accumu- 
late to very high levels in cells, leading to 
hypertonicity, alteration of ion permeability, 
and eventual cell death with consequent tissue 
changes such as cataract formation. Treatment 
of diabetic or galactosemic rats with potent 
AR inhibitors such as sorbinil or tolrestat 
decreases the accumulation of polyols, which 
in turn appears to prevent the formation of 
cataracts in lenses, defective healing in 
scraped corneas, thickening of basement 
membranes in retinal capUlaries, and de- 
creased conduction velocity in nerves. 

We have shown for the first time that the 
rat can be a good model for human diabetic 
retinopathy and that demonstration of early 
lesions can be improved by using a novel 
vessel preparation method. Pericjiie loss, 
endothelial cell proliferation, microaneurysms, 
shunts, occlusions, dilations, and all the other 
microangiopathies that we found in the galac- 
tose-fed rat are identical to the histopatholo- 
gies that characterize human background 
diabetic retinopathy. Until now, the only 
other experimental animal model has been the 
diabetic or galactosemic dog. We have shown 
for the first time that diabetic-Uke retinopathy 
in galactosemic rats can be prevented with an 
AR inhibitor. 

Proposed Course 

The following studies are proposed for fiscal 
year 1995. We will manipulate the rat diets to 
shorten the time when diabetic-Uke retinal 
angiopathies appear, thus improving the rat as 
a model for diabetic retinopathy. As needed, 
we wdll extend the intervention studies to 
determine how late one can interrupt the 
disease process and still obtain beneficial 
results by treatment wdth various AR inhibi- 
tors. Also, we wUl examine the early forma- 



tion of intracellular vacuoles, cell transport 
systems, the mechanism of basement mem- 
brane synthesis, and the relations of these 
changes to AR in isolated retinal cells grown 
under diabetic conditions. 

NEI Research Program 

Retinal Diseases — Diabetic Retinopathy, Sickle 
CeU Retinopathy, and Other Vascular Abnor- 
maUties 

Publications 

HaUfrisch J, Xue Q, MichaeUs OEIV, Robison 
WG Jr: Carbohydrate copper interactions in 
the development of cataracts [abstract]. 
FASEB meeting, Anaheim, California, April 
24-28, 1994, vol. 8, issue 5, p A945. 

Jacot JL, O'NeUl T, ScandUng DM, West SD, 
McKenzie JE: Role of nitric oxide in modulat- 
ing retinal, choroidal, and anterior uveal blood 
flow in piglets [abstract]. Invest Ophthalmol 
Vis Sci 35(4):1288, 1994. 

Laver NM: Diabetic retinopathy: Laboratory 
investigations. Washington Society of Pathol- 
ogists Residents' Night. Washington, DC 
January 20, 1994. 

Laver NM, Robison WG Jr, Calvin HI, Fu S- 
CJ: Early epitheUal lesions in cataracts of 
GSH-depleted mouse pups. Exp Eye Res 
57(4):493-498, 1993. 

Laver NM, Robison WG Jr, Hansen BC: 
Spontaneously diabetic monkeys as a model 
for cUabetic retinopathy [abstract]. Invest 
Ophthalmol Vis Sci 35(4):1733, 1994. 

Lazarous DF, Scheinowitz M, Shou M, Hodge 
E, Rajanayagam S, Hunsberger S, Robison WG 
Jr, Stiber JA, Correa R, Epstein SE, Unger EF: 
Effects of chronic administration of basic 
fibroblast growth factor on coUateral develop- 
ment in the cainine heart. Circulation, in press. 

Robison WG Jr: AR inhibition and retinopa- 
thy. Diabetes 43(2):337-338, 1994. 



197 



FY 1994 NEI Annual Report 



Robison WG Jr, Jacot JL, Laver NM: Diabetic Robison WG Jr, Laver NM, Lou MF, Kinoshita 
retinopathy: Pathogenesis and prevention JH: The role of AR in diabetic retinopathy: 
with various inhibitors of AR. Medico Intera- Prevention and intervention studies, in 
mericano, in press. Osborne NN, Chader GJ (eds): Progress in 

Retinal and Eye Research. Oxford, Pergamon, in 
Robison WG Jr, Laver NM: Sorbinil preven- press, 
tion of cataracts and retinopathy in the galac- 

tose-fed rat model of diabetic compUcations Xue Q, Hallfrisch J, Michaelis OE IV, Robison 
[abstract]. Invest Ophthalmol Vis Sci 35(4):1586, WG Jr: Cataract development and glycation 
1994. in rats fed galactose and fructose with margin- 

al copper [abstract]. FASEB meeting, Ana- 
heim, California, April 24-28, 1994. FASEB J 
8(5):A945, March 18, 1994. 



198 



Laboratory of Molecular and 
Developmental Biology 



Report of the Chief 
Laboratory of Molecular and Developmental Biology 

Joram Piatigorsky, Ph.D. 



In its 13th year, the Laboratory of Mo- 
lecular and Developmental Biology 
(LMDB) has continued to explore the 
molecular basis and consequences of gene 
expression in the eye and especially the lens. 
As always, our work strives to understand the 
eye from a molecular and cellular perspective 
while using this extraordinary organ as a 
model for general processes of evolution and 
development. We have also continued to 
exploit our findings that the lens crystalUns 
are expressed outside of the eye, where they 
perform nonrefractive functions, to link gene 
expression in the eye with that in other parts 
of the body and with noneye and systennic 
diseases. The LMDB now comprises five 
sections, and their major accompUshments this 
year are detailed below. In addition to per- 
forming basic research, which is the primary 
function of each of the groups, the Transgenic 
Animal and Genome Manipulation Section, 
estabUshed last year, also produces transgenic 
mice as a service for the rest of the National 
Eye Institute (NEI). The development of this 
service has been a great success story, as can 
be gleaned from its report below. 



Section on Molecuur Genetics 

This section, headed by Dr. Joram Piatigorsky, 
has continued to investigate the molecular 
basis of crystaUin gene expression in the lens 
and other tissues. An important new develop- 
ment this year was the discovery that a-crys- 
tallins can be autophosphyrylated in vitro. 
This Unks these ubiquitous crystaUins with the 
enzyme-crystaUins and raises the possibUity 



that they are involved in signal transduction 
pathways. Thus, the a-crystallins, which are 
molecular chaperons, can now be considered 
in a metaboUc as weU as a structural role. 
This provides new avenues of approach for 
investigating the functions of a-crystaUin in 
the lens as well in nonlens and diseased 
tissues, for which there is a large and growing 
literature. A great deal of progress has been 
made on identifying the cis- and trans-regala- 
tory controls for crystallin gene expression. 
Shared and tissue-specific ds-elements use 
have been identified for numerous crystallin 
genes. "Regulatory tissues prints," defined as 
the patterns of czs-elements used for expres- 
sion of genes in different tissues, have been 
determined for the mouse aB-crystaUin gene 
in lens, skeletal muscle, heart, and lung by 
performing experiments in cultured cells. 
Apart from its intrinsic interest, this informa- 
tion may be useful for designing promoters 
for gene therapy in the future. A particularly 
exciting development has been the finding 
that Pax-6, a paired-box homeodomain tran- 
scription factor, can act as a lens-specific 
activator for the chicken and mouse aA- and 
chicken 61-crystallin genes. Because numer- 
ous eye mutations in species ranging from 
Drosophila, which have compound eyes, to 
humans, who have complex eyes, are due to 
lesions in Pax-6 or its homologue, it seems 
possible that the diverse crystaUins may be 
linked by one or more common transcription 
factors. Pax-6 will clearly be investigated 
thoroughly next year. Many more findings on 
the molecular events of crystallin gene regula- 
tion have been made that wiU also be fol- 
lowed up, and these can be found in the 
annual reports form this section. 



201 



FY 1994 NEl Annual Report 



Finally, the presence of Dr. Joseph 
Horwitz, who has been on sabbatical leave 
from Jules Stein Eye Institute, UCLA School of 
Medicine, since January 1994, has led to an 
increased attention to biochemistry. One 
important development from this has been the 
finding of pB2-crystaUin in nonlens tissues 
(retina, testis, brain). This was complemented 
by the finding that pB2-crystaUin, like a-crys- 
taUin, can autophosphorylate in vitro. These 
data suggest strongly that the p-crystalUns, 
and possibly their Y-crystaUin relatives, have 
metaboUc nonlens functions in addition to 
their refractive role in the lens. The second 
important development in protein biochemis- 
try has been the discovery that the ratio of the 
two duck 8 crystaUin/argininosuccinate lyase 
polypeptides in the tetrameric native protein 
affects enzyme activity. Kinetic studies indi- 
cated a cooperative interaction between the 81 
and 62 polj^eptides. This provides a regula- 
tory role for the inactive 81 polypeptide and 
explains why it continues to be S3mthesized 
outside of the lens. 



Transgenic Animal and Genome 
Manipulation Section 

This section, headed by Dr. Eric Wawrousek, 
has continued to provide the NEl investigators 
with transgenic mice in support of their re- 
search efforts. This year, 155 new transgenic 
founder mice were generated from 34 deoxjrri- 
bonucleic add (DNA) constructs. More than 
400 matings of transgenic mice were set up, 
and more than 4,000 mice were weaned, 
tagged, tail biopsied, and their DNA isolated 
and analyzed. This year also marked the 
successful launching of the group's embryo 
cryopreservation and banking program, pro- 
viding the NEl with a reliable mechanism for 
long-term storage of valuable mouse Unes. So 
far, 1,326 embryos from five transgenic Unes 
have been frozen. 

This section has also been conducting 
independent research in two areas, generating 
transgenic mouse models of ocular disease 
and probing the function of lens crystallins by 



deleting these proteins in "gene knockout" 
mice. In the first area, the section has created 
a transgenic mouse model of progressive 
ocular inflammation by expressing a modified 
form of human interleukin-ip in the eye 
under transcriptional control of the mouse 
aA-crystaUin promoter. The animals are 
healthy, reproductively capable, and exhibit a 
consistent and reproducible pattern of ocular 
disease. In neonates, the eye is relatively 
normal, but inflammation with accompanying 
neovascularization of aU eye tissues becomes 
evident and progresses to phthisis bulbi in 
adult animals. This will be a useful model for 
investigating many of the processes involved 
in chronic ocular inflammatory disease such as 
cytokine levels, receptor levels, arachidonic 
acid metaboUte levels, cellular adhesion mole- 
cule expression, and systemic effects of the 
localized inflammation. 

Sigiuficant progress has been made toward 
functionally deleting the a-crystaUin gene 
family. Knockout vectors have been generated 
for mouse aA- and aB-crystallin and mouse 
aACRYPB-1, and approximately 20 embryonic 
stem ceU clones have been isolated in which 
one allele of the aA-crystaUin gene has been 
disrupted. Toward generating knockout mice, 
the group has mastered the skills for inserting 
ES cells into mouse embryos to generate 
chimeric mice. Many chimeric mice have been 
produced from unmodified ES cells, and it is 
expected that chimeric mice containing the 
aA-crystaUin knockout ES cells will be pro- 
duced very soon. The knockout mice will 
provide a unique resource for stud5mig the 
function of the a-crystaUins in lens and eye 
development and shed some light on their 
function in nonlenticular tissues. Develop- 
ment of the technology may one day also be 
a valuable resource for other researchers in the 
NEL 



Section on Regulation of Gene 
Expression 

This section, headed by Dr. Ana Chepelinsky, 
has continued to study the expression of genes 



202 



Laboratoiy of Molecular and Developmental Biology 



encoding lens fiber membrane channels, which 
are of utmost importance for maintaining lens 
transparency. They have mapped several cis 
regulatory elements in the 5' flanking se- 
quence of the human major intrinsic protein 
(MIP) gene. Negative regulatory elements 
were found within the sequence -2840/ -254, 
and positive regulatory elements were re- 
vealed between the sequence -70/ -40 of the 
MIP gene. 

A successful collaboration between the 
LMDB and the Laboratory of Immunology at 
the NEl allowed the development of trans- 
genic mice and rats with constitutive expres- 
sion of interferon gamma (IFN-y) and major 
histocompatibility complex (MHC) class 11 in 
their eyes. These animals provide a compre- 
hensive transgenic model system for elucidat- 
ing the Unkage between aberrant MHC class II 
expression and predisposition to autoimmuni- 
ty, the role of IFN-y in the treatment of in- 
flammatory eye diseases, and cytokine signal- 
ing during embryonic eye development. 



Section on Molecular Structure 
AND Function 

This section headed by Dr. Graeme Wistow, 
has continued to investigate the variety of 
proteins that can serve as functional crystal- 
Uns. Work on ^-crystaHin has revealed a 
natural example of a consensus paired-domain 
binding site for Pax-6. This site is essential for 
lens-specific promoter function. Other projects 
have led to discoveries with significance 
beyond the lens. /i-CrystaUin, originally 
discovered in marsupial lens, is expressed 
abundantiy in retinal photoreceptors of hu- 
mans and rodents, probably as an enzyme of 
glutamate or ornithine metabolism. Migration 
inhibitory factor (MIF), which was isolated as 
a developmental marker in chick lens, has 
been shown to have a general role in cell 
proliferation. It interacts with the retinoblas- 
toma protein and is essential for progression 
through the cell cycle. Antisense suppression 
of MIF halts ceU growth even in transformed 
mouse cells. 



Section on Cellular 
Differentiation 

This section, headed by Dr. Peggy Zelenka, 
has made progress in several areas in the past 
year. Several steps have been taken to follow- 
up the surprising discovery that postmitotic, 
differentiating lens fiber ceUs contain cycUn B, 
an activator of the cycUn-dependent kinase, 
p24cdc2^ that is normally associated with mito- 
sis, hnmunocytochemistry has shown that 
cyclin B is concentrated in the fiber cell nuclei 
as early as developmental day E6, and bio- 
chemical analysis indicates that cycHn B and 
p2^cdc2 complex may play a role in lens fiber 
cell denucleation, transgenic mice that carry 
the weel gene under control of the y-crystallin 
promoter. The product of the weel gene is a 
kinase that inactivates p34'"''^. Other studies 
carried out during this year have probed the 
similarities and differences between lens fiber 
cell differentiafion and apoptosis. These have 
shown that cycUn B may be induced during 
apoptosis in PC12 ceUs as well as during lens 
fiber cell difierenfiation. Moreover, in apopto- 
tic cells, cyclin B is complexed with a novel 
partner that is immunologically related to 
PCTAIRE-1, a member of the cycUn-dependent 
kinase family whose function is unknown. 

Another similarity between lens fiber 
differentiation and apoptosis that was discov- 
ered is that the proto-oncogene, c-jun is acti- 
vated during both processes. However, in 
differentiating cells, c-jun is downregulated 
after a few hours, while in apoptotic cells, it 
remains elevated until the ceUs die. Using a 
retrovirus vector to introduce c-jun into cul- 
tured cells, it was shown that elevated levels 
of c-jun promote cell death in the absence of 
growth factors. These studies open new 
opportunities for investigating the relation- 
ships between cell division and terminal lens 
fiber ceU differentiation. 



Z03 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00238-09 LMDB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

P roto-onco g ene Expression During Lens Differentiation and D evelopment 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: Peggy S. Zelenka Ph.D. Head, Section on Cellular LMDB, NEI 



Others: 



Chun Yun Gao 
Emmanuel Vacchiano 
Anuradha Rampalli 
Jaspreet Arora 
Vijay Chauthaiwale 
Graeme Wistow 





Differentiation 


M.D., Ph.D. 


IRTA Fellow 


Ph.D. 


IRTA Fellow 


Ph.D. 


IRTA Fellow 


Ph.D. 


Visiting Fellow 


Ph.D. 


Visiting Fellow 


Ph.D. 


Head, Section on Molecular 




Structure and Function 



LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 



COOPERATING UNITS (if any) 



Department of Surgery and Department of Anatomy and Cell Biology, New Jersey Medical and Dental College 
(Thomas Lysz, Ph.D.) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Cellular Differentiation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



5.6 



PROFESSIONAL 



5.6 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project investigates the expression of proto-oncogenes and other cell cycle regulatory genes in the 
embryonic chicken lens to determine their relationship to cell growth, quiescence, and differentiation. The 
normal developmental profiles of six nuclear proto-oncogene messenger ribonucleic acid (mRNAs) (c-myc, 
N-myc, c-fos, c-jun, Rb, and p53) and the cell cycle regulatory protein, cyclin B, have been completed. The 
unexpected finding that cyclin B is present in postmitotic lens fiber cells and that it is complexed with p34"''^^ 
suggests that lens fiber cell differentiation may involve aberrant progression through the cell cycle. To test 
this hypothesis, a line of transgenic mice has been produced that overexpresses Weel (a protein kinase that 
inhibits cyclin B/p34"''^^) under control of the y-crystallin promoter to target expression to postmitotic lens 
fiber cells. Preliminary analysis of lenses from this transgenic line indicates that lens fiber denucleation may 
be abnormal. Similarities between apoptosis and lens fiber differentiation prompted us to compare proto- 
oncogene expression in differentiating and apoptotic cells. The earliest difference we have detected between 
these two processes is deregulation of c-fos, c-jun, and c-myc expression during apoptosis. We have 
demonstrated that expression of these proto-oncogenes is strictly regulated by 12(S)HETE synthesis and are 
exploring the mechanism of this effect through analysis of the c-fos promoter. Candidate genes that may be 
regulated by these proto-oncogenes are being explored through transfection of cultured cells and 
deoxyribonucleic acid (DNA) binding studies. We have demonstrated that cyclin B is expressed during 
apoptosis of postmitotic PC12 cells, where it complexes with p34"'''^ and with a novel, 40K member of the 
PCTAIRE family. The identity and function of this 40K protein are being investigated. 



204 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biology 



Project Description 

Additional Personnei 



VuBui 



Graeme Wistow PhX). 



IRTA Summer Fellow, 
LMDB, NEl 
Head, Section on 
Molecular Structure 
LMDB, NEI and 
Function 



Objectives 

This project seeks to determine whether the 
expression of specific proto-oncogenes is 
altered during lens cell differentiation, and, if 
so, to determine the mechanism of gene regu- 
lation and the function of the corresponding 
proto-oncogene products in the developing 
lens. The objective is a greater understanding 
of the mechanisms underlying lens ceU growth 
and differentiation. 

Methods 

Techniques of molecular biology are used in 
conjunction v^th traditional techniques of cell 
biology. Conventional methods are used for 
analysis of proteins and nucleic acids, includ- 
ing polyacrylamide gel electrophoresis, ribo- 
nucleic acid (RNA) and deoxyribonucleic acid 
(DNA) isolation, polymerase chain reaction 
(PCR), nucleic acid hybridization, in vitro 
transaction, in situ hybridization, immunocy- 
tochemistry, and immunoblotting. DNA /pro- 
tein interactions are studied using DNase I 
footprintimg, electiophoretic mobility shift 
assays, and ultraviolet crossUnking. 

Studies use lens epithelia and lens fibers 
of embryonic chickens, explants of embryonic 
chicken lens epithelia, primary cultures of 
embryonic chicken lens epithelial cells, and 
other avian and mammaUan cell Unes. In 
addition, tiansgenic mice are produced to test 
the function of proto-oncogenes and cell cycle 
regulatory proteins in the lens in vivo. 



Major Findings 

Terminal differentiation of lens fiber cells is 
marked by chromatin condensation, abrupt 
dissolution of the nuclear lamina, vesiculariza- 
tion of the nuclear membrane, and complete 
degradation of the nucleus and other organ- 
elles. Because these events resemble the 
chromosomal condensation and nuclear enve- 
lope breakdown associated with mitosis, we 
have investigated whether a similar biochemi- 
cal mechanism may be involved by testing for 
the expression and activity of cycUn B/p34'' ^ 
complexes in differentiating lens fiber cells. A 
coupled reverse transcription /PCR (RT/PCR) 
using RNA from embryonic chicken lens fibers 
amplified a product of the expected size, 
which was identified as cycHn B2 by sequenc- 
ing. In situ hybridization localized cycUn B 
messenger RNA (mRNA) to the superficial 
nucleated fiber cells, and immunocytochemis- 
try with antichicken cycHn B2 antiserum 
showed positive staining in lens fiber cell 
nuclei. Immunoblotting of lens fiber proteins 
purified by affiiuty chromatography on pl3- 
agarose beads identified both p34'"''^ and 
cyclin B, indicating that these proteins are 
present as a complex. 

Moreover, fiber cell proteins purified on 
pl3-agarose beads showed histone HI kinase 
activity, which was enhanced by phosphatase 
digestion. These results demonstrate that 
active cyclinB/p34'"''^ complexes are present in 
differentiating lens fiber cells before denuclea- 
tion and support the possibility that phos- 
phorylation of specific nuclear substrates by 
p34'"^'^ may be responsible for the denucleation 
of lens fiber cells during terminal differentia- 
tion. 

Although denucleation of lens fiber cell 
differentiation resembles mitosis, other fea- 
tures such as degradation of DNA to oUgonu- 
cleosomes resemble apoptosis. This similarity 
and our discovery of cyclin B in differentiating 
lens fibers led us to explore the involvement 
of cyclin B in apoptosis. These studies were 
performed using neuronaUy differentiated 
PC12 cells forced to undergo apoptosis by 
withdrawal of nerve growth factor. Cyclin B 



205 



FY 1994 NEI Annual Report 



mRNA increased approximately 10-fold four 
days after NGF withdrawal, as indicated by 
competitive RT/PCR. Cyclin B protein also 
increased during this period, as indicated by 
immunoblotting. Immunoprecipitation with 
anticycUn B antibody demonstrated that cyclin 
B was associated with HIK activity, which 
reached a maximum five days after NGF 
withdrawal. When proteins immunoprecipi- 
tated with anticyclin B antibody were immu- 
noblotted with anti-PCTAIRE antibody, pro- 
teins with apparent molecular weights of 
34kDa and 40kDa were detected. The 34kDa 
protein was identified as pSA"'^'^ on the basis 
of immunoreactivity with antibody against the 
C-terminal portion of p34^'''=^. The 40kDa 
protein reacted strongly with an antibody 
against the C-terminal region of PCTAIRE-1 
and failed to bind to pl3-agarose beads, 
suggesting that it is a member of the 
PCTAIRE family. Kinase assays of anticyclin 
B immunoprecipitates from apoptotic cell 
lysates that were first rmmunodepleted of 
p34'"^^ indicated that significant HIK activity 
was associated with the cyclin B/40kDa pro- 
tein complex. These findings suggest that 
certain members of the PCTAIRE family may 
function as cyclin-dependent kinases in apop- 
totic cells. We are currentiy investigating 
expression of the 40kDa protein in differentia- 
ting lens fibers and in apoptotic retinal cells. 

The relationship between lens differentia- 
tion and apoptosis has been further probed by 
Dr. Anu Rampalli, who has compared expres- 
sion of several proto-oncogenes and cell cycle 
markers in embryonic chicken lens epithelial 
explants during differentiation or apoptosis in 
vitro. Interestingly, proto-oncogenes c-fos, c- 
jun, c-myc, and -pSi are sequentially upregu- 
lated during both processes. However, in 
differentiating cells, these increases in mRNA 
expression are transient, although in apoptotic 
cells they are not. Loss of regulation of c-fos 
and c-jun mRNA expression, which occurs 
within five hours in culture, is the earliest 
difference detected between differentiating 
and apoptotic cells. Moreover, Dr. Emanuel 
Vacchiano has demonstrated that overexpres- 
sion of c-jun in chicken lens epithelial cells in 
the absence of growth factors greatly increases 



the rate of cell death due to apoptosis. Thus, 
deregulated expression of c-jun appears to be 
an early step in the pathway leading to apop- 
tosis of lens epithelial cells. 

Two experimental approaches have been 
used to explore the functional role of proto- 
oncogenes such as c-fos, c-jun, and c-myc in the 
lens. Dr. Vijay Chauthaiwale has used a 
candidate gene approach to determine wheth- 
er c-myc is involved in regulation of the a- 
enolase/x-crystaUin gene. The promoter of 
this gene contains a potential c-myc binding 
site that is conserved in both duck and hu- 
man. Dr. Chauthaiwale has demonstrated 
that c-myc binds to this site by immunoblot- 
ting with anti-(c-mi/c) antibody following 
electrophoretic mobility shift assay of lens 
nuclear proteins bound to an oligonucleotide 
containing the potential binding site. Immu- 
noreactive bands were detected by enhanced 
chemHuminescence. Interestingly, a mutated 
oligonucleotide containing substitutions at the 
two central nucleotides of the potential bind- 
ing site showed unimpaired binding of c-myc 
but greatiy diminished binding of a faster 
migrating band. Transfection studies using a 
a-enolase/x-crystaUin promoter construct 
bearing this same mutation had previously 
shown that this mutation increased basal 
transcription of the promoter and abolished 
inducibility by c-myc. Together these results 
suggest that c-myc competes with a negative 
regulatory factor for occupation of the same 
site. Dr. Chauthaiwale has now demonstrated 
that the fast migrating band that is diminished 
by the mutation comigrates with a band 
produced by in vitro translated max proteins, 
suggesting that max /max homodimers may be 
the negative regulatory agent. This possibility 
is being explored by means of cotransfection 
studies with c-myc and c-max plasmids. 

The other experimental approach used to 
study the targets of proto-oncogenes in the 
lens is overexpression of these genes and 
negative dominant mutations using retroviral 
vectors. We have used the avian retroviral 
vector RCAS to overexpress wild-type chicken 
c-jun, or a deletion mutant of chicken c-jun 
(junAT) lacking the DNA binding region, to 



206 



Laboratory of Molecular and Developmental Biology 



investigate the possible role of c-jun in lens 
epithelial cell proliferation and differentiation. 
Both constructs were efficiently expressed in 
primary cultures of embryonic chicken lens 
epithelial cells. Overexpression of c-jun in- 
creased the rate of cell proliferation and great- 
ly delayed the appearance of "lentoid bodies," 
structures that contain differentiated cells 
expressing fiber cell markers. Excess c-jun 
expression also significantiy decreased the 
level of PA3/ArcrystaUin mRNA without affect- 
ing aA-crystallin mRNA. In contrast, the 
mutated pvotein junA7 had no effect on proUf- 
eration or differentiation but markedly in- 
creased the level of aA-crystallin mRNA in 
proliferating cell cultures. These results sug- 
gest that c-jun or ;Mn-related proteins may be 
negative regulators of aA- and pA3/Al- 
crystallin genes in proUferating lens cells. 

Studies of the role of 12-lipoxygenase in 
regulating lens epithelial cell proliferation 
have made significant advances in the past 
year. To determirie whether products of this 
pathway are involved in lens cell proliferation, 
we measured the effect of 12-Upoxygenase 
inhibitors on endogenous 12-hydroxyeicosa- 
tetraenoic acid (HETE) production and epider- 
mal growth factor (EGF)/insuUn-stimulated 
DNA synthesis and proto-oncogene expression 
in cultured neonatal rat lens epithelial cells. 
Incubation of neonatal rat lenses in EGF plus 
insulin, which stimulated endogenous 12- 
HETE production eightfold to 10-fold, also 
produced a transient induction of c-fos and c- 
myc mRNAs after two to three hours, followed 
by a round of DNA synthesis approximately 
20 hours later. The lipoxygenase inhibitor 
cinnamyl 3,4 dihydroxy-a-cyanocinnamate 
(CDC) strongly inhibited both the endogenous 
12-HETE synthesis and growth-factor stimulat- 
ed DNA sjmthesis with half-maximal inhibi- 
tion between 10-20 fiM. CDC (10/xM) also 
inhibited the expression of c-fos and c-myc 
mRNA and to a lesser extent, c-jun mRNA. 
The inhibitory effects of CDC on proto-onco- 
gene expression and DNA synthesis were 
prevented by 0.3 jiM 12-HETE but not by 
equivalent concentrations of either 5- or 15- 
HETE. These findings suggest that endoge- 
nously synthesized 12-HETE may mediate 



EGF/insuUn-stimulated DNA synthesis in 
neonatal rat lens epitheUal cells by regulating 
proto-oncogene expression. 

If 12-HETE also regulates human lens 
epitheUal cell proUferation, inhibition of 12- 
Upoxygenase activity may provide a means of 
preventing unwanted proUferation following 
cataract extraction. We have, therefore, exam- 
ined expression of this enzyme in human lens 
epitheUal ceUs. Presence of 12-Upoxygenase 
mRNA was demonstrated in epitheUal ceUs of 
adult and neonatal human lenses by RT/PCR 
and sequencing of the PCR product. The 
presence of the corresponcUng protein was 
demonstrated in cultured neonatal human lens 
epitheUal ceUs by immunoblotting with an 
antibody raised against human platelet 12- 
Upoxygenase. A medium derived from hu- 
man lens epitheUal cell cultures was shov^m to 
contain 12-HETE by radio immunoassay. 
Experiments are in progress to test whether 
inhibition of 12-Upoxygenase blocks DNA 
synthesis in cultured human lens epitheUal 
ceUs as in the neonatal rat lens. Dr. Jaspreet 
Arora has also demonstrated the presence of 
the 12-Upoxygenase pathway in cultures of 
rabbit lens epitheUal ceUs (N/N10003 ceUs). 
This system wUl be used to investigate the 
mechanism of the 12-HETE efiect on proto- 
oncogene expression in the lens. 

Proposed Course 

The foUovkdng studies are in progress or are 
proposed for fiscal year 1995: 

(1) The hypotiiesis that the cycUnB/p34^''^ 
kinase is responsible for nuclear loss in differ- 
entiating lens ceUs will be tested by analysis of 
several Unes of transgenic mice that express 
the weeV kinase in lens fiber ceUs. This kinase 
inactivates the cycUnB/p34'"''^ kinase and 
would be expected to delay or prevent nuclear 
loss if, indeed, cycUnB/p34'"''^ is required. 

(2) The finding that cycUn B binds to a 
novel 40kDa PCTAIRE-Uke protein in apop- 
totic ceUs wUl be explored further. The identi- 
ty of the protein will be examined by protein 
microsequendng, and an attempt will be made 



207 



FY 1994 NEI Annual Report 



to clone the corresponding complementary 
DNA. Expression of this protein will be 
examined in the lens during differentiation 
and in a variety of ocular cell types during 
apoptosis. 

(3) The effect of c-myc on transcription of 
the T-crystallin/a-enolase gene wiU be exam- 
ined further by cotiansfection studies with 
max and myc plasmids. 

(4) The collaborative effort with Dr. 
Thomas Lysz, from the University of Medicine 
and Dentistry of New Jersey, will be contin- 
ued to determine whether the 12-lipoxygenase 
pathway plays a role in regulating DNA 
synthesis in human lens epithelial cells. 

(5) The possible role of posttranslational 
modifications of Rb and p53 proteins in lens 
cell differentiation will be explored using 
differential extraction techniques and specific 
antibodies. The relationship between migra- 
tion inhibitory factor and modified forms of 
Rb will be examined in differentiating cells. 



NEI Research Program 

Lens and Cataract — Molecular Genetics 
Publications 

Dash A, Chung S, and Zelenka PS: Expres- 
sion of HSP70 mRNA in the embryonic chick- 
en lens: association with differentiation. Exp 
Eye Res 58, 381-387, 1994. 

Gao CY, Bassnett S, Zelenka PS: Cyclin B, 
p34cdc2^ and Hl-kinase activity in terminally 
differentiating lens fiber cells. Develop Biol, in 
press. 

Lysz TW, Arora JK, Lin C, Zelenka PS: 12(S)- 
Hydroxyeicosatetraenoic acid regulated DNA 
synthesis and protooncogene expression 
induced by epidermal growth factor and 
instdin in rat lens epithelium. Cell Growth Dijf, 
in press. 



208 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00126-13 LMDB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Crystallin Genes: Structure, Organization , Express ion , and Evolut ion 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 



PI: 
Others: 



Joram Piatigorsky 
Sharmilla Basu 
James B. Brady 
Sambath Chung 
Ales Cvekl 
Melinda K. Duncan 
Peter Frederikse 
Rashmi Gopal-Srivastava 
John I. Haynes, II 
John G. Ilagan 



Ph.D. 
Ph.D. 
Ph.D. 
B.A. 
Ph.D. 
Ph.D. 
Ph.D. 
Ph.D. 
Ph.D. 
B.A. 



Chief 



IRTA 
Technician 
Visiting Fellow 
IRTA 

Senior Staff Fellow 
Staff Fellow 
IRTA 

Howard Hughes Medical Institute/ 
NIH Fellow 
(Additional peisonnel listed under Program Description.) 



LMDB, NEI 

LMDB. NEI 
LMDB. NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 



COOPERATING UNITS (if any) 

Jules Stein Eye Institute, UCLA School of Medicine (J. Horwitz, Ph.D.); University of Waterloo (Jacob Sivak, Ph.D. and Judith West 
Mays, Ph.D.); University of Nijmegen (Wilfried de Jong, Ph.D.); National Institute of Diabetes and Digestive and Kidney Diseases, NIH 
(Emery H. Bresnick, Ph.D.); National Cancer Institute, NIH (John N. Brady, Ph.D. and Fatah Kashanchi, Ph.D.) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Molecular Genetics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



15.70 



PROFESSIONAL: 



13.15 



2.55 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The structure, expression, and evolution of the crystallin genes of vertebrates and invertebrates are being 
studied. The following advances have been made. Pax-6 activates the chicken and mouse aA-crystallin 
promoter and the chicken 61 -crystallin enhancer. The chicken aA promoter contains a composite element 
that suppresses activity in fibroblasts by binding USF and AP-1 proteins (JunD and Fra2) and activates 
promoter activity in lens by binding USF and CREB\CREM proteins. USF also acts as a negative regulator 
of the chicken aA promoter by binding to another, downstream element. USF also binds to the 6EF1 site 
of the chicken 61 -crystallin enhancer, where it probably contributes to the activation of this gene. 
CREB\CREM cooperates with aA-CRYBPl and Pax-6 to activate the mouse aA promoter in lens cells. 
Binding studies suggest that HSF may also be involved in chicken aA and mouse y¥ promoter activity. 
Expression of the mouse aB-crystallin gene in different tissues utilizes both shared and tissue-specific control 
elements, the exact pattern being called its "regulatory tissue print." Elements for lens-specific expression have 
been narrowed to positions -101/+30 in the chicken PBI gene and -143/+22 in the chicken PBA3/A1 gene 
in transgenic mouse experiments. aA, aB and PB2 were shown to be able to autophosphorylate, raising the 
possibility that they are involved in a signal transduction pathway. Close linkage was found for chicken pBl 
and PA4, and evidence was obtained indicating that mammalian PB2 is expressed in nonlens cells. A CRl 
element was found in the intergenic spacer of duck 6-crystallin; the duck 61 and 62 polypeptides were shown 
to interact cooperatively to modulate argininosuccinate lyase activity of the tetramer. The jellyfish J3-crystallin 
gene has been cloned and shown to contain at least six introns. 



209 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 



Joseph Horwitz 


Ph.D. 


Jules Stein Eye Institute 


Cynthia J. 


Ph.D. 


Staff FeUow, LMDB, 


Jaworski 




NEI 


Marc Kantorow 


PhD. 


Staff Fellow, LMDB, 

NEI 


Xuan Li 


Ph.D. 


Visiting Associate, 
LMDB, NEI 


Joan B. 


PhD. 


Biologist, LMDB, NEI 


McDermott 






Barbara Norman 


M.S. 


Chemist, LMDB, NEI 


Christina M. Sax 


Ph.D. 


Senior Staff Fellow, 
LMDB, NEI 


Stanislav I. 


PhD. 


Visiting Scientist, 


Tomarev 




LMDB, NEI 



Objectives 

The objective of this project is to understand 
the structure, organization, expression, and 
evolution of the gene families encoding the 
lens crystallins in the animal kingdom. Partic- 
ular attention is given to the regulation of 
crystallin gene expression in the developing 
lens and, in the case of multifunctional 
crystallins and enzyme-crystaUins, in nonlens 
tissues. 

Methods 

Conventional methods used for analysis of 
proteins and nucleic acids include polycry- 
lamide and agarose gel electrophoresis, ribo- 
nucleic acid (RNA) and deoxyribonucleic acid 
(DNA) isolation, molecular hybridization 
(Southern and Northern blots), complementary 
DNA (cDNA) and gene cloning, DNA se- 
quencing, recombinant DNA (rDNA) construc- 
tion and mutagenesis, in situ hybridization, 
expression of recombinant DNAs in trans- 
fected cells and transgenic mice, polymerase 
chain reactions (PCRs), primer extension and 
SI protection experiments, in vitro and in vivo 
footprinting, gel mobility shift analysis, chro- 
matographic purification of proteins, and 
Western immunoblotting. 



Major Findings 

a-Crystallins. -aA-CRYBPl is a zinc-finger 
transcription factor that binds to the mouse 
aA-crystallin gene at positions -66/ -57 of its 5' 
flanking sequence. We have previously 
cloned a partial cDNA encoding this protein. 
This year it was shown that the aA-CRYBPl 
protein exists in different sizes in lens and 
fibroblasts, suggesting the possibility that 
posttranscriptional mechanisms regulate its 
binding or functional activity in a tissue- 
specific fashion. Moreover, we have estab- 
lished the full-length sequence for this 9.5 kb 
messenger RNA (mRNA). The deduced 
protein contains two sets of consensus C2H2 
zinc-fingers (one set at the N-terminal region 
and one set at the C-terminal region) as well 
as a nonconsensus zinc-finger motif in the 
center. The 70 kbp gene has also been cloned 
and its 10 exon structure established. aA- 
CRYBPl cDNAs were isolated from adult 
mouse brain and testis as well as from cell 
lines derived from mouse lens and muscle. 
An alternatively spUced aA-CRYBPl cDNA 
was demonstrated in the muscle cell line, and 
a unique, smaller cDNA was found in testis. 
An antisense expression construct derived 
from an aA-CRYBPl cDNA was introduced 
into the aTN4-l transformed mouse lens cell 
line, and this significantiy inhibited expression 
from a transfected heterologous promoter that 
used the aA-CRYBPl binding site. This 
provides the first direct evidence (apart from 
mutagenesis tests) that aA-CRYBPl is a posi- 
tive transcription factor contributing to expres- 
sion of the aA-crystallin gene in the mouse. 

Transgenic mice experiments have estab- 
Ushed that the DE-1 (-111\-106) and aA- 
CRYBPl (-66\-57) regulatory elements of tiie 
mouse aA-CRYBPl are functionally redun- 
dant. Elimination of either one alone does not 
affect the lens-specific activity of the promoter; 
how^ever, elimination of both simultaneously 
eliminates promoter activity. Footprinting 
experiments have shown that these regulatory 
sites are occupied in lens ceUs that are ex- 
pressing the aA-crystallin gene and in fibro- 
blasts that are not expressing the gene. It is 
not yet known whether the proteins binding 



210 



Laboratory of Molecular and Developmental Biology 



to these regions are identical in the two cell 
types or whether cofactors are involved. 
Mutagenesis, cotransfections, and immunoshift 
experiments have provided strong evidence 
that DE-1 binds the cAMP responsive tran- 
scription factor CREB/CREM and is thus a 
functional CRE site. Consequently it has been 
called DE-1 /CRE. These footprinting experi- 
ments have also revealed the binding of 
proteins to two other putative regulatory 
regions called PEl (involving the TATA box 
and immediately downstream thereof; -35 /-1 9) 
and PE2 (+24\+43). Mutagenesis, transfection, 
and protein-binding experiments have shown 
that PEl and PE2 are indeed important regula- 
tory regions for the expression of this gene. A 
particularly unexpected finding is that site- 
spedfic elimination of the TATA box of the 
mouse aA-crystallin promoter fused to the 
chloramphenicol acetyltransferase (CAT) gene 
did not prevent this transgene from being 
expressed specifically in the lens of transgenic 
mice. Finally, negative-acting and protein- 
binding elements have been identified within 
the -1556 /-1 165 sequence of the mouse aA- 
crystallin 5' flanking sequence. 

Protein binding, mutagenesis, cotransfec- 
tion, and immunoshift experiments have 
indicated that Pax-6, a paired-box/ homeo- 
domain protein expressed highly in the lens in 
the early stages of development of the mouse 
and chicken, is an important transcription 
factor affecting the lens-specific expression of 
the mouse and chicken aA-crystaUin gene. 
Pax-6 also appears to activate the chicken 61- 
crystallin enhancer. A number of studies have 
implicated Pax-6 as a universal regulator of 
eye development in both vertebrates and 
invertebrates, including both complex and 
compound eyes. Our studies provide the first 
evidence for specific target genes activated by 
Pax-6. 

The chicken aA-crystallin promoter \ en- 
hancer (-153 \ +77) has been divided into at 
least five functional regions (A-E) by protein 
binding, mutagenesis, immunoshift, and 
transfection experiments. This region has 
been shown to be composed of a complex 
array of positive and negative elements in- 



volving Pax-6, USE, CREB/CREM and API 
proteins. Sites A (-148\-139, binds USE) and B 
(-138\-132, binds CREB\CREM in lens and 
Fra-2 and \ or JunB in fibroblasts) comprise a 
composite element that activates transcription 
in lens and represses transcription in fibro- 
blasts. Sites C (-128\-101) and E (-56/-41) 
bind Pax-6 only in lens, which appears to be 
of central importance for the lens-specific 
expression of this gene. Site D (-102 \ -93) 
binds USF and is a negative element. 

Finally, electrophoretic mobility shift 
experiments using specific antibodies have 
provided evidence that heat shock transcrip- 
tion factors bind to 5' flanking sequences that 
are involved in the regulated expression of the 
chicken aA-crystaUin and mouse YF-crystaUin 
genes in the lens. These regions include 
positions -157/-136 of the chicken aA gene 
and positions -53/ -23 of the yF gene. Indirect 
evidence for the use of heat shock factors for 
crystallin gene expression has also been ob- 
tained in stably tiansformed and transfected 
K562 cells treated with hemin, which is 
known to induce HSF2, a heat shock transcrip- 
tion factor known to be expressed in the lens. 

The aB-crystallin gene contrasts with the 
aA-crystallin gene in that, although lens- 
preferred, it is also expressed to a relatively 
high degree in numerous other tissues and is 
inducible by heat and other physiological 
stiesses. In 1991, we identified a strong mus- 
cle and weak lens enhancer between positions 
-426 and -257 of the 5' flanking region of the 
gene. Last year, we identified by site-spedfic 
mutagenesis experiments four regulatory 
elements (aBE-1, aBE-2, aBE-3m and muscle 
regulatory factor [MRF]) v^thin this enhancer. 
The MRF site is an E-box and was shown by 
tiansfection and transgenic mouse experiments 
to be muscle specific and to be activated by 
MyoD, myogenin, or another member of this 
family of transcription factors. 

This year, we showed by DNase I foot- 
printing experiments that there is another 
regulatory element (aBE-4) located between 
aBE-1 and aBE-2 that is used spedfically in 
the heart. A series of footprinting, mutagene- 



211 



FY 1994 NEI Annual Report 



sis, and transfection experiments have resulted 
in the following conclusions: aBE-1 and aBE- 
2 are used for expression in muscle, heart, 
lung, and lens; aBE-4 is heart-specific and 
binds a protein identical or related to the 
serum response factor; aBE-3 is used by 
muscle, lens, and heart; MRF is used by mus- 
cle and heart and maybe by lung. Recent 
evidence suggests that MRF may bind differ- 
ent proteins in the different tissues. 

Last year, we reported on another se- 
quence called LSR (for lens specific region), 
that is located downstream of the enhancer at 
position -147\-118 of the mouse aB-crystaUin 
gene. This element binds nuclear proteins that 
appear specific to the lens and may account 
for the high expression of the aB-crystallin 
gene in the lens. Site-specific mutagenesis and 
transfection tests have supported the impor- 
tance of this sequence for lens expression. 
Experiments with tiansgenic mice have shown 
that the -164 \ +44 sequence, which contains 
the LSR, fused to the CAT reporter gene is 
sufficient to direct lens-specific expression of 
CAT; by contrast, the -426 \ +44 sequence 
containing the enhancer directs CAT expres- 
sion to the lens, skeletal muscle, heart, and, to 
a much smaller extent, brain and lung. Thus, 
the differential expression of the aB-crystaUin 
gene is regulated by a combination of tissue- 
specific and shared cr's-control elements. 
Moreover, the shared control elements do not 
necessarily bind the same nuclear proteins in 
the different tissues. We have called the 
combination of regiilatory elenients used for 
expression of the aB-crystallin gene in any 
specific tissue its "regulatory tissue print" and, 
apart from its intrinsic interest, has obvious 
importance for gene therapy. 

We have discovered an unexpected auto- 
phosphorylation ability of the aA- and aB- 
crystaUin pol5q3eptides. This raises the possi- 
bility that these crystaUins are involved in a 
signal transduction pathway that tiltimately 
covdd affect expression of crystaUin genes in 
the lens. We have also shown that the deter- 
gent deoxycholate generates dimers and 
tetiamers of the aA-crystallin polypeptides 
and that these small aggregates have approxi- 



mately 10 times more autophosphorylation 
abihty than the larger molecular weight aggre- 
gates present without the detergent. Interest- 
ingly, the aB-crytaUin polypeptides also form 
dimers and tetramers in the presence of de- 
oxycholate, but their autophosphorylation 
abUity is not increased, suggesting a funda- 
mental difference between the structure and 
possibly function of the aA and aB-crystallin 
polypeptides. Recent collaborative studies 
with Dr. WUfried de Jong, from the University 
of Nijmegen, The Netherlands, have shown 
that the autophosphorylation of aA does not 
occur on serine 122, the amino acid that is 
phosphorylated in the cAMP-dependent 
reaction occurring on this polypeptide. This is 
consistent with our recent finding that the 
chicken aA-crystallin polypeptide is also 
autophosphorylated although it has an alanine 
substituted for serine at position 122. 

^-crystaUins. Our previous fransfection 
and footprinting experiments have identified 
numerous confrol elements in the -152 \ +30 
sequence of the chicken pBl -crystaUin gene. 
Transgenic mice have now been produced that 
have established that the -152 \ +30 sequence is 
sufficient for lens-specific expression. Further 
deletions have shown that even the -101 \ +30 
sequence is sufficient for lens-specific expres- 
sion of the fransgene in fransgenic mice. By 
confrast, the -52\+30 sequence is not capable 
of expressing the fransgene. Transgenic mice 
carrying various mutations in the 152 \ +30- 
CAT transgenes are being produced to identi- 
fy specific confrol elements. 

Analysis of the 5' flanking region of the 
cloned chicken pBl-crystaUin genomic frag- 
ment revealed, unexpectedly, the presence of 
the pA4-crystaIlin gene. This gene is arranged 
head to head with the pBl-crystallin gene, 
with approximately 2 kbp of spacer DNA 
separating the two genes. The pA4 gene was 
sequenced and shown to contain, Hke the pBl 
gene, six exons, with the first being noncod- 
ing. A pA4-crystallin cDNA has been isolated 
from the embryonic chicken lens, indicating 
that this gene is expressed. The close head-to- 
head linkage of the pBl and pA4 crystallin 
genes raises interesting possibilities with 



212 



Laboratory of Molecular and Developmental Biologi/ 



respect to the regulatory mechanisms used for 
their expression. 

We have shown previously that transcrip- 
tional activating sequences lie upsteam of the 
chicken pA3/Al-crystaUin gene between 
positions -382 and -143, which contains a 
complex transcriptional enhancer between 
positions -287 and -254. This year we have 
performed DNase 1 footprint analysis reveal- 
ing extensive protein binding in this region. 
FuU enhancer activity requires sequences 
upstream and downstream of -270, although 
sequence -270 \ -254 has some enhancer activity 
by itself. There is an AP-1 consensus binding 
site at position -264 \ -258. Multiple proteins in 
lens nuclear extracts, including members of 
the AP-1 and ATF\CREB families, bind oligo- 
nucleotide -271 \ -251 in gel shift assays. 
Finally, the -143 \ +22 sequence, which lacks 
the enhancer, fused to the CAT gene in trans- 
genic mice is sufficient for lens-specitic expres- 
sion. Thus, the -287/ -254 enhancer is not 
required to direct expression of the PA3/A1- 
crystallin gene in the lens. 

During his sabbatical year in the LMDB, 
Dr. Joseph Horwitz, from the Jules Stein Eye 
Institute, UCLA, has been exploring the possi- 
bility that pB2-crystallin is expressed in non- 
lens tissues. He had obtained stiong prelimi- 
nary evidence that this was the case before 
starting his sabbatical here. This would be of 
great interest because there is no unequivocal 
evidence that any of the p/y-crystallins have 
nonlens functions as do the a-crystallins or 
the enzjmie-crystallins. At present. Western 
immunoblotting of retina, testes, and brain 
from rats support the idea that pB2-crystalltn 
is present outside of the lens. Amino acid 
sequencing of the putative pB2 polypeptide 
from bovine retina provided strong support 
for the expression of this protein in that tissue. 
The only caveat that must be kept in mind is 
the possibility that the retinal pB2-crystallin 
was derived by contamination from the lens. 
PCR data support the existence of |3B2 mRNA 
in testes and brain of the rat, and Northern 
blots are presentiy being performed. Initial 
experiments on purified bovine |3B2-crystaUin 
indicate that this polypeptide is capable of 



autophosphorylation hke the cx-crystaUin 
polypeptides; this autophosphorylation reac- 
tion has not been characterized yet. Auto- 
phosphorylation ability would open the possi- 
bility that pB2-crystallin is involved in some 
type of metaboUc or signal transduction path- 
way, providing an explanation for its nonlens 
expression as well as introducing new consid- 
erations for the role(s) of this polypeptide in 
the lens. 

b-aystallin/argininosuccinate lyase (ASL). 
There are two Unked ASL/6-crystallin genes 
in the chicken, with the 5' gene being special- 
ized for expression in the lens. The 5' gene 
encodes a polypeptide that lacks ASL activity; 
the 3' gene encodes a polypeptide that has 
ASL activity. A similar situation exists in the 
duck, with the exception that the two ASL/ 
6-crystaUin genes are equally expressed in the 
lens in this species. The 5' 61 gene is ex- 
pressed to a limited extent in nonlens tissues 
in both species. This year, cloning experi- 
ments have shown that the 4.6 kbp intergenic 
spacer of the 6-crystallin genes in the duck is 
79 percent identical to the 4 kbp spacer in the 
chicken, except for the existence of a 615 bp 
CRl element, highly reiterated in the duck 
genome. Further sequence analysis revealed 
that inti-on 3 of the duck ASL/ 62 gene is 80 
percent identical to intron 3 of the chicken 61 
and ASL/ 62 genes; the identity rises to 93 
percent in the region of the chicken 61 en- 
hancer core located within the third intron. 
These findings raise the speculation that the 
CRl repetitive element in the duck intergenic 
spacer plays a role in the high expression of 
the ASL/ 62 gene in the lens in this species, 
perhaps by diminishing the effectiveness of a 
silencer element that remains active in the 
chicken. 

Footprinting and transfection experiments 
have shown that the chicken 61-crystallin 
enhancer has at least two functional Pax-6 
binding sites. The finding that Pax-6 activates 
the 81-crystallin enhancer shows for the first 
time that the a- and 6-crystaUin genes may be 
expressed by similar mechanisms. This idea 
is further supported by another recent finding, 
namely that USF binds to the 6EF1 site in the 



Z13 



FY 1994 NEI Annual Report 



81 enhancer. USF participates in the regula- 
tion of the chicken aA-crystallin gene. The 
6EF1 site binds the negative regulator, 6EF1, 
in nonlens tissues to suppress gene activity. 
We envision that USF binding to the 8EF1 site 
activates 81 expression in the lens, and 8EF1 
binding to this site represses 81 expression. 

Native 8-crystallin is a tetrameric protein. 
In 1979, we showed by SDS-polyacrylamide 
gel electrophoresis that the five major isoelec- 
tric forms of duck native 8-crystallin result 
from differences in the relative amounts of the 
two major polypeptide bands (about 49kD and 
50kD). Current protein sequencing experi- 
ments have established that the 49kD poly- 
peptide is encoded by the 61-crystallin gene 
and the 50kD polypeptide is encoded by the 
ASL/62-crystallin gene. Moreover, we have 
demonstrated that the ASL activity of the 
isolated isoelectric forms of duck 8-crystallin 
is directiy related to the relative amount of 82 
pol3^eptide present in the native tetrameric 
protein. Our new data indicate that the two 
8-crystaIlin polypeptides interact cooperatively 
to modulate ASL activity. This provides a 
dominant negative role to the 81-crystaUin 
pol5rpeptide in the regulation of cellular ASL 
activity within tissues. 

Interesting cDNAs. We have cloned a 
partial cDNA for proxl from an embryonic 
chicken lens library. This homeodomain 
protein is knov^m to be expressed at high 
concentrations in the developing mouse lens; 
its Drosophila homologue, prospero, is ex- 
pressed in the lens secreting cone cells of the 
developing compound eye of this invertebrate. 
Thus, proxl /prospero is another candidate 
transcription factor for controlling the high 
expression of eye genes throughout the animal 
kingdom, as Pax-6. A genomic clone for 
proxl has also been cloned from humans that 
is in the process of being characterized. 

A zinc-finger cDNA has been cloned from 
a newborn mouse lens library. The mRNA for 
this gene is also present in numerous other 
tissues. It encodes a 555-amino add protein 
that contains riine C-terminal zinc-fingers and 
an N-terminal domain found in a subset of 



C2H2 zinc-fingers known as the Kruppel- 
associated box (KRAB). The amino acid 
sequence located between the KRAB domain 
and the zinc-finger shows an unexpected 
similarity to human profilagrin, a protein 
expressed in differentiating epidermal cells. 
Sequences that hybridized to this cDNA are 
detectable in 10 other mammalian species. 

Invertebrate crystallins. We are continuing 
to explore the structure and expression of the 
squid S-crystaUin genes, which are descended 
from glutathione S-transferase. A collabora- 
tive study with Dr. Richard N. Armstrong, 
fiom the University of Maryland, has led to 
the preliminary x-ray structure of squid gluta- 
thione S-transferase. We have cloned this 
cDNA from the digestive gland and its respec- 
tive gene of the squid last year. One of the 
interesting aspects of the squid glutathione 
S-transferase is that it is approximately 100 
times more active than its mammalian homol- 
ogues in in vitro experiments. The three- 
dimensional structure of squid glutathione 
S-transferase is relevant to lens in view of the 
family relationship between this metabolic 
enzyme and the S-crystallins that make up 
approximately 90 percent of the squid lens 
protein. 

A collaborative immunolocaBzation study 
with Dr. Jacob Sivak, fiom the University of 
Waterloo, Canada, has demonstrated that 
during development S-crystalHns accumxilate 
first in the posterior lens primordium and 
subsequently in the anterior lens primordium 
and their respective lentigenic cells and con- 
necting lentigeruc processes. Examination of 
adult lens and lentigenic cells of the squid 
suggested that squid lens crystallins are syn- 
thesized in the mature squid. 

Another major interest of this section is 
the crystallins of the cubomedusan jellyfish. 
These primitive metazoan organisms have 
well developed eyes called oceUi, which con- 
tain cellular lenses. We have previously 
reported that the cubomedusan lenses contain 
three crystallins (Jl, J2, and J3) v^th molecular 
sizes near 35kDa, 20kDa, and 19kDa, respec- 
tively. Last year, we cloned the cDNAs and 



214 



Laboratory of Molecular and Developmental Biology 



genes for Jl-crystallins, which make up a 
family of three very closely similar polypep- 
tides, each encoded by a separate intronless 
gene. This year, we have cloned the cDNA 
and gene for JS-crystaUin. In contrast to Jl- 
crystaUin, J3-crystallin appears to contain only 
a single gene that has at least six introns. 
Another new and exciting development is the 
cloning of a genomic fragment, obtained by 
PCR, for cubomedusan Pax-6. 

Significance to Biomedical Research and tfie 
Program of tlie Institute 

The crystaUins comprise a diverse family of 
differentially expressed proteins that are re- 
quired for the optical and functional proper- 
ties of the transparent lens. Understanding 
the structure, function, and evolution of these 
protein families and their genes contributes to 
our knowledge of embryonic development, 
eukaryotic gene expression, cell differentiation, 
molecvdar evolution, cataract, and the visual 
system. That crystaUins are multifunctional 
proteins expressed in lens and nonlens tissues 
adds another dimension of interest and has 
implications for metaboUsm, ceU biology, and 
drug and gene therapy. 

Proposed Course 

The following studies are proposed for fiscal 
year 1995. 

(1) Continue studying czs-acting elements 
and frans-factors used for expression of crys- 
taUin genes in the lens and other tissues. 

(2) Give particular attention to Pax-6 and 
proxl transcription factors, especially with 
respect to their possible roles in crystallin gene 
expression. 

(3) Continue investigations concerning the 
possible role(s) of a-crystallins in metabolic 
reactions such as the regulation of transcrip- 
tion. 

(4) Continue the cloning and characteriza- 
tion of the cubomedusan jellyfish crystallin 
genes and their putative regulatory sequences. 



(5) Continue to investigate the structure 
and possible functions of the cephalopod S- 
crystaUins and the expression of their genes. 

NEI Research Program 

Lens and Cataract — Molecular Genetics 

Publications 

Brady JP, Piatigorsky J: A mouse cDNA en- 
coding a protein with zinc fingers and a 
KRAB domain shows similarity to human 
profUaggrin. Gene, in press. 

Cvekl A, Sax CM, Bresnick EH, Piatigorsky J: 
Complex array of positive and negative ele- 
ments regulates the chicken aA-crystaUin 
gene: involvement of Pax-6,USF, CREB/CREM 
and AP-1 proteins. Mol Cell Biol 14:7363-7376, 
1994. 

Gopal-Srivastava R, Piatigorsky J: The murine 
aB-crystaUin/ small heat shock protein en- 
hancer: identification of aBE-1, aBE-2, aBE-3, 
and MRP control elements. Mol Cell Biol 
13:7144-7152, 1993. 

Gopal-Srivastava R, Piatigorsky J: Identifica- 
tion of a lens-specific regulatory region (LSR) 
of the murine aB-crystaUin gene. Nucleic 
Acids Res 22:1281-1286, 1994. 

Hejtmancik JF, Kaiser MI, Piatigorsky J: Mo- 
lecular biology of inherited disorders of the 
eye lens, in Scriver CR, Beaudet AL, Sly WS, 
VaUe D (eds): The Metabolic Basis of Inherited 
Disease, ed 7. New York, McGraw-Hill Pub- 
lishing Co, in press. 

Hejtmancik JF, Piatigorsky J: Molecular and 
cell biology of the transparent and cataractous 
eye lens, in Bittar EE (ed): Fundamentals of 
Medical Cell Biology. Greenwich, JAI Press Inc, 
in press. 

Kantorow M, Piatigorsky J: a-CrystaUin/ small 
heat shock protein has autokinase activity. 
Proc. Natl Acad Sci USA 91:3112-3116, 1994. 



215 



FY 1994 NEI Annual Report 



Piatigorsky J: The twelfth Frederick H. 
Verhoeff Lecture: gene sharing in the visual 
system. Trans Am Ophthalmol Soc LXXXI: 283- 
298, 1993. 

Piatigorsky J, Kantorow M, Gopal-Srivastava 
R, Tomarev SI: Recruitment of enzymes and 
stress proteins as lens crystallins, in Jansson B, 
Jornvall H, Ryberg U, Terenius L, VaUe B 
(eds): Toward a Molecular Basis of Alcohol Use 
and Abuse. 86th Nobel Symposium. Basel, 
Birkhauser Verlag, 1994. 

Sax CM, Cvekl A, Kantorow M, Sommer B, 
ChepeUnsky AB, Piatigorsky J: Identification 
of negative-acting and protein-binding ele- 
ments in the mouse aA-crystaUin -1556/-1165 
region. Gene 144:163-169, 1994. 

Sax CM, Piatigorsky J: Expression of the a- 
crystallin/ small heat shock protein /molecular 
chaperone genes in the lens and other tissues, 
in Meister A (ed): Advances in Enzymology and 
Related Areas in Molecular Biology. New York, 
John WUey and Sons Inc, 1994, vol 69, pp 155- 
201. 



Tomarev SI, Zinovieva, Piatigorsky J: Primary 
stiucture and lens-specific expression of genes 
for an intermediate filament protein and a P- 
tubulin in cephalopods. Biochim Biophy Acta 
1216:245-254, 1993. 

Tomarev SI, Duncan MK, Roth JH, Cvekl A, 
Piatigorsky J: Convergent evolution of crystal- 
Un gene regulation in squid and chicken: the 
AP-l/ARE connection. ] Mol Evol 39: 134-143, 
1994. 

West JA, Sivak JG, Pasternak J, Piatigorsky J: 
Immunolocalization of S-crystallins in the 
developing squid {Loligo opalescens) lens. Dev 
Dynamics 199:85-92, 1994. 



226 



'^ I PROJECT NUMBER 

DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00259-05 LMDB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Biology of th e Cornea 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Joram Piatigorsky Ph.D. Chief LMDB, NEI 

Others: W. Todd Kays Ph.D. IRTA LMDB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Molecular Genetics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

1.05 



PROFESSIONAL: 

1.05 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects Q (b) Human tissues [x] (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Aldehyde dehydrogenase class 3 (ALDH3) comprises approximately 40 percent of the cellular protein of the 
mammalian corneal epithelial cells, an amount reminiscent of an enzyme-crystaUin in the lens. Consequently, 
we are investigating the molecular basis for the high expression of the ALDH3 gene in the corneal epithelial 
cells. The results will be compared with those obtained for crystallin genes in the lens and will provide a 
foundation for eventual gene therapy in the cornea. The complete mouse ALDH3 protein has been deduced 
from a cloned corneal complementary deoxyribonucleic acid (cDNA). Three 16-18 kbp mouse genomic 
fragments for ALDH3 have been cloned: one comprises the entire ALDH3 gene, one contains about 13 kbp 
of 5' flanking sequence, exon 1, intron 1 (3.2 kbp) and part of exon 2, and the third is being analyzed. 
Northern blots have established that ALDH3 messenger ribonucleic acid (mRNA) is at least 100 times more 
prevalent in the cornea than in the stomach, bladder, and lung, the only other tissues showing a trace of this 
gene product. Transfection and transgenic mouse experiments using the chloramphenicol acetyltransferase 
(CAT) reporter gene have shown that 1050 bp of 5' flanking sequence of the ALDH3 gene gives low-level 
expression in the liver, but is inactive in all other tissues tested, including the cornea. New promoter/CAT 
constructs containing intron 1 of the ALDH3 gene have been made and are being tested. The 5' flanking 
sequence of the ALDH3 gene contains numerous potential control elements, including antioxidant response 
elements, which will be tested for function. 



217 



PHS 6040 (Rev. 5/92) 



FY 2994 NEI Annual Report 



Project Description 

Objectives 

The project objectives are to identify and 
characterize the genes that are preferentially 
expressed in the epitheUum and endotheUum 
of the cornea and to study the molecular basis 
for their expression in this transparent eye 
structure. 

Methods 

Conventional molecular biology methods of 
cloning, sequencing, recombinant deoxyribo- 
nucleic acid (rDNA) construction, transfection, 
and transgenic mouse production are used. 

Major Findings 

Aldehyde dehydrogenase class 3 (ALDH3) 
comprises approximately 40 percent of the 
soluble protein of the corneal epithelial cells in 
the mouse and other mammals, including 
humans. Such abundance suggests that this 
metaboUc enzyme may have a refractive 
function Uke an enzyme-crystaUin in the lens. 
It is also possible that the high concentration 
of ALDH3 protects the eye from ultraviolet 
absorption and /or protects the cornea from 
oxidative damage. Moreover, the extremely 
high expression of the ALDH3 gene in the 
corneal epithelial cells provides a potential 
source of regulatory sequences for directing 
gene expression to this tissue, which could be 
of great value for gene therapy. • 

Last year, we created transgenic mice 
carrying a transgene comprising the P-galacto- 
sidase reporter gene fused to 1.1 kbp of the 
cloned mouse ALDH3 gene that we thought 
contained the promoter. However, no expres- 
sion of the transgene was observed in any of 
the tissues of the transgenic mice that were 
examined, including the cornea. We thus 
performed rapid amplification of complemen- 
tary DNA (cDNA) ends (RACE) experiments 
using corneal ribonucleic acid (RNA) and 
showed the existence of an additional 5' exon, 
indicating that our construct lacked the cor- 



neal promoter. This 5' exon does not appear 
to be present in the human ALDH3 gene on 
the basis of the published literature. 

This year, we have characterized a new 16 
kbp mouse ALDH3 genomic fragment that 
was shown to contain approximately 13 kbp 
of 5' flanking sequence, exon 1 (40 bp), intron 
1 (about 3.2 kbp), and part of exon 2 (40 bp). 
Two additional mouse ALDH3 genomic clones 
were obtained that were 16 kbp and 18 kbp, 
respectively. The 18 kbp fragment appears to 
contain the whole gene on the basis of se- 
quencing with exon specific primers; the 16 
kbp fragment is in the process of being ana- 
lyzed. The entire mouse ALDH3 sequence 
was deduced from a fuU-length cDNA that we 
isolated from a corneal library. 

The high expression of the ALDH3 gene in 
the mouse and bovine cornea was corifirmed 
by Northern blot experiments. A trace of 
ALDH3 messenger RNA (mRNA) was present 
in the stomach, bladder, and lung (at least 100 
times less than the cornea), and no ALDH3 
mRNA was observed in the lens, adrenal 
gland (except a dubious smear of higher 
molectilar weight), brain, and Hver. 

Constructs were made that contain the 
-1050/ +21 fragment of the ALDH3 gene fused 
to the chloramphenicol acetyltransferase (CAT) 
gene in both the forward and backward orien- 
tation. Transfection tests using corneal SIRC 
cells and lens N/N1003A cells (both from 
rabbit) showed that this plasmid has low but 
detectable promoter activity leading to CAT 
expression when the putative ALDH3 promot- 
er is in the correct orientation. Unexpectedly, 
however, this transgene did not express in 
transgenic mice, except for limited amounts in 
the Hver. The cornea was painfully devoid of 
CAT in the transgenic mice. Consequently, 
new constructs have been made that contain 
1050 bp of 5' flanking sequence, exon 1, the 
3.2 kbp intron 1 and the first 5 bp of exon 2 
(excluding the ATG translation start codon) of 
the ALDH3 gene fused to the CAT gene to 
test the possibility that a corneal enhancer is 
present in intron 1. At the time of writing, 
these constructs are being tested for promoter 



218 



LaboratoTy of Molecular and Developmental Biology 



activity in transfected cells and will be used to 
generate transgenic mice. 

Finally, analysis of the 5' flanking se- 
quence of the ALDH3 gene has revealed 
numerous potential regulatory elements, 
including NF-kB, AP-1, NFI, AP-2, SP-1, GRE, 
EivF/CREB, ARE, and XRE sites. The pres- 
ence of ARE and XRE sites supports the idea 
that the high expression of this gene may be 
connected to an ultraviolet responsive or 
antioxidant response. 

Significance to Biomedical Research and the 
Program of the Institute 

The molecular biology of corneal epithelium 
and endothelium has not advanced to the 
same extent as that of the collagenous stroma; 
consequentiy, it should be investigated. The 
cornea is a tiansparent ectodermally derived 
tissue like the lens. Thus, comparative studies 
between it and the lens are of special interest 
with respect to tiansparency and the regula- 
tion of gene expression. 

Moreover, because of our finding several 
years ago that corneal epithelial cells show 
taxon-specific gene sharing of metaboUc en- 
zymes, as does the lens, comparative studies 
on the cornea and lens are important from 
developmental and evolutionary perspectives. 
Finally, the cornea is particularly accessible for 
gene therapy on account of its exposure to the 
surface and its association with numerous 
hereditary diseases. The identification of a 
functioned promoter with corneal specificity 
will open the door to gene therapy and genet- 
ic manipulation in the cornea as isolation of 
crystallin promoters with lens specificity did 
in that tissue. 



Proposed Course 

The following investigations are planned for 
fiscal year 1995: 

(1) Identify the corneal-specific regulatory 
elements in the ALDH3 gene of the mouse by 
transfection and transgenic mouse experi- 
ments. 

(2) Establish a convenient culture system 
to analyze the expression of ALDH3 and other 
corneal genes by transfection. 

(3) Begin to analyze the hans-factors used 
for expression of the mouse ALDH3 gene in 
corneal epithelial and, perhaps, endothelial 
cells. 

(4) Obtain a human ALDH3 gene for 
comparison with the mouse gene and to 
provide the foundations for gene therapy in 
human corneas. 

NEI Research Program 

Corneal Diseases — Structure and Function, 
Stroma 



219 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00255-06 LMDB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Biology and Functions of Lens Proteins 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI; Graeme J. Wistow Ph.D. Chief, Section on Molecular LMDB, NEI 

Structure and Function 



Others: 



Vishwas Paralkar 
Caroline Graham 
Lorenzo Segovia 
Jill Richardson 
Ronit Frilling 
Cynthia Jaworski 



Ph.D. 

B.S. 

Ph.D. 

Ph.D. 

Ph.D. 

Ph.D. 



Visiting Associate 
Biologist 
Visiting Fellow 
Visiting Fellow 
Visiting Fellow 
Chemist, Section on 
-Molecula r Gen et i c s — 



LMDB, NEI 
LMDB, ^fEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 



COOPERATING UNITS (if any) 

National Institute of Allergy and Infectious Diseases (Christine Kozak, Ph.D.) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Molecular Structure and Function 



INSTITUTE AND LOCATION 

NEI. NIH. Bethesda. MD 20892 



TOTAL STAFF YEARS: 



6.9 



PROFESSIONAL: 



5.9 



1.0 



CHECK APPROPRIATE BOX(ES) 

r~| (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Crystallins are the major components of the normal eye lens. In a novel evolutionary process crystallins have 
arisen by the gene recruitment of stress-proteins and enzymes without prior gene duplication. In the lens- 
specific alternative promoter of guinea pig NADPH:quinone oxidoreductase/^-crystallin we have identified 
an element that is essential for function. We now find that this element contains a binding site for Pax-6, a 
"master gene" for eye development. Mutation of the Pax-6 site abolishes promoter function and Pax-6 is 
required for formation a lens-specific complex. Futhermore, Pax-6 is expressed in the mature lens. Pax-6, 
thus, has a continuing role in maintenance of lens-specific gene expression and a key role in the gene 
recruitment of a crystallin. //-crystallin, which was discovered in marsupial lenses, is a novel NADPH-binding 
protein related to enzymes of ornithine and glutamate metabolism. In humans, in which it has not been 
recruited as a crystallin, it is expressed most abundantly in photoreceptor outer segments, suggesting an 
unexpected role in the visual process. Other proteins are essential for normal development in lens. Migration 
inhibitory factor (MIF), a small protein expressed in differentiating lens cells, is essential for progression 
through the cycle. Antisense to MIF abolishes proliferation of growth factor stimulated and cancer cells and 
halts them at the Gl/S boundary. Promoter analysis of the human MIF gene has identified a region required 
for growth factor response. We have also found that LP2, a lipid/retinoid binding protein we have cloned 
from bovine lens, is expressed preferentially in differentiated fiber cells. 



220 



PHS 6040 (Rev. 5/92) 



LaboratoTy of Molecular and Developmental Biologi/ 



Project Description 

Additional Personnel 



Ph.D. Visiting Fellow, MG, 
LMDB, NEI 
Summer Program 



Ales Cvekl 
Steven Puopolo 

Objectives 



We are investigating the molecular basis of 
normal lens structure and function. This 
includes the characterization of crystaUins, 
their lens and nonlens function, and their 
mechanisms of expression and recruitment. 
This has entailed the identification of factors 
responsible for regulation of lens cell differen- 
tiation and gene expression. The interplay of 
such factors is an essential part of normal lens 
development and function. 

Methods 

A wide range of molecular biology techruques 
are used, including messenger ribonucleic acid 
(mRNA) analysis, gene and complementary 
deox5aibonucleic acid (cDNA) cloning and se- 
quencing, functional gene promoter analysis in 
cultured cells and in transgenic mice, polymer- 
ase chain reaction, and antisense technology. 
We perform some protein analysis and make 
use of commercial facilities for protein se- 
quencing. We make extensive use of com- 
puter-based sequence analysis and molecular 
modeling. 

Major Findings 

(1) Gene Recruitment: Enzyme CrystaUins. 
Guinea pig ^-crystallin is a taxon-specific crys- 
tallin, an enz5ane that has achieved high 
expression in lens through an alternative, lens- 
specific promoter. This promoter contains an 
element (ZPE) that binds tissue-specific com- 
plexes in electrophoretic mobility shift assay. 
Jill Richardson has shown that both lens- 
spedfic complex formation and promoter 
function depend on an intact binding site for 
Pax-6 in the ZPE. Pax-6 is expressed in em- 
bryoruc eye and central nervous system and is 



implicated in the establishment of lens compe- 
tence. Antisera to Pax-6 specifically abolish 
lens complex formation by the ZPE. Protein 
and mRNA for Pax-6 are present in mature 
mouse lens and in cells that support t,-crystal- 
lin lens promoter expression but not in other 
cell types. This suggests that L,-crystallin is a 
target gene for Pax-6 and supports the idea 
that Pax-6 is a "master gene" factor for tissue- 
specific gene expression in the lens. Ronit 
Frilling has shown that the isolated fragments 
covering the ZPE region of the t-crystallin 
promoter can confer lens-preferred expression 
on a heterologous promoter, the thymidine 
kinase minimal promoter driving a reporter 
gene. Experiments in transgenic mice show 
that sequences upstream of the Pax-6 site, 
between -498 and -385, are required for sup- 
pression of promoter function in brain, which 
is also a site of Pax-6 expression. 

Although some taxon-specific crystaUins 
are famihar enz5anes, others were discovered 
first as crystaUins. ju,-Crystallin is the major 
component of the eye lens in several Austia- 
Uan marsupials. In other mammals, including 
humans, it is expressed at enzymatic levels in 
lens, but Lorenzo Segovia has shown that it is 
most strikingly expressed in retinal photore- 
ceptor ceU outer segments. /x-Crystallin has 
sequence similarity with bacterial ornithine 
cyclodeaminases and glutamyl-transfer RNA 
reductases, suggesting a role in amino-acid 
metaboUsm that probably involves a gluta- 
mate-related pathway. This is particularly 
significant because glutamate is the neuro- 
transmitter of the photoreceptors. The gene 
for /i-crystallin has been cloned from both 
kangaroo, where it has undergone gene re- 
cruitment, and from human, where it serves 
only as an enzyme. The human gene for ix- 
crystallin maps close to a locus for congenital 
cataract and microphthalmia. /x-Crystallin has 
also been mapped in the mouse genome. 

Another major enzyme crystallin (up to 25 
percent of total protein) in mammals is i~\- 
CTystallin found in elephant shrews. Caroline 
Graham has cloned the complete cDNA se- 
quence for this protein from two different 
species. Current results suggest that T|-crystal- 



221 



FY 1994 NEI Annual Report 



lin is very closely related to aldehyde dehy- 
drogenase 1 (ALDHI) and is expressed both in 
lens and liver. However, a second, closely 
related ALDHI is also expressed in liver, 
suggesting that the gene recruitment of 
Ti-crystallin has led to gene dupUcation and 
specialization, perhaps as a consequence of 
adaptive conflict. The elephant shrew genome 
also contains several processed pseudogenes 
for Tj-crystallin/ALDHl-like sequences. 

aB-crystallin is multifunctional, serving as 
both a major structural protein in the lens and 
as a small heat shock protein in other tissues 
in mammals. The gene for aB-crystallin in a 
bird {Anas platyrhynchos) has been cloned and 
sequenced. Because only the most important 
functional features of a gene are conserved 
among distantly related species, comparison of 
this sequence with those of mammals can be 
very informative. Although several function- 
ally defined elements in mammals are con- 
served in the duck gene, consensus stress- 
response elements are not well conserved. 
Current experiments are aimed at determining 
whether the bird gene does indeed have the 
stiess role suggested for mammalian genes. 

(2) Molecular Markers of Differentiation. 
Macrophage migration inhibitory factor (MIF) 
was originally identified as a l5anphokine. 
However recent work strongly suggests a role 
for MIF beyond the immune system. We have 
found that it is expressed specifically in the 
differentiating cells of the immunologically 
privileged eye lens and in many other tissues. 
Now Vishwas Paralkar has shovwi that MIF is 
an essential part of the mitogenic response to 
growth factors operating through two differ- 
ent receptor systems. Both platelet-derived 
growth factor and transforming growth factor 
pi stijnulate expression of MIF in NIH 3T3 
cells in a delayed early response. When cells 
treated with either growth factor are grown in 
the presence of antisense oligo to mouse MIF, 
MIF protein synthesis and cellular prolifera- 
tion are abolished. Control oUgos have no 
effect under identical conditions. When anti- 
sense oligo is removed the cells regain the 
ability to proliferate in response to mitogenic 
signals. Analysis of levels of cycUns E, A, and 



B suggests that the effect of MIF is localized 
close to the Gl/S transition in the cell cycle 
and that MIF is essential for progression into 
S phase. In promoter analysis of the human 
MIF gene a region required for induction by 
growth factors has been localized. This MIF 
gene region also contains an E-box, a potential 
myc family-binding site. The gene for MIF has 
been mapped in human and mouse. 

LP2 is a member of the lipid /retinoic add 
binding family of P2 proteins. Cynthia 
Jaworski has cloned bovine LP2 and has 
shown that its mRNA is expressed preferen- 
tially in differentiated fiber cells. Because 
retinoic acid has now been implicated in fiber 
cell specific y-crystaUin gene expression, this 
protein could have a direct role in mediating 
lens gene expression. Molecular modeling 
shows that LP2, unlike adipocyte P2, myelin 
P2 or other relatives, contains two close cyste- 
ine residues capable of disuilfide cross-Unking. 
LP2 is thus a potential target for oxidative 
damage to the lens in aging and cataract. 

Significance to Biomedical Research and the 
Program of the Institute 

We are uncovering fundamental mechanisms 
in the evolution and differentiation of the lens 
that may be generalizable to other complex 
tissues. We have shown how a single gene 
can have multiple functions and how it can 
acquire tissue-specific patterns of expression. 
In examining proteins that have been recruited 
as crystaUins, we have discovered a novel 
enzyme that may have an important role in 
human retinal photoreceptors. Our work in 
lens has also revealed important markers for 
cellular differentiation. In particular one of 
these proteins, MIF, has an essential role in 
cell proliferation, and antisense treatment to 
suppress MIF halts proliferation, even of 
transformed ceUs. 

Proposed Course 

(1) To continue to examine the molecular 
mechanisms for lens preferred expression and 
gene recruitment of crystaUins. 



222 



Laboratory of Molecular and Developmental Biology 



(2) To characterize the function and 
nonlens role of /x-crystallin, particularly its 
role in photoreceptor outer segments. 

(3) To define the role of MIF in the cell 
cycle and to test the potential for antisense 
treatment to suppress the growth of trans- 
formed ceUs. 

(4) To determine the function of LP2 pro- 
tein, a marker for differentiation in the lens. 

NEI Research Program 

Lens and Cataract — Molecular Genetics 



Publications 

Graham C, Wistow G: The predominant 
cadherin in fetal human lens is identical to 
N-cadherin and is not a candidate locus for 
the Marner cataract. Exp Eye Res 59, in press. 

Lee DC, Gonzalez P, Wistow G: Zeta-crystal- 
lin: a lens-specific promoter and the gene 
recruitment of an enzyme as a crystaUin. / Mol 
Biol 236:669-678, 1994. 

Paralkar V, Wistow G: Cloning the human 
gene for macrophage migration inhibitory 
factor (MIF). Genomics 19:48-51, 1994. 



Patent 

G.J. Wistow. US Patent 5,328,990: "Isolation of 
macrophage migration inhibition factor from 
ocular lens." 



223 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00251-07 LMDB 



PERIOD COVERED 

October 1 , 1993 to Septembe r 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Gene tically Engineer ing the Eye with the aA-Cr ystal lin Promoter 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI 



Others: 



Devonne M. Parker 
Charles Egwuagu 
Chi-Chao Chan 

Robert B. Nussenblatt 
Jorge Sztein 



B.S. 

Ph.D. 

M.D. 

M.D. 
D.V.M. 



Regulation of Gene 
Expression 

Biologist 
Scientist, PHS 
Head, Section on 
Immunopathology 
Clinical Director 
Visiting Associate 



LMDB, NEI 
LLNEI 
LI, NEI 

LI, NEI 
VRRS, NEI 



COOPERATING UNITS (if any) 

Department of Cell Biology, Baylor College of Medicine, Howard Hughes Medical Institute (Paul Overbeek, Ph.D.; Michael Robinson, 
Ph.D.); Imperial Cancer Research Fund, London, England (Clive Dickson, Ph.D.); Gerontological Research Unit, National Institute of 
Health and Medical Research, Paris, France (Yves Courtois, Ph.D.; Maryvonne Laurent, Ph.D.) 



LAB/BRANCH 



Labo r atory of Molecular and Developmental Biology 



SECTION 

Section on Regulation of Gene Expression 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.8 



PROFESSIONAL: 



0.5 



0.3 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

We generated transgenic mice and rats expressing interferon (IFN)-y in their lenses to investigate the possible 
role of this lymphokine in ocular pathogenesis. Embryonic lens and retina differentiation were affected in the 
ciA-crystallin/IFN-Y transgenic mice resulting in microphthalmia, microphakia, retinal detachment, and 
persistent hyperplastic primary vitreous in the adult mice. Major histocompatibility complex (MHC) class II 
messenger ribonucleic acid (mRNA) levels were significantly increased in the transgenic eyes, and MHC class 
II proteins were expressed in their cornea, iris, ciliary body, choroid, lens, and retinal pigment epithelial (RPE). 
Constitutive expression of IFN-y and its induction of MHC class II molecules in the eye provide a useful 
model to study the linkage between aberrant MHC class II expression and predisposition to autoimmunity and 
the role of IFN-y in the treatment of inflammatory eye diseases and cytokine signaling during embryonic eye 
development. 

Fibroblast growth factor (FGF)-3 expression was directed to the eye to investigate how the aberrant expression 
of this growth factor would affect the developmental program of the eye. The aA-crystallin/FGF-3 transgenic 
mice presented exophthalmia and aberrant elongation of central lens epithelia at 15 days of embryonic 
development. The hypertrophic lens mass was extruded through the cornea at 16 days of embryonic 
development, resulting in cornea perforation. Postnatal microphthalmia was characterized by intraocular 
hyperplastic glandular structures replacing the normal iris, ciliary body, and lens. 



224 



PHS 6040 (Rev. 5/92) 



Lahoraton/ of Molecular and Developmental Biology 



Project Description 

Objectives 

The objective of this project is to understand 
how aberrant genetic expression of interferon 
gamma (IFN-y) or fibroblast growth factor-3 
(FGF-3) under the control of the aA-crystallin 
promoter perturbs normal eye development in 
transgeruc mice. 

Methods 

Recombinant deox5Tibonucleic acid (rDNA) 
techniques used in this study include plasmid 
construction, oUgonucleotide sequencing. 
Southern and Northern hybridizations, DNA 
sequencing, primer extension, polymerase 
chain reaction (PCR), reversed transcription 
PCR. Other molecular biology techniques 
used in the study include immunohistochem- 
istry, in situ hybridization, and production and 
analysis of transgenic mice. 

Major Findings 

(1) IFN-y. This project is conducted in collab- 
oration with Drs. Charles Egwxiagu, Jorge 
Sztein, Chi-Chao Chan, and Robert B. 
Nussenblatt from the Laboratory of Immunol- 
ogy (LI) at the NEI. The IFN-y gene is specifi- 
cally expressed in activated T lymphocytes 
and natural killer cells. It plays a crucial role 
in the ontogenesis and phenomenology of the 
immune response and has potent immunomo- 
dulatory and antiproliferative effects on tumor 
cells. The ectopic expression of IFN-y in the 
lens of transgeruc mice allowed us to study its 
effect on eye development and its regulation 
of major histocompatibility complex (MHC) 
class n gene expression in a nonlymphoid 
tissue such as the lens. 

We previously generated FVB/N and 
BALB/c transgenic mice containing as a 
transgene the murine aA-crystaUin promoter 
(-3667+46) fused to the murine IFN-y coding 
sequence. In both aA-crystaUin/ IFN-y trans- 
genic mouse Lines, ectopic expression of IFN-y 
in the lens affected the growth of the whole 



eye, resulting in microphthalmia and blephar- 
ophimosis. Lens differentiation was severely 
affected resulting in microphakia, impairment 
of lens fiber formation and cataract, thickening 
of the anterior lens capsule, and rupture of the 
posterior capsule. Retardation of retinal 
differentiation into inner and outer neuroblas- 
tic layers was observed in the transgenic eyes. 
Serous retinal detachment with presence of 
macrophages in the subretinal space, persis- 
tent hyperplastic primary vitreous, corneal 
vascularization, and absence of a normal 
anterior chamber were observed in the adult 
transgenic mice. MHC class 11 messenger 
ribonucleic acid (mRNA) levels were sigiufi- 
cantly increased in the eyes of transgenic mice, 
and MHC class 11 proteins were expressed in 
the corneas, irises, ciliary bodies, choroids, 
lenses and retinal pigment epitheUa. Expres- 
sion of genes coding for IFN-y-inducible 
transcription factors, interferon-consensus- 
sequence-binding protein, and interferon 
response factor 2, absent in the normal eye, 
were induced in the transgenic eyes. These 
results indicate that the ectopically expressed 
transgenic IFN-y is biologically active in vivo. 

We also derived a transgenic Sprague 
Dawley rat line using the same murine 
aA-crystallin /IFN-y construct. The transgenic 
rat eyes, similar to those from the transgenic 
mice, present microphthalmia and micropha- 
kia with cataract formation. However, in 
contrast to the transgenic mouse, the anterior 
chamber of the transgenic rat eye is weU 
formed, and the architecture of the retina is 
intact with focal retinal serous detachment. 
The aA-crystaUin/ IFN-y transgenic rat, the 
first transgenic rat strain generated for vision 
research, is of particular interest because the 
rat is a well-characterized species for experi- 
mental autoimmune uveitis studies. 

(2) FGF-3. This is a collaborative project 
with Drs. Paul Overbeek and Michael Robin- 
son, from Baylor CoUege of Medicine, and Dr. 
Clive Dickson, from the Imperial Cancer 
Research Fund. FGF-3 /int-2 is a member of 
the fibroblast growth factor (FGF) family. To 
assess whether ectopic expression of FGF-3 
would perturb normal lens development, we 



225 



FY 1994 NEI Annual Report 



directed its expression to the eyes of trans- 
genic mice using the murine aA-crystallin 
promoter. We obtained three lines of trans- 
genic mice expressing the aA-crystallin /FGF-3 
transgene. The anterior lens epithelia of the 
transgenic mice undergo premature cell elon- 
gation at day 14 of embryonic development 
concomitant with major intrinsic protein (MIP) 
synthesis. The lens mass was extruded 
through the cornea, resulting in cornea perfo- 
ration at 16 days of embryonic development. 
At postnatal day four, intraocular glandular 
structures that replaced the normal irises, 
ciliary bodies, and lenses and stained positive- 
ly for FGF-3 and Muc-1 (a marker for secreto- 
ry epitheUa) and negatively for connexin 46 
and MIP were observed. We observed a 
marked increase in Muc-1 mRNA levels, while 
MIP, connexins 46 and 50 mRNA levels were 
drastically reduced in the adult transgenic 
mouse eyes. The ectopic expression of the 
FGF-3 during the embryonic development of 
the lens induces prematvure differentiation of 
the central lens epitheUa, expulsion of the lens 
through the cornea, and postnatal appearance 
of intraocular secretory glandular epithelia. 

(3) Acidic FGF. In collaboration with Drs. 
Overbeek and Robinson and Dr. Yves 
Courtois, from the Institute for Gerontological 
Research (INSERM), we obtained three lines of 
transgenic mice carrying as a transgene the 
aA-crystaUin promoter fused to the bovine 
addic FGF complementary DNA. These mice 
do not present any particvilar phenotype. We 
are currently studying whether the lens cells 
derived from these mice are able to give rise 
to a lens epithelia cell line with differentiated 
properties. 

Significance to Biomedical Research and the 
Program of the institute 

Constitutive expression of IFN-y, and its 
induction of MHC class 11 molecules in the 
eye, provides a useful model to study the 
linkage between aberrant MHC class n expres- 
sion and predisposition to autoimmunity and 
the role of EFN-y in the treatment of inflam- 
matory eye diseases and cytokine signaling 
during embryonic eye development. The 



ectopic expression of FGF-3 will allow us to 
elucidate the mechanisms underlying eye 
development. 

Proposed Course 

The following studies wiU continue during 
fiscal year 1995: 

(1) The effect of IFN-y on the regulation 
of gene expression in the eyes of the trans- 
geruc mice will be further characterized. 

(2) The effect of FGF-3 on gene expression 
in the eyes of transgenic mice will continue to 
elucidate the role of FGF-3 in premature lens 
epithelia differentiation and the appearance of 
intraocular secretory epithelia. 

NEi Research Program 

Lens and Cataract — Molecular Genetics 

Pubiications 

ChepeUnsky AB, Robinson M, Overbeek PA, 
Parker DM, Chan C-C, Jamieson S, Dickson C: 
FGF-3 ectopic expression induces differentia- 
tion of central lens epithelia and appearance of 
secretory epithelia in the eyes of transgenic 
mice. Invest Ophthal Vis Sci 35(suppl):1988, 
1994. 

Egwagu CF, Sztein J, Chan C-C, Reid W, 
Mahdi R, Nussenblatt RB, ChepeUnsky AB: 
Ectopic expression of gamma interferon ex- 
pression in the eyes of transgenic mice induc- 
es ocular pathology and MHC class 11 gene 
expression. Invest Ophthal Vis Sci 35:332-341, 
1994. 

Egwagu CF, Sztein J, Chan C-C, Mahdi R, 
Nussenblatt RB, ChepeUnsky AB: Gamma 
interferon expression disrupts lens and retinal 
differentiation in transgenic mice. Dev Biol, in 
press. 

Egwagu CF, Sztein J, Chan C-C, Mahdi R, 
Nussenblatt RB, ChepeUnsky AB: Transgeruc 
rat and mouse models for the study of intraoc- 



226 



Laboratory of Molecular and Developmental Biology 



ular effects of IFN-y and autoimmunity. Sax CM, Cvekl A, Kantorow M, Sommer B, 
Invest. Ophthal Vis Sci 35 (suppl):1987, 1994. Chepelinsky AB, Piatigorsky J: Identification 

of negative-acting and protein-binding ele- 
Egwagu CF, Sztein J, Chan C-C, Mahdi R, ments in the mouse aA-crystalUn -1556/-1165 
Nussenblatt RB, Chepelinsky AB: Transgenic region. Gene 144:163-169, 1994. 
rat and mouse models for studying the role of 
gamma interferon and MHC class II in intra- 
ocular diseases and autoimmunity. Proceed- 
ings of the 6th International Symposium on the 
Immunology and Immunopathology of the Eye, in 
press. 



PROJECT NUMBER 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



ZOl EY 00253-06 LMDB 



PERIOD COVERED 

Octob er 1, 1993 to Septe mber 30, 1994 



TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) 

Regulation o f E x pres sion o f Lens Fiber Membrane Genes 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI 

Regulation of Gene 
Expression 



Others: Chiaki Ohtaka-Maruyama Ph.D. Visiting Fellow 

Xiaoyan Wang M.D. IRTA Fellow 

Devonne M. Parker B.S. Biologist 

Chris Chon Summer Student 



LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 



COOPERATING UNITS (if any) 

Harvard University (David Paul, Ph.D.); Columbia University (Jorge Fischbarg, M.D.); Tokio Medical and 
Dental University, Japan (Sei Sasaki, M.D.) 



Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Regulation of Gene Expression 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.37 



PROFESSIONAL: 



2.5 



0.87 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x| (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project studies the regulation of expression of genes encoding lens fiber membrane channel proteins. We 
are presently focusing on the regulation of expression of the gene encoding, the major intrinsic protein (MIP) 
of the lens fiber membrane, that is specifically expressed in the ocular lens fibers and belongs to an ancient 
superfamily of transmembrane channel proteins. 

We are studying the cis regulatory elements responsible for the lens specificity and developmental regulation 
of the MIP gene. We are fusing 5' flanking sequences of the human MIP gene to the reporter 
chloramphenicol acetyltransferase (CAT) gene and analyzing CAT gene expression in transient assays in 
primary cultures of lens cells. Deletion analysis of the 5' flanking sequence of the human MIP gene suggests 
that negative regulatory elements are located within the sequence -2840/-254. The human MIP sequence -70/- 
40 appears to contain an important regulatory element for the activity of the MIP promoter. This sequence 
contains one of the domains that interacts with Spl and AP2 transcription factors by DNase I footprinting 
analysis; it also forms a complex with chicken lens nuclear extracts that appear as a retarded band in mobility 
shift assays. Mutations at positions -49/-51 affect binding of this nuclear factor in vitro, and deletion of the 
sequence -70/-49 abolishes promoter activity in transfected lens cells. These studies will further our 
understanding of the regulation of the MIP gene expression in the lens. 



228 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biology 



Project Description 

Oblectives 

The objective of this project is to elucidate the 
mechanisms involved in the regulation of 
expression of fiber membrane genes involved 
in cell-cell communication in the lens. The 
identification of the cis regulatory elements of 
these genes and their interaction with trans- 
acting factors are essential for understanding 
the regulation of gene expression in the lens. 

Methods 

Recombinant deoxyribonucleic acid (rDNA) 
techniques used in this study include screen- 
ing genomic libraries; subclonirig; plasmid 
construction; oligonucleotide synthesis; South- 
em and Northern hybridizations; DNA se- 
quencing; primer extension; polymerase chain 
reaction (PCR); reversed transcription PCR; gel 
mobUity shift assays; DNA footprinting; meth- 
ylation interference; preparation of nuclear 
extracts by subcellular fractionation; in vitro 
transcription; tissue culture techniques, includ- 
ing transfection of primary lens explants and 
cell Unes; analysis of transgenic mice; in situ 
hybridization. 

Major Findings 

(1) Cis Regulatory Elements of the Human Major 
Intrinsic Protein (MIP) Gene Promoter. We have 
characterized 2840 bp of 5' flanking sequence 
of the human MIP gene to identify the cis 
regulatory elements responsible for the tissue 
specificity and developmental regulation of 
the MIP gene. We found that a DNA frag- 
ment containing the human MIP sequence 
-253/ +42 fused to the reporter chlorampheni- 
col acetyltransferase (CAT) gene activates CAT 
gene expression when transfected into lens 
cells. However, the -2840 / +42 sequence does 
not activate CAT gene expression, suggesting 
that negative regulatory elements are located 
v^thin the sequence -2840/ -254. Deletion 
mutants experiments indicated that -70/ +42 
sequence contains an active promoter in 
transfected lens ceUs. However, when se- 



quences -70/ -49 are deleted, the promoter 
activity is lost. The human MIP sequence -70/ 
-40 appears to contain an important regulatory 
element for the activity of the MIP promoter. 
An oUgonucleotide corresponding to sequence 
-67/ -38 forms a complex with chicken lens 
nuclear extracts that appear as a retarded 
band in mobility shift assays. Mutations at 
positions -49/ -51 affect binding of this factor. 
This sequence corresponds to one of the 
domains that interact with Spl and AP2 
transcription factors by DNase I footprinting 
analysis. 

We have generated two lines of transgenic 
mice containing 2840 bp of the human MIP 
gene 5' flanking sequence and 42 bp of exon 
one fused to the p-galactosidase gene as a 
tiansgene. We are presently analysing p- 
galactosidase expression in these transgenic 
mice to determine whether cis regulatory 
elements responsible for lens-specific expres- 
sion of the MIP gene are located in that do- 
main. 

(1) Cloning of the Mouse MIP Gene. We 
have isolated one MIP genomic clone from a 
PI phage mouse genomic library. Subcloning 
and characterization of this clone will allow us 
to study the regulation of expression of the 
murine MIP gene. 

(3) Cloning of the Connexin 46 Gene. The 
connexin 46 gene encodes one of the lens fiber 
gap junction proteins. To be able to study 
how its expression is regulated in the lens, we 
are sequencing, in collaboration with Dr. 
David Paul, the mouse cormexin 46 gene from 
a clone isolated from a mouse genomic library. 

(4) Aquaporin Gene Expression in Cornea 
Endothelial Cells. Although the physiological 
basis for the maintenance of corneal transpar- 
ency has been extensively studied, the molecu- 
lar mechanisms involved in maintaining 
cornea transparency are poorly understood. 
Swelling of the corneal stroma is involved in 
the loss of cornea transparency. In collabora- 
tion with Dr. Jorge Fischbarg, from Columbia 
Uruversity, we are characterizing the member 
of the MIP family responsible for the CHIP28- 



229 



FY 1994 NEl Annual Report 



like water channels observed in primary 
cultures of bovine cornea endothelial cells. 
Sequencing data indicate that it has a high 
degree of identity with CHIP28, suggesting 
that it may be a new member of the MIP 
family derived from CHIP28 by gene conver- 
sion. 

Significance to Biomedical Research and the 
Program of the Institute 

Proper lens fiber membrane biosynthesis and 
physiology are of upmost importance for 
maintairung lens transparency. Membrane 
protein synthesis is temporal and spatially 
regulated during lens development. Lens 
membranes undergo biochemical and structur- 
al changes during cataractogenesis and aging. 
Therefore, studying the regulation of genes 
encoding lens membrane channels should 
further our understanding, not only of the 
mechanisms involved in the regulation of gene 
expression in the normal lens but also of its 
disruption during disease. 

Proposed Course 

The following studies will continue during 
fiscal year 1995: 

(1) Characterization of the cis regulatory 
elements of the mouse MIP gene promoter 
and comparison with its human homologue. 

(2) Interaction of the mouse MIP gene cis 
regulatory elements with transcription factors. 

(3) Sequencing of connexin 46 genomic 
clones to locate the 5' end of this gene. 



NEl Research Program 

Lens and Cataract — Molecular Genetics 
Publications 

ChepeUnsky AB: The MIP transmembrane 
channel family, in Peracchia C (ed): Handbook 
of Membrane Channels. San Diego, Academic 
Press, 1994, pp 413-432. 

Chepelinsky AB, Ohtaka-Maruyama C, Wang 
X: General transcription factors and lens 
specific expression of the MIP gene. / Cellular 
Biochem 18(B):388, 1994. 

Ohtaka-Maruyama C, Wang X, Chepelinsky 
AB: AP2 transcription factor is involved in 
the transcription of the lens MIP Gene. / 
Cellular Biochem 18(C):50, 1994. 

Saito F, Sasaki S, Chepelinsky AB, Fushimi K, 
Marumo F, Ikeuchi T: Human AQP-2 and 
MIP genes, two members of the MIP family, 
map within chromosome band 12ql3 using 
two-color FISH. Cytogen Cell Genetics, in press. 

Wang X, Ohtaka-Maruyama C, Chepelinsky 
AB: Cis regulatory elements of the human 
MIP gene promoter. Invest Ophthalmol Vis Sci 
35(suppl):1997, 1994. 



230 



PROJECT NUMBER 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



ZOl EY 00285-02 LMDB 



PERIOD COVERED 

October 1, 1993 to September 30, 199 4 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

NEI Central T ransgeni c Animal Pr oduction F acility 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI 



Others: Susan DiCamillo B.S. 

R. Steven Lee B.S. 

Mariana Gonzalez-Baez 



Chemist 
Biologist 

Biological Science 
Lab Aide, Stay-in- 
School Program 



LMDB, NEI 
LMDB, NEI 
LMDB, NEI 

LMDB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Transgenic Animal and Genome Manipulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL: 



0.5 



2.5 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The NEI Central Transgenic Animal Production Facility is a research support facility for all NEI intramural 
researchers requiring the use of transgenic mice in their research programs. We are currently providing 
transgenic animal support to researchers from four laboratories in the NEI (Laboratory of Immunology, 
Laboratory of Mechanisms of Ocular Diseases, Laboratory of Molecular and Developmental Biology, and 
Laboratory of Retinal Cell and Molecular Biology). In our program, there are currently 84 deoxyribonucleic 
acid (DNA) constructs that are at various stages of completion. NEI researchers using molecular biology 
techniques to study the eye submit DNA constructs to our section for production of transgenic mice. We 
create transgenic mice by standard procedures, then biopsy and perform DNA analyses on the mice that are 
bom from these procedures to identify positive mice. At researchers' request, we mate positive transgenic 
mice, wean litters, biopsy and analyze DNA from successive generations of transgenic mice, and provide the 
transgenic animals to researchers for use in their experiments. During the year, we have generated 155 
transgenic founder mice from 34 DNA constructs; set up 456 matings of transgenic mice; weaned, tagged, and 
tail-biopsied 4,527 mice; isolated DNA from 4,708 samples; and performed 4,912 DNA analyses. This year 
we began an embryo cryopreservation and banking program to provide long-term storage of important 
transgenic lines without the need to maintain live mice. A total of 1,326 embryos from five transgenic lines 
have been frozen. In addition to service functions, we also collaborate with NEI researchers on transgenic 
animal projects. 



231 



PHS 6040 (Rev. 5/92) 



FY 1994 NEl Annual Report 



Project Description 

Objectives 

This project is to produce transgenic animals 
for use in eye research in the NEI, supply 
ancillary services related to maintenance of 
transgenic animals, and provide advice and 
expertise in matters of transgenic animal 
projects to all NEI intramural researchers 
using this technology in their research. In 
addition, we act as a central fadlity for aU 
transgenic animal work conducted in the NEI 
intramural research program to coordinate 
and conserve resources and use severely 
Umited animal housing space with maximum 
efficiency. We provide a comprehensive 
program for short- and long-term storage of 
transgenic animal lines for both live aivimals 
and frozen embryos. 

Methods 

Standard methods are used for microinjecting 
deoxyribonucleic add (DNA) into the pronu- 
cleus of one-celled mouse embryos and surgi- 
cally reimplanting the injected embryos into 
foster mothers to enable development. Con- 
ventional molecular biology techniques are 
used to isolate and analyze DNA from biopsy 
samples of transgeruc mice. Data on all trans- 
genic mice are maintained in a computerized, 
relational database accessed by programs 
written within our group. 

Major Findings 

Production of New Transgenic Mouse Lines. We 
have generated 155 new transgenic founder 
mice from 34 constructs submitted by re- 
searchers in four NEI intramural laboratories. 
These constructs are quite diverse in nature 
and reflect the diversity of research being 
performed in the NEI. Some of the general 
categories of constructs are: (1) pro- 
moter/reporter constructs in which the pro- 
moter of an eye gene is fused to a reporter 
gene to assess transcriptional activity in the 
transgenic mouse, (2) eye-specific or ubiqui- 
tous promoters driving expression of genes 



beUeved to be involved in eye pathologies to 
assess their roles in pathological conditions in 
a transgenic mouse model, and (3) other 
constructs for probing normal eye function 
and pathological conditions in the mouse. 

Maintenance of Transgenic Mouse Lines. 
Transgenic mouse Hnes are derived by mating 
of the original transgenic founder niice and 
derivation of successive generations of proge- 
ny, which are then used in biomedical re- 
search. To generate lines of transgenic mice 
from our transgenic founder mice, we have set 
up 456 mouse matings and weaned, tagged, 
and biopsied 4,527 mice resulting from mat- 
ings and microinjection procedures. 

DNA Analyses. Approximately 15 to 30 
percent of mice bom from microinjected 
embryos are transgenic, and approximately 50 
percent offspring from a transgenic mating are 
transgenic sensitive. A rapid, efficient, and 
reliable method of identifying transgene 
positive and negative mice is in place in our 
group. We have processed 4,708 biopsy 
samples to obtain DNA and performed 4,912 
analyses to determine whether the mice were 
transgene positive. 

Embryo Cryopreservation and Banking. We 
have begun freezing mouse embryos for 
banking of important lines of transgenic mice. 
Between 200 and 300 embryos must be frozen 
for each line of mice banked. This year, we 
have frozen and banked 1,326 embryos from 
five lines of transgenic mice. Successful recon- 
stitution of these transgenic lines has been 
accomplished by thawing a small portion of 
the banked embryos and transferring them 
into the oviducts of pseudopregnant foster 
mothers. 

Significance to Biomedical Research and the 
Program of the Institute 

Transgenic mice are currently the only readily 
attainable system for studjdng gene expression 
in the context of an entire, intact animal. Al- 
though tissue culture can yield a great deal of 
information in many studies, a true under- 
standing of how a particular gene affects an 



232 



Laboratory of Molecular and Developmental Biology 



organ (such as the eye) or an entire orgarusm 
can only be obtained by studying that gene in 
the intact organism. We play a pivotal role in 
many NEI intramural research projects by 
providing the technology and expertise to 
insert into the mouse genes related to normal 
eye development and pathological conditions 
of the eye. 

Proposed Course 

(1) Continue producing new transgenic mice 
for NEI researchers as required for their 
research projects. 

(2) Continue breeding and maintaining 
existing transgenic mouse lines needed for 
ongoing research in the NEI. 



(3) Continue freezing and banking embry- 
os from important lines of transgenic mice. 
This will free some of our limited animal 
housing space and ensure that important Unes 
of mice will not be lost due to aging and loss 
of fertility. 

NEI Research Program 

Lens and Cataract — Molecular Genetics 

Publications 

As a service organization, we are generally 
not included as authors on publications result- 
ing from research performed on the transgenic 
animals we produce and maintain. 



233 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00286-02 LMDB 



PERIOD COVERED 

October 1, 1993 t o September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

g-Crystallin Gene Disr u ption in the Mouse 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI 



Others: James P. Brady 



Ph.D. 



IRTA Fellow 



LMDB, NEI 



COOPERATING UNITS (if any) 



University of Maryland Medical School (Nicholas Ambulos, Ph.D.) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Transgenic Animal and Genome Manipulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.8 



PROFESSIONAL: 



0.8 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neitiier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The a-crystallins compose a large fraction of the soluble protein in the vertebrate lens, where they are believed 
to function as structural proteins; are the first crystallins to be expressed in the developing mouse lens; and 
are a relatively small family of crystallins encoded by only two genes, the aA- and aB-crystallin genes. The 
a-crystallins exhibit molecular chaperon activity and kinase activity and, at least in the case of aB-crystallin, 
have been shown to be expressed in a variety of nonlenticular tissues, where their function is unknown. 
Toward understanding the role of the a-crystallins in lens and nonlens tissues, we are attempting to 
functionally delete a-crystallins by disrupting their genes in mice. We are using the technique of homologous 
recombination in pluripotent mouse embryonic stem cells followed by generation of chimeric mice containing 
the altered stem cells. We have isolated and mapped 15 kb clones containing the aA- and aB-crystallin gene 
loci from a mouse 129SV library (the same strain as most of the embryonic stem cell lines currently in use). 
Construction of aA-crystallin, aB-crystallin, and aA-CRYBPl knockout vectors is complete. We are 
mastering the technologies to (1) effect homologous recombination iri embryonic stem (ES) cells and (2) 
introduce ES cells into embryos that develop into chimeric mice. In collaboration with Nicholas Ambulos 
(University of Maryland Medical School), we are sequencing the mouse aA-crystallin gene locus. 



234 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biology 



Project Description 

Additional Personnel 



Ellen Liberman PhD. Anterior Segment 

Disease Branch of the 

Extramural 

Research Program, NEl 

Christina Sax Ph.D. Senior Staff Fellow, 

LMDB, NEl 



Objectives 

The objective of this project is to disrupt the 
a-crystallin genes (aA and aB) in the mouse 
to study their effect on normal lens and eye 
development. Disruption of the genes will 
essentially delete these proteins from the 
mouse and enable us to analyze the effects 
these proteins have on expression of other 
lens proteins, developmental regulation and 
morphology of the lens and other eye struc- 
tures, and the role of these proteins in nonlen- 
ticular tissues. 

Methods 

Standard molecular biology techniques were 
used to clone the a-crystaUin genes from a 
mouse 129SV genomic library and construct 
"gene knockout" vectors. Disruption of the 
genes will be accomplished by the now stan- 
dard technology of homologous recombination 
in pluripotent mouse embryonic stem cells, 
followed by insertion of the genetically altered 
cells into blastocyst mouse embryos to gener- 
ate chimeric mice with the gene disruption. 
Chimeric knockout mice wiU be bred to gener- 
ate mice with heterozygous and homozygous 
knockout mutations. 

Major Findings 

Gene Knockouts. Construction of aA-crystaUin, 
aB-crystaUrn, and aA-CRYBPl gene knockout 
vectors has been completed. These vectors 
contain large pieces of the respective gene 
functionally disrupted by insertion of the 
selectable marker for neomycin resistance and 
flanked by the negative selectable marker. 



HSV tk. Use of these positive and negative 
selectable markers to facilitate selection of 
appropriately modified cells is currently 
standard procedure in this field. Following 
electroporation of the aA-crystaUin knockout 
vector into Jl mouse embryonic stem cells and 
selection with G418 and ganciclovir, approxi- 
mately 200 colonies were picked. PCR and 
Southern blot analysis of these clones to detect 
correct homologous recombination is currently 
under way. PreUminary analysis indicates that 
11 of the first 96 clones screened contain the 
appropriate aA-crystaUin gene knockout. 

The technology for incorporating the 
modified cells into intact mice is being mas- 
tered in our laboratory. We have successfully 
generated chimeric mice by microinjecting 
unmodified ES cells into mouse blastocysts 
and allowing the embryos to develop in 
pseudopregnant foster mothers. Several 
chimeras have been bom in which ES ceUs 
contribute 10 to 90 percent of the cells in the 
mouse (estimated by coat color of the mice). 
We are also developing the newer method of 
creating chimeric mice by simple aggregation 
of morula stage embryos with clumps of ES 
cells. 

Sequence of the Mouse oA-crystallin locus. 
Approximately 12.7 kb of the 15 kb aA-crys- 
tallin locus has been sequenced on both 
strands. 

Functional Significance of Sequences Flanking 
the Mouse oA-Crystallin Gene. We are con- 
structing vectors containing portions of the 
aA-crystaUin gene locus to search for possible 
regvdatory elements located far from the 
promoter region. A basic promoter vector 
containing the mouse aA-crystallin promoter 
(-366 to +46) fused to the bacterial chloram- 
phenical acetyltransferase gene (CAT) reporter 
gene has been constructed. In transient trans- 
fection assays with the N/N1003 rabbit lens 
epitheUal cell hne, this construct eUcits signifi- 
cantly higher levels of CAT activity than the 
corresponding promoterless vector. 

Several large pieces of the aA locus have 
been inserted into the basic promoter, and 



135 



FY 1994 NEI Annual Report 



after completion of several additional con- 
sti-ucts, all of the constructs will be tested in 
the transient transfection assay for modulation 
of promoter activity. 

Significance to Biomedical Researcii and the 
Program of tfie Institute 

Deletion of the a-crystallin proteins, individu- 
ally or together, will provide a fundamental 
understanding of how these proteins function 
during normal lens development and how 
they may influence the structure and function 
of the lens and the entire eye. Additionally, 
it would give us insight into the function of 
these proteins in nonlenticular tissues that in 
turn could help us understand some of their 
more subtie roles in the eye. 

Proposed Course 

(1) Continue the gene knockout experiments 
in embryonic stem ceUs wdth the two addition- 
al knockout vectors (aB-crystaUin and aA- 
CRYBP). Produce chimeric mice from appro- 
priately modified ES cells for each of our three 
selected genes, and mate these mice to pro- 
duce lines of knockout mice for investigation. 
Deletion of a single allele of either aA or aB 
wdll be useful in assessing gene dosage effect 
(50 percent reduction of protein), and breeding 
to homozygosity (deletion of both alleles) will 
allow us to study the consequences of com- 
plete absence of the individual protein. It will 
be extremely interesting to mate eventually 
aA and aB knockout mice to produce mice 
which are totally devoid of a-crystallin. 



(2) Continue collaborative sequencing the 
aA-crystallin gene locus. Although a consid- 
erable amount is known about regulation of 
the mouse aA-crystallin gene, the complete se- 
quence of the gene has not yet been deter- 
mined. The complete sequence of the gene 
and flanking regions will be beneficial for 
designing and interpreting experiments with 
this gene. 

(3) Continue construction of vectors that 
wiU be used in transient transfection assays to 
locate regulatory elements spatially distant 
from the promoter of the aA-crystallin gene. 
This along with the sequence of the locus will 
help to identify potential sites influencing 
levels of gene expression. 

NEI Research Program 

Lens and Cataract — Lens Development and 
Aging 



236 



PROJECT NUMBER 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



ZOl EY 00291-01 LMDB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Transgenic Animal Models 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Eric Wavi^rousek Ph.D. Research Biologist LMDB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Molecular and Developmental Biology 



SECTION 

Section on Transgenic Animal and Genome Manipulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.2 



PROFESSIONAL: 



0.2 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Transgenic mice offer a unique tool for studying normal and pathological states associated with expression, 
overexpression, or misexpression a particular gene. Aberrant expression of genes believed to be involved in 
disease can be easily achieved with the well-established transgenic methodologies. The resulting transgenic 
mice often may be used as models for studying diseases associated with the respective gene. We have 
developed a line of transgenic mice that expresses a modified form of mature human interleukin (IL)-ip in 
the ocular lens. The afflicted mice exhibit a progressive inflammation of the eye and neovascularization of 
many eye tissues resulting in the eventual destruction of the eye. IL-ip messenger ribonucleic acid (mRNA) 
and protein have been detected at high levels in the eye. There is upregulation of vascular adhesion molecules 
and significant influx of inflammatory cells, predominantly macrophages. A systemic unresponsiveness to IL- 
1-mediated events has been observed in these mice. 



237 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 



Chi-Chao Chan 



Igal Gery 



James Lai 



MD. 



Ph.D. 



Chief, 

Immunopathology 
Section, Immunology 
Section, NEI 
Chief, Experimental 
Immunology Section, 
NEI 

Howard Hughes 
Medical Institute 



Objectives 

The objective of this project is to create trans- 
genic mouse models of ocular disease by aber- 
rantiy expressing proteins believed to be in- 
volved in maintenance of normal state or 
initiation and potentiation of a pathological 
condition. Our first model involves expres- 
sion of human interleukin (hIL)-l(3 in lens of 
transgenic mice to generate an abundant 
supply of identical mice afflicted with an 
ocular inflammatory disease of defined origin. 
These mice can be used to study the many 
parameters associated with progression of the 
inflammation and test therapeutic regimens 
for controUing the inflammation and conse- 
quent ocular damage. 

Methods 

Standard methods were used to construct the 
transgene with the murine aA-crystallin 
promoter driving expression of a cassette 
containing the human tissue plasminogen 
activator secretion signal fused in frame to the 
coding region of mature human IL-ip. Trans- 
genic mice were made by microinjecting the 
transgene construct into FVB/N single cell 
embryos. Mice used in this study were Fj 
hybrids of the transgenic FVB/N and DBA/ 2. 
These mice are heterozygous for a congenital 
retinal degeneration found in the FVB/N 
strain and have histologically normal retinas. 



Major Findings 

One line of transgenic mice was generated 
containing the IL-ip construct. This Une con- 
tains a single complete copy of the transgene. 
Large amounts of hIL-ip have been detected 
in the eyes of these mice both at the messen- 
ger ribonucleic acid (mRNA) and protein 
levels but has not been detected in other 
tissues (mRNA by Northern analysis) nor in 
serum (protein by enzyme-Unked immuno- 
sorbent assay). The afflicted mice exhibit a 
progressive ocular inflammation accomparued 
by neovascularization, resulting in the destruc- 
tion of the eyes in adult mice. The inflamma- 
tory infiltrate consists predominanfly of mac- 
rophages with some polymorphonuclear 
neutrophils and Ijonphocyte involvement. 
Increased expression of the vasctilar adhesion 
molecule intercellular adhesion molecule 1 
was evident in eyes of these nuce. Nontrans- 
genic litter mates exhibit none of these charac- 
teristics. The transgenic mice are otherwise 
healthy exhibiting normal reproductive capaci- 
ty and longevity. 

A systemic unresponsiveness to IL-1 
mediated events was observed in the trans- 
genic mice. They showed a decreased suscep- 
tibility to the toxic effects of Upopolysaccha- 
ride injection. Injection of 40;u-g per gram 
body weight resulted in lethaUty in only 6.3 
percent of transgenic mice compared with 86 
percent lethality in nontransgenic mice. 
Thymocj^es isolated from the transgenic mice 
were also less responsive to IL-1 in culture 
than those isolated from normal litter mates. 
The mechanism by which this systemic effect 
is induced is currentiy unknown. 

Significance to Biomedical Research and the 
Program of the Institute 

This model is the first instance of successful 
creation of a transgenic mouse containing the 
potent cytokine IL-1. This model provides a 



238 



readily accessible stock of identical mice 
exhibiting a consistent pattern of ocular in- 
flammation beginning with nearly normal eyes 
at four days of age, progressing with age, and 
resulting in destruction of the eye in adults. 
The roles of other cytokines, cellular adhesion 
molecules and arachidonic acid metabolites in 
progression of the ocular inflammation can be 
studied as can the estabUshment of the sys- 
temic unresponsiveness to IL-1. 

Proposed Course 

(1) Continue characterization of the IL- 
ip transgenic line. Quantitate mRNA levels 
for mouse IL-1, IL-1 receptor and receptor 
antagonist in the eyes of affected mice. Study 
alterations in cytokine expression patterns 
and arachidonic acid metabolites in these 
mice. 



Laboraton/ of Molecular and D evelopmental Biology 

(2) Attempt to ameUorate the inflamma- 
tion in this model by administration of anti- 
inflammatory drugs or antibodies to cytokines 
or cellular adhesion molecules. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 



239 



Laboratory of Ocular Therapeutics 



Report of the Chief 
Laboratory of Ocular Therapeutics 



Peter F. Kador, Ph.D. 



The Laboratory of Ocular Therapeutics 
(LOT) focuses on the development, 
evaluation, and mechanism of action of 
new ophthalmic drugs to treat eye diseases. 
The LOT research team is examining aldose 
reductase inhibitors (ARl) and anticataract 
agents. In pursuing the development of more 
effective and less toxic ARls, the efforts are 
progressing the development of an inhibitor 
urvrelated to previous ARIs. At present, a new 
inhibitor is being developed using biochemi- 
cal, pharmacological, and computer molecular 
design techniques. Studies designed to eluci- 
date the specific mechanism (s) of how ARIs 
diabetic compUcations are also being conduct- 
ed. In studies using galactose-fed dogs, LOT 



investigators have estabUshed that retinal 
changes associated with diabetic retinopathy 
progressed to the proliferative stage and that 
the dog represents the first animal model to 
demonstrate clinical and histological changes 
found in all stages of retinopathy. Studies are 
now focused on the development of prolifera- 
tive retinopathy in long-term galactose-fed 
dogs. Biochemical changes observed in these 
in vivo studies are being investigated using in 
vitro tissue culture techniques. Biochemical 
turnover in these cells is being monitored 
through nuclear magnetic resonance. Magnet- 
ic resonance imaging techniques are also being 
used to measure in vivo ARI efficacy. 



243 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00003-21 LOT 



PERIOD COVERED 



October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit or} one line between the borders.) 

Phar macology of Ocular Complications 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 

Others: 



Peter F. Kador 
Ping Ding 
William Greentree 
Petra Lachner 
Yong Lee 
Martin Lizak 
Heike Neuenschwander 
Irina Obrosova 
Libaniel Rodriguez 
Katsumi Sugiyama 



Ph.D. 

Ph.D. 

D.V.M. 

D.V.M. 

Ph.D. 

Ph.D. 

M.D. 

Ph.D. 

Ph.D. 

Ph.D. 



Chief 

Visiting Fellow 
IRTA 
IRTA 

Staff Fellow 
Staff Fellow 
Special Volunteer 
Visiting Scientist 
Staff Fellow 
Staff Fellow 



LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Ocular Therapeutics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



7.69 



PROFESSIONAL: 



7.69 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



Ix| (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Events leading to the onset of various ocular complications are being investigated. Specifically, the role of 
the enzymes aldose reductase and aldehyde reductase in the onset and progression of retinopathy, cataract, 
keratopathy, pupil function changes, and iris and ciliary process structure changes associated with diabetes and 
galactosemia are being studied. In addition, methods for either delaying or preventing the onset and 
progression of these complications through the pharmacological control of these enzymes are being developed. 

Events leading to the formation of several types of cataracts are also being investigated, as well as methods 
for controlling the onset of these cataracts through pharmacological intervention. 



244 



PHS 6040 (Rev. 5/92) 



Laboraton/ of Ocular Therapeutics 



Project Description 

Additional Personnel 



Robert Balaban 



Duane Miller 



PhD. 



Ph.D. 



National Heart, Lung, 
and Blood Institute 
University of Tennessee 
College of Pharmacy, 
Memphis, Tennessee 



Objectives 

To gain insight into the mechanisms by which 
polyol-induced ocular diabetic complications 
and cataracts are formed and to develop 
methods for their regulation. 

Methods 

Diabetes can be experimentally induced in 
animals by injecting streptozotocin. Diabetes- 
related complications linked to the sorbitol 
pathway can also be induced in animals such 
as rats and dogs by feeding them a 
galactose-enriched diet. Cataract formation 
and clinical retinal changes in experimental 
animals can be monitored through fundus 
photography. Biochemical studies used for 
the purification of enzymes include column 
chromatography, polyacrylamide gel electro- 
phoresis, isoelectric focusing, chromatofocus- 
ing, and high-pressure Uquid chromatography. 
Polyol levels were determined by gas-Uquid 
chromatography. Immunological analyses 
include the use of enzyme-linked immuno- 
sorbent assay, radioimmunoassay. Western 
blots, and immunohistochemical techniques 
using the coupled antibody DAB-PAP tech- 
nique. Computational methods for enzyme 
analysis, inhibitor structure-activity-studies, 
and pupil-function changes require the use of 
the National Institiates of Health (NIH) 
PROPHET computer system and Charm and 
Quanta Computer Systems from Molecular 
Design. 

Major Findings 

Biochemical Studies. Studies on defining the 
inhibitor site of aldose reductase and aldehyde 



reductase and the development of new aldose 
reductase inhibitors (ARI) are continuing. To 
facilitate the rational development of more 
specific and potent ARJs, more specific knowl- 
edge of the structural and pharmacophoric 
requirements of the site at which ARIs interact 
is required. It has recently been reported that 
cocrystallization of human placental aldose 
reductase with the inhibitor zopolrestat result- 
ed in a complex where the inhibitor was 
almost completely sequestered in the large, 
deeply eUiptical hydrophobic pocket that 
forms the substrate site. Zopolrestat's ob- 
served location, which makes the active site 
pocket inaccessible to solvent or further pro- 
ductive binding of substrate, does not support 
stiucture activity relationships (SAR), observa- 
tions, and kinetic results, which indicates that 
ARIs such as zopolrestat are either noncom- 
petitive or uncompetitive inhibitors. Using an 
5-iodoacetamido analog of the alrestatin as an 
affinity-labeled ARI, we have located an 
alternative site of interaction on aldose reduc- 
tase. The affinity-labeled ARI was irreversibly 
bound to purified rat lens aldose reductase, 
and the bound residues were identified by 
mass spectrometry. 

ModeUng studies of the identified site 
were subsequentiy conducted on the protein 
structure of aldose reductase, calculated by 
Charm force field using the crystal structure 
coordinates of human placental aldose reduc- 
tase pubhshed in the Brookhaven Protein Data 
Bank. Potential interactions of inhibitors at 
this site were estimated through docking and 
binding studies using Quanta 3.3. The appar- 
ent ARI site is composed of a single region 
independent of the substrate binding site. It 
contains a number of pharmacophoric ele- 
ments previously proposed for the inhibitor 
site. These include the amino acids arginine, 
serine, and tyrosine; proline; and a lipophilic 
area containing hydrogen bonding substi- 
tuents. Multiple three-point attachments that 
include possible nucleophiUc interactions with 
either serine or tyrosine are possible. Also 
present are two carboxylate binding groups 
that can orient the inhibitor on the hpophUic 
binding site. This proposed inhibitor site 
contains a number of pharmacophoric ele- 



245 



FY 1994 NEI Annual Report 



ments previously proposed for the inhibitor 
site, and its location and composition are 
consistent with reported kinetic data, SAR 
observations, stereochemical reqmrements, 
and quantum chemical calculations. 

Several reports suggest that a metabolite 
of S-88-0773 (4-[4-N,N,-Dimethylsulfamoyl)- 
piperazino] -2-methylpyrimidine) increases 
sugar alcohol levels in normal and diabetic 
rats and, as a result, speeds up the appearance 
of the polyol-induced complications. Others 
report that the compound slows down the 
appearance of complications by altering redox 
states of NAD (P) -couples through inhibition of 
sorbitol dehydrogenase. We have used this 
compound to investigate its effect on cataract 
formation. For these studies, young (50g) 
streptozotocin-induced diabetic rats received 
normal diet with or without 0.06 percent S-88- 
0773 while similar-aged nondiabetic rats 
received either normal, 20 percent, 30 percent, 
or 50 percent galactose diets with or without 
0.06 percent S-88-0773. 

Cataract progression was monitored week- 
ly by portable slit-lamp microscopy. Arumals 
were periodically killed and polyol levels in 
the lenses evaluated. Polyol levels were 
increased in both galactosemic and diabetic 
rats, and no difference in lens polyol levels 
between each corresponding group of rats 
tieated with or without drug was observed in 
the galactose-fed groups. Increases in tissue 
polyol levels associated with sorbitol dehydro- 
genase inhibition were not anticipated in 
galactose-fed rats because galactitol is not 
metaboUzed by sorbitol dehydrogenase. 
Nevertheless, sugar cataract formation was 
delayed in both diabetic and galactose-fed rats 
treated with S-88-0773. These results suggest 
that S-88-0773, or its active metaboUte, delays 
sugar cataract formation through a biochemi- 
cally unknown mechanism not related to 
either sorbitol dehydrogenase or ARI. 

Magnetization transfer contrast (MTC) 
enhancement, which generates high-contrast 
images based on tissue characteristics result- 
ing from the interaction of water and macro- 
molecvdes, was applied to document in vivo 



cataract formation and other structural chang- 
es associated with diabetic eye compUcations. 
For these studies, male beagles ranging from 
six to 24 months of age were fed a diet con- 
taining 30 percent galactose. All dogs were 
then placed in a General Electric 2-T Omega 
magnetic resonance imaging system under 
anesthesia with muscle relaxant at four-week 
intervals. M^ and Mq images were acquired 
using gradient-recalled-echo sequences, with 
and without the saturation pulses, respective- 
ly, consisting of rf -irradiation 10 kHz off- 
resonance from the free-water proton signal. 
The Tj33, images were calculated from the data 
using a one-shot Tj-imaging sequence. 

The acqviired images were compared with 
photographs obtained with sUt-lamp microsco- 
py and retioillumination photography. Excel- 
lent images detailing the fine structures of the 
anterior segment that includes the lens, cor- 
nea, iris, dliary body, choroid membrane, and 
Schlemm's canal were obtained by MTC. In 
these images the progression of osmotic cata- 
ract formation in the galactose-fed dogs could 
be followed from the initial appearance of 
distinct cortical vacuoles. Distinct fluctuations 
in lens size and shape were observed as lenses 
progressed to the more advanced cataractous 
stages. 

"F NMR spectroscopy is also being used 
to measure in vivo aldose reductase activity in 
the dog lens by measuring the conversion of 
3-deoxy-3-fluoroglucose to 3-deoxy-3-fluoro- 
sorbitol. This work is an extension of the in 
vivo evaluation of aldose reductase activity in 
rabbit lenses. Iiutial spatial coordinates for 
lenses are calctilated from ^H-images deter- 
mined on a 2.0 Tesla GE Omega-CSI spec- 
trometer. The SLOOP-technique (Spectral 
Localization with Optimal Pointspread func- 
tion) is then used vdth a proton decoupler to 
measure the accumulation of sorbitol in the 
rabbit lens. A double spin-echo sequence is 
used with selective excitation and refocusing 
pulses and with optimized phase-encoding 
gradient pulses using one-second repetition 
times and 25 millisecond echo times. SLOOP 
experiments indicate that 3-deoxy-3-fluorosor- 
bitol can be observed in spectra of the anterior 



246 



hahoratory of Ocular Therapeutics 



portion of the lens when adequate amounts of 
3-deoxy-3-fluoroglucose are administered. 

Retinal Studies. Vascular changes associ- 
ated with diabetic retinopathy can be experi- 
mentally produced in beagles fed a 30 percent 
galactose diet. In studies designed to clarify 
the initiating lesions and progression of dia- 
betic retinopathy, we have documented the 
progression of retinal lesions from background 
through the proliferative stage in the dog with 
ophthalmoscopic, fluorescein angiographic, 
and histopathologic findings. Initial retinal 
changes include aldose reductase-Unked 
formation of pericyte ghosts and subsequent 
development of aceUular capillaries, micro- 
aneurysms, and intraretinal hemorrhages. This 
early retinopathy progresses to include the 
appearance of occluded vessels, areas of 
nonperfusion, and intraretinal microvascular 
abnormalities. Finally, proUferative retinopa- 
thy develops, including the formation of 
fibrovascular membranes seen histologically 
on both the retinal surface and the posterior 
hyaloid membrane. 

Diabetes-like microvascular changes in 
galactosemic beagles have been shown to be 
arrested in a dose-dependent manner by ARIs. 
To determine if retinal changes also can be 
reduced through the marked reduction of 
galactitol production after early retinal lesions 
have developed, galactose diet was removed 
after either the appearance of pericyte ghosts 
or microaneurysm formation. The subsequent 
progression of retinal changes over 24 months 
was then quantitively compared with retinal 
changes in remaining galactose-fed dogs. For 
these studies, 10 control dogs were fed a 
normal diet, while 50 dogs were fed diet 
containing 30 percent galactose. The galactose 
diet was removed from 15 dogs after 24 
months, at which time pericyte ghosts had 
developed; another 15 dogs were removed 
after 31 months when microaneurysms had 
developed. Eighteen remained on galactose 
diet. Beginning at 24 months, four to five 
eyes from each experimental group and two to 
three eyes from the control group were enu- 
cleated at six-month intervals. Isolated retinas 
were quantified as previously described (/ 



Ocular Pharmacol 9:257, 1993). Significant 
increases in the endotheUum/ pericyte ratio 
and decreases in pericyte density were ob- 
served with the duration of galactose-feeding. 
Although no reversal of retinal lesions oc- 
curred, differences in the progression of reti- 
nal lesions between the galactose-fed and 
galactose-removed groups became evident 
after 12 to 24 months. This study suggests 
that reduction of polyol accumulation at the 
initial stages of background retinopathy bene- 
ficially reduces the progression of retinal 
lesions. 

Corneal Studies. Specular microscopic 
studies indicate that the size (polymegathism) 
and shape (pleomorphism) of the hexagonal 
corneal endotheUal cells change in diabetics. 
Similar morphometric changes of the corneal 
endotheUum have also been experimentally 
observed in diabetic rats as well as in diabetic 
and galactose-fed dogs, and concomitant 
administration of ARIs can reduce these 
morphological changes. The purpose of this 
study was to examine whether corneal endo- 
thelial changes in galactose-fed dogs are 
reversible by the marked reduction of galac- 
titol production after stopping prolonged 
galactose feeding. Ten control dogs were fed 
a normal diet, while 48 dogs were fed a diet 
containing 30 percent galactose. The galactose 
diet was removed from 15 dogs after 24 
months, at which time pericyte ghosts in the 
retina had developed; another 15 dogs were 
removed after 31 months when retinal micro- 
aneurysms had developed. Eighteen dogs 
remained on galactose diet throughout the 
study. 

Specular microscopy was conducted on 
members of all groups after 38 months of 
study, and the photographs were analyzed in 
masked fashion on the Bambi image analysis 
systems. The evaluation of the corneal endo- 
theUal ceUs revealed significant differences in 
the ceU size and density among aU galactose- 
fed dogs and normal, age-matched control 
dogs. Corneal endotheUal changes were not 
significantiy reduced in dogs fed galactose for 
either 24 or 31 months and then receiving 
normal diet for 14 and seven months, respec- 



247 



FY 1994 NEI Annual Report 



lively, indicating that amelioration of endothe- 
lial cell changes requires therapy before the 
advent of endothelial morphologic changes. 

Significance to Biomedical Research and tt)e 
Program of the Institute 

Loss of vision from cataract and diabetic reti- 
nopathy is significant; therefore, methods for 
the pharmacological control of these ocular 
compUcations are required. We have devel- 
oped an animal model that demonstrates 
advanced retinal vessel changes that are 
virtually clinically and histologically identical 
to those observed in advanced diabetic reti- 
nopathy. Our present studies in dogs demon- 
strate for the first time that loss of retinal 
pericytes, associated with aldose reductase, 
initiates retinal changes associated with both 
background and advanced diabetic retinopa- 
thy and that administration of ARIs in preven- 
tion studies can ameliorate the loss of peri- 
cytes and subsequent development of micro- 
aneurysms and retinal hemorrhages in a dose- 
dependent manner. The successful develop- 
ment of noninvasive methods for monitoring 
aldose reductase activity by nuclear magnetic 
resonance procedures may directiy affect 
ongoing and plarmed clinical trials where this 
procedure could serve as a quantitative indica- 
tor of drug efficacy. Cataract is also one of 
the major causes oif blindness in the develop- 
ing world. In addition, loss of vision due to 
cataract is one of the major health problems of 
both the diabetic and the aging populations in 
the United States. 

Proposed Course 

These studies will be continued. Discovered 
ARIs will be pharmacologically evaluated and 
developed. The inhibitor site will be further 
probed through the use of affinity labels so 
that more potent and specific inhibitors may 
be developed. Studies on the mechanisms 
through which aldose reductase induces 
diabetic compUcations in various tissues will 
be continued. 



NEI Research Program 

Retinal Disease — ^Diabetic Retinopathy, Sickle 
Cell Retinopathy, and Other Vascular Abnor- 
malities 

Lens and Cataract — Pathogenesis of Cataract 

Publications 

Greentree W, Takahashi Y, Wyman M, Kador 
PF: Quantitative analysis of retinal vessel 
changes in galactose-fed dogs: Intervention 
studies. Invest Ophthalmol Vis Sci 35(4):1589, 
1994. 

Kador PF: Biochemistry of the lens: Interme- 
diary metabolism and sugar cataract forma- 
tion, in Viola E, Dowling J (eds). Principals and 
Practice of Ophthalmology. New York, Basic 
Sciences, 1994, pp 146-167. 

Kador PF, Lee YS, Rodriguez L, Carper D, 
Bartozko-MaHk A, ParmeU L: Characterization 
of the aldose reductase inhibitor site. Invest 
Ophthalmol Vis Sci 35(4):2152, 1994. 

Kador PF, Takahashi Y, Schaffhauser M: 
Vorbeugung diabetischer Komphkationen im 
Auge mit Aldosereduktase-Hemmern. Diabe- 
tes und Stojfwechsel, in press. 

Kador PF, Takahashi Y, Sato S, Wyman M: 
Amelioration of diabetes-Uke retinal changes 
in galactose-fed dogs. Prev Med, in press. 

Kador PF, Takahashi Y, Wyman M, Ferris F 
ni: Diabetes-Uke proliferative retinal changes 
in galactose-fed dogs. Arch Ophthalmol, 
110:1295-1302, 1992. 

Lee YS, Peralstein R, Kador PF: Moleciilar 
modeling of aldose reductase inhibitors. / 

Med Chem 8(6):787-792, 1993. 

Li Q, Lopez JS, Caspi RR, Nussenblatt RB, 
Kador PF, Chan C-C: Suppression of S-anti- 
gen induced experimental autoimmune uveo- 
retinitis in Lewis rats by oral administration 
with cgs-13080, a thromboxane synthetase 
inhibitor. Exp Eye Res 57:601-608, 1993. 



248 



Laboratory of Ocular Therapeutics 



Lizak MJ, Ceckler TL, Balaban RS, Kador PF: 
In vivo measurement of magnetization trans- 
fer in galactosemic dog lens. Proceedings of 
the Society of Magnetic Resonance 1994 1:205, 
1994. 

Lizak MJ, Mori K, Ceckler TL, Kador PF, 
Balaban RS: Magnetic resonance imaging of 
the galactosemic dog eye using magnetization 
transfer contrast enhancement. Invest Ophthal- 
mol Vis Sci 35(4):1948, 1994. 

Mori K, Takahashi Y, Tsuduki S, Kador PF, 
Akagi Y: Significance of aldose reductase to 
experimental corneal epitheliopathy. Invest 
Ophthalmol Vis Sci 35(4):1946, 1994. 

Neuenschwander H, Julia C, Wyman M, 
Kador PF: Corneal endothelial changes in 
galactose-fed dogs. Invest Ophthalmol Vis Sci 
35(4):1601, 1994. 

Obrosova I, Inoue J, Greentree W, Sato S, 
Rodriguez L, Kador PF: Evaluation of s-88- 
0773 on sugar cataract formation. Invest 
Ophthalmol Vis Sci 35(4):1931, 1994. 

Ogawa K, Yamawaki I, Matsusita Y, Nomura 
N, Kador PF, Kinoshita JH: Synthesis of 
substituted2,4-dioxo-thienopyrimidine-l-acetic 
adds and their evaluation as ARIs. Eur J Med 
Chem 28:769-781, 1993. 



Sato S, Kador PF: Retinal changes in arumal 
diabetic models. Diabetes Frontiers 5:108-112, 
1994. 

Sato S, Old S, Carper D, Kador PF: Purifica- 
tion and characterization of recombinant 
human placental and rat lens aldose reduc- 
tases expressed in Escherichia coli. Adv Exp 
Med Biol, in press. 

Secchi EF, Lizak MJ, Sato S, Kador PF: Pres- 
ence of polyol pathway in fibroblast. Invest 
Ophthalmol Vis Set 35(4):1589, 1994. 

Takahashi Y, Augustin W, Wyman M, Kador 
PF: Quantitation of retinal vessel changes 
associated with diabetic retinopathy in galac- 
tose-fed dogs. Ocular Pharmacology 9:257-269, 
1993. 

Takahashi Y, Wyman M, Kador PF: Retinal 
vascularization in galactose-fed dogs. Invest 
Ophthalmol Vis Sci 35(4):1734, 1994. 

WaldbiUig RJ, Jones BE, Schoen TJ, 
Heidersbach S, Bitar MS, Van Kuijk FJGM, de 
Juan E, Kador PF, Chader GJ: Vitreal insulin- 
like growth factor binding proteins (IGFBPs) 
are increased in human and animal diabetics: 
ImpUcations for understanding diabetic reti- 
nopathy. Current Eye Res 13:539-546, 1994. 



249 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00275-03 LOT 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

NADPH Reductases and Polyol Pathway in Ocular Complications 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Sanai Sato M.D., Ph.D. Visiting Scientist LOT, NEI 



Others: 



Peter F. Kador 
E. Filippo Secchi 



Ph.D. 
Ph.D. 



Chief 
Fogarty Fellow 



LOT, NEI 
LOT, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Ocular Therapeutics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



2.05 



PROFESSIONAL: 



2.05 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

In diabetes the increased flux of glucose into the polyol pathway results in the accumulation of the sugar 
alcohol sorbitol that is linked to the onset of various diabetic complications, such as cataract formation and 
retinopathy. The sugar alcohol formation and properties of NADPH-dependent reductases in fibroblasts, the 
key cells in the formation of fibrous proliferative tissues, has been investigated. 



250 



PHS 6040 (Rev. 5/92) 



Laboratory of Ocular Vierapeutics 



Project Description 

Objectives 

Glucose is metabolized into fructose through 
sugar alcohol sorbitol in sorbitol pathway. In 
diabetes the increased flux of glucose into the 
polyol pathway results in the accumulation of 
sugar alcohol sorbitol. Experimental evidence 
has demonstrated the link between the accu- 
mulation of sugar alcohols and the selective 
loss of pericjiies, the initial lesion of diabetic 
retinopathy. In the more advanced stages of 
retinopathy, fibroblasts play a key role in the 
formation of fibrous proliferative tissues. This 
project is designed to investigate whether the 
sugar alcohol accumulation is also linked to 
the formation of proliferative tissues. 

Methods 

Biochemical techniques include affinity chro- 
matography, chromatofocusing, isoelectric 
focusing, electrophoresis, immunoblot and gas 
chromatography for sugar analysis. "F NMR 
with 3-deoxy-3-fluoro-D-glucose (3FDG) is 
used to investigate the metabolism of glucose. 
The National Institutes of Health (NIH) 
Prophet computer system is used for kinetic 
analysis and IC50 calculations. 

Major Findings 

The crude extract of murine fibroblast cell line 
L929 cells displayed reductase activity with 
DL-glyceraldehyde as substrate and dehydro- 
genase activity with D-sorbitol as substrate, 
suggesting that these cells contain two en- 
zymes of the polyol pathway: aldose reduc- 
tase (and /or aldehyde reductase) and sorbitol 
dehydrogenase. A nuclear magnetic reso- 
nance study with 3FDG confirmed the conver- 
sion of glucose into fructose through sorbitol 
in mouse fibroblast ceU line L929. Sugar 
alcohol accumulated in the cells cultured in 
medium containing 30 mM galactose, and this 
accumulation was inhibited by aldose reduc- 
tase inhibitors (ARI). Purification studies 
demonstiated that the dominant reductase in 
L929 cells was aldehyde reductase rather than 



aldose reductase. Two other fibroblast cell 
Unes estabUshed from dog and human also 
appeared to possess aldehyde reductase as the 
dominant reductase in these cells. 

These results confirm the presence of the 
polyol pathway in three different fibroblast 
cell Hnes. However, aldehyde reductase 
rather than aldose reductase predominates in 
these cells. 

Significance to Biomedical Research and the 
Program of the Institute 

Diabetic retinopathy is a leading cause of 
blindness. The development of potent ARIs is 
of clinical significance in preventing bUndness 
associated diabetes. Animal studies have 
demonstiated that ARIs prevent the selective 
loss of pericytes, the initial pathology of 
retinopathy. The observation that the polyol 
pathway is also present in fibroblast suggests 
that excess amounts of sugar alcohol may also 
play a role in the formation of proliferative 
tissues in advanced stages of retinopathy. 
This evidence provides additional rationales 
for the use of ARIs in the prevention and 
intervention of diabetic retinopathy. 

Proposed Course 

The evaluation of the polyol pathway v^ be 
continued with various tissues where diabetic 
changes occur. Cell culture techniques will be 
used to investigate retinal pericytes and 
endothelial cells as key cells in diabetic 
retinopathy. 

NEI Research Program 

Retinal Diseases — ^Diabetic Retinopathy, Sickle 
CeU Retinopathy and Other Vascular Abnor- 
malities 

Publications 

Obrosova I, Inoue J, Greentree W, Sato S, 
Rodrigues L, Kador PF: Evaluation of S-88- 
0773 on sugar cataract formation. Invest 
Ophtlmlmol Vis Sci 24(suppl):1931, 1994. 



251 



FY 1994 NEI Annual Report 



Sato S, Kador PF: Diabetic retinopathy. I. 
Retinal changes in animal models. Diabetes 
Frontier 5:108-112, 1994. 

Sato S, Old S, Carper D, Kador PF: Purifica- 
tion and characterization of recombinant 
placental and rat lens aldose reductases ex- 
pressed in Escherichia coli. Adv Exp Med Biol, 
in press. 



Schaffhauser MA, Sato S, Kador PF: NADPH- 
dependent reductases in dog thyroid: Compar- 
ison of a third enzyme "glyceraldehyde reduc- 
tase" to dog thyroid aldehyde reductase. Adv 
Exp Med Biol, in press. 

Secchi EF, Lisak MJ, Sato S, Kador PF: Pres- 
ence of polyol pathway in fibroblast. Invest 
Ophthamol Vis Sci 34(4 suppl):1589, 1994. 



252 



Laboratory of Retinal Cell and Molecular Biology 



Report of the Chief 
Laboratory of Retinal Cell and Molecular Biology 



Gerald J. Chader, Ph.D. 



Members of the Laboratory of Retinal 
CeU and Molecular Biology (LRCMB) 
elucidate new genes and biochemical 
mechanisms to better understand the underly- 
ing causes of ocular diseases. With this 
knowledge, we hope to intervene better in the 
disease process before substantial damage to 
vision has been done or to apply rational 
methods of gene therapy before the terminal 
stages of the disease have been reached. The 
approaches taken are molecular biological, 
molecular genetic, and studies on candidate 
genes. The following three areas are empha- 
sized: 

• Molecular Biology and Molecular Genetics 

• Gene Therapy of Retinal Diseases 

• Molecular Immunopathology 

Following are specific advances within these 
areas: 

Molecular Biology and Molecular 
Genetics 

(1) Retina-Specific Genes. Several genes that 
are predominantly or exclusively expressed in 
ocular tissues have been identified by subtrac- 
tive cloning. These include an important gene 
HIOMT that is involved in the maintenance of 
circadian rhythms within the eye. Retina- 
specific genes and genes located on the short 
arm of the X chromosome have been pinpoint- 
ed. These genes are being chromosomally 
localized to see if they are linked to eye dis- 



eases. Genomic cloning and sequencing are 
being done such that appropriate restriction 
fragment length polymorphisms and /or 
microsateUite repeats are generated to allow 
for disease testing. 

(2) Retinal pigment epithelium (RPE)- 
Specific Genes. Cloning of genes unique to 
RPE and its functioning is of importance 
because the RPE cell layer is critical for retinal 
homeostasis. A new 65 kDa protein of poten- 
tial importance has been isolated from the 
human RPE. The bovine and mouse genes 
have been cloned, allowing for study of tissue- 
specific expression. The gene is highly con- 
served, and its posttranslational expression is 
tightly regulated. This gene is the first RPE- 
specific gene to be reported and characterized. 
Knowledge of the 5' regulatory sequence 
should facilitate RPE-specific gene transfer and 
gene therapy. 

(3) Interphotoreceptor Retinoid-Binding 
Protein (IRBP). IRBP is an integral part of the 
visual cycle. Collaborative studies with Dr. 
Harris Ripps, from the University of Illinois at 
Chicago, on a model of experimental retinal 
detachment have demonstiated that IRBP is 
intimately involved in rhodopsin regeneration. 
We also have identified a protein homologue 
and cloned the homologous gene of human 
IRBP from the fiuitfly Drosophila melanogaster . 
The gene maps to an area of the Drosophila 
genome that is rich in mutants of ocular 
disease. We hope to pinpoint a specific hu- 
man population with similar characteristics 
and examine the gene for defects in specific 
human families. 



255 



FY 1994 NEI Annual Report 



(4) Pigment Epithelium-Derived Factor 
(PEDF). PEDF is synthesized by human RPE 
cells and may be import:ant in the develop- 
ment of retinal photoreceptors. PEDF induces 
the extension of elaborate neuronal processes 
from cultured retinoblastoma cells and, as 
such, is a neurotrophic protein. Because Y-79 
cells are thought to be derived from photore- 
ceptor cone cells, it is hoped that PEDF can be 
as effective on cone neuron development in 
vivo. Collaborative studies have also shown 
PEDF to be a neuron-survival factor. The 
clinical use of PEDF in retinal transplantation 
is thus a distinct possibility. The molecular 
biology of this potentially very important 
neurotrophic and neuron-survival protein is 
being studied for appUcation to retinal degen- 
erations. 



(2) Gene Therapy. Ribozymes are specifi- 
cally constructed ribonucleic acid species that 
can control expression of proteins v^thin cells. 
By linking these simplified gene forms to 
appropriate promoters and using a suitable 
transfer vector, new therapeutic modaUties can 
be constructed. Gene therapy can then be 
planned to treat autosomal dominant disor- 
ders that are unmanageable. Ribozyme con- 
structs for IRBP have been designed and are 
being studied in a transfected human retino- 
blastoma ceU system. Once perfected, ribo- 
zymes should be useful in conditions such as 
diabetic retinopathy and retinopathy of pre- 
maturity, in w^hich the disorders probably 
involve overexpression of normal proteins 
such as growth factors. 



(5) Fatty Add and Tubulin Defects in 
Retinal Degeneration. In collaborations with 
Dr. Muriel I. Kaiser, fatty acid uptake and 
metaboUsm in Bietti's crystaUine retinopathy 
and a tubulin acetylation defect in a form of 
atypical retinitis pigmentosa are being investi- 
gated in hopes of elucidating the specific 
defects. Sigr\ificant progress has been made in 
pinpointing the metabolic problems expressed 
in both these hereditary conditions. 



Gene Therapy of Retinal Diseases 

(1) Transgenic Studies. Transgenic studies 
can help to uncover factors contiolling gene 
activation in the embryonic period, specifically 
in retinal photoreceptor cells. Gene analysis 
systems in transgenic mice and in tiansient 
transfections in cultured human retinoblasto- 
ma cells have been established for IRBP. 
Much of the 5'-flanking region of IRBP has 
been determined, and enhancer elements 
necessary for expression are being defined 
through target mutagenesis studies. Tissue- 
and stage-specific elements, including TATA 
and CAAT boxes, are being exactiy defined as 
to retinal expression. This work is important 
so that specific molecules can be "gene-target- 
ed" to the retina with precision. 



Molecular Immunopathology 

(1) Immunopathology. Work with Dr. Igal 
Gery continues to study aspects of the IRBP- 
induced uveitis seen in models of human 
uveoretinitis. Studies in the human are also 
under way, with the final goal of contiolling 
or preventing at least some forms of uveitis in 
man. 

(2) Immunogenetics. With Dr. Rachel 
Caspi, an IRBP-mouse model for experimental 
autoimmune uveitis has been established that 
is very useful for studying the genetics of the 
disease and its relapsing characteristics. 

(3) Antigen Presentation. Collaborative 
work with Drs. Mark de Smet and Robert 
Nussenblatt has previously demonstrated the 
presence of a ceU-surface protein of B cells 
that specifically binds the major immunopath- 
ological determinant of IRBP. It is thought 
that this protein may function as a molecular 
chaperone in antigen presentation. Recentiy, 
three intracellular proteins fiom human B cells 
have been uncovered that bind specifically to 
the immunodominant, uveopathological epi- 
tope of the IRBP molecule. Two of these 
proteins are now known to be novel heat 
shock proteins. Elevation of serum antibody 
levels to HSP 70 was found to occur during 



256 



Lahoratou) of Retinal Cell and Molecular Biology 



ociilar inflammatory episodes in patients with 
Beghet's disease. These studies could lead to 
better diagnosis and treatment of forms of 
ocular uveitis and serve as a focus for gene 
therapy. 



157 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00070-17 LRCMB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit orj one line between the borders.) 

Vitamin A and Ocular Tissues 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Barbara Wiggert Ph.D. Head, Section on LRCMB, NEI 

Biochemistry 



Others: Kalpana Rengarajan 
R. Krishnan Kutty 
Todd Duncan 
Geetha Kutty 



Ph.D. 
Ph.D. 
M.S. 
M.S. 



Visiting Fellow 
Senior Staff Fellow 
Biologist 
Visiting Associate 



LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 



COOPERATING UNITS (if any) 

U. Lund, Sweden (T. van Veen, Ph.D.); U. Illinois Coll. of Med., Chicago (D. Pepperberg, Ph.D., T.-I. Okajima, Ph.D., H. Ripps, Ph.D.); 
Med. U.S.C. (R. Crouch, Ph.D., S. Hazard, Ph.D.); SLU Inst. F. Kir, Sweden (K. Narfstrom, D.V.M., Ph.D.); U. Hosp., Utrecht, The 
Netherlands (B. Zonnenberg, M.D., Ph.D.); Medical College of Georgia (S. Smith, Ph.D.); Emory Eye Center (J. Nickerson, Ph.D.) 



Laboratory of Retinal Cell and Molecular Biology 



SECTION 

Section on Biochemistry 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



5.0 



PROFESSIONAL: 



3.0 



2.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

In the toad retinal pigment epithelium (RPE) eyecup it was demonstrated that interphotoreceptor 
retinoid-binding protein (IRBP) promotes the formation, as well as the release, of ll-cis retinal. 

In the experimentally detached skate retina system, the introduction of ligand-free IRBP purified from bovine 
retina to the subretinal space significantly increased the rate of rhodopsin regeneration and more than doubled 
the amount of rhodopsin reformed in the darkness as compared with controls in which IRBP content of the 
subretinal space was diluted as a consequence of detachment and no purified IRBP was added back. 

In the vitiligo mouse model of retinal degeneration, elevated retinoid levels were found in both the RPE and 
the liver. These results represent the first evidence of biochemical malfunction in this mutant and suggest that 
in the eye, the RPE is the primary site of the defect that leads to retinal degeneration. 

A -70 kDa glycoprotein binding both retinoids and fatty acids was purified to homogeneity from Drosophila 
melanogaster heads. 

A method was developed, using reverse transcriptase-polymerase chain reaction (RT-PCR), for the quantitation 
of IRBP message in small amounts of tissue such as a single mouse pineal gland. 

Three intracellular human B-cell proteins (-40, 72, and 74 kDa) that bind specifically to peptide 1169-1191, 
an immunodominant, uveitopathogenic determinant of bovine IRBP, were partially characterized by 
immunoblotting and microsequencing. The 40 kDa protein was identified as actin, and the 72 and 74 kDa 
proteins were determined to be members of the hsp 70 family. The 72 kDa protein had 40 percent homology 
with human hsp 72 from lung fibroblasts, and the 74 kDa protein had a high degree of homology with human 
hsp 78 glucose-regulated protein from liver. Elevation of serum antibody levels to hsp 70 were found to occur 
during ocular inflammatory episodes in patients with Behcet's disease. 
258 



PHS 6040 (Rev. 5/92) 



Laboratory of Retinal Cell and Molecular Biology 



Project Description 

Additional Personnel 

Igal Gery PhI5. Head, Section on 

Experimental 
Immunology, LI, NEI 

Rachel Caspi PhD. Visiting Associate 

LI, NEI 

Tatiana Putilina PhD. Visiting Associate 
LRCMB, NEI 

Mark de Smet MD. Visiting Scientist 

LI, NEI 

Objectives 

The purpose of this research project is to 
investigate the role of specific retinoid-binding 
proteins such as interphotoreceptor retinoid- 
binding protein (IRBP) in mediating the action 
of retinoids in both normal and diseased 
ocular tissues. 

Methods 

Affinity chromatography, fluorescence spec- 
troscopy, high-performance liquid chroma- 
tography (HPLC), SDS-polyacrylamide gel 
electrophoresis. Western blotting. Northern 
blotting, reverse transcriptase-polymerase 
chain reaction (RT/PCR), slot-blotting, and the 
enzyme-Unked immunosorbent assay were 
used to study retinoid-binding proteins. 

Major Findings 

The formation of ll-czs retinal in the retinal 
pigment epitheUum (RPE) and its release to 
extracellular medium containing IRBP were 
studied in the RPE-eyecup of the toad. The 
results not only indicate that IRBP promotes 
the formation (from a]l-trans precursor) as well 
as the release of ll-cis retinal but also suggest 
the preferred use of recentiy incorporated and 
esteritied aR-trans retinol in the 11-ds retinal 
synthesis in a "last in /first out" manner. 

It has been shown that, compared with a 
normal eyecup preparation, the amount of 
rhodopsin regenerated and the rate at which 
it was resynthesized after bleaching were 



reduced about 50 percent when the skate 
retina was detached from its RPE and re- 
placed immediately on the apical surface of 
the RPE. 

In current studies, the detachment proce- 
dure was performed under fluid to dilute the 
IRBP content of the interphotoreceptor matrix. 
Fundus reflectrometry showed that allowing 
fluid to enter the subretinal space exposed by 
the detachment procedure caused profound 
deficits in both the rate and amount of rho- 
dopsin that regenerated after bleaching. The 
introduction of Ugand-free IRBP purified fiom 
bovine retina to the subretinal space signifi- 
cantiy increased the rate of rhodopsin regener- 
ation and more than doubled the amount of 
rhodopsin reformed in darkness. It appears 
that one important consequence of retinal 
detachment is the dilution of IRBP in the 
subretinal space, so that its replacement, 
particularly in cases of extensive rhegmato- 
genous detachments, may be beneficial for the 
recovery of retinal function in human patients. 

The vitiUgo mouse (C57BL/6-mi"7mi"") 
model of retinal degeneration exhibits a slow- 
ly progressing loss of photoreceptor cell nu- 
clei, gradual loss of rhodopsin, and unevenly 
pigmented RPE. Analyses of retinoid levels 
and distribution between the neural retina and 
RPE in these mutant mice showed that retinyl 
palmitate levels were significantiy higher in 
the mutant RPE than in control mice. Further- 
more, ah-trans retinol was elevated approxi- 
mately fourfold above controls in the RPE of 
vitUigo mice. To assess possible systemic 
involvement in vitiligo mice, retinoids were 
evaluated in liver and plasma. Plasma retinol 
levels were normal, but mean Hver total vita- 
min A levels in affected mice were approxi- 
mately 1.7 times greater than controls. Analy- 
sis of esterified and unesterified retinoids in 
Uver showed that retinyl palmitate levels were 
elevated. These results showing elevated 
retinoid levels in both the RPE and hver of the 
vitiligo mouse represent the first evidence of 
biochemical malfunction in this mutant and 
suggest that, in the eye, the RPE is the prima- 
ry site of the defect that leads to photorecep- 
tor cell degeneration. This study provides the 



259 



FY 1994 NEI Annual Report 



first evidence of altered systemic retinoid 
metabolism in vitiligo mice that is occurring, 
significantly, under normal dietary conditions. 

A glycoprotein binding both retinoids and 
fatty acids has been purified to homogeneity 
from Drosophila melanogaster heads. This 
protein, which has an apparent molecular 
mass of ~70kDa on SDS-PAGE, is similar to 
the 140 kDa IRBP in that both proteins are 
glycosylated, both exhibit endogenous cova- 
lent and noncovalent fatty acid binding, and 
both exhibit similar binding affinities for 16-[9- 
anthryloxy] palmitic acid. The Drosophila 
protein has a higher affinity for retinol than 
that of IRBP. 

A method for the radioanalytic estimation 
of amplification products generated by reverse 
transcription coupled to the polymerase chain 
reaction of the IRBP message in mouse retina 
and pineal using [a-^^P] was developed. This 
method allows the detection of the IRBP 
message in small amounts of tissue such as a 
single mouse pineal gland. Quantitation of 
the IRBP message using the G3PDH message 
as an internal standard was demonstrated. 

Peptide 1169-1191 is an immunodominant, 
uveitopathogenic determinant of bovine IRBP 
that causes severe ocular inflammatory disease 
in the Lewis rat. Three intraceUular-binding 
proteins, with apparent molecular masses of 
72 and 74 kDa, from Epstein-Barr virus-trans- 
formed human B cells (from both normal 
subjects and Beh^ets patients) and stimulated 
with Upopolysaccharide bind specifically to 
this peptide. These binding proteins could be 
released fiom the peptide with adenosine 
triphosphate (ATP), showing that all three 
contain an ATP-binding site. 

Partial characterization of these proteins 
by immunoblotting and microsequencing of 
peptides obtained by in situ digestion of 
specific protein bands demonstiated a 40 kDa 
protein to be actin and the 72 and 74 kDa 
proteins to be members of the heat shock 
protein (HSP) 70 family. The 74 kDa protein 
has a high degree of homology with human 
HSP 78 glucose-regulated protein from human 



Uver, whereas the 72 kDa protein has 40 
percent homology with a human HSP 72 from 
human lung fibroblasts and probably repre- 
sents a new member of the HSP 70 family. It 
was also shown in a corollary study that 
elevation of serum antibody levels to HSP 70 
occurred during ocular inflammatory episodes 
in Behgets patients. Large-scale purification of 
IRBP was continued for studies on the pro- 
duction of experimental autoimmune uveitis 
(EAU) in rats and mice and possible modes of 
suppression of the disease. 

Significance to Biomedical Researcti and the 
Program of the Institute 

Because of its importance in normal photore- 
ceptor cell physiology, i.e., in facilitating the 
transport of retinoids during the visual cycle 
as well as transport of fatty acids that are 
essential to normal function, abnormalities in 
IRBP function resulting from changes in 
concenfration, distribution, or affinity for 
retinoids or fatty acids could be important 
either directiy or indirectiy in visual cell 
pathogenesis. 

Proposed Course 

The physiological role of IRBP in the visual 
cycle, in particular, the mechanism by which 
it promotes the formation and release of 11 -cfs 
retinal, will continue to be probed. We will 
also be seeking to identify the epitopes on the 
IRBP molecule that bind retinoids and fatty 
acids as well as those that are required for 
eliciting the release of 11-ds retinal from the 
RPE. Continued studies on the vitiligo mouse 
model of retinal degeneration will assess 
retinoid turnover in Uver and RPE as well as 
the consequences of manipulation of dietary 
vitamin A on levels of IRBP and retinoids and 
on photoreceptor degeneration in the eye. 
The Drosophila head retinoid and fatty acid- 
binding protein will be further characterized 
by cloning and sequencing and compared 
with IRBP. 

The 72 kDa protein, which binds the uvei- 
togenic peptide 1169-1191 of IRBP and appears 
to be a new member of the HSP 70 family of 



260 



Laboraton/ of Retinal Cell and Molecular Biology 



HSPs will be investigated further by cloning 
and sequencing. Antibodies to this protein 
will also be obtained to study its possible role 
in antigen presentation. We will continue to 
conduct large-scale purification of IRBP pro- 
tein for studies of EAU. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and 
Other Inherited Disorders 

Publications 

Caspi RR, Chan C-C, Grubbs BG, SUver PB, 
Wiggert B, Parsa CF, Bahmanyar S, BiUiau A, 
Heremans H: Endogenous systemic interfer- 
on-gamma has a protective role against ocular 
autoimmunity in mice. J Immunol 152:890-899, 
1994. 

Duffy M, Sun Y, Wiggert B, Duncan T, Chader 
GJ, Ripps H: Interphotoreceptor retinoid 
binding protein (IRBP) enhances rhodopsin 
regeneration in the experimentally detached 
retina. Exp Eye Res 57:771-782, 1993. 

Duncan T, Kutty G, Chader GJ, Wiggert B: A 
glycoprotein binding retinoids and fatty adds 
is present in Drosophila. Arch Biochem Biophys 
312:158-166, 1994. 

Kutty RK, Kutty G, Duncan T, Nickerson J, 
Chader GJ, Wiggert B: Radioanalytic estima- 
tion of amplification products generated by 
reverse transcription PCR using [a-^^P] deoxy- 
ribonucleoside triphosphate. Biotechniques 
15:808-812, 1993. 

Kutty RK, Nagineni CN, Kutty G, Hooks JJ, 
Chader GJ, Wiggert B: Increased expression 
of heme oxygenase-1 in human retinal pig- 
ment epithelial cells by transforming growth 
factor-p. / Cell Physiol 159:371-378, 1994. 

Kutty RK, Kutty G, Rodriguez IR, Chader GJ, 
Wiggert B: Chromosomal localization of the 
human heme oxygenase genes: Heme oxy- 
genase-1 (HMOXl) maps to chromosome 
22ql2 and heme oxygenase-2 (HM0X2) maps 



to chromosome 16pl3.3. Genomics 20:513-516, 
1994. 

Kutty G, Duncan T, Nickerson JM, Si JS, van 
Veen T, Chader GJ, Wiggert B: Light depriva- 
tion profoundly affects gene expression of 
interphotoreceptor retinoid-binding protein in 
the mouse eye. Exp Eye Res 58:65-75, 1994. 

Kutty RK, Kutty G, Nagineni CN, Hooks JJ, 
Chader GJ, Wiggert B: RT-PCR assay for 
heme oxygenase-1 and heme oxygenase-2: A 
sensitive method to estimate cellular oxidative 
damage. Ann N Y Acad Sci, in press. 

Okajima TL, Wiggert B, Chader GJ, 
Pepperberg DR: Retinoid processing in the 
retinal pigment epitheUum of the toad (Bufo 
Marinus). / Biol Chem 269:21983-21989, 1994. 

Pepperberg DR, Okajima TL, Wiggert B, Ripps 
H, Crouch RK, Chader GJ: Interphotoreceptor 
retinoid-binding protein (IRBP). Molecular- 
biology and physiological role in the visual 
cycle of rhodopsin. Mol Neurobiol 7:61-85, 
1993. 

Rajagopalan S, Rodrigues MM, Wiggert B, 
Advani SH, Nair CN, Nickerson JM: Retino- 
blastoma: Interphotoreceptor retinoid-binding 
protein mRNA analysis by polymerase chain 
reaction. Ophthalmic Paediatr Genet 14:117-125, 
1993. 

Rengarajan K, de Smet MD, Chader GJ, 
Wiggert B: Identification of heat shock pro- 
teins binding to an immunodominant uveito- 
pathogenic peptide of IRBP. Curr Eye Res 
13:289-296, 1994. 

Sasamoto Y, Kawano YI, Wiggert B, Chader 
GJ, Gery I: Induction of unresponsiveness in 
adult rats by immunodominant and nondomi- 
nant peptides. Cell Immunol 152:286-292, 1993. 

Smith MA, Kutty RK, Richey PL, Chader GJ, 
Wiggert B, Petersen RB, Perry G: Heme oxy- 
genase-1 is associated with the neurofibrillary 
pathology of Alzheimer Disease. Am J Pathol 
145:42-47, 1994. 



161 



FY 1994 NEI Annual Report 



Smith SB, Duncan T, Kutty G, Kutty RK, 
Wiggert B: Elevation of retinyl palmitate in 
eyes and livers and of IRBP in eyes of vitiligo 
mutant mice. Biochem J 300:63-68, 1994. 



Wiggert B, van Veen T, Kutty G, Lee L, 
Nickerson J, Si JS, Nilsson EG, Chader GJ, 
Narf Strom K: An early decrease in interphoto- 
receptor retinoid-binding protein gene expres- 
sion in Abyssinian cats homozygous for he- 
reditary rod-cone degeneration. Cell Tissue 
Res, in press. 



262 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00297-01 LRCMB 



PERIOD COVERED 

October 1, 1993 to Sep t ember 30, 1 994 



TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) 

Microtubule Stability as a Factor in Retinal Degenerations 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Susan Gentleman Ph.D. LRCMB, NEI 



COOPERATING UNITS (if any) 

Genetics & IVF Institute, Fairfax, VA (R. Sherins, M.D.) 



LAB/BRANCH 

Laboratory of Retinal Cell and Molecular Biology 



Section on Gene Regulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.0 



PROFESSIONAL: 



1.0 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Instability of the microtubules of the connecting cilia has been suggested as a cause or complicating factor 
in some cases of retinitis pigmentosa. These microtubules are normally highly acetylated on Lys40 of a 
tubulin, a posttranslational modification that appears to be restricted to polymerized microtubules; the function 
of this acetylation is as yet unknown. We have described the case of an infertile male subject with a rod- 
dominant retinal degeneration whose sperm flagella show severe morphological abnormalities and greatly 
reduced acetylation. Currently, the project is directed toward characterization of tubulin acetyl transferase 
(TAT) using bovine retina and brain as the source. TAT has been purified about 1,000-fold and is in a 
complex of about 300 kDa. SDS-PAGE analysis of the complex shows four major and several minor bands 
ranging from 30 to 120 kDa. The major bands are N-terminal blocked and will require digestion and 
purification of peptides before sequencing can be done. Arrestin (S-antigen) copurifies with the TAT complex 
from retina and is a substrate for the acetyltransferase activity. A 70 kDa band of x-immunoreactive material 
is also found in the TAT complex from both brain and retina. The partially purified TAT complex from Y-79 
retinoblastoma cells is under evaluation for use in future studies of the molecular biology of the TAT complex. 



263 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 



Muriel I. Kaiser 


MD. 


Chief, OGCSB, NEI 


W. Gerald 


Ph.D. 


Chief, Section on 


Robison 




Pathophysiology, 
LMOD, NEI 



Objectives 

Characterization of the components of the 
microtubule-associated acetyl transferase 
activity will be used to obtain molecular 
probes for analysis of deoxyribonucleic acid 
(DNA) from human subjects with retinal 
degenerations in which microtubule instability 
is suspected. 

Methods 

An assay of acetyl transferase activity in crude 
tissue fractions has been developed. An acetyl 
coenzjone A agarose affinity column, with 
attachment through the phosphate using 
phosphoramidate chemistry, has been made. 
Conventional techniques of protein chemistry 
and purification are used. 

Major Findings 

(1) Sperm flagella from an infertile male 
subject with a rod-dominant retinal degenera- 
tion were evaluated for alpha tubulin iso- 
forms. The level of tubulin acetylation was 30 
percent if the normal population, although 
tyrosinated tubulin level was normal. The 
tubulin acetyl transferase-specific activity of 
these flagella was also significantly lower than 
normal. 

(2) The acetyl transferase activity from 
bovine brain and retina has been purified 
about a thousandfold from the subcellular 
fraction containing cold-stable microtubules 
(27,000 xg pellet) by high-salt extraction, 
anion-exchange chromatography, gel filtration 
by high-performance liquid chromatography, 
and affinity chromatography on AcCoA-aga- 
rose. SDS-polyacrylannide gel electrophoresis 



(PAGE) analysis of the purified fractions 
shows four major bands and several minor 
bands from both brain and retina. The major 
bands from brain have been eluted from gels 
for N-terminal analysis. These bands appear 
to be homogeneous but all are N-terminally 
blocked and cannot be sequenced without 
further processing. 

(3) One band in the retinal complex is 
acetylated in vitro by incubation of the puri- 
fied complex with AcCoA. This protein has 
been identified as arrestin (S-antigen) both by 
N-terminal sequencing and by immuno- 
blotting. 

(4) The purified complexes from both 
brain and retina were analyzed by immuno- 
blotting with a panel of monoclonal antibodies 
against a variety of microtubule binding 
proteins (MAPs). A 70 kDa band of x immu- 
noreactivity was found in both preparations. 
Treatment of the complex with alkaline phos- 
phatase appeared to increase the immunoreac- 
tivity of the 70 kDa band to the antibody x-l, 
which specifically recognizes the unphos- 
phorylated form of x. 

(5) The acetyl fransferase complex from 
Y-79 retinoblastoma cells was partially puri- 
fied and characterized by immunoblottrng. 
Arrestin immunoreactivity copurified with the 
complex. The 70 kDa band of x immunoreac- 
tivity in the complex from these cells also 
reacted with the MAP2 monoclonal antibody 
HM-2 and did not show an increase in x-Uke 
immunoreactivity with dephosphorylation. 

Significance to Biomedical Research and the 
Program of the Institute 

The microtubule acetyl fransferase complex is 
a mvdtifunctional complex containing the 
enzyme with apparentiy a broad specificity 
and microtubule-binding proteins as well as 
other proteins, some of which are tissue spe- 
cific. The single band of x immunoreactivity 
suggests that a particular isoform is used by 
this complex. Therefore, a genetic defect in 
one splice site of x might have major effects 
on the function of the complex. However, the 



264 



Laboraton/ of Retinal Cell and Molecular Biology 



data from Y-79 cells indicate that other MAPs 
may substitute for the x-like form, although 
possibly not as weU. The association of arres- 
tin with the complex in retina also suggests 
that it may play a role in the trafficking of 
arrestin in photoreceptor ceUs. Therefore, the 
retina might be particularly sensitive to mal- 
function of the complex. 

Proposed Course 

The current direction of the project is to con- 
tinue the characterization of the components 
of the acetyl transferase complex. With this 
information, we intend to obtain molecular 
probes suitable for screening human material. 

(1 ) Protein sequence of components of the 
complex will be obtained by digestion and 
purification of peptides from the proteins 
eluted from SDS-PAGE. The N-terminal 
blockade in most of the proteins of the com- 
plex requires that initial protein sequence data 
be obtained from internal peptides. Sequences 
will be matched to those in the available 
protein databases {e.g., Swiss Prot) for identifi- 
cation. 

(2) Peptide sequences not matching se- 
quence in current data banks wiU be used to 
design degenerate oUgos for polymerase chain 
reaction probing of complementary cDNA 
libraries with the objective of obtaining in- 
ferred protein sequence for the complete 
proteins. 



(3) From the sequence information ob- 
tained, molecular probes will be made to 
examine human genes. Family pedigrees of 
type 2 Usher's syndrome are of particular 
interest. Several reports from various labora- 
tories have demonstrated axonemal abnormal- 
ities at a higher than normal frequency in 
some of these people. 

NEI Research Program 

Retinal Diseases — Photoreceptors and Pigment 
Epithelium 

Publications 

Lloyd RA, Gentieman S, Chader GJ: Assay of 
tubulin acetyl tiansferase activity in subcellu- 
lar tissue fractions. Anal Biochem 216:42-46, 
1994. 



265 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00196-11 LRCMB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Genetics of the Eye and Ocular Diseases 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Diane E. Borst Ph.D. Senior Staff Fellow LRCMB, NfEI 

Steven Bernstein Ph.D., M.D. Senior Staff Fellow LRCMB, NEI 



COOPERATING UNITS (if any) 

University of Texas, Dallas (R. Hammer, Ph.D.); University of Illinois (H. Ripps, Ph.D.) 



Laboratory of Retinal Cell and Molecular Biology 



SECTION 

Section on Gene Regulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



2.0 



PROFESSIONAL: 



2.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Antisense refers to a nucleic acid molecule that is complementary to an expressed messenger RNA sequence 
present in an organism. We are evaluating the use of various forms of antisense molecules, both catalytic 
(ribozymes) and noncatalytic in nature, to evaluate the function of specific gene expression in the eye. 
Ribozymes complementary to interphotoreceptor retinoid-binding protein mRNA have been evaluated in vitro 
and in vivo. Antisense to aldose reductase is currently being evaluated in vivo. 



266 



PHS 6040 (Rev. 5/92) 



Lahoratory of Retinal Cell and Molecular Biology 



Project Description 

Additional Personnel 



Eric Wawrousek PhD. Research Biologist, 

OSD, NEI 
Susan DiCamillo B.S. Chemist, OSD, NEI 



Objectives 

Antisense and catalytic antisense are being 
used to downregulate specific functions in 
ocvdar tissues for the purposes of both basic 
research and clinical application. 

This research is designed to define the as- 
acting elements in the mouse interphotorecep- 
tor retinoid-binding protein (IRBP) promoter 
that controls the regulation of the IRBP gene 
expression. Other factors such as deoxyribo- 
nucleic acid (DNA) methylation may be in- 
volved in the regulation of IRBP gene expres- 
sion; another objective is to define the DNA 
methylation state of the IRBP promoter in 
different tissues. 

Methods 

This study uses in vitro transcription and 
assay methodology as well as transgenic 
expression of antisense constructs in mice and 
rats. Conventional techniques for clorung and 
analysis of nucleic acids are used. 

Transgeruc mice are being used to study 
the cfs-acting elements of the IRBP promoter. 
The transgene contains varying lengths of the 
mouse IRBP promoter fused to the reporter 
gene, chloramphenicol acetyl transferase 
(CAT). The levels of CAT activity in these 
mice show which DNA sequences are impor- 
tant for the tissue and developmentally specif- 
ic expression of the IRBP gene. DNA was 
isolated from different tissues, digested with 
Msp I or Hpa II, Southern blotted, and probed 
with fragments of the IRBP promoter to study 
its DNA methylation state. 



Ma'ior Findings 

Most attempts at using ribozymes for down- 
regulation and "gene therapy" in human 
immunodeficiency virus have used ribozymes 
of varying complement length; this comple- 
ment length gives specificity to the target site 
of the message. The optimal length for this 
enzyme-target interaction is unknown, and 
this is partially confirmed by the high failure 
rate and exceedingly variable activity rates for 
engineered molecules against various targets. 
Using i?j vitro partial duplex transcription, 
cloned ribozyme templates, and substrate 
fragments, I have studied the effect of site 
specificity and varying complement length on 
ribozyme activity in vitro. I have found that 
ribozyme activity can be "tuned" in vitro by 
varying complement length and that this 
tuning is unique and target-site specific. The 
current computer-based programs for predict- 
ing site sensitivity are inadequate to this task; 
it must be performed empirically. This study 
is completed. 

With the help of the above technique, I 
have generated two constructs containing 
ribozymes targeted against different sites in 
the messenger ribonucleic add (mRNA) for 
IRBP, which have the highest demonstrated in 
vitro activity and have generated transgenic 
founder lines expressing these ribozymes in 
ocvdar and other tissues. The data from this 
study is preliminary, but there are apparently 
significant differences in the embryonic sur- 
vival and tissue-specific expression of 
transgene and IRBP mRNA in the different 
constructs. We are currently doing ocular 
histology studies on outbred animals (Fl 
generation). 

With the collaboration of Dr. Harris Ripps, 
from the Uiuversity of Illinois at Chicago, we 
have performed electrophysiological studies 
on these mouse lines. There are apparent 
differences in some of the transgenic lines in 
electroretinogram responses, and these appar- 
ently correlate with transgene expression. 
Animal Unes apparently expressing the high- 
est levels of the transgene also demonstrate 
high embryonic fataUty. Thus, IRBP expres- 



267 



Pi' 1994 NEI Annual Report 



sion mav be important in fetal development. 
This work correlates weU with earlier reports 
of IRBP expression in fetal tissue of both rats 
and cows. 

Aldose reductase (AR) is believed to play 
a kev role in the development of diabetic 
retinopathy, neuropathy, and nephropathy. 
Additionally, AR is apparently involved in 
cataractogenesis in diabetic humans and 
galactose-fed rats. Research to date has fo- 
cused on AR inhibitors in animal systems, 
these inhibitors must be administered chroni- 
callv and are expensive; in addition, in one of 
the most \\'idely studied animal systems, the 
dog, development of retinopathy requires an 
extended period of time. Studies in mice are 
hampered bv the fact that they do not develop 
histologically documented retinopathy or 
neuropathy, although they apparently exhibit 
ner\'e conduction velocity changes. 

Rats also develop diabetic changes and are 
an alternative, relatively low-cost mammalian 
system in which to study progression of 
diabetic patholog^^ Rat transgenic model 
svstems are no^v available. Because the 
mRNA sequence of rat AR is kno\\Ti, we have 
designed gene constructs containing antisense- 
based sequence to rat AR. Expressed m vivo, 
AR deficient rats should show delayed galac- 
tose-induced cataractogenesis and delayed 
onset of diabetic histopatholog^^ In collabora- 
tion with Dr. Robert Hammer, from the 
Howard Hughes Medical Research Institute in 
Houston, Texas, 1 am generating strains of rats 
that express antisense for AR in a variet}^ of 
tissues. This work is in the animal production 
stage. 

Tissue culture correlates of retinoblastoma. 
We have characterized two mouse retinoblas- 
toma cell lines in terms of photoreceptor- 
spedfic gene expression. These Hnes may be 
useful in the future for in vitro transfection 
studies using antisense technology' without the 
need for transgenic mice production. This 
study is completed. 

Retinopathy of prematurity (ROP) is one 
of the leading causes of early childhood blind- 



ness in the United States. Pathology in this 
disease results from the development of reti- 
nal neovascularization in premature or low 
birth weight infants that can then progress, in 
its most severe form, to invasion of the vitre- 
ous body, and either during the rapid period 
of vessel growth or following involution, to 
retinal detachment. The only effective treat- 
ment is surgical, with 25 to 35 percent of the 
most severely affected infants maintaining 
vision of 5/200 or less. 

Dr. Lois Smith, from the Massachusetts 
Eye and Ear Infirmary, Boston, Massachusetts, 
has developed a reproducible model of ROP 
in newborn mice. In collaboration with Dr. 
Smith, 1 have prepared a series of antisense 
molecules that I am testing for efficacy of 
specific inhibition of mitosis. These com- 
pounds will then be used to determine effica- 
c}^ of inhibition of ROP in vivo. 

Msp I and Hpa 11 are isoschizomers that 
are methylation dependent. Hpa n will not 
digest the recognition sequence if the 3' 
cytosine is methylated. Msp I sites of the 
IRBP 5' flanking region were studied: five in 
the cow and two in the mouse. One of these 
sites is in the homologous location and is 
hypomethylated only in the retina. Similar 
results have been found for the other Msp I 
sites that have been studied. Perhaps these 
regions of the IRBP promoter are important in 
the regulation of IRBP expression either by 
being a direct protein-binding site or by some- 
how influencing the secondary structure of 
this region. 

Significance to Biomedical Research and the 
Program of the Institute 

hi vitro testing of ribozyme activity may 
enable the selection of unique ribozymes, with 
the possibihty of enhanced in vivo action, for 
use in both gene therapy and basic research. 
The transgenic animal results may teU us 
much about the role of IRBP in embryonic 
development as well as the function of the 
retina during relative IRBP deficiency^ Gener- 
ation of transgenic rats expressing antisense 
for AR may be useful in evaluating the direct 



Laboratory of Retinal Cell and Molecular Biology 



pathophysiological Unk between AR activity 
and pathology in diabetes. Evaluation of 
antisense molecules with antimitotic activity 
will likely be useful in direct pharmacological 
treatment of retinopathy of prematurity. 

IRBP in adults is expressed only in the 
retina and pineal gland. Understanding the 
mechanisms and the nucleotide elements that 
control IRBP expression is fundamental to 
understanding retinal development and func- 
tion. This understanding could help us design 
drugs that would specifically target the retina 
and perhaps would also elucidate abnormal 
retinal function. 

Proposed Course 

Evaluation of mouse retinoblastoma ceUs: 
completed. In vitro evaluation of hammerhead 
ribozyme activity: completed. 

Transgenic animal studies/expression of ribo- 
zyme activity. I am breeding transgenic animal 
lines expressing the active ribozyme constructs 
to select for those lines with the highest homo- 
zygous expression of ribozyme activity. I 
anticipate sending some of these adult animals 
to Dr. Ripps for further electrophysiological 
studies. In addition, 1 am attempting to 
determine the time of onset of lethality of the 
ribozyme expression in the prenatal animal. 



AR antisensel transgenic rats. I am awaiting 
initial confirmation that Dr. Hammer has 
indeed generated a strain of rats expressing 
the AR antisense construct that I sent him. 
When this is completed, I will begin evalua- 
tion of in vivo expression of the construct. 
Iiutial feedings with high galactose will be 
performed to determine the speed of onset of 
cataractogenesis vis-a-vis control rat strains. 
Further analysis depends of the initial delivery 
on these animals. 

Determination of antineovascular activity of 
antisense compounds. Irutial studies determin- 
ing the specific antimitotic activity are under 
way. The compounds possessing the highest 
antimitotic activity wiU be evaluated for their 
pharmacotherapeutic index in vitro. These 
specific compounds wdll be evaluated further 
for their relative antineovascular activity in 



NEI Research Program 

Retinal Diseases — Photoreceptors and Retinal 
Pigment Epithelium 



269 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00124-14 LRCMB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Biol og y of the Retina and Pigment Epithelium 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI 

Others: R. Theodore Fletcher 
Joyce Tombran-Tink 
S. Patricia Becerra 
Timothy Schoen 



M.S. 


Chemist 


LRCMB, NEI 


Ph.D. 


IRTA Fellow 


LRCMB, NEI 


Ph.D. 


Visiting Scientist 


LRCMB, NEI 


M.S. 


Biologist 


LRCMB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Retinal Cell and Molecular Biology 



Section on Gene Regulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



4.0 



PROFESSIONAL: 



2.5 



1.5 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x| (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The title of this project has been changed to more accurately reflect the thrust of the science. The retina and 
pigment epithelium are neuroepithelial tissues that work in close cooperation. Specific growth and 
differentiating factors are found in the eye that guide development and interactions of individual ocular tissues 
to form a functional visual system. Studies on this project are focused on an understanding of the molecular 
biology and molecular genetics of the retina and discovering new genes that are candidates for hereditary 
retinal degenerations. For example, ocular tissues synthesize a number of growth factors. There now appear 
to be several systems present that could self-regulate growth and metabolic activity in the retina-pigment 
epithelium complex and be involved in eye diseases. In this regard, we have cloned and characterized a 
unique protein secreted from fetal human pigment epithelial cells, called pigment-epithelial derived factor 
(PEDF), that is "neurotrophic" to cultured human retinoblastoma cells and may affect neural retinal 
development in vivo. This protein also is a potent "neuronotrophic" agent in that it promotes neuronal cell 
survival of cultured cerebellar granule cells. Finally, PEDF is "gliastatic" in that it markedly retards glial cell 
growth. Along with being a candidate gene in retinal degenerations, the uses of PEDF in neuronal transplant 
in retina and other CNS areas are obvious. 



270 



PHS 6040 (Rev. 5/92) 



Laboratory of Retinal Cell and Molecular Biology 



Project Description 

Additional Personnel 

Joan Schwartz 



PhD. Chief, Molecular 

Genetics Section, DIR, 
NINDS 



Objectives 

Our objective is to obtain a better understand- 
ing of the molecular biology and molecular 
genetics of ocular tissues in health and dis- 
ease. The study of growth and differentiation 
factors, be they protein {e.g., pigment epitheli- 
um-derived factor [PEDF]) or polypeptide 
{e.g., insulin-like growth factor [IGF-1]) is 
critical in obtaining a view of the events that 
control the early development of the eye and 
also in maintaining normal function in the 
adult. 

Methods 

Molecular biological, genetic, and immunocy- 
tochemical techniques are used. Tissue cul- 
ture is performed on cultured cells. In partic- 
ular, the human retinoblastoma cell line Y-79 
is used as a test system for differentiating 
agents. 

Major Findings 

Hereditary diseases are often caused by de- 
fects in genes that are important in cell divi- 
sion and differentiation; PEDF seems to be 
such a gene product. It is secreted from fetal 
pigment epithelial ceUs and is present in 
normal adtilt interphotoreceptor matrix. The 
protein migrates at approximately 50 kDa on 
sodium dodecyl-sulfate-polyacrylamide gels. 
Importantly, PEDF causes marked differentia- 
tion of human Y-79 retinoblastoma ceUs in 
culture (neurotrophic effect). This is charac- 
terized by an extensive elongation of neurite- 
like processes and a gathering of cells into 
"rosette-Uke" aggregates. Immunocytochemi- 
caUy, the expression of specific neuronal 
markers is also enhanced. Thus, PEDF is a 
unique protein, synthesized and secreted by 



retinal pigment epitheUal cells, that could 
direct early development even in early em- 
bryogenesis. PEDF is also present after the 
important developmental period and may 
help to maintain retinal cell viability (neuron 
survival) in the adult retina. PEDF is also a 
candidate gene in the retinal dystrophy ob- 
served in the Royal College of Surgeons rat. 

After the cloning of the complementary 
deoxyribonucleic acid for the PEDF gene, we 
have determined that the protein is a member 
of the SERPIN (serine protease inhibitor) 
superfamily of genes. One other member of 
this family is known to promote neuronal 
differentiation so it makes it more probable 
that PEDF has a major, and similar, role in the 
retina. The recombinant protein (rPEDF) has 
now been expressed in Escherichia coli cells 
and has been shown to be an active neuro- 
trophic agent. The availabihty of relatively 
large amounts of PEDF should allow for more 
direct studies on its role(s) in ocular develop- 
ment and disease. We also find PEDF mes- 
senger ribonucleic acid in ciliary body and 
PEDF protein in vitreous humor, indicating 
that PEDF could be of importance in ocular 
tissues other than the retina. 

In work with Dr. Joan Schwartz, National 
Institute of Neurological Disorders and Stroke 
(NINDS), we have evidence demonstrating 
that PEDF is also a potent "neuronotrophic 
agent," i.e., neuron survival factor in a cul- 
tured cerebellar granule cell (CGC) system. 
Very small amounts of rPEDF added to the 
CGCs are effective in keeping the cells alive 
for prolonged periods of time. Surprisingly, 
we have found that PEDF has "gliastatic" 
properties as well, in that gUal cells in the 
cerebellar cultures are markedly inhibited. 
Importantly, this is a long-term effect; a one- 
time exposure to nanogram quantities of 
rPEDF inhibits growth of cultured cerebellar 
gUa for up to 12 weeks. 

Significance to Biomedical Research and the 
Program of the Institute 

Determining the genes that control normal 
ocular growth, differentiation, and function 



171 



FY 1994 NEl Annual Report 



and studying them on a molecular biological 
and molecular genetics level will aid us in 
understanding diseases of the eye, especially 
those of a hereditary, early developmental 
nature. With such knowledge, rational meth- 
ods of gene therapy can be appUed to ocular 
diseases. Because of its potent neurotrophic, 
neuronotrophic, and gUastatic effects, PEDF 
seems to be of potential use in retinal and 
central nervous system transplantations. 

Proposed Course 

The molecular biology and molecular genetics 
of ocular development will be further exam- 
ined. The factors that affect normal and 
abnormal growth will be investigated. The 
fuU PEDF gene will be examined and ana- 
lyzed to help elucidate its presumptive role(s) 
in retinal development. The rPEDF protein 
will be used to elucidate the role of the novel 
new protein in retinal diseases processes. 

NEl Research Program 

Retinal Diseases — Retirutis Pigmentosa and 
Other Inherited Disorders 

Publications 

Arnold DR, Moshayedi P, Schoen TJ, Jones BE, 
Chader GJ, WaldbiUig RJ: Distribution of IGF- 
I and -n, IGF binding proteins (IGFBPs) and 
IGFBP mRNA in ocular fluids and tissues: 
Potential sites of synthesis of IGFBPs in aque- 
ous and vitreous. Exp Eye Res 56:555-565, 
1993. 

Becerra SP, Palmer I, Kumar A, Steele F, 
Shiloach J, Notario V, Chader GJ: Overexpres- 
sion of fetal human pigment epitheUum-de- 
lived factor in Escherichia coU: A functionally 
active neurotrophic factor. / Biol Chem 
268:23148-23156, 1993. 



Gaudet SJ, TsUou E, Chader GJ: Identification 
and characterization of arylamine N-acetyl- 
transferase activity from the bovine retinal 
pigment epithelium. Curr Eye Res 12:271-278, 
1993. 

Li A, Lane WS, Johnson LV, Chader GJ, 
Tombran-Tink J: Neuron-specific enolase: A 
neuronal survival factor in the retinal extracel- 
lular matrix? / Cell Biol, in press. 

Poggi L, Melchiori A, Pellegrini R, Defilippi P, 
Noonan D, Campbell MA, Gentieman S, 
Chader GJ, Albini A: Laminin-induced Y-79 
retinoblastoma ceU differentiation occurs in 
the absence of "classic" laminin adhesion 
molecules, in Fassina GF, Percaiio M (eds): 
Cell Adhesion Molecules in Cancer and Differenti- 
ation. London, Harwood- Academic Publish- 
ers, in press. 

Seigel GM, Becerra SP, Chader GJ, Diloreto 
DA Jr, del Cerro C, Lazar ES, del Cerro M: 
Differentiation of Y79 retinoblastoma cells 
with pigment epitheUal-derived factor and 
interphotoreceptor matrix wash: Effects on 
tumorigenicity. Growth Factors, in press. 

Tombran-Tink J, Pawar H, Swaroop A, Rodri- 
guez I, Chader GJ: Localization of the gene 
for pigment epitheUum-derived factor to 
chromosome 17pl3.1 and expression in cul- 
tured human retinoblastoma. Genomics 19:266- 
272, 1994. 

WaldbiUig RJ, Jones BE, Schoen TJ, 
Heidersbach S, Bitar MS, van Kujik F, de Juan 
E, Kador P, Chader GJ: Vitreal insuUn-Uke 
growth factor binding proteins (IGFBPs) are 
increased in human and animal diabetics. 
Curr Eye Res 13:539-546, 1994. 



del Cerro M, Seigel GM, Lazar E, Grover D, 
del Cerro C, Brooks DH, DiLoreto D, Chader 
GJ: Transplantation of Y79 cells into rat eyes: 
An in vivo model of human retinoblastomas. 
Invest Ophthalmol Vis Sci 34:3336-3345, 1993. 



272 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



1 



ZOl EY 00148-21 LRCMB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Visual Contro l Mechanistns and Hereditary Defeneration 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI 



Others: 



Paul Wong 
Tatiana Putilina 
Ignacio Rodriguez 
June Lee 
Timothy Schoen 



Ph.D. 
Ph.D. 
Ph.D. 
M.D. 
M.S. 



Visiting Fellow 
Visiting Associate 
Staff Fellow 
Visiting Associate 
Biologist 



LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 



COOPERATING UNITS (if any) 

School of Veterinary Medicine, Cornell University (G. Aguirre, D.V.M., Ph.D.); Department of Zoology, 
University of Lund, Lund, Sweden (T. van Veen, Ph.D.); Institute Nazionale per la Ricera sul Cancro, Geneva, 
Italy (A. Albini, Ph.D., D. Noonan, Ph.D.) _^___ 

LAB/BRANCH 

Laboratory of Retinal Cell and Molecular Biology ^ 

SECTION 

Section on Gene Regulation . 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



5.0 



PROFESSIONAL: 



4.5 



0.5 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The title of this project has been changed to more accurately reflect the thrust of the science. We are studying 
the expression of specific gene products that could be related to hereditary diseases of the retina. If normal 
genetic control mechanisms fail, hereditary diseases of the retina such as retinoblastoma or retinitis pigmentosa 
will resuh. We have now developed new techniques to clone and sequence retina-specific genes at a higher 
efficiency. We have found several genes that are either expressed exclusively or predominantly in the retina 
and are using these as candidate genes in specific blinding diseases. Among these are hydroxy-0-methyl 
transferase, an important gene on the X chromosome involved in circadian control in the retina and a new gene 
mapping to chromosome Uq close to four important genetic retinal diseases. Similarly, we are investigating 
the properties of known retina-specific genes such as the interphotoreceptor retinoid-binding protein (IRBP) 
and their involvement in retinal disease processes. Progress has also been made in identifying apoptosis as 
a primary and unifying mechanism for cell death in several hereditary retinal degenerations. All these factors 
and processes could lead to more efficient gene therapy of the diseased neural retina. 



273 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 

Muriel I. Kaiser M.D. Chief, OGCSB, NEI 

Objectives 

Expression of genes in the retinal photorecep- 
tor neuron in a normal manner is crucial to 
visual function in the adult. Thus, the factors 
that code for normal gene control and expres- 
sion in human retina and in animal models of 
retinal degeneration are of primary interest. 
We also have started a major effort to develop 
new molecular biological techniques such that 
unique retinal and retinal pigment epithelium 
(RPE) genes can be identified, cloned, and 
sequenced to be used ultimately in screening 
human populations with inherited diseases of 
the visual system. 

Methods 

Standard molecular biological, biochemical, 
and neurocheirucal techniques are used. 
Histochemical techniques are used when 
necessary. 

Major Findings 

(1) We have developed new molecular biolog- 
ical techniques that allow for more efficient 
identification of highly expressed genes of the 
retina-PE complex. Each tissue of the body 
expresses a unique complement of genes that 
are trai scribed and translated at a high level. 
In the retina and pigment epithelium, several 
very specific proteins are highly expressed 
such that photoreception and the visual pro- 
cess can take place. In a similar vein, it is 
often a genetic defect in these tissue-specific 
genes that results in a hereditary degeneration 
such as retinitis pigmentosa. We have devel- 
oped and are using new methods for rapid 
polymerase chain reaction-based construction 
of specifically enriched libraries fiom very 
small retinal samples. This is especially im- 
portant because tissue samples are limited in 
studying early development and rare patholo- 



gy samples. A significant methodological 
advance we have made involves subtractive 
cloning on an immobilizing Dynabead base®. 
In this way, several new genes have been 
found that are being used as candidate genes 
for hereditary retinal degenerative diseases. 

(2) In collaboration with National Eye 
Institute (NEI) clinicians, we are now applying 
these techniques to the study of the role of 
several proteins and genes in retinal disease. 
Among these are: 

(a) Fatty acid-binding proteins that may 
be involved in normal and degenerating 
retinas such as in Bietti's crystalline dys- 
trophy. 

(b) A newly discovered gene on chro- 
mosome llq that is a prime candidate 
gene in Bardet-Biedl's syndrome. Best's 
disease, familial exudative vitreoretino- 
pathy, and one form of Usher's syndrome. 

(c) The hydroxjdndole-O-methyl trans- 
ferase (HIOMT) gene that maps to the 
short arm of the X-chromosome and is a 
key enzyme in melatonin production in 
the retina. 

(3) Finally, we are beginning to under- 
stand the mechanism by which photoreceptor 
ceUs die in a number of hereditary retinal 
diseases. This process is called "apoptosis" or 
programmed ceU death. We have found induc- 
tion of the marker gene TRPM-2 (clusterin) in 
aU cases of retinal degenerations studied. It 
thus appears that programmed cell death may 
be a common mechanism by which many 
hereditary defects initiate photoreceptor cell 
death. 

Significance to Biomedical Research and the 
Program of the Institute 

One approach to studying a hereditary disease 
process in a tissue and its reversal through 
gene therapy is to first identify the normal 
complement of unique genes expressed in that 
tissue. This is also applicable in an early 
degenerative process, e.g., retinitis pigmentosa. 



Lnhoratonj of Retinal Cell and Molecular Biology 



and in other hereditary diseases such as 
Bietti's crystalline dystrophy or Best's disease 
in which the disease may be systemic, but the 
disease primarily affects the retina. In paral- 
lel, if one knows the common mechanism by 
which photoreceptor cells actually die in the 
various retinal degenerations, it may be possi- 
ble to design strategies by which retinal cells 
are spared, at least long enough for transplan- 
tation or gene therapy experiments to be 
successful. Thus, studying apoptosis and 
similar processes in the retina wiU lead to 
better methods for gene therapy in the neural 
retina. 

Proposed Course 

We will continue to study molecular biological 
and developmental control mechanisms in the 
retina and pigment epitheUum. In particular, 
we will investigate gene expression in normal 
retinas and in retinas affected with specific 
genetic diseases and focus on subtiactive 
cloning as a prime method for identif5dng 
defective or missing genes in retinal diseases. 
Apoptosis wiU continue to be an important 
area for study because future gene therapy in 
retinal degenerations may depend on under- 
standing how to prevent death of the photore- 
ceptor neuron. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and 
Other Inherited Diseases 

Publications 

Chader GJ: Retinal degenerations of heredi- 
tary, viral and autoimmune origins: Studies 
on opsin and IRBP, in Osborne N, Chader GJ 
(eds): Progress in Retinal and Eye Research. 
Oxford, Pergamon Press Ltd, 1994, vol 13, pp 
65-99. 

Duffy M, Sun YF, Wiggert B, Duncan T, 
Chader GJ, Ripps H: Interphotoreceptor 
retinoid-binding protein (IRBP) enhances 
rhodopsin regeneration in the experimentally 
detached retina. Exp Eye Res 57:771-782, 1993. 



Duncan T, Kutty G, Chader GJ, Wiggert B: A 
glycoprotein binding retinoids and fatty acids 
is present in Drosophila. Arch Biochem Biophys 
312:158-166, 1994. 

Fassina G, PagUalunga G, Noonan DM, 
Chader GJ, Albini A: Modulation of Y-79 
retinoblastoma cell differentiation and IRBP 
expression by dibutyryl cyclic AMP and 
laminin. M ] Oncol 2:745-751, 1993. 

Hershfield B, Chader G, Aguirre G: Cloning 
of a polymorphic canine genetic-marker, 
which maps to human chromosome-9. Anim 
Genet 24:293-295, 1993. 

Kutty G, Duncan T, Nickerson J, Si JS, van 
Veen T, Chader GJ, Wiggert B: Light depriva- 
tion profoundly affects gene expression of 
interphotoreceptor retinoid-binding protein in 
the mouse eye. Exp Eye Res 58:65-75, 1994. 

Kutty RK, Kutty G, Duncan T, Nickerson J, 
Chader GJ, Wiggert B: Radioanalytic estima- 
tion of amplification products generated by 
reverse tianscription PCR using [alpha-33P] 
deoxyribonucleoside triphosphate. Biotech- 
nicjues 15:808-812, 1993. 

Kutty RK, Kutty G, Nagineni CN, Hooks JJ, 
Chader GJ, Wiggert B: RP-PCR assay for 
heme oxygenase-1 and heme oxygenase-2: A 
sensitive method to estimate ceUvdar oxidative 
damage. Ann NY Acad Sci, in press. 

Kutty RK, Kutty G, Rodriguez IR, Chader GJ, 
Wiggert B: Chromosomal localization of the 
human heme oxygenase genes: Heme oxy- 
genase-1 (HMOXl) maps to chromosome 
22ql2 and heme oxygenase-2 (HM0X2) maps 
to chromosome 16pl3.3. Genomics 20:513-516, 
1994. 

Kutty RK, Nagineni CN, Kutty G, Hooks JJ, 
Chader GJ, Wiggert B: Increased expression 
of heme oxygenase-1 in human retinal pig- 
ment epitheUal cells by transforming growth 
factor-beta. / Cell Physiol 159:371-378, 1994. 

Lloyd RA, Gentieman S, Chader GJ: Assay of 
tubulin acetyl-transferase activity in subcellu- 



FY 1994 NEI Annual Report 



lar tissue fractions. 
1994. 



Anal Biochem 216:42-46, 



Okajima TL, Wiggert B, Chader GJ, 
Pepperberg DR: Retinoid processing in the 
retinal pigment epithelium of the toad (Bufo 
marinus). / Biol Chem 269:21983-21989, 1994. 

Pineda R, Chang CC, Ni M, Hayden BJ, John- 
son MA, Nickerson J, Chader GJ: Human 
retinoblastoma ceUs express beta-crystaUin in 
vivo and in vitro. Curr Eye Res 12:239-245, 
1993. 

Putilina T, Sittenfeld D, Chader GJ, Wiggert B: 
Study of a fatty-acid binding-site of interpho- 
toreceptor retinoid-binding proteins using 
fluorescent fatty adds. Biochemistry 32:3797- 
3803, 1993. 

Rajagopalan S, Rodrigues M, Polk T, Wilson 
D, Chader GJ, Hayden BJ: Modulation of 
retinoblastoma cell characteristics by hexa- 
methylene bis-acetamide and other differenti- 
ating agents in culture. / Histochem Cytochem 
41:1331-1337, 1993. 



Sasamoto Y, Kawano YI, Wiggert B, Chader 
GJ, Gery I: Induction of unresponsiveness in 
adult rats by immunodominant and nondomi- 
nant peptides. Cell Immunol 152:286-292, 1993. 

Wiggert B, van Veen T, Kutty G, Lee L, 
Nickerson J, Si JS, Nilsson SE, Chader GJ, 
Narfstrom K: An early decrease in tnterphoto- 
receptor retinoid-binding protein gene expres- 
sion in Abyssinian cats homozygous for he- 
reditary rod-cone degeneration. Cell Tissue 
Res, tn press. 

Wong P, Putilina T, Chader GJ, Tenniswood 
M: The human gene encoding TRPM-2 exists 
as a single gene locus on the short arm of 
chromosome 8. Am J Human Genet, in press. 

Wong P, Taillefer D, Lakins J, Pineault J, 
Chader G, Tenniswood M: Molecular charac- 
terization of human TRPM-2 /clusterin, a gene 
associated with sperm maturation, apoptosis 
and neurodegeneration. Eur J Biochem 
221:917-925, 1994. 



Rengarajan K, de Smet MD, Chader GJ, 
Wiggert B: Identification of heat shock pro- 
teins binding to an immunodominant uveito- 
pathogenic peptide of IRBP. Curr Eye Res 
13:289-296, 1994. 



276 



PROJECT NUMBER 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



ZOl EY 00260-05 LRCMB 



PERIOD COVERED 

October 1, 1993 to S eptember 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Biology of Out er Retin a-Specific Proteins 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: T. Michael Redmond Ph.D. Research Biologist LRCMB, NEI 



Others: Suyan Liu 



M.D., Ph.D. Visiting Fellow 



LRCMB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Retinal Cell and Molecular Biology 



SECTION 

Section on Gene Regulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



2.0 



PROFESSIONAL: 



2.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Retinal pigment epithelium (RPE) cells and photoreceptor cells are functionally and developmentally closely 
integrated. Derangements of the RPE are involved in certain retinal diseases. However, the RPE is poorly 
understood at the molecular level. We have cloned and characterized RPE65, a novel developmentally- 
regulated conserved 65 kDa RPE-specific microsomal membrane-associated protein. The cDNA sequence is 
being used to overexpress RPE65 protein for functional studies. The potential role of the protein in inducing 
uveitis will also be studied using recombinant protein. 

The RPE65 protein is not expressed in cultured RPE even though its mRNA is abundant. In characterizing 
this example of posttranscriptional regulation, we have identified distinct sequences in the 3' untranslated 
region of the RPE65 mRNA that control the stability and the efficiency of translation of the RPE65 message. 
We have cloned and are sequencing a full-length human genomic clone for RPE65. It is at least 40 kilobases 
in length. Sequence analysis shows that the RPE65 protein is highly conserved between human and cow. We 
have also cloned the mouse gene. The human gene for RPE65 is localized to chromosome lp31, and the 
mouse homolog to distal chromosome 3. These do not correspond to any ocular disease gene localized so far. 
Nonetheless, RPE65 remains a candidate gene for RPE-involved disease. 



277 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The retinal pigment epithelium (RPE) and the 
photoreceptor cell layer of the neural retina 
form a functionally and developmentaUy 
interdependent complex. Dysfunction of the 
RPE, accordingly, is deleterious to the photo- 
receptors and, hence, to vision itself. In spite 
of these important considerations, Uttle is 
known about the RPE at the molecular level. 
In this laboratory, we are cloning proteins 
specifically or preferentially expressed in the 
RPE with a view to understanding mecha- 
rusms important to the RPE. At present, our 
major emphasis is on a 65 kDa protein that we 
have named RPE65. We are also studying 
other RPE-expressed proteins. 

Methods 

Molecular cloning and biochemical and pro- 
tein chemistry techniques are used in this 
study. Additionally, we are using automated 
fluorescent deoxyribonucleic acid (DNA) 
sequencing and gene mapping techniques. 

Major Findings 

(1) RPE65 is a developmentaUy regulated, 
membrane-associated, nonglycosylated 65 kDa 
protein restricted to and conserved in verte- 
brate RPE and is the major protein of the RPE 
microsomal fraction. The protein displays an 
affinity for phosphoUpids that is calcium 
independent. We have cloned a composite 
3,115 bp complementary DNA (cDNA) for this 
protein. 

(2) We have found that distinct sequences 
in the 3' untranslated region (UTR) of the 
RPE65 messenger ribonucleic acid (mRNA) 
regulate the stability of the RPE65 message 
and the efficiency of its translation. This is 
the first example of 3' UTR-mediated regula- 
tion in an ocular gene. 

(3) We have isolated a human genomic 
done for RPE65. It is at least 40 kilobases in 



length. Much of the gene has been sequenced. 
Preliminary sequence analysis indicates that 
the RPE65 protein is highly conserved in 
evolutionary terms. 

(4) We have localized the gene for the 
RPE65 to human chromosome lp31 and to the 
far distal end of mouse chromosome 3. Nei- 
ther of these loci matches that of a known 
ocular disease or phenotjqse. The mouse gene 
has been cloned to allow us to pursue trans- 
genic and homologous recombination ap- 
proaches for studying RPE65 gene regulation 
and function in the mouse. 

Significance to Biomedical Researcli and the 
Program of the Institute 

The RPE is poorly characterized at the molec- 
ular level. This is in spite of its pivotal role in 
the maintenance of photoreceptor function 
and, hence, of vision itself. We have identi- 
fied RPE65 as a conserved RPE-spectfic mole- 
cule that is developmentaUy expressed. cDNA 
sequencing demonstrates that it is a novel 
protein. The function of this protein, although 
not yet clear, may be related to its affinity for 
phospholipids. Elucidation of the basis for its 
posttianscriptional regulation in vitro may 
have significant bearing on the cvdture of RPE 
ceUs. This is of some clinical significance 
because RPE ceU transplantation is receiving 
much attention as a possible mode of inter- 
vention in treating some retinal diseases. In 
addition, because of its RPE specificity, the 
RPE65 gene can be considered a potential 
candidate gene for retinal disease. 

At present, however, neither its human 
nor mouse chromosomal locations match those 
of any mapped disease loci. This may change 
as more disease loci are matched. Again, in 
view of its RPE-specific expression, elucidation 
of its gene structure may uncover RPE-spedfic 
regulatory elements. The high degree of 
conservation in the RPE65 protein suggests an 
evolutionarily important function for this 
protein. FinaiUy, in view of the involvement of 
the RPE in uveitis, it is possible that RPE65 is 
uveitogenic. Because we have cloned the 



278 



Laboraton/ of Retinal Cell and Molecular Biologi/ 



cDNA, it will now be possible to overexpress 
the protein to test this hypothesis. 

Proposed Course 

(1) The translational efficiency regulating 
sequence in the 3' UTR will be characterized, 
as will the mRNA instabihty region. This wUl 
provide insights into translational regulation 
in the outer retina. 

(2) Analysis of the structure of the human 
RPE65 gene will be continued. The remainder 
of the gene wiU be sequenced. Due to its 
importance as an RPE-specific gene, regulatory 
regions as well as the promoter will be identi- 
fied and analyzed. 

(3) The mouse RPE65 gene will be com- 
pared with the human RPE65 gene, especially 
with regard to the promoter region. Trans- 
geruc and homologous recombination "knock- 
out" studies win be pursued to understand 
RPE65 function and regulation as well as to 
provide potential new models for human 
retinal disease. 

(4) RPE65 will be tested as a possible RPE 
autoantigen. RPE65 protein will be overex- 
pressed for this purpose. 



(5) Elucidation of the structure and func- 
tion of the RPE65 protein will continue. This 
will involve use of a variety of approaches. 
We will pay special attention to its possible 
role in retinoid and /or hpid trafficking. 

NEI Research Program 

Retinal Diseases — Photoreceptors and Pigment 
Epithelium 

Publications 

Hamel CP, Jenkins NA, Gilbert DJ, Copeland 
NJ, Redmond TM: The gene for the retinal 
pigment epitheUum-specific protein RPE65 is 
localized to human lp31 and distal mouse 3. 
Genomics 20:509-512, 1994. 

Redmond TM, Jenkins NA, Gilbert DJ, 
Copeland NJ, Hamel CP: The gene for the 
retinal pigment epithelium-specific protein 
RPE65 is localized to human lp31 and distal 
mouse 3. Invest Ophtlialmol Vis Sci 
35(suppl):1312, 1994. 



279 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00250-07 LRCMB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Biology of Experimental Autoimmune Uveitis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI 

Molecular Biology 



Others: 



Dhirendra Singh 
Shirley Yu 



Ph.D. 
B.S. 



Visiting Associate 
Biologist 



LRCMB, NEI 
LRCMB, NEI 



COOPERATING UNITS (if any) 

Department of Ophthalmology, Miami University, Miami, PL (D. Hamasaki, Ph.D.); Department of Anatomy, 
Nagoya University School of Medicine, Tsuramai, Showa-ku, Nagoya, Japan (Jiro Usukura, M.D.) 



Laboratory of Retinal Cell and Molecular Biology 



SECTION 

Section on Molecular Biology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



2.5 



PROFESSIONAL 



1.5 



1.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

We have previously determined amino acid sequences of human, mouse, rat, and bovine retinal S-antigen and rat pineal 
gland S-antigen. Immunogenic sites and four uveitopathogenic sites of S-antigen were also determined. Two of the 
immunogenic sequences were highly conserved among these species. 

Many proteins that have a similar sequence with a uveitopathogenic site are in the National Biomedical Research 
Foundation database. We chemically synthesized many peptides, and some of them induced experimental autoimmune 
uveitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis rats. In addition, native yeast histone H3 was 
also capable of inducing EAU. 

To understand the role in autoimmunity of infectious micro-organisms that have cross-reactive antigens, we injected Lewis 
rats with peptide M together with one of six different killed bacteria, either with or without incomplete Freund's adjuvant 
(IFA). The rats injected with IFA developed EAU. To assess the impact of infection by live micro-organisms, low doses 
of live E. coli expressing S-antigen and baker's yeast, with a cross-reactive antigen, were injected several times into the 
rats. The rats injected with either live E. coli or live yeast developed EAU. We conclude that infection by micro- 
organisms that have cross-reactive antigens can break immune tolerance to self-antigens and induce inflammatory 
autoimmune diseases. 

As an extension of our previous EAU research, we speculated that some types of cataracts may be induced by 
autoimmune insults. To investigate this issue we carried out similar experiments. Three groups of four rats were injected 
three times with lens homogenate, p-crystallins or a P-crystallin (P-Al) emulsified with complete Freund's adjuvant 
(CFA). All the animals developed severe damage in lens epithelial cells after 5 weeks from the date of the first injection. 
The rats injected with a synthetic peptide derived from Salmonella typhimurium protein that has five amino acid residues 
identical to rat p-crystallin (P-B2) also induced similar damage. Infection of microbes having homologous antigens to 
the lens antigens can induce the auto-antibodies at high levels that provoke damage to the lens epithelial cells. Thus, 
autoimmune insult in the lens epithelial cells may be an etiology of an initial stage of cataractogenesis. Direction of our 
future research will be to focus more on the autoimmunity in the lens cataractogenesis. 

280 



PHS 6040 (Rev. 5/92) 



Lahoratoni of Retinal Cell and Molecular Biology 



Project Description 

Objectives 

The objectives of this project are to understand 
the basic etiology of autoimmune inflamma- 
tion, including uveitis, and to find possible 
treatments for human uveitis. 



Methods 

Conventional methods for analysis of proteins 
and nucleic acids are used. These include 
protein purification, ribonucleic acid (RNA) 
and deoxyribonucleic acid isolation, character- 
ization and sequencing, molecular cloning, 
screening of clones, in situ hybridization, 
immunocytochemistry, and chromosome 
mapping. We also synthesized and used 
oligopeptides and oligonucleotides. Bovine, 
murine, primate, and human materials are 
used. Arumal experiments were carried out 
with Lewis rats and monkeys. T-ceU response 
and adaptive transfer were done with lymph 
node or spleen cells of rat. 

Major Findings 

(1) Local sequence homology was found 
between peptide M and several other foreign 
proteins, including potato proteinase inhibitor 
Ha, Escherichia coli hjrpothetical protein. 
Hepatitis B virus probable DNA polymerase, 
Moloney murine sarcoma virus gag-polypro- 
tern, Moloney murine leukemia virus Gag-pol 
polyprotern. Baboon endogenous virus gag-pol 
polyprotein, and Baker's yeast histone H3. 

(2) The synthetic peptides of the above- 
mentioned proteins induced experimental 
autoimmune uveitis (EAU) in Lewis rats v^dth 
similar pathology to EAU induced by peptide 
M or native S-antigen (S-Ag). 

(3) For the first time, we proposed and 
showed the evidence that molecular mimicry 
plays a role in the process of pathogenesis of 
EAU and perhaps in autoimmune diseases in 
general. 



(4) Oral administiation of histone H3 
peptide suppressed EAU in the Lewis rats. 

(5) The suppression of EAU by histone 
H3 was also found in the EAU induced by the 
S-Ag. Thus, the tolerance also crossreacted 
between the peptide that has molecular mim- 
icry. 

(6) The T-lymphocytes obtained from rats 
immunized with peptide M or yeast histone 
H3 transferred disease, EAU, in the naive rats 
(adoptive transfer) when stimulated either 
with peptide M or histone H3. In addition, 
oral tolerance was also adoptively transferred 
from rats fed peptide M or histone H3 to the 
naive rats. 

(7) Infection by microorganisms that have 
crossreactive antigens can break immune 
tolerance to a self-antigen and induce inflam- 
matory autoimmune diseases. 

Significance to Biomedical Research and the 
Program of the institute 

Uveitis is a leading cause of visual handicap 
in the United States and throughout the 
world. Some types of uveitis were suspected 
to be induced by bacterial and viral infections 
by many physicians for many decades. How- 
ever, there is no clear link between infection 
and disease. 

Autoimmune processes are thought to 
play a significant role in the pathogenesis of 
disease. Molecular mimicry, a process by 
which an immune response directed against a 
nonself protein crossreacts with a normal host 
protein, may play a role in autoimmunity. 
Here, we have proposed the idea of molecular 
mimicry and showed evidence that molecular 
mimicry may play a role in the pathogenesis 
of EAU. In addition, we have shown evidence 
that infection is a possible cause of autoim- 
mune iiiflammation. These findings provide 
an important clue for understanding the 
etiology of autoimmune inflammatory diseases 
in humans. 



ZSl 



FY 1994 NEI Annual Report 



Proposed Course 

This project will be terminated. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 



Publications 

Li Q, Abe T, Kikuchi T, Nussenblatt RB, 
Shinohara T, Chan C-C: Corticosteroids 
enhance S-antigen expression in non-retinal 
ocular tissue of rats with experimental autoim- 
mune uveitis. Exp Mol Pathol 60:27-38, 1994. 

Singh DP, Kikuchi T, Singh VK, Shinohara T: 
A single amino acid substitution in core resi- 
dues of S-antigen peptide confers the capacity 
to prevent experimental autoimmune uveitis 
(EAU). 7 Immunol 152:4699-4705, 1994. 



282 



I PROJECT NUMBER 
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE ' 

NOTICE OF INTRAMURAL RESEARCH PROJECT i ZOl EY 00132-13 LRCMB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Molecular Biolog y of Phototran sduction 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute afniiatlon) 

PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI 

Molecular Biology 

Others: Takanobu Kikuchi Ph.D. Visiting Associate LRCMB, NEI 



COOPERATING UNITS (if any) 

Mount Sinai Hospital, Toronto, Canada (Martin Breitman, Ph.D.); Department of Anatomy, Nagoya University 
School of Medicine, Tsurumai, Showa-Ku, Nagoya, Japan (J. Usukura, M.D.) 



LAB/BRANCH 



Laboratory of Retinal Cell and Molecular Biology 



SECTION 



Section on Molecular Biology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 

1.5 



PROFESSIONAL 

1.5 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects □ (b) Human tissues [x] (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

We have characterized the S-antigen genes from human and mouse, 33K protein genes from mouse and 
human. The S-antigen genes were approximately 50 kbp in length, contained 16 exons and 15 introns. and 
were composed of 97 percent intron and 3 percent exon. The 5'-flanking regions of the genes, approximately 
1.5 kbp long, had no known regulatory elements for transcription such as TATA, GC, or CCAAT boxes. 

Regulatory sequences and nuclear factors governing tissue-restricted expression of the mouse arrestin gene 
were investigated. The results showed that while proximal promoter sequence -38 to +304 are sufficient to 
direct low levels of retina-specific gene expression, sequences extending upstream to position -209 support 
higher levels of expression in the retina, as well as detectable expression in the lens, pineal gland, and brain. 
Within the interval between positions -205 and -185 is a region that contains two direct repeats of the 
hexamer, TGACCT. The proximal promoter binds three apparently retina-specific nuclear factors, Bpl, Bp2, 
and Bp3, through overlapping sequences centered between positions -25 and -15. Bpl and Bp3 also recognize 
a closely related sequence found in the promoter regions of several other vertebrate photoreceptor-specific 
genes. Moreover, the consensus binding site for Bpl, designated PCEl, is identical to RCSl, an element 
known to play a critical role in eliciting photoreceptor-specific gene expression in Drosophila melanogaster . 
The results suggest that PCEl and RCSl are functionally, as well as structurally, similar and, that despite 
marked differences in the fly and vertebrate visual system, the transcriptional machiner\' involved in 
photoreceptor-specific gene expression has been strongly evolutionarily conserved. 



283 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

The objective of this project is to understand 
the basic mechanism of phototransduction in 
the retina and to understand the structure, 
function, and evolution of the proteins present 
in photoreceptor rod cells and pinealocytes. 

Methods 

Conventional methods for analysis of proteins 
and nucleic acids are being used. These 
include protein purification, ribonucleic acid 
and deoxyribonucleic acid (DNA) isolation, 
characterization, and sequence determination. 
Various recombinant DNA techniques are also 
being used, including a Baculovirus expres- 
sion vector system, S5mthesis of point muta- 
tion clones, characterization of promoters and 
transgenic animals. We have also sjnithesized 
and used purified oligopeptides and oligo- 
nucleotides. 

Major Findings 

(1) The gene sequences of S-antigen (S-Ag) 
from human and mouse were determined. It 
is 50 Kbp in length and has 15 introns and 16 
exons. The smallest exon encodes for three 
amino acids. 

(2) The intron-exon map sequence of the 
mouse S-Ag gene has been well conserved. 
Approximately 97 percent of the S-Ag gene is 
intron, and 3 percent is exon. 

(3) The human and mouse S-Ag comple- 
mentary DNAs (cDNA) have been subcloned 
into two expression vectors and have been ex- 
pressed. The products of S-Ag cDNA were 
purified by column chromatography and were 
prepared for crystallization. 

(4) The 5'-flanking sequence of the human 
and mouse S-Ag genes were determined and 
demonstrated promoter activity in the in vivo 
and in vitro transcriptional assays. 



(5) Although the S-Ag promoter sequenc- 
es are highly conserved between human and 
mouse, promoter activity was found at differ- 
ent locations of the 5'-flanking region in the 
human and mouse genes. This result suggests 
that the promoter activity is highly specific to 
tissues and species. 

(6) The mouse S-Ag promoter, 1,300 bp in 
length, was fused with the chloramphenicol 
acetyl transferase (CAT) gene, and this gene 
was introduced into transgenic mice. The 
transgenic animals expressed CAT activity 
only in the retina and pineal gland. This 
result indicates that the promoters have a 
tissue-spedfic enhancer and promoter activity. 

(7) The opsin promoter was fused with a 
diphtheria toxin gene. This fusion gene was 
introduced into transgenic mice, which subse- 
quentiy lost only the photoreceptor rod cell 
layer. 

(8) Several cDNAs of Shuzin, a retinal 
photoreceptor protein, were isolated from 
human and cow retinal cDNA libraries 
(lambda-gtU). The entire DNA sequences 
were determined. The deduced protein has 
sequence similarity with TFIID. Its gene was 
also isolated from a genomic library, and the 
DNA sequence was determined. It is com- 
prised of two introns and three exons. 

(9) Two genes of 33 kDa ROS-specific 
proteins have been isolated from the retinal 
libraries of human and mouse. The entire 
DNA sequence of these genes has been de- 
termined, and they have four exons and 
three introns. 

(10) The proximal promoter sequence 
positions -38 to +304 are sufficient to direct 
low levels of retina-specific gene expression. 

(11) The proximal promoter binds three 
retinal-specific nuclear factors, Bpl, Bp2, and 
Bp3, through overlapping sequences cen- 
tered between posifions -25 and -15. 



284 



Lahoraton/ of Retinal Cell and Molecular Biology 



(12) The distal promoter sequence posi- 
tions -205 to -185 is a region that contains 
two direct repeats of the hexamer TGACCT. 

(13) We found a consensus retinal pho- 
toreceptor-specific site (PCEl). 

(14) The transcriptional machinery in- 
volved in photoreceptor-specific gene ex- 
pression has been strongly and evolutionar- 
ily conserved. 

Significance to Biomedical Research and the 
Program of the Institute 

Eyes have remarkable properties in function- 
ing efficiently over a wide range of illumina- 
tions. Rod cells having photosensitive rho- 
dopsin are more sensitive to dim Hght and 
adapt in the dark to increase their sensitivity. 
However, rod cells cease their sensitive photo- 
transduction in bright Ught. Cone ceUs in 
contrast do not operate in dim Ught but are 
operative in bright Ught. Rhodopsin, trans- 
ducin, phosphodiesterase, rhodopsin kinase, 
and S-Ag have been known to be associated 
with the phototransduction cascade. Rhodop- 
sin kinase and S-Ag are considered to be the 
important proteins for Ught-dependent modu- 
lation of phototransduction. To understand 
this Ught-dependent modulatory mechanism 
in rod outer segments, we have characterized 
S-Ag, Shuzin, and 33 K protein as well as their 
genes. Interestingly, other signal transduction 
systems have cascades similar to that of 
phototransduction (one of the best character- 
ized receptor-mediated signal transduction 
processes). In the phototransduction cascade, 
the shutoff mechanism appears to be modu- 
lated by the phosphorylation and dephos- 
phorylation of rhodopsin. Studying this 
modulation mechanism is important for un- 
derstanding phototransduction as weU as for 
imderstanding signal transduction in general. 
In addition, we think that the night blindness 
of vision may in part be associated with Ught 
adaptation. 



Proposed Course 

This project will be terminated. 
NEI Research Program 

Retinal Diseases — Photoreceptors and Pigment 
EpitheUum 

Publications 

Abe T, Kikuchi T, Chang T, Shinohara T: The 
sequence of the mouse phosducin gene and its 
5'-flanking region. Gene 133:179-186, 1993. 

Abe T, Kikuchi T, Shinohara T: The sequence 
of the human phosducin gene and its 5' flank- 
ing region. Genomic 19:369-372, 1994. 

Shinohara T, Kikuchi T, Tsuda M, Yamaki K: 
A family of retinal S-antigens (arrestins) and 
their genes: Comparative analyses of human, 
mouse, rat, bovine, and Drosophila. Comp 
Biochem Physiol 103:505-509, 1993. 

Usukura J, Khoo W, Abe T, Shinohara T, 
Breitman M: Cone ceUs fail to develop nor- 
maUy in transgenic mice ablated of rod pho- 
toreceptor ceUs. Tissue Cell 275:79-90, 1994. 



1S5 



Laboratory of Sensorimotor Research 



Report of the Chief 
Laboratory of Sensorimotor Research 



Robert H. Wurtz, Ph.D. 



Research in the Laboratory of Sensorimo- 
tor Research concentrated on the con- 
trol of movement by the visual system. 
These projects covered a range of topics ex- 
tending from visual processing to the control 
of specific eye movements. My group has 
studied how visual motion filling the entire 
visual field (optic flow) might be organized to 
modify the movement of observers as they 
move through the visual world. Dr. Lance 
Optican looked at an earUer step in the visual 
pathway and investigated how visual informa- 
tion might be encoded in the patterning of 
neuronal impulses in visual cortex. Dr. Op- 
tican also developed a model of the control of 
rapid or saccadic eye movements based on 
recent work done in the laboratory on a brain- 
stem structure, the superior colliculus (SC). 
Dr. David Lee Robinson investigated how 
these eye movements could be modified by 
changing activity in the coUiculus during the 
generation of the eye movements. Dr. Michael 
E. Goldberg also studied saccadic eye move- 
ments but in relation to the question of how 
our spatial stability is maintained in spite of 
the eye movements that move our fixation 
from part of the field to another. Dr. Freder- 
ick A. Miles concentrated on a very different 
type of eye movement, the vergence eye 
movement, that allows us to fixate on objects 
at varying distances from us. A summary of 
each of the projects is presented below. 



Section on Visual Motor 
Integration 

WhUe moving through the environment, the 
visual world streams past observers in a 
pattern depending on their movement. These 
optic-flow fields provide visual information 
that can guide self-motion, stabilize posture, 
and reveal the structure of the environment. 
My group has studied neurons in the dorsal 
region of the medial superior temporal area 
(MSTd) of monkey extrastriate cortex that 
previously have been shown to respond to the 
expanding radial motion that occurs with 
forward movement of an observer. Different 
directions of observer motion are accompanied 
by centers of motion located in different areas 
of the visual field, and, in our current experi- 
ments, we tested to see whether MSTd neu- 
rons responded best when centers of motion 
were located in different parts of the field. 
About 90 percent of the neurons studied 
responded differentiy to at least one stimulus 
with a shifted center of motion as compared 
with those positioned in the middle of the 
visual field. The preferred centers of motion 
were always limited to one area of the visual 
field for a given cell, and all parts of the 
visual field were represented. We found that 
many cells prefer a center of motion in the 
middle of the field, and those cells respond to 
motion over a more limited region of the field. 



289 



FY 1994 NEI Annual Report 



Using these experiments as a basis, we 
suggest that each of the MSTd neurons has a 
center of motion field with a gradient of 
preferred centers of motion and that there is 
an orderly arrangement of these neurons, with 
each region of the visual field being represent- 
ed by a set of neurons. The responses of 
individual neurons would be graded accord- 
ing to proximity of the center of motion of a 
stimulus to the preferred center of motion for 
the neuron, just as other visual cortical neu- 
rons have gradients for the direction of planar 
motion or for the location of a spot of light. 
The role of MSTd neurons in interpreting the 
optic flow fields would not be one of qualita- 
tive feature matching but rather one of re- 
sponding to visual motion according to the 
degree of match between the visual input and 
the preferred optic flow field of the neuron. 
These MSTd neurons could contribute to the 
determination of heading of observers moving 
through the environment. The organization of 
this area might also be relevant to the control 
of posture because the influence of full-field 
visual motion on posture is well recognized. 



Section on Neural Modeling 

In the past year. Dr. Optican's section has 
worked on two distinct problems. The first 
problem was how the brain represented visual 
information. The second problem was how 
the SC helped control saccadic eye 
movements. 

Neuronal Encoding of Visual Parameters 



information about form and color is grouped 
into separate "channels" in cortex but rather 
suggests that all neurons participate in visual 
processing irrespective of the type of visual 
parameter involved. 

Control of Two-Dimensional Saccadic Eye 
Movements 

Recenfly, Drs. Douglas Munoz and Robert H. 
Wurtz identified three types of neurons (fixa- 
tion, burst, and buildup) in the SC that were 
involved in controUing saccadic eye move- 
ments. During a saccade, activity seems to 
spread through the buildup neurons. Classic 
models, which control saccades in one dimen- 
sion, have a resettable integrator within a local 
feedback loop. Dr. Optican has proposed a 
new, two-dimensional model of the saccadic 
system that places the SC inside the local 
feedback loop. His principal new hj^othesis 
is that the spread of activity in the buildup 
neurons represents the resettable integrator of 
the local feedback loop. 

One complication in this model is that the 
SC is not mapped in cartesian coordinates. 
Rather, there is a retinotopic map in polar 
coordinates that is warped logarithmically 
with eccentricity. Because polar coordinates 
do not follow the rules of vector addition, it is 
not simple to figure out how the activity in 
the buildup neurons should spread during the 
saccade to represent accurately the resettable 
integrator. Dr. Optican has shown that a 
vector field can provide the appropriate motor 
updates for the buildup neurons. 



Dr. Optican and his colleagues used a visual 
discrimination task to study the encoding of 
color and form information in cortical neu- 
rons. It has been proposed that color and 
form information is divided into separate 
channels {e.g., cytochrome oxidase blob and 
interblob regions) in the cortex. A cluster 
method of information calculation was devel- 
oped that made it possible to show that infor- 
mation about color and pattern rises over time 
in all neurons in cortical areas V1-V4. Such a 
result is not consistent with the idea that 



Section on Visual Behavior 

Within the Section on Visual Behavior, Dr. 
David Lee Robinson and his colleagues have 
been studjdng the changes within the oculo- 
motor pathways during the course of saccadic 
eye movements. Most models of the oculomo- 
tor system hypothesize that the signal for 
retinal error undergoes an integration before 
its effect on the oculomotor neurons. Our 
recent models have proposed that this integra- 



290 



Lahoratoiif of Sensorimotor Research 



tion actually takes place within the SC. By 
electrically stimulating the colliculus before 
and during eye movements. Dr. Robinson's 
group members have provided data that 
support this idea. They have demonstrated 
that stimulation at any time during periods of 
fixation leads to rather stereotyped eye move- 
ments; these have been termed fixed vector 
saccades because they are constant in terms of 
direction and amplitude. 

In contrast, when the colliculus was stimu- 
lated during the course of a saccade, the direc- 
tions of these evoked saccades were systemati- 
cally shifted. Just as the eye began to move, 
the movement evoked by electrical stimulation 
was rotated in a direction opposite to the 
primary saccade by as much as 80 degrees. 
Excitation applied at progressively later times 
led to progressively smaller shifts. These data 
are of considerable importance in understand- 
ing the neural contiol of saccadic eye move- 
ments and specifying the collicular contribu- 
tions to them. 



Section on Neuro- 
Ophthalamologic Mechanisms 

Dr. Goldberg and his collaborators have 
studied neurons in the lateral intraparietal that 
have visual responses. The visual responses 
are sometimes predictive: they occur before a 
saccade that will bring the spatial location of 
a visual target into the receptive field of the 
neuron. There are two possibilities to explain 
the phenomenon: In one, there is a general 
increase in retinal excitability, basically an 
expansion of the retinal receptive field; in the 
other, the receptive field moves on the retina, 
like a spotUght of excitability. 

To distinguish between these two hypoth- 
eses, stimuli were flashed in the presaccadic 
receptive field or the postsaccadic receptive 
field at various times before and after the 
saccade. When a monkey is programming a 
saccade, neuronal excitability at the old recep- 
tive field decreases. Just before the saccade, 
many cells are unresponsive to visual stimuU 



that would be expected to drive them during 
normal fixation. At the same time, stimuli 
that would be expected to drive the neuron 
after the saccade drive the neuron before the 
saccade. This result suggests that the locus of 
excitability moves like a spotlight across the 
retina, so that the spatial location that will 
excite the cell after the movement excites it 
before the movement. The disappearance of 
excitability ftom the retinal location that will 
excite the neuron after the saccade prevents 
the cell from providing an inaccurate message 
after the saccade and enables spatially accu- 
rate processing across saccades. 

Monkeys with deep cerebellar nuclear 
lesions have a battery of deficits in their 
oculomotor performance. The crude dynamics 
of their saccadic eye movements are normal; 
the main sequence of saccade peak velocity 
versus ampUtude is normal, but the accuracy 
of visually guided saccades is diminished. 
Monkeys make larger saccades than normal, 
and this deficit is a function of initial orbital 
position. Normal monkeys can adjust the 
ampUtude of saccades for target motion, but 
after deep cerebellar lesions monkeys do not 
compensate for target motion. They therefore 
relatively overshoot centripetally moving tar- 
gets and relatively undershoot centrifu gaily 
moving targets. Normal monkeys can change 
the amplitude of their saccadic eye move- 
ments in response to intrasaccadic target steps; 
postiesion monkeys cannot. Finally, the 
variability of the amplitude of visually guided 
eye saccades is increased in the lesioned 
monkey. These data all suggest that the role 
of the cerebellum in the generation of saccadic 
eye movements is to fine-tune the transforma- 
tion from visual stimulus to movement, a 
fine-tuning that requires an ongoing monitor- 
ing of the saccadic motor signal during the 
saccade. 

Three patients with the unusual finding of 
torsional nystagmus during vertical pursuit 
were examined. Magnetic resonance imaging 
studies in these patients show small arteriove- 
nous malformations in the region of the mid- 
dle cerebellar peduncle. Eye movements 
recorded in one patient demonstrate torsional 



191 



FY 1994 NEI Annual Report 



nystagmus that scales linearly in velocity with 
increasing vertical pursuit velocity and is not 
present during saccades, horizontal pursuit, or 
horizontal vestibulo-ocular reflex cancellation. 
The torsional velocity is decreased when the 
patient pursues an afterimage target, a case 
where there is no retinal sUp signal present. 
These findings suggest that the problem in 
these patients is in the visual-to-motor-signal 
coordinate transformation. The linear relation- 
ship between torsional and vertical pursuit 
velocity suggests that these findings arise fi:om 
an inappropriate cross-coupling between 
vertical visual (or motor) signals, which are 
used in vertical pursuit, and the torsional eye 
movement system. 



Section on Oculomotor Control 

Using monkeys. Dr. Miles and his collabora- 
tors have demonstrated that the vergence eye 
movements resulting from sudden changes in 



the binocular disparity of large textured 
scenes have almost machine-Uke consistency 
and a latency of 52-53 msec, which is less than 
one-third of the value commonly cited in the 
Uterature (which was obtained with small 
targets). They suggest that these disparity 
vergence responses are mediated by neurons 
that have binocular receptive fields that are 
spatially selective (in terms of size, shape, and 
orientation) and offer new insights into the 
properties of such (cortical) neurons. These 
short-latency-vergence eye movements were 
sfrongly affected by an antecedent saccadic 
eye movement, whereby disparity steps ap- 
pUed in the immediate wake of a saccade 
were much more effective than identical steps 
delivered only 200 milliseconds later: fran- 
sient postsaccadic enhancement. This postsac- 
cadic enhancement should normally help to 
speed binocular realignment when gaze is 
redirected to (large?) objects in different depth 
planes. 



292 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00045-16 LSR 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Visuomotor Properties of Neurons in th e Tha lamus 



PRINCIPAL INVESTIGATOR (List ottier professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: David Lee Robinson Ph.D. Section Chief LSR, NEI 



Others: Alexander A. Kustov 



Ph.D. 



Visiting Fellow 



LSR, NEI 



COOPERATING UNITS (if any) 

Department of Anatomy, Howard University (Robert J. Cowie, Ph.D.) 



LAB/BRANCH 

Laboratory of Sensorimotor Research 



SECTION 

Visual Behavior Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.3 



PROFESSIONAL: 



2.0 



1.3 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

With each movement of the eyes, there are changes in the state of the neural processes that generate eye 
movements and changes in the perceptual frame of reference. We have electrically stimulated the superior 
coUiculus at various times around visually guided eye movements to understand the collicular contribution to 
these changes. When the colliculus is stimulated during periods of fixation, the amplitude and direction of the 
saccadic eye movement is rather constant. When the same stimulation is applied while an eye movement is 
in progress, there is a systematic shift in the direction of the evoked eye movement. Just as the eye begins to 
move toward a visual target, the eye movement evoked by stimulation of the coUiculus is rotated in a direction 
opposite to the primary visual saccade by as much as 80 degrees. Electrical stimulation that is applied at 
progressively later times during the visually guided eye movement will evoke eye movements with 
progressively less shift. These data suggest that the colliculus participates in the changes in spatial reference 
during eye movements or that there is a gradual decay in the integration process that the colliculus provides 
to the oculomotor system. 



293 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

Within the oculomotor pathways, there are 
changes before, during, and after each move- 
ment of the eyes. These are related to the 
underl5dng neural processes that generate the 
movement, and they are related to the plan- 
ning that must take place before the next eye 
movement. It has been known for a number 
of years that the superior coUiculus (SC) plays 
an important role in the generation of saccadic 
eye movements. The present studies were 
conducted to understand how the colliculus 
relates to the preparation for saccadic eye 
movements and the compensation for neural 
computations after their initiation. 

Methods 

To localize specific regions of the brain and 
reliably test these sites, adult monkeys w^ere 
trained to enter a primate chair, sit quietly, 
and fixate on a spot of light. They also 
learned to make rapid, saccadic eye move- 
ments when the fixation spot was turned off 
and another Hght appeared. The timing 
between these two events was varied to 
change the temporal properties of the eye 
movements and also modify the direction of 
the animal's attention. After the animals were 
adapted to these situations, they were im- 
planted during sterile surgery with several 
devices for recording eye movements, electri- 
cal activity within the brain, and instruments 
for head restraint. Once the monkeys had 
recovered from the surgery, we positioned 
fine wire electrodes into specific brain sites, 
recorded the discharge patterns of individual 
cells, and electrically excited these loci. The 
timing of the electrical stimulation was varied 
in relation to the onset of target Ughts as well 
as the time course of the evoked saccadic eye 
movements. The evoked eye movements and 
other behavioral responses were recorded and 
quantified. 



Major Findings 

As the oculomotor system plans and generates 
each eye movement, there are many changes 
within its pathways and those systems that 
begin to calculate the next saccade. We want- 
ed to learn how the SC interacted with these 
changing requirements for saccade program- 
ming. 

When an electrode enters the superficial 
layers of the SC, the first neurons encountered 
are very visually responsive. These neurons 
have relatively small visual receptive fields 
that have a fixed position relative to the fovea. 
The location of the receptive field moves with 
the direction of the eye. At these dorsal sites 
within the coUiculus, the threshold for evoking 
saccadic eye movements by electrical stimula- 
tion is at its highest (100 /xA). If stimulation 
of these cells occurs while the monkey fixates 
a central target, then the evoked eye move- 
ment has a fixed vector; the eye moves in a 
set direction with a set amplitude. If the 
stimulation is appUed while the monkey 
fixates targets in different locations, then there 
is a slight effect of the initial eye position on 
the evoked eye movement. Eye movements 
that start on the side of the stimulating elec- 
trode are slightly larger than those that start 
opposite the stimtilation site. 

Next, we wanted to learn if the planning 
or execution of an eye movement would alter 
the movement evoked by electrical stimula- 
tion. If the same site were stimulated during 
fixation, after the disappearance of the fixation 
point, or shortly after the appearance of the 
eye movement target, then the same, fixed 
vector saccade was also elicited. Eye move- 
ments that were evoked by stimulation just 
before the visually guided saccade had a sUght 
shift in their evoked direction. If the electrical 
stimulation was applied during the course of 
a visually guided eye movement, then the 
threshold (120 fiA) and latency were in- 
creased. The characteristics of these evoked 
saccades were also modified but wiU be con- 
sidered in a later section. 



294 



Laboratory of Sensorimotor Research 



As we advanced the microelectrode deep- 
er within the SC, we reached a transition point 
where the neurons began to respond with a 
brisk burst of activity that was before and 
time-locked with the saccadic eye movement. 
The amount of visual excitability of these 
neurons was less than for the cells above 
them. When we electrically stimulated at the 
sites of these cells, the threshold was much 
less (20 fiA). In addition, the latency of the 
evoked eye movements was less; they began 
at least 15 milliseconds (msec) sooner. Just as 
for the more dorsal sites, the eye movements 
evoked during periods of fixation had fixed 
vectors, and there was the same subtie influ- 
ence of irutial eye position. Stimulation ap- 
plied at points long before the visually guided 
saccade evoked the same reproducible eye 
movement. Eye movements that were evoked 
by stimulation just before the visually guided 
saccade to a contralateral target had a sHght 
shift in direction. 

When we stimvilated the coUiculus at the 
sites of eye movement cells during the course 
of an eye movement, the threshold did not 
increase as it had at the more dorsal, visual 
sites. Also, the latencies of the movements 
were shorter. The directions of these eye 
movements shifted in a systematic fashion. At 
the instant of the start of the eye movement to 
a visual target, the evoked eye movements 
had a direction that was shifted from the fixed 
vector by as much as 90 degrees; the shifted 
direction was opposite to that of the primary 
visual saccade. As the time of stimulation 
occurred later and later during the visually 
guided saccade, the amount of rotation from 
the fixed vector systematically diminished. 
The final position of these electrically evoked 
saccades was on a line parallel to the plane of 
the visually guided eye movements. The time 
period during which the evoked saccades 
were shifted lasted about 80 msec longer than 
the duration of the executed visually guided 
saccade. Thus, the absolute duration of the 
whole process could last as long as 120 msec. 

As we advanced the microelectrode still 
deeper, we encountered neurons that had 
more prolonged activity between the appear- 



ance of the saccade target and the saccade to 
acquire it. Electrical stimulation here evoked 
fixed vector saccades during periods of fixa- 
tion, shght influences due to initial position, 
latencies and thresholds similar to the sites 
just above, and systematic shifts in the direc- 
tion of movements when they were evoked 
during the course of the eye movement. 

These data show that the eye movements 
elicited by electiical stimulation are systemati- 
cally influenced by the preparation and occur- 
rence of eye movements. There are two 
possible mechanisms for these effects. The 
direction of gaze changes with each eye move- 
ment, and the visual scene is also modified; 
thus, there is the need to establish a new 
frame of reference. The systematic shift in 
evoked eye movements may reflect this slower 
change in the oculomotor frame of reference. 
Certain studies of spatial localization during 
the course of saccadic eye movement provide 
perceptual data to support this interpretation. 
Alternatively, the systematic shift may reflect 
the resetting of the neural integrator. In most 
models of the oculomotor system, it has been 
hypothesized that the signal for the retinal 
error is integrated before its effect on the 
motoneurons of the eye muscles. After this 
integration has taken place, the neural ele- 
ments responsible will take some time to 
return to their state of rest. Recent data 
suggest that some neurons within the SC 
participate in the integration; the systematic 
shift in electrically evoked saccades may 
reflect the resetting of the integrative functions 
of these neurons. 

Significance to Biomedical Research and the 
Program of the Institute 

One of the major goals of system neuroscience 
is to understand how the brain controls move- 
ment. A thorough understanding of this 
function of the brain is necessary for any 
attempts to rehabilitate patients with cential 
nervous system lesions. Considerable prog- 
ress has been made in this area from studies 
of the control of the oculomotor system. The 
SC is known to be a major participant in the 
transformation of visual signals to the oculo- 



295 



FY 1994 NEI Annual Report 



motor system. The ideas presented here help 
to clarify the types of signals that the collicu- 
lus sends to the oculomotor system and the 
impact of these signals on the control of 
saccadic eye movements. 

Proposed Course 

The most studied aspect of the SC is the 
presaccadic discharge of neurons within its 
intermediate layers. Most modeling of this 
activity describes descending SC projections to 
preociilomotor centers. However, previous 
anatomical data have shown that this region 
also projects to the thalamus. Preliminary 
data obtained whUe extending our study of 
coUicular control of eye movement have 
demonstrated that the region of the thalamus 
that receives a projection from the coUicular 
burst neurons contains cells that discharge 
before eye movements. Our future studies 
will involve extensive recording from these 
neurons, characterizing their discharge pat- 
terns, testing the effects of microstimulation of 
these ceUs, and evaluating the effects of local- 
ized inactivations of this region. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Process- 
ing — Visual Processing and Functional Organi- 
zation, Structure and Function of Central 
Visual Pathways 

Publications 

Cowie RJ, Robinson DL: Head and eye move- 
ments eUcited from the superior coUiculus of 
the macaque. Soc Neurosci Abstr 18:788, 1993. 

Cowie RJ, Robinson DL: Subcortical contribu- 
tions to head movements in macaques. I. 
Contrasting effects of electrical stimulation of 
a medial pontomeduUary region and the 
superior coUiculus. / Neurophysiol, in press. 



Cowie RJ, Smith MK, Robinson DL: Subcorti- 
cal contributions to head movements in ma- 
caques, n. Connections of a medial ponto- 
meduUary head-movement region. / Neuro- 
physiol, in press. 

Kustov AA, Robinson DL: Changes in sac- 
cade trajectory from stimulation of the coUicu- 
lus of the macaque. Soc Neurosci Abstr 
20(1):141. 

Robinson DL, Bowman EM, Kertzman C: 
Covert orienting of attention in macaques. E. 
Contributions of parietal cortex. / Neurophy- 
siol, in press. 

Robinson DL, Cowie RJ: Attentional engage- 
ment and the pulvinar. Behav Brain Sci 
16:586-587, 1993. 

Robinson DL, Cowie RJ: Influences of subcor- 
tical centers on head movements, in Buttner 
U, Brandt T, Fuchs A, Zee D (eds): Contempo- 
rary Ocular Motor and Vestibular Research: A 
Tribute to David A. Robinson. International 
Meeting Eibsee, 1993. Stuttgart, New York, 
Georg Thieme Verlag and New York, Thieme 
Medical PubUshers. 

Robinson DL, Cowie, RJ: Visual contributions 
of the pulvinar, in McCormick DA, Jones EG, 
Steriade M (eds): Thalamus. Elsevier Science 
Ltd., in press. 

Robinson DL, Kertzman C: Covert orienting 
of attention in macaques. HI. Contributions 
of the SC. / Neurophysiol, in press. 



296 



-DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00049-16 LSR 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Cerebral Cortical Mech anisms foi^e MovemeDts and Visual Attention 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Michael E. Goldberg M.D. Senior Medical Research LSR, NET 

Officer 



Others: Edmond J. FitzGibbon M.D. Medical Officer 

Carol L. Colby Ph.D. Senior Staff Fellow 

Makoto Kusunoki Ph.D. Visiting Fellow 

Marc Umeno M.A. pre IRTA 



LSR, NEI 
LSR, NEI 
LSR, NEI 
LSR, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Sensorimotor Research 



SECTION 

Neuro-Ophthalmologic Mechanisms Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



5.2 



PROFESSIONAL 



4.0 



1.2 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Three lines of inquiry were followed to determine how the cerebral cortex and its efferent regions control eye 
movements and visuospatial attention. 

In the first single, a neuron recording was used to probe the mechanisms whereby the parietal cortex of the 
monkey achieves spatial accuracy. It is well known that parietal neurons respond to stimuli in a certain 
location on the retina, the receptive field. The receptive fields of parietal neurons transiently change before 
a saccadic eye movement, so that they respond, before the eye movement, to stimuli that will be in their 
receptive fields after the movement. At the same time, the current retinal location becomes unresponsive. 
These data establish that there is a specific shift of retinal receptive field, rather than a general change of 
excitability. 

In the second, the oculomotor performance of rhesus monkeys was assessed while the monkeys performed an 
oculomotor task before and after electrolytic lesions of the deep cerebellar nuclei. A number of deficits were 
found that were consistent with the cerebellum playing a role in adjusting the amplitude of saccadic eye 
movements but not in initiating them: monkeys had increased variability in the amplitude of their saccadic eye 
movements, they had tended to overshoot targets and this inaccuracy was dependent upon the initial position 
of the eye in the orbit; they were unable to adjust the amplitude of their saccadic eye movements for target 
motion; and they could not adjust the amplitude of their saccadic eye movements in a model of muscle 
weakness. 

In the third, the performance of humans with superior cerebellar peduncle lesions was studied in a smooth 
pursuit task. Cross-coupling between the torsional and vertical pursuit systems was demonstrated. 



297 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

This section has concentrated on three aspects 
of the physiology and phenomenology of 
higher visual and oculomotor processing in 
monkey and human, especially as these func- 
tions relate to the parietal and frontal regions 
of the cerebral cortex, their afferent regions, 
and their efferent targets. Previous work in 
this section has shown that neurons in the 
parietal cortex discharge in response to visual 
stimuU and before saccadic eye movements. 
The neurons contain a predictive aspect to 
their visual responses: neurons respond to 
stimuli whose spatial location wiU be brought 
into their visual receptive fields by impending 
saccadic eye movements. Work in the labora- 
tory for this period has concentrated on un- 
derstanding the mechanism of this predictive 
response by studying the time course of 
changes in retinal excitability before and after 
saccadic eye movements. 

Although the cerebellum has been known 
to participate in the guidance of saccade eye 
movements, its exact contribution has not 
been clear. To estabUsh the role of the cere- 
bellum in the guidance of saccadic eye move- 
ments, and to chart a path for future research, 
comprehensive lesions were made in the deep 
cerebellar nuclei of rhesus monkeys; the per- 
formance of these monkeys was studied in a 
number of tasks before and after the lesions. 

Human disease can often provide insights 
into normal processes. In the course of evalu- 
ating oculomotor performance in patients with 
neurological disease, three patients were 
discovered with torsional nystagmus on up- 
ward smooth pursuit. AU three had lesions of 
the superior cerebellar peduncle. The magnet- 
ic search coil technique was used to study 
their eye movements quantitatively. 

Methods 

Monkeys were implanted with magnetic 
search coils for the measurement of eye posi- 



tion, along with devices for temporary re- 
straint and electrophysiological recording and 
stimulation. They were trained to perform a 
number of visuomotor tasks, including fixa- 
tion, saccades, and smooth pursuit. Microelec- 
trodes were placed in the lateral intiaparietal 
area, and single neurons were studied while 
the monkey performed various visuomotor 
tasks. In a second set of experiments, micro- 
electrodes were placed in the deep cerebellar 
nuclei of the monkeys, neurons recorded, and 
saccades evoked by electrical stimulation. 
Electrolytic lesions were made by passing 
current through the recording microelectrodes 
until saccades could no longer be evoked from 
the area. Oculomotor performance was evalu- 
ated after the lesion, using the same battery of 
tasks for which normal data had been estab- 
lished before the lesion. 

Magnetic search coil limbic rings were 
placed on one patient with torsional nystag- 
mus during vertical smooth purstiit, and his 
eye movements were measured while he 
performed pursuit eye movements at several 
velocities. 

Major Findings 

Neurons in the lateral intiaparietal have visual 
responses. The visual responses are some- 
times predictive: they occur before a saccade 
that wiU bring the spatial location of a visual 
target into the receptive field of the neuron. 
There are two possibilities to explain the 
phenomenon: in one, there is a general in- 
crease in retinal excitability, basically an 
expansion of the retinal receptive field; in the 
other, the receptive field moves on the retina, 
like a spotlight of excitability. To distinguish 
betw^een these two hypotheses, stimuli were 
flashed in the presaccadic receptive field or 
the postsaccadic receptive field at various 
times before and after the saccade. 

When a monkey is programming a sac- 
cade, neuronal excitability at the old receptive 
field decreases. Just before the saccade, many 
cells are unresponsive to visual stimuli that 
would be expected to drive them during 
normal fixation. At the same time, stimuli 



298 



Laboratory of Sensorimotor Research 



that were expected to drive the neuron after 
the saccade drive the neuron before the sac- 
cade. This result suggests that the locus of 
excitabihty moves like a spotUght across the 
retina, so that the spatial location that will 
excite the cell after the movement excites it 
before the movement. The disappearance of 
excitability from the retinal location that will 
excite the neuron after the saccade prevents 
the cell from providing an inaccurate message 
after the saccade and enables spatially accu- 
rate processing across saccades. 

Monkeys with deep cerebellar nuclear 
lesions have a battery of deficits in their 
oculomotor performance. The crude dynamics 
of their saccadic eye movements are normal; 
the main sequence of saccade peak velocity 
versus amplitude is normal, but the accuracy 
of visually guided saccades is diminished: 
morJ<eys make larger saccades than normal, 
and this deficit is a function of initial orbital 
position. Normal monkeys can adjust the 
ampHtude of saccades for target motion, but, 
after deep cerebellar lesions, monkeys do not 
compensate for target motion, and therefore 
relatively overshoot centripetally moving 
targets and relatively undershoot centrifugally 
moving targets. Normal monkeys can change 
the amplitude of their saccadic eye move- 
ments in response to intrasaccadic target steps; 
postiesion monkeys cannot. Finally, the 
variability of the ampUtude of visually guided 
eye saccades is increased in the lesioned 
monkey. These data all suggest that the role 
of the cerebellum in the generation of saccadic 
eye movements is to fine-tune the tiansforma- 
tion from visual stimulus to movement, a 
fine-tuning that requires an ongoing monitor- 
ing of the saccadic motor signal during the 
saccade. 

Three patients with the unusual finding of 
torsional nystagmus during vertical pursuit 
were examined. Magnetic resonance imaging 
studies in these patients show small arteriove- 
nous malformations in the region of the mid- 
dle cerebellar peduncle. Eye movements 
recorded in one patient demonstrate torsional 
nystagmus that scales linearly in velocity with 
increasing vertical pursuit velocity and is not 



present during saccades, horizontal pursuit, or 
horizontal vestibulo-ocular reflex cancellation. 
The torsional velocity is decreased when the 
patient pursues an afterimage target, a case 
where there is no retinal slip signal present. 
These findings suggest that the problem in 
these patients is in the visual-to-motor-signal- 
coordinate transformation. The hnear relafion- 
ship between torsional and vertical pursuit 
velocity suggests that these findings arise ftom 
an inappropriate cross-coupHng between 
vertical visual (or motor) signals that are used 
in vertical pursuit and the torsional eye move- 
ment system. 

Significance to Biomedical Research and the 
Program of the Institute 

Understanding how the cerebral cortex and its 
afferent regions guide eye movements and 
modulate visual attention and learning is 
useful both as a model for the neural contiol 
of other, more compUcated behaviors and as 
a key to understanding and developing treat- 
ments for disorders of the neural control of 
vision, eye movements, and attention. 

Proposed Course 

Lesions in the superior coUiculus wiU be made 
in monkeys with deep cerebellar lesions, to 
see if the deficit previously seen in monkeys 
with combined frontal and coUicular lesions 
can be dupUcated in monkeys with combined 
cerebellar and coUicular lesions. Frontal eye 
field and parietal neurons will be studied in a 
paradigm that more closely resembles a nor- 
mal visual environment, to see if the activity 
demonstrated in a sparse visual environment 
exists under more normal circumstances. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Process- 
ing — Visual Processing and Functional Organi- 
zation, Structure and Function of Central 
Visual Pathways 



199 



FY 1994 NEI Annual Report 



Publications 

Colby CL, Duhamel JR, Goldberg ME: Oculo- 
centric spatial representation in parietal cortex. 
Cerebral Cortex, in press. 

Goldberg ME, Musil SY, Colby CL, Duhamel 
JR, Olson, CR: Cortical mechaiusms for vol- 
untary and involuntary attention: Posterior 
cingulate and lateral tntraparietal areas in the 
monkey, in Albowitz B, Albus K, Kuhnt U, 
Nothdurft HC, Wahle P (eds): Structural and 
Functional Organization of the Neocortex. New 
York, Springer- Verlag, 1994, pp 267-278. 

Olson CR, Musil SY, Goldberg ME: Posterior 
cingulate cortex and visuospatial cognition: 
properties of single neurons in the behaving 
monkey, in Vogt BA, Gabriel M (eds): Neuro- 
biology of Cingulate Cortex and Limbic Thalamus: 
A Comprehensive Handbook. Boston, Birkhauser, 
1993, pp 366-380. 



Segraves MA, Goldberg ME: Effect of stimu- 
lus position and velocity upon the mainte- 
nance of smooth pursuit eye velocity. Vision 
Res, in press. 

Walker MF, FitzGibbon EJ, Goldberg ME: 
Predictive visual responses in monkey superi- 
or coUiculus, in Buttner U, Brandt T, Fuchs A, 
Zee D (eds): Contemporary Ocular Motor and 
Vestibular Research: A Tribute to David A. 
Robinson. International Meeting Eibsee, 1993. 
Stuttgart, New York, Georg Thieme Verlag 
and New York, Thieme Medical PubUshers. 



300 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00153-12 LSR 



PERIOD COVERED 

October 1, 1993 to September 30, 1 994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Visual Motion and the Stabilization of Gaze 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Frederick A. Miles D.Phil. Senior Research LSR. NEI 

Physiologist 

Others: Richard J. Krauzlis 
Geoffrey A. Bush 
Claudio Busettini 



Ph.D. 


IRTA Fellow 


LSR, NEI 


Ph.D. 


IRTA Fellow 


LSR, NEI 


Ph,D. 


Visiting Scientist 


LSR, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Sensorimotor Research 



SECTION 

Oculomotor Control Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



3.8 



PROFESSIONAL; 



2.6 



1.2 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Using monkeys, we have demonstrated that the vergence eye movements resulting from sudden changes in 
the binocular disparity of large textured scenes have almost machine-like consistency and a latency of 52-53 
msec, which is less than one-third of the value commonly cited in the literature (which was obtained with 
small targets). For small steps (less than about 2 degrees) initial vergence accelerations were generally in the 
correct direction — convergent with crossed disparities and divergent with uncrossed — and the initial vergence 
acceleration increased with increases in disparity. However, as disparities exceeded about 2 degrees, initial 
vergence accelerations gradually regressed to a low, albeit nonzero (convergent) level. In fact, disparities in 
excess of 4 to 5 degrees — regardless of whether crossed, uncrossed, or vertical — resulted in the same initial 
(weakly convergent) responses. This was not due to an esophoria or to accommodative convergence because 
there were no early vergence responses when the image seen by the left eye was blanked rather than simply 
shifted. Further, these default convergence responses were not simply due to the disruption of retinal image 
stability consequent to shifting the retinal images because they were not seen with conjugate steps: they must 
have resulted specifically from the change in binocular disparity. That responses peaked with disparities of 
about 2 degrees (or less) suggests that the neurons providing the primary drive for these early vergence 
responses only decode small disparities. We suggest that these disparity vergence responses are mediated by 
neurons that have binocular receptive fields that are spatially selective (in terms of size, shape, and orientation) 
and offer new insights into the properties of such (cortical) neurons. The default responses might represent 
the net output of such neurons to uncorrelated patterns, a phenomenon recently observed in monkey visual 
cortex. 

These short-latency-vergence eye movements were strongly affected by an antecedent saccadic eye movement, 
whereby disparity steps applied in the immediate wake of a saccade were much more effective than identical 
steps delivered only 200 ms later: transient postsaccadic enhancement. Further, this enhancement was due in 
part to the visual stimulation elicited by the saccadic eye movement and could be simulated by shifting the 
visual scene in a saccade-like way. We suggest that this postsaccadic enhancement will normally help to speed 
binocular realigimient when gaze is redirected to (large?) objects in different depth planes. 301 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

Good binocular vision requires that the two 
eyes be aligned on the same object, and this 
necessitates disconjugate (vergence) eye move- 
ments. Neurons that sense the slight differ- 
ences in the images seen by the two 
eyes — resulting from the sUght difference in 
their viewpoints ("binocular disparity") — are 
thought to have an important role in this 
process. The advent of new techniques for 
recording eye position with high precision in 
both monkeys and humans has for the first 
time permitted detailed studies of the relative 
alignment of the two eyes on objects at vary- 
ing distances in a three-dimensional world. 
We have used these high-resolution tech- 
niques (the electromagnetic search coU) to 
examine the vergence eye movements that 
result from sudden changes in the binocular 
disparity of a large, textured scene (more than 
40 degrees x 40 degrees). 

Methods 

Subjects (three rhesus monkeys) faced a tan- 
gent screen onto which were projected two 
superimposed images, each of which was 
visible only to one of the two eyes. This was 
achieved using a special screen and polarizing 
filters so that one of the images was polarized 
in a plane orthogonal to the other, and, when 
viewed through goggles with cross-polarizing 
filters, each eye saw only one of the images. 
The images were irregular geometrical forms 
or computer-generated random dot patterns. 
The positions of both eyes were recorded 
using the electromagnetic search coil method, 
and disparity steps were appUed to the pat- 
terned background after subjects made saccad- 
ic eye movements from a target located 10 
degrees right of center to one straight ahead. 
The targets were extinguished during the 
centering saccade so that only the background 
patterns were visible when the eyes arrived at 
the center of the screen. 



Major Findings 

The vergence eye movements resulting from 
sudden changes in the binocular disparity of 
a large textured scene had almost machine-like 
consistency and ultrashort latency: 52-53 
millisecond (msec). This is less than one-third 
of the latency commonly cited in the Uterature 
(about 160 msec). Most previous studies have 
used small targets viewed against a dark or 
featureless background, necessitating that 
animals be trained to perform appropriately. 
This is not necessary with large textured 
scenes: Responses were obligate, and mon- 
keys were neither trained to tiack the steps 
nor reinforced for doing so. For small steps 
(less than about two degrees), initial vergence 
accelerations were generally in the correct 
direction — convergent with crossed disparities 
and divergent with uncrossed — and the initial 
vergence acceleration increased with increases 
in disparity. However, as disparities exceeded 
about two degrees, initial vergence accelera- 
tions gradually regressed to a low, albeit 
nonzero (convergent) level. In fact, disparities 
in excess of four to five degrees, regardless of 
whether crossed, uncrossed, or vertical, result- 
ed in the same initial (weakly convergent) 
responses. This was not due to an esophoria 
or to accommodative convergence because 
there were no early vergence responses when 
the image seen by the left eye was blanked 
rather than simply shifted. Further, these 
default convergence responses were not sim- 
ply due to the disruption of retinal image 
stabUity consequent to shifting the retinal 
images because they were not seen with 
conjugate steps: They must have resulted 
specifically from the change in binocular 
disparity. We suggest that the default re- 
sponses represent the net output of spatially 
tuned disparity detectors to uncorrelated 
patterns, a phenomenon recentiy observed in 
monkey visual cortex. 

These short-latency-vergence eye move- 
ments were stiongly affected by an antecedent 
saccadic eye movement, whereby disparity 
steps applied in the immediate wake of a 
saccade were much more effective than identi- 
cal steps delivered only 200 msec later: tran- 



302 



Laborato7-i/ of Sensorimotor Research 



sient postsaccadic enhancement. Further, this 
enhancement was due in part to the visual 
stimulation elicited by the saccadic eye move- 
ment and could be simulated by shifting the 
visual scene in a saccade-like way. The post- 
saccadic enhancement of disparity vergence 
resembles the postsaccadic enhancement of 
early ocular following that we described some 
years ago. We suggest that this enhancement 
will normally help to speed binocular realign- 
ment when gaze is redirected to large objects 
in different depth planes. 

We have recently obtained preliminary 
data that indicate that humans also show 
short-latency disparity vergence responses 
wdth large textured patterns: The mean laten- 
cy (±SD) for five subjects was 79 (±5) msec, 
which is almost exactly one-half the value 
usually cited in the Uterature. Human dispari- 
ty vergence also showed a limited range of 
sensitivity to disparity, with default (conver- 
gence) responses for disparities exceeding a 
few degrees, regardless whether crossed, 
uncrossed, or vertical, as well as postsaccadic 
enhancement. 

Significance to Biomedical Researcli and the 
Program of ttie Institute 

Despite their ultrashort latency, it is highly 
likely that these vergence eye movements are 
mediated by cortical pathways because binoc- 
ular disparity is thought to originate from 
cortical processing. (It has been reported that 
a human subject with a sectioned corpus 
callosum could not initiate vergence eye 
movements to images presented to opposite 
hemispheres, that is, to objects bounded by 
the two lines of sight. This points to the 
cortical mediation of vergence eye movements 
in general, not merely disparity-driven ver- 
gence.) That responses peaked with disparities 
of approximately two degrees (or less) sug- 
gests that the neurons providing the primary 
drive for these early vergence responses only 
decode small disparities. We suggest that the 
disparity of extensive patterns such as those 
that we have used can only be sensed by 
neurons that have binocular receptive fields 
that are spatially selective (in size, shape, and 



orientation). These short-latency vergence eye 
movements elicited by large textured patterns 
provide a new tool for exanuning the tem- 
porospatial properties of spatially selective 
binocular cortical neurons. That the responses 
were enhanced by an antecedent saccade or 
saccade-like shift of the field points to a visu- 
ally mediated boost of vergence at the very 
time that binocular aUgnment is commonly 
most challenged — in the wake of saccades to 
a new target in a new plane. 

Proposed Course 

Future studies will examine the temporo- 
spatial properties of these short-latency ver- 
gence eye movements in monkeys and hu- 
mans. 

NEI Research 

Strabismus, Amblyopia, and Visual Process- 
ing — Image Formation and Stabilization, Ocu- 
lar Motility 

Publications 

Busettini C, Krauzlis RJ, Miles FA: 
Short-latency vergence responses, in Buttner 
U, Brandt T, Fuchs AF, Zee DS (eds): Contem- 
porary Ocular Motor and Vestibular Research: A 
Tribute to David A. Robinson. International 
Meeting Eibsee, 1993. Stuttgart, New York, 
Georg Thieme Verlag and New York, Thieme 
Medical Publishers. 

Busettini C, Miles FA, Schwarz U, Carl }R: 
Human ocular responses to translation of the 
observer and of the scene: dependence on 
viewing distance. Exp Brain Res, in press. 

Bush GA, Van der Steen J, Miles FA: When 
the two eyes see patterns of unequal size they 
produce saccades of unequal ampUtude, in 
Delgado-Garcia JM, Godaux E, Vidal PP (eds): 
Information Processing Underlying Gaze Control. 
Tarrytown, NY, Pergamon Press, in press. 

Kawano K, Inoue Y, Takemura A, Miles FA: 
Effect of disparity in the peripheral field on 



303 



FY 1994 NEI Annual Report 



short-latency ocular following responses. 
Visual Neurosci 11:833-837, 1994. 

BCrauzlis RJ, Miles FA: Similar changes in the 
latency of pursuit and saccadic eye move- 
ments observed with the "gap paradigm," in 
Delgado-Garcia JM, Godaux E, Vidal PP (eds): 
Information Processing Underlying Gaze Control. 
Tarrytown, NY, Pergamon Press, in press. 



MUes FA: Stimulus specificity in the primate 
optokinetic system, in Delgado-Garcia JM, 
Godaux E, Vidal PP (eds): Information Process- 
ing Underlying Gaze Control. Tarrytown, NY, 
Pergamon Press, in press. 

Miles FA: The sensing of optic flow by the 
primate optokinetic system, in Findlay JM, 
Kentridge R (eds): Eye Movement Research 
Mechanisms, Processes and Applications. New 
York, Elsevier, in press. 



304 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00109-14 LSR 



PERIOD COVERED 

October 1, 1993 to September 30 , 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Visuomotor Processing in the Pri mate Brain 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: Robert H. Wurtz Ph.D. Chief LSR, NEI 



Others: Hiroshi Aizawa 

Gregg H. Recanzone 
Charles J. Duffy 



Ph.D. 
Ph.D. 
M.D., Ph.D. 



Visiting Fellow 
Guest Researcher 
Senior Staff Fellow 



LSR, NEI 
LSR, NEI 
LSR, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Sensorimotor Research 



Visuomotor Integration Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



4.6 



PROFESSIONAL: 



3.2 



1.4 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Neurons in the dorsal region of the medial superior temporal area (MSTd) of monkey extrastriate cortex have 
previously been shown to respond to the expanding radial motion that occurs as an observer moves through 
the environment. In previous experiments, the MSTd neurons have been shown to respond to radial and 
circular motion when the center of that motion was in the middle of the visual field. Different directions of 
observer motion are accompanied by centers of motion located in different areas of the visual field, and in our 
current experiments we tested to see whether MSTd neurons responded best when centers of motion were 
located in different parts of the field. About 90 percent of the 245 neurons studied responded differently to 
at least one stimulus with a shifted center of motion as compared with those positioned in the middle of the 
visual field. The preferred centers of motion were always limited to one area of the visual field for a given 
cell, and all parts of the visual field were represented. There was a preference among the sample of neurons 
for centers of motion located closer to the middle of the visual field, and neurons preferring this part of the 
field also responded to centers of motion over a more limited region of the field. Based on these observations, 
we suggest that each of the MSTd neurons has a center of motion field with a gradient of preferred centers 
of motion and that there is an orderly arrangement of these neurons with each region of the visual field being 
represented by a set of neurons. This neuronal organization creates the potential for graded responses from 
individual neurons for different directions of heading as an observer moves through the environment. 



305 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Objectives 

We have continued our studies of the visual 
control of movement through the investigation 
of several questions about the organization of 
the cerebral cortex and brainstem of the rhe- 
sus monkey. This year, we have studied the 
control of saccadic eye movements by the 
brainstem (the superior coUiculus), the modu- 
lation of visual processing by attention in 
extrastriate areas of cortex, and the role of 
activity in extrastriate areas in providing 
visual information concerning the heading of 
an observer moving through the environment. 
This report summarizes work on this latter 
topic. 

While moving through the environment, 
the visual world streams around observers in 
a pattern that reflects their motion. These 
optic-flow fields combine the effects of all 
observer movements in three-dimensional 
space to provide visual information that can 
guide self-motion, stabUize posture, and reveal 
the structure of the environment. Previous 
efforts to investigate the neuronal processing 
related to this optic flow has concentrated on 
the medial superior temporal area (MST) of 
the monkey cerebral cortex. Neurons in this 
region respond to visual motion, and neurons 
in the dorsal part of this area (MSTd) have 
large receptive fields that are most responsive 
to the movement of correspondingly large 
patterns of motion. Previous work in our 
laboratory has demonstrated that many of 
these neurons respond selectively to compo- 
nents of optic flow stimuli, including radially 
expanding stimuU. In addition, in our last 
annual report, we provided evidence that 
monkeys use the optic flow stimuli in the 
stabilization of their posture. 

In our previous experiments, and in those 
of others, we have generally tested the neu- 
rons to radial motion when the center of 
motion was in the center of the monkey's 
visual field. One possibility is that these 
neurons might respond better if that center of 



motion were shifted to difterent regions of the 
visual field, as would be the case if the ob- 
server were moving in different directions in 
the visual field under certain conditions. To 
test this possibility, we studied the MSTd 
neurons with large-field optic flow stimuU 
with centers of motion shifted to different 
regions of the visual field. 

Methods 

Our experiments use the rhesus monkey, 
Macaca mulatta, for three reasons. First, our 
experiments frequentiy require the monkey to 
perform specific behaviors in the course of the 
experiments, and the monkey is easily trained 
and becomes a cooperative subject in experi- 
ments requiring high levels of visual perfor- 
mance. Second, to study the ceUs related to 
such a complex function as optic flow, we 
need to know where the characteristics appro- 
priate for analysis of this type of visual mo- 
tion are in the brain cells. The extensive study 
of the cerebral cortex in the rhesus monkey 
over the past two decades and the identifica- 
tion of specific areas that contain the appropri- 
ate neurons, make it possible to study the 
phenomenon of optic flow. Third, the behav- 
ior in response to optic flow in the monkey is 
similar to that studied extensively in humans, 
and what is learned in the monkey is directiy 
relevant to problems in humans. 

An online computer controls the experi- 
ment; it rewards the monkey, collects the 
neuronal and behavioral data, and stores these 
data for later analyses. A second computer, 
under control of the first, generates the optic 
flow stimuli that are projected on a screen in 
front of the monkey by a television projector. 
During the experiment, the monkey faces the 
screen and fixates on a small spot of Ught in 
difterent areas of the screen for a period of 
several seconds to obtain a reward. 

Major Findings 

We studied the responses of aU neurons we 
encountered in the MSTd area and concentrat- 
ed on 245 ceUs that responded to radial and 
circular motion, with the center of this motion 



306 



Laboratory of Sensorimotor Research 



in the middle of the visual screen. We next 
tested each of these cells with the stimulus to 
which it responded best, radial motion or 
circular motion, with the center of motion 
moved away from the middle of the screen. 
We found that about 90 percent of the neu- 
rons had significantly different responses to 
one or more of these shifted centers of motion. 

Thus, a large fraction of the neurons gave 
a different response to the stimulus with a 
shifted center of motion than they did to the 
centered radial or circular stimuU, although 
the magnitude of the response changes varied 
widely. We found a distribution of neuron 
types. Some showed the best response to the 
centered motion in the middle of the visual 
field with all other responses being significant- 
ly smaller; some showed the best responses to 
a center of motion that was eccentric from the 
center; others responded best to a planar 
stimulus, one that we regarded as having a 
peripheral center of motion off the screen. AU 
the areas of the visual field that we tested (17 
sites) had at least one neuron that responded 
to a center of motion in that area, so that we 
conclude that the population of neurons in 
MSTd are likely to be capable of responding 
to a wide variety of different centers of mo- 
tion. 

The most frequently preferred individual 
stimulus was one centered in the middle of 
the visual field. Furthermore, when we exam- 
ined over what region of the field a given cell 
would respond to centers of motion, we found 
that neurons preferring a center of motion in 
the middle of the field responded to centers of 
motion over the smallest regions of the field. 
Thus, many ceUs prefer a center of motion in 
the middle of the field, and those cells re- 
spond to motion over a more limited region of 
the field. In contiast, fewer ceUs prefer cen- 
ters of motion that are more eccentric from the 
center; when they prefer such centers, they 
prefer the centers over a much larger region of 
the field, frequenfly a quadrant of the visual 
field. 

In our complete survey of neurons, we 
used 17 stimuU with different centers of mo- 



tion randomly presented. On a smaller num- 
ber of neurons we used more stimuU (25 or 
more), and with this finer grain analysis for 
the centers of motion, we found neurons that 
selectively preferred the location of the added 
centers of motion that we presented. These 
studies with more refined stimuU suggest that 
the preferred center of motion, rather than 
being a simple segment of the visual field, has 
a more complex structure that our relatively 
Umited stLmuU are only beginning to tap. 

Thus, we found that MST neurons are 
sensitive to shifts in the centers of motion: at 
least one of the shifted center of motion stimu- 
U produced a response significantly different 
from the centered stimulus in more than 90 
percent of the neurons. More than one-half of 
our neurons gave sigruficant responses to 
three or fewer stimuU, indicating that the 
preferred centers of motion were clustered in 
one part of the visual field and not scattered 
about. The neurons studied preferred centers 
of motion throughout all parts of the visual 
field, with the largest number preferring 
centers of motion in the middle of the field. 
These neurons responded to centers of motion 
over a more Umited area of the field. These 
observations suggest that a center of motion 
and the accompanjdng shift in the pattern of 
that motion throughout the visual field are 
stimuU characteristics to which the MST neu- 
rons respond. 

We suggest that each of the MSTd neu- 
rons can be regarded as having a preferred 
center of motion. The responses of individual 
neurons would be graded according to prox- 
imity of the center of motion of a stimulus to 
the preferred center of motion for the neuron, 
just as other visual cortical neurons have 
gradients for the direction of planar motion or 
for the location of a spot of Ught. The role of 
MSTd neurons in interpreting the optic flow 
fields would not be one of quaUtative feature 
matching but rather one of responding to 
visual motion according to the degree of 
match between the visual input and the pre- 
ferred optic flow field of the neuron. The 
population of neurons would have a gradient 
in the density of the centers of motion with 



307 



FY 1994 NEI Annual Report 



those near the middle of the field more highly 
represented and responding to centers of 
motion over a more limited area of the field. 

Significance to Biomedical Research and the 
Program of the Institute 

We think that the MSTd neurons that respond 
to radial motion with centers of motion ar- 
rayed across the visual field could contribute 
to the determination of heading of observers 
moving through the environment. Although 
heading may be determined by a combination 
of visual cues, our findings support the con- 
clusions of a number of psychophysical exper- 
iments that suggest the existence of brain 
mechanisms that might determine heading 
from optic flow. The simplification in our 
visual stimuU limit the conditions under 
which we can apply our observation to head- 
ing judgments. But within those limits, the 
activity of the cells we observed could be used 
to determine heading. Their activity would be 
relevant, for example, in determining heading 
with a fixation point at a distance, as in a 
visual landing of an airplane where the optic 
flow conditions closely approximate the condi- 
tions in our experiment. 

Our experiments are also relevant to a 
growing number of neural models that at- 
tempt to show how directionaUy sensitive 
elements in the brain might produce heading 
judgments. 

Although we have concentrated in our 
discussion on the relevance of these neurons 
for influencing the heading of an observer 
moving through the environment, the organi- 
zation of this area is also relevant to the 
control of posture. Such fuU-field stimulus 
control of posture is well recognized, and the 
visual control of posture is an area only re- 
centiy receiving more intense investigation. 
An understanding of the neuronal mecha- 
nisms underlying the visual processing is 
essential for full understanding of how vision 
modulates posture. 



Proposed Course 

In the experiments described, the optic flow 
stimuU were deUberately made as simple as 
possible to allow study of as many neurons as 
possible and to have as simple an interpreta- 
tion as possible. Future experiments are 
needed to expand the nature of these stimuli 
to more closely approximate those encoun- 
tered in the environment, particularly by 
including depth in the stimulus instead of the 
single-plane stimuli used in the present exper- 
iments. In addition, experiments in this 
laboratory have shown that many of the cells 
studied in the present experiments are also 
sensitive to binocular disparity, that is, they 
give information by binocular mechanisms 
about depth just as optic flow gives informa- 
tion about movement. Combining experi- 
ments using both binocular depth information 
and optic flow information might reveal a 
sensitivity of these neurons beyond what these 
initial experiments have revealed. 

NEI Research Program 

Strabismus, Ambylopia, and Visual Process- 
ing — Visual Processing and Fxmctional Organi- 
zation, Structure and Function of Central 
Visual Pathways 

Publications 

Munoz DP, Wurtz RH: Fixation ceUs in 
monkey superior coUiculus. I. Characteristics 
of ceU discharge. / Neurophysiol 70:559-575, 
1993. 

Munoz DP, Wurtz RH: Fixation cells in 
monkey superior coUiculus. U. Reversible 
activation and deactivation. / Neurophysiol 
70:576-589, 1993. 

Munoz DP, Wurtz RH: Saccade-related activi- 
ty in monkey superior coUicvdus. I. Charac- 
teristics of burst and bviildup cells. / Neuro- 
physiol, in press. 

Wurtz RH, Munoz DP: Organization of sac- 
cade related neurons in morikey superior 
coUiculus, in Buttner U, Brandt T, Fuchs A, 



308 



Lahoratory of Sensorimotor Research 



Wurtz RH, Munoz DP: Role of monkey Zee D (eds): Contemporary Ocular Motor and 
superior colliculus in control of saccades and Vestibular Research: A Tribute to David A. 
fixation, in Gazzaniga MS (ed): The Cognitive Robinson. International Meeting Eibsee, 1993. 
Neurosciences. Cambridge, MIT Press, 1994. Stuttgart, New York, Georg Thieme Verlag 

and New York, Thieme Medical Publishers. 



509 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00256-06 LSR 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Information Processing by Visual System Neurons 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

Chief, Neural Modeling Section LSR, NEI 

Staff Fellow LSR, NEI 

Electronics Engineer LSR, NEI 

Computer Programmer LSR, NEI 

Visiting Associate LSR, NEI 

Visiting Associate LSR, NEI 



PI: 


Lance M. Optican 


Ph.D. 


Others: 


John W. McClurkin 


Ph.D. 




Arthur V. Hays 


B.A. 




Brad J. Zoltick 


M.A. 




Merk Na Chee-Orts 


Ph.D. 




Marc H. Cohen 


M.S.E. 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Sensorimotor Research 



SECTION 

Neural Modeling Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



3.2 



PROFESSIONAL: 



2.6 



0.6 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x| (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Our studies indicate that different visual areas in the brain may communicate via temporally modulated 
messages. We showed previously that neurons in different areas of the brain encode and transmit information 
about stationary, two-dimensional pictures that vary in form, brightness, and duration. We also showed that 
information about remembered visual features was also carried by a temporal code. Now we have extended 
those studies to show that neurons in visual cortex (areas VI, V2, and V4) carry information about the form 
and color of a stimulus in a temporally modulated code. Our results suggest that cortical neurons are able to 
convey information about many different features without confounding them. The mechanism for encoding 
these multiple messages uses temporal modulation to multiplex the different messages together on the neuron's 
response in a separable way. 

It has been proposed that color and form information are divided into separate channels (e.g., cytochrome 
oxidase blob and interblob regions) in the cortex. In a visual discrimination task studying the encoding of color 
and form information in cortical neurons, we showed that information about color and pattern rises over time 
in all neurons in cortical areas V1-V4. Such a result is not consistent with the idea that information about form 
and color is grouped into separate "channels" in cortex, but rather suggests that all neurons participate in visual 
processing, irrespective of the type of visual parameter involved. 



310 



PHS 6040 (Rev. 5/92) 



Laboiaton/ of Sensorimotor Research 



Project Description 

Objectives 

Perception and recognition of complex visual 
pictures depend on the normal function of 
interconnected brain regions extending from 
the retina through inferior temporal cortex. 
The properties of these regions are derived 
from the function of the single neurons within 
them. Thus, to understand how visual per- 
ception occurs, we must learn how informa- 
tion is encoded by the neurons in each stage 
of processing. If we could understand this 
neuronal code, it would become possible to 
distinguish between information related to the 
physical properties of a stimulus {e.g., form, 
luminance, color, size) and information related 
to its behavioral significance {e.g., leading to a 
reward). Individual neurons in aU the visual 
areas studied thus far (retinal ganglion ceU 
fibers; lateral geniculate nucleus neurons; 
pulvinar neurons; cortical neurons in visual 
areas VI, V2, and V4; and inferior temporal 
cortical neurons) encode and transmit infor- 
mation about stationary, two-dimensional 
pictures that vary in form, color, brightness, 
and duration. The neurons use a multidimen- 
sional temporal code to represent and transmit 
their stimulus-dependent messages. We have 
now shown that visual neurons convey com- 
plex messages about both a stimulus' physical 
parameters and its behavioral significance. 
Thus, using information theory, we can begin 
to explore how physical and behavioral com- 
ponents of a neuron's response contribute to 
higher visual cognitive functions such as 
perception, attention, and memory. 

Major Findings 

We have developed a new approach to study- 
ing single neurons in which they are treated 
as communication channels that transmit 
information about visual pictures in their 
responses. This approach has allowed us to 
apply methods from signal processing, statis- 
tics, systems analysis, and information theory 
to understand single neurons. 



According to a commonly held view of 
neuronal function, the strength of a neuron's 
response represents how closely the stimulus 
matches the receptive field's characteristics 
{e.g., orientation or color). Thus, if response 
strength were the only parameter a neuron 
could use to encode information, different 
stimulus features would be confounded by 
individual neurons. Using an informational 
analysis, we have shown that information 
about different stimulus parameters is not 
confounded but is carried across the different 
parts of the multidimensional neuronal code. 

In recent experiments, responses of neu- 
rons in three visual cortical areas (VI, V2, and 
V4) were recorded from a monkey trained to 
choose one of three parafoveal stimuH based 
on whether their color or pattern matched that 
of a cue stimulus. These responses were 
modulated by the pattern and color of the 
stimulus on the receptive field and by the 
pattern or color of the preceding cue. In other 
experiments, stimuh consisted of colored bars 
that were isoluminant with the background or 
black or white bars that varied in size. Infor- 
mation about stimulus features developed 
continuously, but not uniformly, throughout 
the time-course of neuronal responses. Most 
of the information was encoded in the initial 
50 to 60 milliseconds (msec) of the response. 
Some neurons also encoded a large amount of 
information in a second 50 msec interval, 
beginning 20 to 30 msecs after the first. 

These results show that neurons in VI -V4 
carry information about the color, pattern, 
contrast, and size of stimuh. Finally, the 
development of information over time in 
different areas suggests that temporally modu- 
lated waves of activity may form a code for 
visual information. In fact, the response to 
each stimulus could be represented as the 
product of two waveforms that were specific 
for the features paired in each stimulus {e.g., 
color and pattern, color and orientation, con- 
trast and orientation, or size and orientation). 
Feature-specific waveforms for each color, 
pattern, contrast, orientation, and size were 
isolated from the neuronal responses by a 
neural net. The product of these feature 



311 



FY 1994 NEI Annual Report 



waveforms predicted the neuronal responses 
to stimuli with feature combinations not used 
to train the neural net {e.g., novel-colored 
patterns). 

Code waveforms were often similar for all 
neurons within a cortical area. Waveforms 
encoding pattern were strikingly similar across 
aU areas, irrespective of the behavioral task. 
These results suggest that neurons convey 
information about compound visual features 
by multiplexing feature-specific messages 
together. The invariance of the code wave- 
forms suggests that information about a stim- 
ulus feature is represented similarly in aU 
visual areas. 

Significance to Biomedical Research and the 
Program of the Institute 

This project studies how visual information is 
encoded and transmitted by neurons. Knowl- 
edge of these fundamental processes is impor- 
tant for understanding deficits of visual pro- 
cessing, such as occur in amblyopia, and for 
developing visual prosthetic devices to com- 
pensate for field defects or bUndness. 

Proposed Course 

Discovering that the responses of visual sys- 
tem neurons are multidimensional led to the 
discovery that information about multiple 
stimulus features may not be corvfounded by 
single neurons, a result with important — even 
revolutionary — consequences. We now know 
that a substantial part of the temporal modu- 
lation arises after visual information has left 
the retina. Our latest results show that the 
neural code arises due to the influence of 
feedback. 



Ever since we found evidence of a neural 
code, and saw a possible structure for it, we 
have been trying to delineate it. The proper- 
ties of the code should give clues about the 
functions performed by the neurons. Now 
that we have shown that some of the temporal 
codes are invariant across cells, and even 
across areas, a new theory of visual informa- 
tion processing is required that will treat the 
visual system more as a concurrent processing 
system rather than as a hierarchical cascade of 
independent areas. Both these issues are 
being pursued. 

Our findings suggest a completely new 
conceptional framework in which to investi- 
gate neuronal function. One presumed reason 
for the huge number of single neurons has 
been the necessity to unconfound stimulus 
features. However, we propose that the 
simultaneous messages about different fea- 
tures can be used as tags, so that the messages 
that arise in different processing regions of the 
visual system can be reunited into a unified 
percept. This would provide the mechanism 
to build a whole perception across many 
processing regions. With the use of new 
computational equipment, this hypothesis is 
being explored both experimentally and theo- 
retically. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Process- 
ing — Visual Processing and Functional Organi- 
zation, Structure and Function of Central 
Visual Pathways 



312 



Ljiboratory of Sensorimotor Research 



Publications 

McClurkin JW, Zarbock JA, Optican LM: 
Temporal codes for colors, patterns, and 
memories, in Peters A, Rockland KS (eds): 
Cerebral Cortex, Vol. 10, Primary Visual Cortex of 
Primates. New York, Plenum, 1994, pp 
443-467. 



Optican LM: Control of saccade trajectory by 
the superior colliculus, in Buttner U, Brandt T, 
Fuchs A, Zee D (eds): Contemporary Ocular 
Motor and Vestibular Research. A Tribute to 
David A. Robinson. International Meeting 
Eibsee, 1993. Stuttgart, New York, Georg 
Thieme Verlag and New York, Thieme Medi- 
cal Publishers. 



McClurkin JW, Optican LM, Richmond BJ: 
Cortical feedback increases visual information 
transmitted by monkey parvocellular lateral 
geruculate nucleus neurons. Vis Neurosci 
11:601-617, 1994. 



313 



Ophthalmic Genetics and Clinical 
Services Branch 



i 



Report of the Chief 
Ophthalmic Genetics and Clinical Services Branch 



Muriel I. Kaiser-Kupfer, M.D. 



The Ophthalmic Genetics and CUnical 
Services Branch (OGCSB) within the 
National Eye Institute (NET) Intramural 
Research Program has been operational since 
February 1989. The branch is divided into 
four sections: Ophthalmic Genetics, Acting 
Chief Dr. Muriel I. Kaiser-Kupfer; Cataract 
and Corneal Diseases, Chief Dr. Manuel B. 
Datiles; Ophthalmic Pathology, Acting Chief 
Dr. W. Gerald Robison, Jr.; Clinical Services, 
Acting Chief Dr. Rafael C. Caruso. 

The purpose of the OGCSB is to conduct 
clinical and laboratory research on gene ex- 
pression and molecular interactions important 
to the eye and to apply clinically relevant 
research findings to the prevention, diagnosis, 
and treatment of diseases affecting the eye 
and the visual system. Such disorders include 
corneal and retinal diseases, cataract, and 
visual pathway abnormalities. 

The OGCSB is responsible for the essential 
psychophysical and electrophysiological diag- 
nostic tests of visual function required by all 
clinical intramural research programs of the 
National Institutes of Healtii (NIH). In addi- 
tion, it processes ocular clinical biopsy and 
autopsy materials. The OGCSB differs from 
other NEI laboratories engaged in molecvdar 
investigations because its emphasis is on 
translating the appropriate research findings 
dirertly to the clinical setting. Thus, OGCSB 
is also a point of focus for the trans-NIH 
emphasis on research in genetics, more effec- 
tively aligning its organizational structure 
within the NEI's Intramural Research 
Program. 



Since beginning its operation, the OGCSB 
has shown considerable growth and produc- 
tivity. 



Section on Cataract and Corneal 
Diseases 

The Section on Cataract and Corneal Diseases 
continued to pursue research of the anterior 
segment, especially on the short-term and 
long-term effects of contact lens wear on the 
cornea. Analysis of the data may be helpful 
in understanding the dynamics of contact 
lens-cornea interaction, the risk to corneal 
tissues, and how systemic or local ocular 
disorders may increase the risk of wearing 
contact lenses. Corneal endothelial morpholo- 
gy is being studied by specular microscopy to 
compare the endotheUal status in patients 
wearing different types of lenses. The devel- 
opment of automated computer analysis is 
under way to facilitate data analysis currentiy 
done by hand, which is time consuming and 
laborious. 

This section has been particularly produc- 
tive in studies using different systems to 
develop objective and subjective methods of 
monitoring and documenting opacities in the 
human lens. Objective systems being tested 
and developed include the Scheimpflug sys- 
tems (Zeiss and Oxford) and the retroiUumina- 
tion camera (Neitz and Oxford). Subjective 
systems or methods such as the LOCS 11 
grading system and the effects of cataracts on 
visual field, contrast sensitivity, and glare are 
also being studied and refined. These systems 



317 



FY 1994 NEI Annual Report 



are now being used to study the natural 
history of various cataracts such as presenile, 
senile or age-related, steroid induced, radia- 
tion, diabetic, retinitis pigmentosa, gyrate 
atrophy (GA), and neurofibromatosis 2 (NF2). 
Monitoring and documenting human cataract 
development is a crucial step toward the 
ultimate testing of several medications that 
might be helpful in preventing or reversing 
human cataracts. A large U.S. family has been 
ascertained, and the autosomal dominant 
cataracts with interocular variabihty has been 
identified. A second family has been identi- 
fied in Hyderabad, India, and linkage has 
been established using micro markers. 

Research in cataractogenesis has been 
hampered by the extreme scarcity of tissue 
and an abrupt shift in surgical technique from 
intracapsular (intact lens) to extracapsular 
(fragmented lens) extraction. Through the 
collaborative efforts of cataract surgeons and 
basic researchers, efforts have been under way 
to develop and modify techniques to study 
materials that become available at surgery and 
can be well documented clinically. We are 
presently performing careful documentation of 
the cataract in patients preoperatively using 
clinical aphotographic LOGS II grading and 
Zeiss Scheimpflug and Oxford retroiUumina- 
tion video photography and image analysis. 
Gataracts are extracted extracapsularly fol- 
lowed with implantation of an intraocular 
lens. Specimens obtained are examined histo- 
logically using Ught and electron microscopy 
and biochemically using two-dimensional gel 
electrophoresis (PHAST and LSB systems). 
Gataractous specimens are compared with 
normal tissues obtained from eye-bank eyes. 
Abnormal proteins are identified using im- 
munoblotting techniques as well as by protein 
sequencing. 

It has been demonstrated that with aging 
there is an acidic shift of proteins and an 
increased number of polypeptide species in 
the molecular weight range of the crystaUins. 
It is of interest that these changes occur main- 
ly in the lens nucleus and may be more for 
protection of lens proteins and preservation of 
lens clarity rather than leading to cataract 



formation. These studies are helping differen- 
tiate changes due to aging from changes 
occurring in cataract formation. 

Investigators in this section have been in 
the forefront of recognizing the role of the 
neural crest in normal and abnormal develop- 
ment of the anterior segment. Studies contin- 
ue on anterior chamber abnormalities and 
iridocorneal endotheHal syndrome patients. 



Section on Ophthalmic Genetics 

Studies by the Section on Ophthalmic Genetics 
have emphasized retinal degeneration and 
ophthalmic involvement in systemic genetic 
diseases. This section has been a leader in 
stud5dng GA of the choroid and retina. The 
accumulation of natural history data and the 
work on definition of the genetic abnormah- 
ties have been unique. Evidence for biochemi- 
cal, cUnical, and molecular heterogeneity 
continue to be confirmed. There appear to be 
many different single point mutations in the 
ornithine aminotransferase gene in GA pa- 
tients. Dietary intervention studies using an 
arginine-deficient diet have been very promis- 
ing, especially in young patients in whom a 
delay in the onset of pathologic changes has 
been demonstrated. GA is a condition that 
may be amenable to gene therapy; preliminary 
laboratory studies are under way. Usher's 
syndrome, congenital deafness, and retinitis 
pigmentosa patients are being studied using 
molecular techniques to map the gene and to 
identify the responsible mutation. Bietti 
crystaUine retinopathy, a tapetoretinal degen- 
eration with marufest corneal dystrophy is a 
rare condition apparentiy more common in 
Asians than in other ethnic groups. Linkage 
studies are under way to attempt to locate the 
gene responsible for this rare condition. 

Foveal cone sensitivity (assessed by mea- 
surements of increment thresholds) and orien- 
tation (estimated with measurements of the 
Stiles-Grawford effect) were found to be 
abnormal in a group of patients with GA. 
These results suggest that foveal cones are 



318 



Ophthalmic Genetics and Clinical Services Branch 



altered in their orientation and sensitivity 
before the encroachment on the foveal area by 
the atrophic lesions of GA. Genetic linkage 
studies are under way to pursue the gene(s) of 
this congenital cataract in this family. 

Albinism in arumals has been associated 
with an anatomic anomaly of the visual path- 
ways characterized by an excessive crossing of 
the retino-geniculate fibers with two different 
modes of geniculo-cortical projection. In 
humans there is indirect evidence of the same 
anomaly demonstrated by asymmetry in 
visual evoked potentials (VEP) elicited by 
pattern reversal stimulation. Recent studies 
using appearing-disappearing patterns claim 
VEP asymmetry to be diagnostic and propose 
a uniform type of asymmetry. We used the 
same recording conditions to determine the 
diagnostic value of VEP in albinism and to 
attempt to correlate the VEP results with 
cUnical features. This study shows that there 
are two different patterns of VEP asymmetry 
in albinism that may be explained by the 
differences in the reorganization of the genicu- 
lo-cortical pathway. VEP asymmetry is fre- 
quent but may not be constant in this condi- 
tion. However, its value is decreased in some 
cases where the low amplitude of the respons- 
es makes the interpretation difficult. Further- 
more, there is no correlation between the type 
of the asymmetry with any other feature of 
albinism. 

Collaboration with the Interinstitute Ge- 
netics Program has continued with active 
participation by the Genetics CUnic. During 
the past year, approximately 200 individuals 
representing approximately 60 different dis- 
ease categories have been seen. Because of the 
high frequency of ocular involvement in these 
cases, almost all of these patients were evalu- 
ated by the OGCSB staff. 

NF2, otherwise known as bilateral acoustic 
neuroma, is inherited as an autosomal domi- 
nant disorder. Multiple members of several 
large pedigrees as well as a large number of 
unrelated families have been studied in collab- 
oration with Dr. Dilys Parry, from the Nation- 
al Cancer Institute (NCI). An important 



original observation was the striking frequen- 
cy (80 to 85 percent) of posterior capsular 
cataract in patients with NF2. In addition, 30 
percent of patients have shown associated 
cortical cataracts. These findings are helpful 
in estabUshing a diagnosis of NF2 in at risk 
patients. The etiology of the cataract is un- 
clear; however, it is interesting that the gene 
locus for bilateral acoustic neuromas is on 
chromosome 22 as is the gene for p-B-crystal- 
hn. Combined pigmented retinal harmarto- 
mas appear to be another ocular marker for 
some patients with severe NF2. 

Finally, the results from the continuing 
double-masked control cUnical trial of topical- 
ly administered cysteamine to patients with 
nephropathic cystinosis are very exciting. 
After confirming the usefulness of 0.5 percent 
cysteamine eye drops in the young patients, 
we have expanded our study to include older 
patients, with similarly striking results. Partic- 
ularly important is the fact that these patients 
have shown dramatic relief from their ocular 
symptoms with a decrease in crystals in the 
treated eye and a significant improvement of 
their quality of life. 



Section on Clinical Services 

The Section on CUnical Services has been very 
active in characterizing psychophysical and 
electrophysiological findings in patients with 
diseases that affect the eye and the visual 
system. Continued documentation by nonin- 
vasive techniques has shown that more and 
more refined and accurate classification of 
diseases is possible. Psychophysical and 
electrophysiological information is particvdarly 
helpful in understanding the pathogenesis of 
disease as well as being available for use as a 
marker in various treatment modaUties. 

Results of VEP studies have shown that a 
summation of two waveforms, which fre- 
quentiy show the same surface-positive polari- 
ty and are generated by stimulation of each 
hemifield, combine to generate the peaks of 
the fiill-field VEP. 



329 



FY 1994 NEI Annual Report 



Our results indicate that the sum of the 
asymmetrical contribution of either hemi- 
sphere and either eye are responsible for the 
symmetrical VEP elicited by binocular stimula- 
tion with a full-field stimulus. An asymmetri- 
cal full-field VEP may be seen in normal 
subjects and does not imply an abnormality in 
the visual pathways. 

Studies of dark adaptation (DA) in pa- 
tients with retinal dystrophies have indicated 
that a complete evaluation of DA should 
include, in addition to this measurement, the 
time constant of adaptation, that provides 
information about the rate at which this final 
threshold is reached. The time constant serves 
as a clinically relevant parameter in both the 
diagnosis of retinopathies and in the followup 
of individual patients over time. 



Section on Ophthalmic Pathology 

The Section on Ophthalmic Pathology has 
provided technical support services to investi- 
gators involved in cliriical and basic research 
as well as to those performing routine pathol- 
ogy. Careful monitoring of the volume of 
material handled shows a steady increase in 
processing by the laboratory with excellent 
results. Considerable savings to the NEI have 
resulted from the elimination of costiy contract 
services. 



320 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00187-11 OGCSB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

The Effects of Corneal Contact Lenses on the Cornea 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 
PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI 



Others: 



Gregory P. Kracher 
Lessie M. McCain 
Louella Lopez 
Doretha Leftwood 
Anup Mahurkar 



O.D. 
R.N. 
M.D. 
B.A. 
B.S. 



Expert 

Nurse Specialist 
Visiting Associate 
Computer Specialist 
Visiting Associate 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 

Image Processing and Analysis Laboratory, Division of Computer Research and Technology, NIH 
Vivino, B.S., Staff Engineer) 



(Mark 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.00 



PROFESSIONAL 



0.00 



0.00 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



THIS PROJECT HAS BEEN TERMINATED 



321 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00188-11 OGCSB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Documentation and Monitoring of Opacities in the Human Lens 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI 



Others: 



Benjamin V. Magno 


M.D 


Anup Mahurkar 


B.S. 


Doretha Leftwood 


B.A. 


Joan Lee 





Visiting Associate 
Visiting Associate 
Computer Specialist 
Clinic Coordinator 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 

Image Processing and Analysis Laboratory, Division of Computer Researcii and Technology (DCRT), NTH (Benes Trus, Ph.D., Chief; 
Mark Vivino, B.S.); Biomedical, Engineering and Instrumentation Branch, DCRT, NIH (Michael Unser, Ph.D., Biomedical Engineer); 
Division of Epidemiology Qinical Trials, NEI (Valerie Friedlin, Ph.D. and Marvin Podgor, Ph.D.) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.775 



PROFESSIONAL: 



2.325 



1.45 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project uses different systems to develop objective and subjective methods to monitor and document 
opacities in the human lens. We are actively recruiting patients with and without cataracts for reproducibility 
and foUov^Tip studies on the objective systems — the Scheimpflug cameras (Zeiss and Oxford) and 
retroillumination (Neitz and Oxford) cameras. Our study of subjective systems or methods such as the LOCS 
II grading system and the effects of cataracts on visual perception, contrast sensitivity, and glare may be useful 
in identifying additional parameters for monitoring cataract presence, progression, or regression. In addition, 
we are modifying the systems to improve their accuracy and usefulness as well as developing outright 
improved systems and methods of documenting and monitoring progression of cataracts. We are now using 
these systems to study the natural history of various cataracts such as presenile, senile, or age-related, steroid- 
induced, radiation, diabetic, retinitis pigmentosa, gyrate atrophy, and neurofibromatosis 2 cataracts. This study 
will prepare the way for eventual clinical trials of anticataract drugs. 

Genetic linkage studies under way are pursuing the gene(s) of congenital cataract. 



322 



PHS 6040 (Rev. 5/92) 



Ophtfwlmic Genetics and Clinical Services Branch 



Project Description 

Additional Personnel 



Yvonne Duglas- 


B.S. 


Biologist, OGCS, NEl 


Tabor 






Marvin Podgor 


Ph.D. 


Epidemiologist, DEE, 
NEl 


Rita Hiller 


M.S. 


Epidemiologist, DEE, 
NEl 


Robert Sperduto 


MJD. 


Chief, EE EE, NEl 


Laura Wozencraft 


M.S. 


Genetic Counselor, 
OGCS, NEl 


J. Fielding 


MX)., 


Medical Officer, 


Hejtmancik 


PhD. 


LMOD, NEl 



Clinical Protocol Number 

84-EI-0132 

Objectives 

The objective of this project is to formulate a 
means of documenting cataract formation and 
progression. This is an important step to take 
before undertaking clinical trials of drugs 
purported to prevent cataract and cataract 
progression. Family studies are involved in 
looking for the gene for congenital cataract via 
linkage studies. 

Methods 

Complete ophthalmologic examination, includ- 
ing contrast sensitivity, glare testing, and 
potential acuity testing, will be performed for 
each person in the study. Techniques used to 
measure and evaluate cataracts include 
Scheimpflug photography, retroUlumination 
photography, specular microscopy, and laser 
light-scanning spectroscopy. 

Major Findings 

We have found that clinical grading of cata- 
racts using the LOGS n system and photo- 
graphic analysis of cataracts using the NEl 
Scheimpflug system can quantitatively detect 
the progression of age-related cataracts within 
one year. In addition, we found that in vari- 
ous types of cataracts, glare and contrast 



sensitivity testing showed abnormal results 
only in the severe or more advanced grades. 
The only exception was in posterior subcapsu- 
lar cataracts, which showed an abnormality in 
contrast and glare sensitivity in the early 
stages based on LOGS II grading. In a study 
of pure nuclear cataracts, we found a signifi- 
cant correlation between lens nuclear density 
(using either LOGS 11 grading or Scheimpflug 
photography) and contrast sensitivity loss of 
intermediate and high spatial frequencies. 

In our continued development of objective, 
semiautomated methods of detecting and 
following cataracts, we now are able to quick- 
ly perform densitometry of Scheimpflug 
nuclear cataract images and compare it with 
previous images to detect significant changes 
expressed in optical density units. For posteri- 
or subcapsular and cortical cataracts we have 
also developed a semiautomated method of 
quantitating the cataracts in mm" using retioU- 
lumination photographs. 

Significance to Biomedical Research and the 
Program of the Institute 

Monitoring and documenting human cataract 
progression is a crucial step toward the ulti- 
mate testing of several medications believed 
capable of preventing or reversing human 
cataracts. This step is also important in cate- 
gorizing types of cataracts in various parts of 
the world and correlating them with the 
physical and genetic factors within specific 
geographic regions. 

Subjective methods of determining visual 
function are also important to determine the 
handicap cataract patients have in coping with 
daily activities. In our studies none of the 
subjective methods could demonstrate subjec- 
tive experiences in early cataracts; therefore, 
research is needed to develop more sensitive 
techniques. 

Proposed Course 

We will continue the study and development 
of subjective and objective methods of docu- 
menting and morutoring human cataracts. We 



323 



FY 1994 NEI Annual Report 



will pursue the improvement and automation 
of the present system of lens photography 
(e.g., Scheimpflug, retroUlumination, and laser- 
Ught spectroscopy) as well as exploration of 
possible applications of new technological 
advances. Appropriate population groups for 
study will be identified. 

NEI Research Program 

Lens and Cataracts — Epidemiology of Cataract 
Publications 

DatUes M: Progress in development and 
testing of anticataract medications. Ophthal- 
mology 100:9A:70, 1993. 

Datiles M, Lasa S, Podgor M, Hemandez- 
GaleUa E, Magno B: Reproducibility of the 
NEI cortical cataract retroillumination system. 
Curr Eye Res, in press. 

Datiles M, Magno B, Leftwood D, Friedlin V, 
Vivino M: Longitudinal study of age-related 
nuclear cataracts using the NEI Scheimpflug 
Imaging System. Invest Ophthalmol Vis Sci 
(Suppl) ARVO Absti-act 34(4):943, 1993. 

Kashima K, Trus B, Unser M, Datiles M, 
Edwards P, Sibug M: Aging studies on nor- 
mal volunteer lenses using the Scheimpflug 
slit-lamp camera. Invest Ophthalmol Vis Sci 
34:263-269, 1993. 

Kashima K, Unser M, Datiles M, Trus B, 
Edwards P: Minimum views required to 
characterize cataracts when using the 
Scheimpflug camera. Graef Arch Ophthalmol 
231:687-691, 1993. 

Lasa S, Datiles M: Longitudinal study of age- 
related cortical cataracts using retroillumina- 
tion photographs. Invest Ophthalmol Vis Sci 
34(4):943, 1993. 



Lasa S, Datiles M, Friedlin V: Potential vision 
tests in patients with cataracts. Invest Ophthal- 
mol Vis Sci 35(4a):1962, 1994. 

Lasa S, Podgor M, Datiles M, Magno B: Glare 
sensitivity in early cataracts. Brit J Ophthalmol 
77:489-491, 1993. 

Lopez L, Datiles M: Longitudinal study of 
age-related posterior subcapsular cataracts 
using retroUlumination photographs. Invest 
Ophthalmol Vis Sci 34(4) :943, 1993. 

Lopez L, Datiles M, Podgor M, Vivino M, 
Mahurkar A, Lasa S: ReprodudbUtiy study on 
the NEI retroillumination analysis system. 
Invest Ophthalmol Vis Sci 35(4a):1962, 1994. 

Magno B, Datiles M, Lasa S: Cataract progres- 
sion rates using the lens opacity classification 
system. Invest Ophthalmol Vis Sci 34:2138-2141, 
1993. 

Magno B, Friedlin V, Datiles M: Comparison 
of Unear, multilinear and mask microdensito- 
metric analysis of Scheimpflug images of the 
lens nucleus. Curr Eye Res, in press. 

Magno B, FriedHn V, DatUes M: Reproducibil- 
ity of the NEI Scheimpflug Imaging System. 
Invest Ophthalmol Vis Sci 35:3078-3084, 1994. 

Vivino M, Chintalagiri S, Trus B, Datiles M: 
Development of a Scheimpflug slit lamp 
camera system for quantitative densitometric 
analysis. Eye 7:791-798, 1993. 

Vivino M, Mahurkar A, Trus B, Lopez L, 
Datiles MB: Quantitative analysis of retroillu- 
mination images. Invest Ophthalmol Vis Sci 
35(4a):1947, 1994. 



324 



I PROJECT NUMBER 
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00212-09 OGCSB 

. \ — 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on arte line between the borders.) 

Use of Human Lens Mate rial f or D etermining Possible Causes of Cataracts^ 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Irtstltute affiliation) 

PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI 

Others: Susan M. Lasa M.D. Visiting Associate OGCSB, NEI 

Benjamin V. Magno M.D. Visiting Associate OGCSB, NEI 

Yvonne Tabor B.S. Biological Technician OGCSB, NEI 

Louella Lopez M.D. Visiting Associate OGCSB, NEI 

Pushpa Sran M.D. Medical Officer OGCSB, NEI 



COOPERATING UNITS (if any) 

Laboratory of Mechanisms of Ocular Diseases, NEI (Donita Garland, Ph.D.); Laboratory of Immunology, NEI 
(Miguel Bumier, Jr., M.D.) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 

3.05 



PROFESSIONAL: 

1.75 



1.3 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects Q (b) Human tissues □ (c) Neither 

□ (a1) Minors 

□ (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

There is an extreme scarcity of properly documented and classified human cataract material because of an 
abrupt shift of cataract surgical technique from intracapsular (intact lens) to extracapsular (fragmented lens) 
with the advent of the use of intraocular lens. We are exploring ways by which fragmented lens materials can 
be maximally used in cataract basic research through close collaboration with cataract surgeons and basic 
researchers and through modification of techniques by both groups. 

We are now carefully documenting the cataracts preoperatively, using clinical and photographic LOGS 11 
grading and Zeiss Scheimpflug and Oxford retroillumination video photography and image analysis. Cataracts 
are extracted extracapsularly with implantation of an intraocular lens. Specimens obtained are examined 
histologically, using light and electron microscopy, and biochemically, using two-dimensional gel 
electrophoresis (PHAST and LSB systems). Cataractous specimens are compared with normal tissues obtained 
from eye bank eyes. Abnormal proteins are identified using immunoblotting techniques as well as protein 
sequencing. 



325 



PHS 6040 (Rev. 5/92) 



FY 1994 NEI Annual Report 



Project Description 

Additional Personnel 



Samuel Zigler 


PhD. 


Head, Section on 
Cataracts, LMOD, NEI 


Muriel I. Kaiser- 


MD. 


Chief, OGCS, NEI 


Kupfer 






J. Fielding 


M.D., 


Medical Officer, 


Hejtmancik 


PhD. 


LMOD, NEI 


Clinical Protocol Number 




84-EI-0194 







Objectives 

In light of the present and future scarcity of 
intact human cataracts for basic research, this 
study was designed to develop methods in 
which fragmented lens materials can be maxi- 
mally used for biochemical and genetic re- 
search. 

Methods 

We examined patients who have different 
forms of cataracts and documented their 
cataracts as described in the project Documen- 
tation and Monitoring of Opacities in the 
Human Lens (ZOl EY 00188). After extracap- 
sular cataract extraction, fragmented lens 
materials, including the anterior lens capsule, 
lens nucleus, and aspirated lens cortical mate- 
rial, undergo protein and biochemical analy- 
ses. Some of the specimens are frozen for 
future genetic studies. We also have under- 
taken studies to determine the visual results of 
current techniques of cataract surgery and 
how to modify and improve them further. 

Major Findings 

We have found that a successful collaboration 
requires a close professional relationship 
between the chnidan-surgeon and the basic 
researcher. Although the two professionals 
have different immediate priorities (one being 
patient care, the other adequacy of tissue 
sample for laboratory studies), the ultimate 



goal of alleviating human suffering is the 
same. 

The iwo-dimensional gel electrophoresis 
technique may be extremely useful in deter- 
mining lens protein changes using very small 
amounts of tissue (300 meg). Aging results in 
acidic shifting of proteins, an increased num- 
ber of polypeptide species in the molecular 
weight range of the crystallins, and covalent 
cross-linking of crystallins — changes that need 
to be differentiated from changes occurring in 
cataract formation. 

Recently, we found that in normal aging 
these age-related changes in lens proteins are 
confined to the nuclear region of the lens; the 
cortex remains the same. This means that 
these age-related changes are adaptations to 
maintain clarity of the nucleus and are not 
precataract. We also have found that lens 
material aspirated through the irrigator-aspira- 
tor or phakoemulsifier loses some crystallins. 
Optimum samples are those we obtain sepa- 
rately, thus avoiding oxidation. 

Significance to Biomedical Research and the 
Program of the Institute 

A severe setback is being dealt many cataract 
projects because of the lack of human cataract 
material available for basic research studies. 
Whereas the current technique, which involves 
fragmenting the cataract during extraction, is 
extremely successful and effective, there is no 
foreseeable change back to use of the intracap- 
sular method (removal of the lens in toto). 
Hence, it is imperative to modify our basic 
research methodology to adapt to the use of 
fragmented lens materials to continue the 
various basic lens research projects that deal 
with human materials. We are already learn- 
ing about possible differences between normal 
age-related changes in lens protein and precat- 
aract. This knowledge is crucial for the even- 
tual understanding of the cause of cataracts. 

Proposed Course 

We will continue to pursue the development 
of the use of fragmented lens material in basic 



326 



Ophthalmic Genetics and Clinical Services Bratich 



research experiments. Using two-dimensional 
gel electrophoresis, we will study ways in 
which surgeons may modify their surgical 
techniques without compromising patient care 
while providing scientists with critical lens 
tissue for basic research. In addition, we will 
investigate ways in which scientists can work 
with surgeons in handhng lens materials to 
maximize the quality of specimens for basic 
research. 

NEI Research Program 

Lens and Cataract — Pathogenesis of Cataract 



Publications 

Datiles M: Progress in development and 
testing of anticataract medications. Ophthal- 
mology (suppl) 100(9A):70, 1993. 

Garland D, Datiles M, Zigler J, Duglas-Tabor 
Y: Post translational modification of human 
crystalline. Invest Ophthalmol Vis Sci 

35(a):1904, 1994. 



327 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00281-02 OGCSB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Addendum to Use of Human Lens Material for Determining Possible Causes of Cataracts 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI 



Others: 



J. Fielding Hejtmancik 
Laura A. Wozencraft 



M.D., Ph.D. 
M.S. 



Medical Officer 
Genetic Counselor 



LMOD, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



1.325 



PROFESSIONAL: 



1.325 



0.0 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 
\x\ (a1) Minors 
□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Although the etiologies of some secondary cataracts are becoming better understood and certain animal models 
show promise for elucidating the relationships between lens crystalline and hereditary cataract, little is known 
about the causes of congenital cataracts in humans. To date, the classification of different congenital cataracts 
has been cumbersome and imperfect. A better understanding of cataractogenesis will come through an 
understanding of the molecular components of the lens of the eye and the ways in which lesions of these 
components are manifested, structurally and functionally, as opacity of the lens. Animal studies have 
suggested that alterations in lens crystallins can cause hereditary cataracts, making them reasonable candidate 
genes for causing hereditary cataracts in humans. In addition, it is apparent that hereditary lesions that mimic 
or contribute additively to environmental stress known to cause cataracts might be candidate genes for causing 
hereditary cataracts. The work in this project is designed to concentrate specifically on congenital and 
hereditary cataracts and to take full advantage of molecular technology developed for linkage analysis. Studies 
performed on informative families will include collecting blood specimens from available family members and, 
when possible, analyzing lens material from patients who undergo cataract surgery. 



328 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 

Clinical Protocol Number 

84 EI-0194A 
Objectives 

The objective of this study is to locaUze, 
identify, and study by linkage analysis and 
other molecular genetic techniques the genes 
that cause hereditary cataracts. 

Methods 

Linkage analysis (gene mapping) will be 
carried out by following the inheritance of 
genetic markers in families with hereditary 
cataracts. Blood specimens will be obtained 
and analyzed for gene marker linkage analysis 
from informative famUies. 

Major Findings 

At present, the process is under way to accu- 
mxilate families with congenital cataracts; a 
large family with known autosomal dominant 
congenital cataract has been analyzed. Of 
note, it has shown for the first time evidence 
of intraocular phenotypic heterogeneity in a 
family with autosomal dominant congenital 
cataract. Studies for markers for gene analysis 
are under way. 



Significance to Biomedical Research and the 
Program of the Institute 

By studying patients with congenital inherited 
cataract, it may be possible to find the specific 
gene that is responsible for the development 
for congenital cataracts. 

Proposed Course 

Additional families will be recruited that have 
congerutal cataract, and linkage analysis will 
be performed on these families. 

NEl Research Program 

Lens and Cataract — Pathogenesis of Cataract 

Publications 

Hejtmancik }F, Kaiser MI, Piatigorsky J: 
Molecular biology and inherited disorders of 
the eye lens, in Scriver CR, Beaudet Al, Sly 
WS, Valle D, (eds): Metabolic Basics of Inherited 
Disease. New York, McGraw-Hill Inc., in 
press. 

Scott MH, Hejtmancik JF, Wozencraft LA, 
Renter LM, Parks MM, and Kaiser-Kupfer MI: 
Autosomal dominant congenital cataract: 
Interocular phenotypic heterogeneity. OphtJml- 
mologij 101:866-871, 1994. 



319 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00084-16 OGCSB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Anterior Chamber Anomalies Associated With Glaucoma or Ocular Hypertension 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Carl Kupfer M.D. Director NEI 

Others: Muriel I. Kaiser-Kupfer 

Lessie M. McCain 

Manuel B. Datiles 

Susan M. Lasa 

Benjamin V. Magno 
Louella Lopez 



M.D. 


Chief 


OGCSB, NEI 


R.N. 


Nurse Specialist 


OGCSB, NEI 


M.D. 


Medical Officer 


OGCSB, NEI 


M.D. 


Visiting Associate 


OGCSB, NEI 


M.D. 


Visiting Associate 


OGCSB, NEI 


M.D. 


Visiting Associate 


OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



.85 



PROFESSIONAL: 



.75 



0.10 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



Recent embryological research has indicated the role of the neural crest in contributing to all connective tissues 
anterior to the lens epithelium. Therefore, the group of developmental anomalies of the anterior chamber with 
glaucoma or ocular hypertension is being reviewed. 



330 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 

Clinical Protocol Number 

77-EI-0119 
Objectives 

The objective of this study is to determine 
whether congenital or developmental anoma- 
lies of the anterior chamber are related to 
faulty migration or terminal differentiation of 
neural crest tissue. 

Methods 

Patients of all ages with congenital or devel- 
opmental anomahes of the anterior chamber 
are being examined to determine involvement 
of cornea, trabecular meshwork, iris stroma, 
lens, and ciliary body. When intractable 
glaucoma cannot be controlled with medica- 
tion, surgery is performed; the specimens are 
examined histologically. When the lenses 
become cataractous, cataract extractions are 
performed; the lens epithelium is grown in 
tissue culture. When the cornea is opaque 
and corneal tiansplantation is indicated, that 
procedure is performed; the corneal specimen 
is examined histologically. 

Major Findings 

It appears that in this group of anomalies of 
anterior chamber development, there are 
pathological changes in one or several tissues 
derived from neural crest. These changes 
include corneal stroma, corneal endothelium, 
anterior iris stroma, Descemet's membrane, 
and trabecular meshwork endothelium. 



We have recentiy performed trabeculec- 
tomies on patients with the irido-comeal- 
endothelial syndrome. HistopathologicaUy, 
we found the presence of a membrane cover- 
ing the trabecular meshwork. That membrane 
may have caused the peripheral anterior 
synechias and glaucoma. 

Significance to Biomedical Research and the 
Program of the Institute 

A better understanding of the pathogenesis of 
this glaucoma may help by improving diagno- 
sis and treatment. The presence of this mem- 
brane may explain glaucoma's progressive 
nature and suggest possible surgical or laser 
treatments as a way to control or prevent the 
progression of the disease. 

Proposed Course 

Patients with other anomalies of the anterior 
chamber, including congenital cataracts, will 
be examined for abnormalities in tissue de- 
rived from neural crests. We will continue to 
study cases of congenital corneal disorders to 
uncover the cause and to determine treatment 
choices for these cases. 

NEI Research Program 

Glaucoma — Basic Science Research, Patho- 
biology and Morphology 



331 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00011-20 OGCSB 



PERIOD COVERED 

October 1, 1993 to September 30, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Pigment Dispersion Willi and Without Glaucoma 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NET 



Others: Lessie M. McCain 



R.N. 



Nurse Specialist 



OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Ophthalmic Genetics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.20 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 
(a1) Minors 
[x] (a2) Interviews 



PROFESSIONAL: 



0.1 



0.1 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The purpose of this project is to determine the risks of patients with pigment dispersion syndrome for 
glaucoma. Comparisons of patients with and vvdthout glaucoma are made on the basis of diagnostic tests, 
genetic screening, and aqueous humor dynamics. The data acquired may enable determination of pigment 
dispersion syndrome patients' risk of developing glaucoma as well as adding to the understanding of the 
pathology of the disease. 



332 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 

Additional Personnel 

Marvin Podgor Ph.D. Statistician, BEP, NEI 

Clinical Protocol Number 

76 EI-0189 

Objectives 

This project was designed (1) to compare 
pat