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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

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.

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

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.

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

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-

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

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.

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.

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.

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

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

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.

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.

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

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.

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.

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.

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.

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

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-

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

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

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.

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

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.

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

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

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.

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

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.

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.

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.

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.

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.

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

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.

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

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-

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

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

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.

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.

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

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.

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

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.

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

PROJECT NUMBER

ZOl EY 00262-05 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.)

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.

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

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.

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.

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.

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

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

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.

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.

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.

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

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.

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.

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

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.

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.

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.

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

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.

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

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

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

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.

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.

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

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

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, 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.

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

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.

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

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

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

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

0.0

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

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.

M.S.

M.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.

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.

D.V.M.

Ph.D.

M.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, 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

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, 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.

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

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

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

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

□ (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

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