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Full text of "Annual report"

,\ A T I O^ L EYE INSTITUTE 




» 1 9 9 




Cover Photo 



Immunofluorescence showing the Purkinje 
cells of a transgenic mouse brain reacting 
with a chloramphenicol acetyltransferase 
antibody. The transgene was driven by the 
promoter and enhancer of the chicken 62- 
crystallin/argininosuccinate lyase gene. The 
photograph was taken by Dr. Steven Bassnet 
(Department of Anatomy and Cell Biology, 
Uniformed Services University of the Health 
Sciences) and is a 3-dimensional reconstruction 
using the Voxel View program on a Silicon 
Graphics Workstation taken with a confocal 
microscope. The photograph was originally 
published on the cover of Developmental 
Dynamics ^%■. 1993. Copynght ®1993, 
Wiley-Liss, a Division of John Wiley and 
Sons, Inc. and is described in Piatigorsky J: 
Puzzle of crystallin diversity in eye lenses. 
Developmental Dynamics ^%■.2Q7, 1993. 
Reprinted by permission of Wiley-Liss, a 
|~ii\':^inn of John Wiley and Sons, Inc. 



NATIONS. rilfSTmiTES OF 
NiHLIBRARy 



ifTHESDA, MO 20892-1150 



National Eye Institute 



Annual Report 
Fiscal Year 1993 



U.S. Department of Health and Human Services 
Public Health Service 

National Institutes of Health 



Re 
I 



■•• :T?v*» i'-y 



Table of Contents 



Statement of the Institute Director i 

Carl Kupfer, M.D. 

Extramural Research 5 

Report of the Associate Director 7 

Jack A. McLaughlin, Ph.D. 

Division of Basic Vision Research 7 

Peter Dudley, Ph.D. 

Retinal and Choroidal Diseases 7 

Corneal Diseases 9 

Lens and Cataract . . . 10 

Glaucoma j j 

Strabismus, Amblyopia, and Visual Processing 12 

Division of Collaborative Clinical Research 14 

Richard Mowery, Ph.D. 

Research Results 14 

Division of Biometry and Epidemiology 19 

Report of the Acting Director 21 

Roy C. Milton, Ph.D. 

Research Highlights 21 

Research Activities 22 

Professional Activities 24 

Publications 25 

International Program Activities 27 

Report of the Acting Assistant Director 29 

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

Research Activities 29 

Activities With International and Multinational Organizations 32 

Science Policy and Legislation 33 

Report of the Associate Director 35 

Michael P. Davis, M.S. 

Policy, Legislation, Planning, and Evaluation Branch 35 

Carmen P. Moten, Ph.D. 

Management Information Systems Branch 37 

David Scheim, Ph.D. 

Scientific Reporting Branch 30 

Judith A. Stein, M.A. 

Office of the Scientific Director 41 

Report of the Scientific Director 43 

Robert B. Nussenblatt, M.D. 



Office of the Scientific Director 

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

Physiological Studies of the Primate Visual System 47 

Anatomical Studies of the Primate Visual System 49 

Helen H. Hess, M.D. 

Biochemistry of Retina and Pigmented Epithelium in Health and Disease 51 

Laboratory of Immunology 55 

Report of the Chief 57 

Robert B. Nussenblatt, M.D. 
Section on Clinical Immunology 

Frangois G. Roberge, M.D. 

Study of Immunosuppressants for the Treatment of Uveitis in Animal Models 60 

Section on Experimental Immunology 

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

Analysis of T Lymphocytes and Cytokines Involved in Experimental 

Autoimmune Uveoretinitis 63 

Ectopic Expression of Interferon-Gamma in the Eyes of Transgenic Mice and 

Rats Induces Ocular Pathology and MHC Class II Gene Expression 66 

Igal Gery, Ph.D. 

Immune Responses to Ocular Antigens 68 

Section on Genetics and Molecular Immunology 

Moncef Jendoubi, Ph.D. 

Gene Therapy for Ocular Genetic Disease 72 

Section on Lnmunology and Virology 

John J. Hooks, Ph.D. 

Interferon System in Cellular Function and Disease 75 

Smdies on the Bioregulatory Aspects of the Retinal Pigment Epithelial Cell 78 

Virus Infections in the Eye 81 

Toxoplasmosis Infections in the Eye 86 

Chandrasekharam N. Nagineni, Ph.D. 

Role of Retinal Pigment Epithelium in Retinal Disorders 88 

Section on Immunopathology 

Chi-Chao Chan, M.D. 

Immunopathology in Eyes With Experimental and Clinical Ocular Diseases 91 

Immunopathology of Ocular Diseases in Humans 96 

Cytokines and Ocular Antigens in the Eye 97 

Scott M. Whitcup, M.D. 

The Diagnosis and Treatment of Human Uveitis 98 

Ocular Toxicity of 2',3'-Dideoxyinosine (ddl) 101 

Cell Adhesion Molecules in Ocular Inflammation 103 

Section on Immunoregulation 

Rachel R. Caspi, Ph.D. 

Cellular and Immunogenetic Mechanisms in Uveitis 107 

Marc D. de Smet, M.D. 

Ocular Manifestations of the Acquired Immune Deficiency Syndrome 112 

Characterization of Immune Responses to S- Antigen 115 

Surgical Management of Uveitis 118 

Robert B. Nussenblatt, M.D. 

Cyclosporine Therapy in Uveitis 122 

Oral Administration of Antigen and tiie Ocular Immune Response 125 



Laboratory of Mechanisms of Ocular Diseases 129 

Report of the Acting Chief 131 

J. Samuel Zigler, Jr., Ph.D. 
Section on Cataracts 

Deborah Carper, PhD. 

Structure and Expression of Polyol Pathway Enzymes 132 

Donita L Garland, Ph.D 

Oxidation of Proteins in Cataractogenesis 134 

James Fielding Hejmancik, M.D., Ph.D. 

Inherited Ocular Diseases 137 

Paul Russell, Ph.D. 

Characterization of the Lens 142 

Cataract in the Philly Mouse Strain 145 

J. Samuel Zigler, Jr., Ph.D. 

Structure and Composition of Lens Crystallins with Respea to Cataractogenesis ........ 146 

Section on Pathophysiology 

W. Gerald Robinson, Jr., Ph.D. 

Ultrastructure and Function of the CelLs and Tissues of the Eye 149 

Laboratory of Molecular and Developmental Biology 153 

Report of the Chief 155 

Joram Piatigorsky, Ph.D. 
Section on Cellular Differentiation 
Peggy S. Zelenka, Ph.D. 

Proto-Oncogene Expression During Lens Differentiation and Development 157 

Section on Molecular Genetics 
Joram Piatigorsky, Ph.D. 

Crystallin Genes: Structure, Organization, Expression, and Evolution 161 

Molecular Biology of the Cornea 167 

Section on Molecular Structure and Function 
Graeme J. Wistow, Ph.D. 

Molecular Biology and Functions of Lens Proteins 169 

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

Genetically Engineering the Eye with the aA-Crystallin Promoter 173 

Regulation of Expression of Lens Fiber Membrane Genes 176 

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

NET Central Transgenic Animal Production Facility 179 

a-Crystallin Gene Disruption in the Mouse 182 

Laboratory of Ocular Therapeutics 185 

Report of the Chief 187 

Peter F. Kador, Ph.D. 
Peter F. Kador, Ph.D. 

Pharmacology of Ocular Complications 188 

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

Role of NADPH-Dependent Reductases in Ocular Complications 193 



ui 



Laboratory of Retinal Cell and Molecular Biology 197 

Report of the Chief 199 

Gerald J. Chader. Ph.D. 
Section on Biochemistry 
Barbara Wiggert, Ph.D. 

Vitamin A and Ocular Tissues 201 

Section on Gene Regulation 
Diane E. Borst, Ph.D. 

Molecular Genetics of the Eye and Ocular Diseases 204 

Gerald J. Chader, Ph.D. 

Metabolism of the Retina and Pigment Epithelium 207 

Visual Control Mechanisms 210 

T. Michael Redmond, Ph.D. 

Molecular Biology of Outer Retina-Specific Proteins 213 

Section on Molecular Biology 
Toshimichi Shinohara, Ph.D. 

Molecular Biology of Phototransduction 216 

Molecular Biology of Experimental Autoimmune Uveitis 219 

Laboratory of Sensorimotor Research 223 

Report of the Chief 225 

Robert H. Wurtz, Ph.D. 
Section on Neural Modeling 
Lance M. Optican, Ph.D. 

Information Processing by Visual System Neurons 228 

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

Cerebral Cortical Mechanisms for Eye Movements and Visual Attention 232 

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

Visual Motion and the Stabilization of Gaze 235 

Section on Visual Behavior 
David Lee Robinson, Ph.D. 

Visuomotor Properties of Neurons in the Thalamus 238 

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

Visuomotor Processing in the Primate Brain , 241 

Ophthalmic Genetics and Clinical Services Branch 245 

Report of the Acting Chief 247 

Muriel I. Kaiser-Kupfer, M.D. 
Section on Cataract and Corneal Diseases 
Manuel B. Datiles, M.D. 

The Effects of Corneal Contact Lenses on the Cornea 250 

Documentation and Monitoring of Opacities in the Human Lens , 252 

Use of Human Lens Material for Determining Possible Causes of Cataracts 255 

Muriel I. Kaiser-Kupfer, M.D. 

Addendum to Use of Human Lens Material for Determining Possible Causes 

of Cataracts 257 

Carl Kupfer, M.D. 

Anterior Chamber Anomalies Associated With Glaucoma or Ocular Hypertension ....... 259 

iv 



Section on Eye Services 

Rafael Caruso, M.D. 

Clinical Psychophysics of the Visual System 261 

Clinical Electrophysiology of the Visual System 264 

Visual Function Diagnosis Service 267 

Section on Ophthalmic Genetics 

Muriel I. Kaiser-Kupfer, M.D. 

Pigment Dispersion With and Without Glaucoma 269 

Visual Function and Ocular Pigmentation in Albinism 271 

Gyrate Atrophy of the Choroid and Retina and Other Retinal Degenerations 273 

NIH Interinstitute Genetics Program: The Genetics Clinic 276 

A Double-Masked Controlled Randomized Clinical Trail of Topical Cysteamine 279 

Usher's Syndrome — Clinical and Molecular Studies 281 

A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine 283 

Mark H. Scott, M.D. 

Characteristics of Macular Scotomas in Patients With Primary Monofixation 

Syndrome 285 

Index 287 



statement of the Institute Director 



statement of the Institute Director 



Carl Kupfer, M.D. 



With the publication this year of our newest 
long-range plan Vision Research — A National 
Plan: 1994-1998 we have taken stock of the ac- 
complishments and current status of vision research 
and have focused once again on the exciting research 
opportunities that lie ahead. Our extramural and our 
intramural laboratories and clinical scientists have 
helped us make excellent progress in accomplishing 
our mission of conducting and supporting research, 
training, health information dissemination, and other 
programs relevant to eye diseases and vision disor- 
ders. About $235 million went to extramural resear- 
chers in the form of grant support and $5 million 
was expended to support research and development 
contracts. Another $7.2 million was used to support 
training awards. This funding has led to a number of 
important findings this year. 

National Eye Instimte (NEI)-supported researchers 
have demonstrated that mutations in several retina- 
specific genes cause photoreceptor degeneration in 
humans and mice. Although the mechanism by 
which these gene mutations lead to photoreceptor 
degeneration is unknown, scientists have suggested 
that there is a common pathway in the disease 
process. Apoptosis, or programmed cell death, 
appears to play a role in all of these retinal 
degenerations through fragmentation of the 
deoxyribonucleic acid (DNA) by intracellular en- 
zymes at specific sites in the genome. If this is the 
case then there is the exciting possibility of an 
intervention for a variety of retinal degenerations 
based on inhibition of these DNA-cutting enzymes. 

Our knowledge of the genetic loci for some of the 
macular degenerations has also been expanded. The 
genes for at least three forms of macular 
degeneration have been localized to specific 
chromosomes by NEI-sponsored investigators. A 
juvenile form of macular degeneration known as 
Best's disease has been mapped to chromosome 11, 
and two other forms of hereditary macular disease 
have been linked to chromosome 6. 

Results from a prospective, double-masked 
clinical trial designed to assess the effectiveness of 



vitamin A and/or vitamin E supplements in halting or 
slowing the progression of retinitis pigmentosa (RP) 
were released. They showed that adults who sup- 
plemented their diets with 15,000 lU of vitamin A 
daily had on average about a 20 percent slower 
annual decline of remaining retinal function than 
those not taking this dose. 

Last year, clinical trial results released by the 
Herpetic Eye Disease Smdy (HEDS) investigators 
showed that oral acyclovir was no better than a 
placebo in treating active herpes simplex stromal 
keratitis. Another part of the HEDS examined the 
effect of steroid eye drops as a treatment for this 
disease. This year, researchers conducting the smdy 
reported rapid improvement of stromal keratitis with 
immediate steroid ther^y, but for those patients 
having their first episode of stromal keratitis, topical 
steroids could be safely deferred. 

Progress has also been made in further under- 
standing of the chaperon functions of a-crystallins. 
In vitro experiments have demonstrated that a- 
crystallin efficiently suppresses the aggregation of p- 
and Y-crystallins. This suggests that a biological role 
of a-crystallins is to prevent posttranslational chan- 
ges in the interactions between lens crystallins and 
hence to maintain the transparent state of the lens. 

NEI-supported scientists have been studying 
affected families in Michigan, the New England 
states, and Iowa in an attempt to identify a 
"glaucoma" gene and ultimately to characterize the 
protein encoded by this gene. Recently, the disease- 
associated gene has been mjqiped to chromosome 1. 
Although the link between juvenile onset glaucoma 
and the more prevalent primary open-angle glaucoma 
is unclear, finding the gene responsible for one form 
of glaucoma is a beginning in the quest for iden- 
tification of at least one of this disease's causative 
factors. 

The scientists conducting the Opuc Neuritis Treat- 
ment Trial (ONTT) reported that a three-day, high- 
dose treatment with intravenous corticosteroids 
followed by a short course of oral corticosteroid 
reduced the rate at which smdy participants 



Statement of the Institute Director 



NEI Annual Report— FY 1993 



developed multiple sclerosis (MS). Last year the 
ONTT scientists reported that this treatment enabled 
patients to recover their vision about two weeks 
sooner than would be the case without treatment but 
that oral prednisone, when used alone, was ineffec- 
tive and actually increased a person's risk for future 
attacks. 

Intramural scientists have continued their research 
efforts to understand the systems within the brain 
that process visual information and produce eye 
movements as well as to understand what h^pens 
when disease or trauma lead these systems to fail. 
Through the use of a superb animal model, these 
researchers have found that the animals respond to 
simulations of motion projected on a screen with 
postural changes similar to those reported in humans. 
This iirformation will allow researchers to investigate 
further the regions of the brain that are known to 
process this type of visual motion information and to 
see whether alterations of these regions affect pos- 
tiu-e. They were also able to locate the approximate 
region of the frontal eye fields in humans and alter 
certain eye movements after first locating this region 
in monkeys. By using information obtained from 
animal models to locate areas in the himian brain that 
perform similar functions, we may gain a better 
understanding of how the intricate mechanisms that 
guide the visual process operate normally and how 
they might be impaired by disease or injury. 

In studies of the regulatory elements required for 
expression of genes in the eye and other tissues, 
intramural scientists have found that these elements 
are quite diverse with each having its own special 
properties. The elements may also be functionally 
redundant, that is, removing one does not necessarily 
eliminate the expression of the gene. A more 
complete understanding of gene expression in the eye 
may one day allow treatments to be directed to 
specific eye tissues. 

Intramural researchers have continued their 
leadership role in the study of gyrate atrophy. 



Dietary intervention studies are continuing in 
families with two affected children. These studies 
have demonstrated a marked decrease in the retinal 
progression of this disorder in the children who 
began the dietary intervention at an early age. It is 
anticipated that this original work will lead to gene 
therapy aimed at preventing this disease. 

Intramural epidemiologists investigated the effect 
of vitamin and/or mineral supplements on the risk of 
developing age-related cataracts in conjunction with 
two National Cancer Institute (NCI) trials using the 
same vitamin and/or mineral interventions in a 
population with chronic nutritional problems and 
high rates of esophageal and stomach cancer. In 
these highly cost-effective studies of populations 
with chronic deficiencies of multiple nutrients, NEI 
investigators and their colleagues found that use of 
the supplements was associated with a decreased risk 
of nuclear cataract. Additional research is underway 
to determine whether these findings apply to less 
nutritionally deficient populations. 

These are only a few highlights of the important 
accomplishments of vision researchers in fiscal year 
(FY) 1993, a year in which we marked the 25 th 
anniversary of the establishment of the NEI. An- 
niversary activities were organized around the theme 
A Celebration of Vision Research and were designed 
to provide the American public with a report on its 
investment in vision research. A traveling science 
museum has been developed that demonstrates the 
progress and accomplishments of vision research 
during these past 25 years. 

It is with great sadness that we must also note the 
passing of our friend and colleague Julian M. Morris. 
Much of what we have accomplished, since his 
selection as the NEI's first information officer in 
1970, we owe to him and his dedication to the field 
of vision research. In recognition of his many 
contributions, we have dedicated Vision Research— A 
National Plan: 1994-1998 to his memory. We will 
miss him. 



Extramural Research 



Report of the Associate Director for Extramural Research 

Jack A. McLaughlin, Ph.D. 



Research activities supported by tiie Exti-amural 
Vision Research Program address the leading 
causes of bUndness and impaired vision in the United 
States, including retinal diseases, corneal diseases, 
cataract, glaucoma, strabismus, and amblyopia. The 
program seeks to increase understanding of the 
normal development and function of the visual 
system; to understand the causes of and better 
diagnose, prevent, and treat sight-threatening con- 
ditions; and to enhance the rehabilitation, training, 
and quality of life of individuals who are partially 
sighted or blind. 

In working to tliis end, the Vision Research 
Program supports vision research tlirough grants, 
cooperative agreements, and research and develop- 
ment contracts; encourages high-quality clinical 
research, including clinical trials and other 
epidemiologic studies; encourages research training 
and career development 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 1993, an estimated total of $235,005,000 
was expended for NEI extramural grants, cooperative 
agreements, and research and development contracts 
in the following categories and amounts: 



Research Grants 
Research Training Awards 
Research and Development 
Contracts 

Total Extramural Support 



$222,735,000 
$7,226,000 

$5.044.000 

$235,005,000 



Concurrent with the reorganization of the NEI, a 
number of personnel changes occurred in the 
Extramural Program during this fiscal year. Among 
these were the appointments of Drs. Lor6 Anne 
McNicol, Peter A. Dudley, and Richard L. Mowery, 
as director of the division of extramural activities, as 
director of the division of basic vision research, and 



as director of the division of collaborative clinical 
research, respectively. 

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



Division of Basic Vision Research 

Peter Dudley, Ph.D., Director 



Retinal and Choroidal Diseases 

Retinal Degeneration and Apoptosis 

Cell death is an important part of normal develop- 
mental programs. For example, the balance of cell 
survival with neuronal cell death is thought to be an 
important mechanism whereby an organism controls 
the interconnections between populations of 
developing cells. Apoptosis, or programmed cell 
death, seems to be an important mechanism used by 
the retina during its development into a functional 
layered tissue. 

Mutations in several retina-specific genes have 
been shown to cause photoreceptor degeneration. 
Mutations in the rd, rds/peripherin, and rhodopsin 
genes cause retinal degenerations in humans and 
mice. The mechanism by which these gene 
mutations lead to photoreceptor degeneration is 
unknown. Recent work by Fulton Wong at Duke 
University shows that although the phenotypes of 
these degenerations are different, there appears to be 
a common pathway in the disease process. Apop- 
tosis appears to play a role in all of these retinal 
degenerations ttirough fragmentation of DNA by 
cleavage at specific sites in the genome. Apoptosis 
has been demonstrated to occur in retinal 
degenerations by observing a characteristic "ladder" 
of DNA fragments using gel electrophoresis. The 



Extramurai Research 



NEI Annual Report— FY 1993 



DNA ladder gel pattern results from a predictable 
fragmentation of genomic DNA at internucleosomal 
sites during the degeneration process. 

Although apoptosis may be a common pathway 
that leads to cell death, the mechanism triggering this 
process is unknown. It seems likely that intracellular 
enzymes called endonucleases are activated to cut 
DNA into small fragments. If this is the case, then 
there is the exciting possibility of an intervention for 
a variety of retinal degenerations based on inhibition 
of these DNA-cutting enzymes. 

Cancer Associated Retinopathy 

Disorders of the retina leading to a loss in vision 
can result from the remote effects of cancer. Certain 
types of tumors at a distant site can set up an im- 
munological reaction that can manifest itself in the 
retina as well as in other tissues of the nervous 
system. When the retina is involved in this 
paraneoplastic syndrome, it is called cancer- 
associated retinopathy or CAR. This syndrome 
develops mainly as a result of a primary tumor in the 
lung, although other tissues may be implicated. 
Metastasis is not involved, but rather molecules of 
the immune system called autoantibodies appear in 
the serum of patients. This immimological reaction 
appears to be, in part, a response of the host or 
patient to the tumor. Thus, the patient appears to be 
producing antibodies as a defense against the tumor 
in the hopes of limiting its growth. Vision loss is 
frequently the first sign of illness leading to subse- 
quent clinical examinations that identify the causal 
cancer. 

Patients experiencing CAR report a sudden loss of 
vision resulting from inactivation of important 
proteins in the retina by the autoantibodies generated 
in response to the tumor. Patients with other types 
of retinopathies do not produce antibodies that react 
with the CAR protein or antigen in the retina, in- 
dicating a high degree of specificity of the im- 
munological reaction. 

Several NEI-supported investigators are looking 
into the role of specific retinal antigens in the 
development of CAR in patients, and further work 
hopefully will uncover the basis of this disease. 
Recent work has shown that one protein involved in 
phototransduction, called recoverin, may be present 
in cancer cells. It is the immune response to this 
protein that results in retinal involvement. Further, 



although recoverin has been found only in rod cells, 
antisera to recoverin label both rods and cones. The 
reason may be that there is a molecule in cones that 
has some sequence homology to recoverin and that 
reacts with antirecoverin antibody. Current inves- 
tigations are focusing on the isolation and cloning of 
the human recoverin gene. The mouse gene has 
recently been cloned, and the deduced amino acid 
sequence is identical to that of human recoverin 
protein. Segregation analysis shows close linkage to 
the tumor suppressor gene p53. The current 
hypothesis being tested is that CAR is the result of 
a single mutational event in a cell that deletes the 
tissue-specific regulatory elements of the recoverin 
gene while joining its coding sequence to an active 
gene. This would delete the function of the p53 
cancer suppressor gene and simultaneously turn on 
synthesis of recoverin. The cell becomes cancerous 
because the p53 protein product is either absent or 
inactive and no longer functions as a cancer 
suppressor. 

These kinds of studies could lead to a method for 
early diagnosis of cancer and the opportunity to 
institute treatment at an early stage. 

Molecular Genetics of Macular Degeneration 

Macular degeneration is the most common cause 
of severe visual impairment in older persons in the 
United States. It robs otheirwise healthy older 
Americans of useful vision, depriving them of the 
ability to read, drive, and enjoy leisure activities. 
Currently, there is no effective tteatment for the vast 
majority of individuals with this condition because 
the basis for the disease is not understood. 

The genes for at least two forms of macular 
degeneration have been localized to specific 
chromosomes by Dr. Richard Stone at the University 
of Iowa. Best's disease is an autosomal dominant 
condition characterized by the accumulation of 
lipofuscin within and beneath the retinal pigment 
epithelium (RPE). It has an earlier age of onset than 
the more prevalent age-related macular degeneration 
(AMD), and there is an absence of drusen. Drusen 
are deposits of extracellular material lying between 
the RPE and Bruch's membraiie. The Best's gene 
has been localized to chromosome llqlS. Dominant 
macular dystrophy with flecks (DMDF) is an 
autosomal recessive degeneration characterized by 
severe vision loss with macular lesions ringed with 



NEI Annual Report— FY 1993 



Extramural Research 



fleck-like deposits of yellow pigment Preliminary 
evidence indicates that the gene for DMDF is 
localized to chromosome 6q. 

Linkage analysis offers the opportunity to map 
human disease genes when the causative agent is 
unknown, as is the case for macular degenerations. 
Gene mapping can lead to actually cloning the gene 
responsible for specific eye diseases, for example, 
AMD, by application of reverse genetics. 

Retinitis Pigmentosa 

RP is a group of hereditary eye diseases with an 
overall incidence of about 1 in 3^00 births in the 
United States. The emotional and economic costs of 
the disease to society are enormous, particularly 
because no effective treatments are known for most 
types of retinal degeneration. RP is genetically 
heterogeneous and can be transmitted as a dominant, 
recessive, or X-linked trait 

NEI-supported research has led to significant 
advances in identifying the molecular defects in 
different forms of RP. Scientists reasoned that a 
gene coding for a structural or a functional protein, 
important in the physiology of the pigment epithelial 
and the photoreceptor cells of the retina, might be 
defective in patients with retinal degenerations. With 
this candidate gene ^proach, it was discovered that 
20 to 30 percent of individuals with autosomal 
dominant RP have a mutation in the rhodopsin gene. 
In autosomal recessive RP, a different rhodopsin 
gene mutation was shown to be present. Mutations 
have also been found in the human homologue of the 
murine rds locus, the photoreceptor-specific 
peripherin/RDS gene, in some families with 
autosomal dominant RP. Although the function of 
this protein is not known, it may serve as an ad- 
hesion molecule, stabilizing the outer segment discs 
through interactions across the intradiscal space. 

Most recentiy. Dr. Ted Dryja at the Harvard 
Medical School has found that a third photoreceptor- 
specific gene is defective in patients with another 
form of autosomal recessive RP. Mutations in the 
gene encoding the beta-subunit of the cGMP 
phosphodiesterase, a key molecule of the visual 
transduction pathway, are present. This is of par- 
ticular interest because the murine homologue of this 
gene is defective in the rd strain of mice with retinal 
degeneration. 

In related research, a two-base pair deletion was 
found in the human peripherin/RDS gene in a family 



with autosomal dominant retinitis punctata albescens, 
an uncommon form of retinal degeneration clinically 
related to RP. A defect in the rhodopsin gene was 
also found in congenital stationary night blindness. 
In studying flie molecular mechanism of this genetic 
defect Dr. Dryja's group found that the mutant 
rhodopsin protein activated transducin without 
binding its natural chromophore, retinal. This 
appears to be caused by abnormal constitutive 
activation of transducin in the phototransduction 
pathway. 

The identification of genetic defects in retinal 
degenerations and dystrophies is an important step in 
developing effective ther^eutic strategies. With this 
information in hand, scientists will now be able to 
explore the molecular mechanisms responsible for 
these diseases and translate this information into 
rational and effective diagnosis, treatment, and 
prevention strategies. 



Corneal Diseases 

The corneal stroma is unique among the col- 
lagenous connective tissues in being transparent 
Understanding the molecular basis of transparency 
requires a detailed knowledge of the regulation of the 
collagen types, proteoglycans, and glycoproteins 
expressed in this organ. The Corneal Diseases 
Program is supporting several laboratories engaged in 
studies of the development and synthesis of specific 
collagen genes. 

Dr. Bjom R. Olsen at Harvard Medical School 
has been investigating the synthesis of type Vin 
collagen, one of the short-chain collagen species. 
This protein had been reported as a component of the 
intima layer of vascular endothelium and as the 
major structural component of Descemet's membrane 
in the cornea. Dr. Olsen has cloned and sequenced 
the two genes encoding type Vlll collagen and has 
found that it exists as a heterotrimer of composition 
[al(VIII)]2[0(2(Vin)]. In vitro hybridization studies 
revealed the surprising observation that the al gene 
is expressed in the lens as well as in the cornea. 
Chromosomal localization studies have shown that 
the a2 gene is located at the region of the defect 
dysgenetic lens (dyl) gene. This is a recessive 
hereditary disorder that shows a persistent coimection 
between the lens and the corneal epithehum as well 



Extramural Research 



NEI Annual Report— FY 1993 



as various degrees of corneal opacity. Dr. Olsen has 
prepared transgenic mice carrying defects in both the 
al and a2 genes. This should permit a more 
detailed examination of the unexpected role of type 
VIII collagen in the embryonic development of the 
cornea and lens. 



Lens and Cataract 

Lens Biochemistry 

The a-crystallins are major structural proteins of 
the vertebrate lens and contribute to its refractive 
mass and transparency. During the past decade, it 
has been shown that in some species "housekeeping" 
enzymes that are found in nonlenticular tissue are 
recruited to serve as structural crystallins in the lens. 
This has led to the concept of "gene-sharing" 
implying that a single gene encodes a protein with 
dual functions. Evidence for a dual function for a- 
crystallins has come from two Unes of investigation. 
First, the a-crystalhns are found in nonlenticular 
tissues, including the heart, lung, spinal chord, brain, 
kidney, and retina oB-crystallin specifically ac- 
cumulates in many neurological disorders. Second, 
the a-crystallins are structurally related to the family 
of heat-shock proteins that can be induced by heat or 
hypertonic stress and accumulate in a number of 
pathological conditions. 

These two lines of evidence have converged with 
the recent finding by Dr. Joseph Horwitz from the 
University of California at Los Angeles that a- 
crystallins function in vitro as molecular chaperons. 
These are a subset of heat-shock proteins that are 
overproduced in response to physiologic "stress" and 
that act by affecting protein-protein interactions. 
They stabilize native protein conformations, mediate 
the folding and correct oligomeric assembly of 
nascent proteins, catalyze the membrane transloctions 
of secretory proteins, and prevent protein aggregation 
under conditions of heat denaturation. In vitro 
experiments have demonstrated that a-crystallin 
efficiently suppresses the aggregation of P- and y- 
crystallins. This suggests that a biological role of a- 



crystallins is to prevent posttranslational changes in 
the interactions between lens crystallins and hence 
maintain the transparent state of the lens. 

Developmental Biology 

In many vertebrate species, proper iris and cornea 
development appears to be coupled to lens growth 
and viability. A new tool in the study of early lens 
development has been the small eye (Sey) mutation 
in mice in which abnormalities in lens development 
are accompanied by other anterior chamber defects. 
This mutation has resulted in a potentially useful 
animal model of aniridia. This condition results 
from defects in PAX-6, a gene encoding paired-box 
and homeobox motifs that are expressed in the 
developing eye. It shares homology with paired box 
genes of Drosophila that conttol the development of 
body segmentation. The homeobox encodes the 
helix-tum-helix motif seen in DNA-binding proteins. 

Aniridia is a human developmental disorder 
closely related to the Sey mutation and is charac- 
terized by hypoplasia of the iris and is conmionly 
associated with other clinical anomalies such as 
cataracts and lens dislocation. The disease is in- 
herited in an autosomal dominant fashion. It is 
frequently cofransmitted with Wilms' nephroblas- 
toma, genitourinary abnormalities, and mental retar- 
dation (termed WAGR complex), demonstrating a 
close linkage with the genes responsible for these 
anomalies. This has greatly facilitated the mapping 
and identification of the human aniridia gene. 

The WAGR complex has been mapped to a large 
interstitial deletion on the short arm of human 
chromosome 11. Dr. Lisa Davis at the Applied 
Genetics Laboratory in Melbourne, Florida and Dr. 
Richard Maas of Harvard University have been 
working independently to fine map and characterize 
the gene. This area is homologous to the region of 
the mouse chromosome 2, which contains the Sey 
gene. Using a mouse PAX-6 clone as a probe, the 
aniridia region has been identified in human cDNA 
libraries. Physical mapping of the aniridia gene 
using DNA isolated from patients with aniridia will 
provide us with new insights into ocular develop- 
ment. 



10 



NEI Annual Report— FY 1993 



Extramural Research 



Glaucoma 

Molecular Genetics 

Glaucoma is a potentially blinding condition as- 
sociated with increased intraocular pressure (lOP) 
and gradual destruction of the optic nerve. Little is 
known about the underlying causes of glaucoma and 
the accompanying nerve degeneration that leads to 
loss of vision. Furthermore, the relative influence of 
genetic and environmental characteristics is poorly 
understood. Fortuitously, juvenile onset glaucoma, 
a form of the disease characterized by early 
adulthood onset and elevated lOP, displays an 
autosomal dominant pattern of inheritance. The 
unambiguous phenotype, high degree of penetrance 
and early age of onset, makes a genetic approach 
ideal for the study of this form of the disease. 

Dr. Julia Richards at tiie University of Michigan 
has been working with a number of families that 
have sufGcient meiotic events to perform genetic 
linkage studies. To date, family histories have been 
collected from families in Michigan, the New 
England states, and Iowa. The ultimate goal is to 
identify a "glaucoma" gene using positional cloning, 
followed by sequencing analysis to characterize the 
encoded protein. Recently, the disease-associated 
gene has been mapped to chromosome 1. Cor- 
roborating data from different laboratories using 
different families have confirmed this locus. In one 
of the Michigan families, linkage analysis has placed 
the gene within a 14 centimorgan region of the 
chromosome, at Iq21-q31. 

The link between juvenile onset glaucoma and 
primary open-angle glaucoma is unclear, but finding 
the gene responsible for one form of glaucoma is a 
beginning in the quest for identification of at least 
one causative factor. 

Ganglion Cell Function 

Currently, there is controversy about whether 
glaucomatous damage is selective for a subset of 
ganglion cells. Each area of the retina has several 
functionally distinct types of ganglion cells serving 
the same photoreceptor cell in parallel pathways. 
The majority of ganglion cells have large cell bodies 
and large dendritic fields and are classified as M or 
magnocellular. The P or parvocellular ganglion cells 



are more numerous, have small cell bodies, restricted 
dendritic fields, and are involved in color vision. 
Early investigations seemed to indicate that glaucoma 
initially damages the large diameter nerve fibers that 
are prevalent in the M pathway. 

More recently, psychophysical studies carried out 
by Dr. Chris Johnson from the University of Califor- 
nia at Davis indicate that early neuropathy involves 
both P and M pathways. Using longitudinal studies, 
he has shown a link between abnormalities detected 
using blue-on-yellow perimetry and temporal 
modulation perimetry. Temporal modulation 
perimetry is a noninvasive psychophysical technique 
used to assess visual sensitivity at various frequen- 
cies of flickering light Short wavelength light 
responses are diagnostic of the P pathway that 
processes spatial and finely detailed information. 
Flicker sensitivity at low frequencies is a good 
monitor of the M pathway. Blue-on-yellow 
perimetry has application for psychophysically 
isolating and measuring the sensitivity of the short- 
wavelength or blue cone pathway. Johnson's work 
suggests tiiat there may be a lack of selectivity for 
any particular ganglionic cell subset in glaucoma. 
Patients tested with blue-on-yellow perimetry show 
deficits that precede standard visual field defects. 
Using temporal modulation perimetry there was an 
overall loss of flicker contrast sensitivity in patients 
with early glaucomatous visual field loss, but this 
deficit was not selective for high frequencies. The 
decrease in sensitivity demonsfrated in these tests 
occurs at the same time that early visual field defects 
are seen. This is consistent with the idea that early 
glaucomatous damage is not limited to a specific 
subset of ganglion cells. 

Ultimately, understanding optic nerve pathology 
can lead to more sensitive predictors for the risk of 
glaucomatous nerve fiber and hence vision loss. 

Aqueous Humor Dynamics 

An important role of the aqueous humor in the 
anterior portion of the eye is to maintain the proper 
lOP. Aqueous humor is derived from plasma in the 
capillaries that feed the anterior portion of the eye 
via a specific tissue — ^the ciliary body epithelium. 
Fluid produced by the epithelial cells (inflow) leaves 
the eye via the frabecular meshwork and Schlemm's 
canal (outflow), reentering the vascular system 



11 



Extramural Research 



NEI Annual Report— FY 1993 



through the venous route. It is the balance between 
inflow and outflow that maintains the lOP. A major 
aim of current glaucoma research is to gain a better 
understanding of the mechanisms that regulate these 
two pathways. 

Our understanding of outflow biology has been 
enhanced by a recent discovery by the laboratory of 
Dr. James Nathanson at Massachusetts General 
Hospital, showing that nitrovasodilators lower lOP. 
Nitrovasodilators are a class of compounds produced 
in response to increased levels of nitric oxide in the 
cell. Nitric oxide has been shown to be an important 
mediator in many physiological functions, including 
muscle relaxation, vasodilation, and transmission of 
neural impulses. All these effects are mediated by 
the second messenger cGMP. This finding adds 
important information to our understanding of 
outflow regulation and opens the door to the pos- 
sibility of new therapeutic strategies. 



Strabismus, Amblyopia, and Visual 
Processing 

Development of Visual Pathways 

One of the primary characteristics of the visual 
system is the precise pattern of connections, a virtual 
map, that exist between the retina and the visual 
centers of the brain. This pattern is established 
during embryonic development and refined during 
early life. Activity mediated by chemical signals 
(neurotransmitters) at the contact points (synapses) 
between nerve cells is suspected to play an important 
role in this developmental process. Research by Dr. 
Steve McLoon at the University of Minnesota has 
shown that the presence of the chemical precursors 
for nitric oxide, a recently discovered neurotransmit- 
ter, in a visual center of the brain coincides with the 
timing of the ingrowing processes fi'om the retina in 
the chick embryo. In fact, the concentration of these 
chemicals reaches a peak just as the initial visual 
map is being established by the terminals of the 
retinal cells. Unlike the results from other studies 
that have implicated a number of chemical signals in 
the establishment of visual maps in the developing 
nervous system, these results demonstrate the 
presence of a chemical signal at the right time and 
the right place needed to establish a precise map. 



In related research, Dr. Carla Shatz from the 
University of California at Berkeley finds that when 
ganglion cells in the retina first make their connec- 
tions with nerve cells in the brain, the pattern is not 
nearly as precise as it is in the adult. Many extra 
connections are made that are later pruned away. 
How do cells "know" which connections to maintain 
and which ones to eliminate? Dr. Shatz examined all 
branches to determine if electrical signals were being 
transmitted from one nerve cell to the next. 
Branches to be eliminated were found to function 
while they were present. This result lead Dr. Shatz 
to envision that perhaps a branch from a nerve in the 
eye confirms that it is in the correct location by 
sending a signal to the nerve cell in the brain to 
verify its location, much like placing a telephone call 
to verify an address. Dr. Shatz found that by block- 
ing the signaling of the nerve cell it is possible to 
stop incorrect branches from being removed. Where 
do these signals originate? The coimections between 
the eye and the brain form very early in fetal life, 
even before vision begins. Dr. Shatz suspected that 
the nerve cells in the eye might be signaling spon- 
taneously to nerve cells in the brain. Dr. Shatz 
discovered that the retinal ganglion cells are spon- 
taneously and repeatedly signaling their target cells 
in the brain during the weeks before vision takes 
over. Thus, in the visual system, and very likely 
elsewhere in the developing brain, nerve cell sig- 
naling before birth plays a crucial role in establishing 
correct connections. 

Plasticity in the Visual Cortex 

The traditional view states that the overall ar- 
chitecture and the connections between nerve cells in 
the adult visual cortex of the brain are fixed fol- 
lowing a period in early postnatal life when these 
connections can be modified. This early period is 
called the critical period, and the ability of nerve 
cells to modify their connections is called plasticity. 
The connections between nerve cells carry visual 
information encoded in signals that are fransformed 
and integrated as they ascend sequentially through 
visual centers in the brain. The transformation that 
occurs in these centers is analyzed in terms of 
receptive fields. These are sets of nerve cells that 
encode features of a visual stimulus falling on the 
retina and connect with other sets of visual nerve 
cells along the visual pathway. In this way receptive 



12 



NEI Annual Report— FY 1993 



Extramural Research 



fields form the building blocks that underlie visual 
p)erception. 

In the adult, the traditional view holds that the 
cortex processes the visual scene through a fixed set 
of receptive fields, handing on information to the 
next stage in the visual pathway. Clearly, our ability 
to store new memories in adulthood requires some 
form of cortical plasticity, but it was generally 
believed that this would only occur in high-order 
association. 

Experiments by Dr. Charles Gilbert at the Rock- 
efeller University have changed our view of cortical 
visual processing. Dr. Gilbert made small, focal 
retinal binocular lesions in monkeys and found that 
the area of cortex receiving input from those parts of 
the retina became initially silenced, as expected. 
However, to his surprise, in adult animals the initial- 
ly silent area of the cortex becomes remapped, 
responding instead to areas outside the lesion. Even 
more surprising was the finding that quite striking 
changes could be observed physiologically within 
minutes after making the lesion, and, finally. Dr. 
Gilbert found that a lesion is not necessary, certain 
patterns of visual stimulation can cause receptive 
fields to expand and contract over a time scale of 
minutes. 

The finding of this degree of cortical plasticity in 
adult animals presents a radically different idea of 
how the cortex works, and the functional 
implications of the findings are closely related to the 
time course over which the changes take place. 
Dynamic changes over a time scale of minutes are 
usefiil for adapting to changes in the sensory en- 
vironment. It is as if the cortex is constantly ex- 
panding and contracting its representation of various 
aspects of the sensory environment in response to the 
amount of input it receives from particular sets of 
stimuli. Future studies may imcover how these 
changes in visual cortex relate to learning and 
memory such as the representation of complex 
images occurring in higher cortical areas. 

Development of Myopia 

More than 25 percent of the adult population of 
the United States is near-sighted (myopic). This 
relractive error usually develops in the vast majority 
of people between the ages of six and 14 years. The 



relationship between accommodation and the 
development of myopia has been a controversial 
topic for a number of years. Animal models of 
myopia are beginning to shed some light on this 
issue. Recent experiments in the tree shrew (a 
mammal closely related to primates), the chicken, 
and the monkey have shown that a biological feed- 
back mechanism controls the shape of the eyeball. 
Under normal circumstances this effect causes the 
focal length of the visual image to fall on the retina 
(i.e., the eye is in good focus). Dr. Thomas Norton 
from the University of Alabama at Birmingham has 
done experiments that indicate that the presence of 
visual images on the retina produces a signal within 
the eye that, through a cascade of events, affects the 
structural nature of the sclera, the outer coat of the 
eye, without involving the central visual nervous 
system. Myopia occurs when this mechanism is 
disrupted, by visual deprivation in animals and by 
unknown perturbations in humans, causing the eye to 
become too long for its focal length. Work by Dr. 
Norton suggests that blurred images, or form 
deprivation, slow the accumulation of proteoglycans 
and collagen, two chemical components of the sclera. 
This in turn may cause the sclera to be less resistant 
to lOP and consequently causes the eye to become 
too long, producing myopia 

Low Vision 

AMD of the retina is a leading cause of low 
vision and poses a particularly difficult problem for 
vision testing because of the central field scotomas 
(blind areas) that commonly result from this disorder. 
Recent developments in eye monitoring technology 
have made it possible to position a target very 
precisely on known locations of the retina This has 
great potential for both documentation of visual loss 
and possible retraining of eye movements to enable 
use of remaining intact parts of the retina. 

Progress has been made in developing new 
devices that aid and assist visually impaired persons, 
e.g., more ergonomically satisfying magnifiers, 
cosmetically acceptable telescopic spectacles, and 
voice control and output for computers. Attention 
now is focused on devices to assist in changes in 
terrain, text navigation for both printed materials and 
computer screens, image processing, and route- 
finding. 



13 



Extramural Research 



NEI Annual Report— FY 1993 



Division of Collaborative Clinical 
Research 

Richard Mowery, Ph.D., Director 

The Division plans and directs a program of 
grant, cooperative agreement, and contract 
support for applied clinical vision research, including 
clinical trials, natural history studies, surveys, cohort 
studies, and studies of cases and controls. The 
Division manages 21 clinical trials, 1 1 epidemiology 
studies, and three eye health education demonstration 
projects with an annual budget of $38.7 million. 



Research Results 

Retinitis Pigmentosa 

RP is a diverse group of hereditary retinal 
diseases that cause a progressive degeneration of the 
rod and cone photoreceptors and loss of visual 
function. RP affects approximately 100,000 people 
in the United States and approximately 1.5 million 
people worldwide. Individuals with RP typically 
begin to lose peripheral vision in adolescence and 
early adulthood, and most lose central vision later in 
life. Results from a prospective, double-masked 
clinical trial designed to assess the effectiveness of 
vitamin A and/or vitamin E supplements in halting or 
slowing the progression of RP showed that adults 
who supplemented their diets with 15,000 lU of 
vitamin A daily had on average about a 20 percent 
slower annual decline of remaining retinal function 
than those not taking this dose. Based on this 
finding, an average patient who started taking a 
15,000 lU vitaniin A capsule at age 32 would retain 
some useful vision until age 70, whereas a patient 
not on this dose would lose useful vision at age 63. 
The study also found that in patients taking high- 
dose vitamin E supplements the disease appeared to 
progress faster on average than in patients taking a 
trace amount of the vitamin. 

Cytomegalovirus Retinitis 

Cytomegalovirus (CMV) retinitis is a potentially 
blinding disease of the retina that affects about 25 
percent of people with acquired immunodeficiency 



virus (AIDS). NEI supports a network of inves- 
tigators with expertise in AIDS clinical research, 
retinal diseases, and clinical trial methodology to 
expedite the testing of treatments for CMV retinitis 
and other ocular complications seen in patients with 
AIDS. This network is called the Studies of Ocular 
CompUcations of AIDS (SOCA). The first clinical 
trial conducted under SOCA was designed to com- 
pare the efficacy and safety of foscarnet and gan- 
ciclovir. The investigators found that patients treated 
with foscarnet lived longer than those who received 
ganciclovir. Foscarnet patients lived an average of 
12.6 months after starting treatment compared with 
8.5 months for patients taking ganciclovir. The 
drugs appeared to be equally effective in halting the 
progression of CMV retinitis and preserving vision. 

VUreoretinopathy 

The most common cause of failure in retinal 
detachment surgery is the development of abnormal 
contractile tissue on the retinal surface. Mild forms 
of this condition can sometimes be treated by exter- 
nal surgery and the retina successfully reattached. 
However, in more severe forms intraocular surgery 
is required. The Silicone Oil Study, a multicenter 
clinical trial, was designed to evaluate the benefits 
and risks of using a long-acting gas or silicone oil as 
an aid in reattaching the retina. The study found that 
use of silicone oil is superior to use of long-acting 
gas, resulting in a higher rate of successfiil retinal 
reattachment. 

Retinopathy of Prematurity 

More than 4,000 infants weighing less than 1,251 
grams at birth underwent sequential ophthalmic 
examinations to monitor the incidence and progres- 
sion of retinopathy of prematurity (ROP) in the 
multicenter Cryotherapy for Retinopathy of 
Prematurity Clinical Trial. Two-thirds of the infants 
developed some degree of ROP. The incidence and 
severity of ROP were higher in lower birth weight 
and gestational age categories. African-American 
infants appeared less susceptible to ROP than did 
Caucasian infants. 

Herpes Simplex 

About a one-half million Americans are affected 
by ocular herpes, which often begins as a relatively 
painful sore on the surface of the cornea. Like 
herpes cold sores, these ocular lesions may 



14 



NEI Annual Report— FY 1993 



Extramural Research 



periodically recur, and the herpes virus can also over 
time cause an inflammation deep inside the cornea. 
This advanced infection is known as herpes simplex 
stromal keratitis, which can lead to severe corneal 
scarring, inflammation of the interior of the eye, and 
even blindness. A randomized, controlled clinical 
trial was conducted as part of the HEDS to evaluate 
whether oral acyclovir, when given to patients with 
steroid and antiviral eye drops, improved the treat- 
ment of active herpes simplex stromal keratitis. 
Researchers randomly assigned 104 patients to take 
either oral acyclovir or a placebo. After a 10-week 
treatment regimen and a six-month followup period, 
oral acyclovir was found to be no better than a 
placebo in successfully clearing the stromal keratitis. 
This indicates that the financial cost and minimal 
potential health risk associated with the use of this 
drug is not warranted. 

The role of acyclovir in the prevention of recur- 
rences of herpetic eye diseases during the course of 
one and one-half years and the role of acyclovir in 
preventing progression of superficial (epithelial) 
keratitis to the more severe stromal keratitis, or iritis, 
is currently being examined by the HEDS research 
group. 

Although many ophthalmologists use steroids to 
control the corneal inflammation associated with 
herpetic stromal keratitis, clinical research has 
yielded mixed results regarding their overall effect. 
For example, some clinicians have reported en- 
couraging results with this treatment, although others 
have indicated that steroid therapy worsens or 
prolongs the corneal lesions and predisposes patients 
to known complications such as glaucoma and 
cataract. A second randomized, clinical trial con- 
ducted as part of the HEDS examined the effect of 
steroid eye drops as a treatment for active herpetic 
stromal keratitis. After 10 weeks of treatment and 
six months of patient followup, corneal inflammation 
was held in check longer and corneal inflammation 
cleared faster in patients treated with steroids. 
However, delaying steroid therapy by one-to-three 
weeks did not significantly influence lesion recur- 
rence or affect visual acuity at six months. Thus, 
rapid improvement of stromal keratitis was achieved 
with immediate steroid therapy, but for those patients 
having their first episode of stromal keratitis topical 
steroids could be safely deferred. 



Corneal Transplantation 

More than 40,000 corneal transplant operations 
are performed annually in the United States. But 
about one in 10 patients receiving a corneal 
transplant is at high risk of rejecting the donor tissue 
or graft because: (1) they have previously rejected 
a corneal transplant or (2) new blood vessels have 
grown into their damaged cornea, introducing im- 
mune cells into this normally avascular region of the 
eye that may later recognize the graft as foreign and 
attack it 

The Collaborative Corneal Transplantation Study 
(CCTS) was designed to evaluate whether donor- 
recipient tissue typing, transplanting a donor cornea 
that has cell-surface proteins (human leukocyte 
antigens [HLA]) that closely resemble those on the 
recipients' s natural cornea, helps to prevent 
transplant rejection. These antigens serve as 
molecular "fingerprints" on every cell in the body 
and allow a person's immune system to distinguish 
its own cells from those belonging to another person. 
Previous studies had suggested that closely matching 
the donor's HLA with those of the recipient might 
increase the likelihood that the immune system 
would accept, rather than reject, the donor tissue. 

After three years of patient followup, CCTS 
researchers foimd that people who received corneal 
transplants with well-matched antigens did not fare 
significantiy better than those with a poor match. 
Each patient group had similar rates of initial im- 
mune reactions, graft rejection, and graft failure due 
to infection or other causes. These findings indicate 
that tissue typing was not an important factor in 
transplant survival. 

If donor-recipient tissue typing were to become 
standard practice in corneal transplantation, it would 
greatiy increase the cost and waiting period for this 
operation. The process of matching antigens is labor 
intensive and would add at least $1,(X)0 to the nearly 
$5,000 cost of a corneal transplant operation. 
Moreover, because there is already a national 
shortage of donor corneas, high-risk patients would 
likely have to wait even longer for a suitably 
matched donor cornea. 



15 



Extramural Research 



NEI Annual Report— FY 1993 



Lens Opacities Case-Control Study 

Cataracts are a leading cause of visual disability 
and blindness, however, little information exists on 
the cause or progression of cataracts. Development 
of each cataract type (nuclear, cortical, mixed, and 
posterior subc^sular) could be influenced by dif- 
ferent risk factors. The Lens Opacities Case-Control 
Smdy (LOCS) evaluated medical, nutritional, 
demographic, familial, environmental, and ocular 
factors that could lead to cataract development. Of 
the 1,380 LOCS study participants, 435 were cataract 
free. Cases of cataract were as follows: 72 with 
posterior subcapsular cataract, 137 nuclear cataract, 
290 cortical cataract, and 446 with mixed types of 
cataract. The results of the study indicate that 
development of all three cataract types was as- 
sociated with a lower educational level; and regular 
use of a multivitamin dietary supplement decreased 
the risk of cataract formation. Low dietary intakes 
of vitamins A, C, and E, riboflavin, niacin, thiamin, 
and iron were associated with development of 
cortical and mixed cataracts (odds ratios .31 to .56). 
Low intake of vitamins, low socioeconomic status, 
diabetes, race, use of some medications, smoking, 
and other factors were associated with development 
of specific types of cataracts. The rate of cataract 
progression and factors affecting this progression are 
currently being evaluated in the Natural History of 
Lens Opacities Study, where individuals will be 
examined aimually for five years. 

Linxian Eye Study 

The Linxian Cataract Studies sought to determine 
whether vitamin and mineral supplements were 
effective in preventing the development of lens 
opacities. In 1985, the NCI launched two nutrition 
intervention trials in Linxian, a county in north 
central China, whose population has chronic 
nutritional problems and high rates of esophageal and 
stomach cancer. Because the vitamins and minerals 
under study might have potential for preventing lens 
opacities, the NEI collaborated with the NCI to 
determine the effects of the supplements on the eye's 
lens. In 1991, the NEI provided support for eye 
examinations, including detailed lens evaluations, for 
participants in both trials. 

In the first trial, participants were randomly 
assigned to either a multivitamin and/or mineral 
supplement or a placebo. Examinations were con- 
ducted on 2,141 participants who were between the 



ages of 45 and 74. The smdy found a 36 percent 
reduction in nuclear opacities among the oldest 
participants (ages 65 to 74) who took a multivitamin 
and/or mineral supplement. 

In the second trial, participants were assigned 
randomly to various combinations of vitamins and 
minerals — a study design that allowed researchers to 
determine the effects of individual nutrients. 
Examinations were conducted on 3,249 participants 
who were also between the ages of 45 to 74. The 
results indicated a significantly lower prevalence of 
nuclear opacities in people taking riboflavin and 
niacin compared with those not taking these 
vitamins. Again, the oldest participants (ages 65 to 
74) showed the greatest reduction, 44 percent, in 
nuclear cataracts. 

Optic Neuritis 

Optic neuritis is an acute debilitating inflam- 
mation of the optic nerve that affects more than 
25,000 Americans each year, primarily women 
between the ages of 18 and 45. People with the 
disease usually have rapid vision loss and ocular 
pain. The ONTT compared oral corticosteroid, 
intravenous steroid followed by oral corticosteroid, 
and placebo for the treatment of new cases of optic 
neuritis. ONTT results showed that oral cor- 
ticosteroid, the most common treatment for the 
disease, when used alone is ineffective in treating the 
disease and actually increases a person's risk for 
future attacks. 

Strabismus 

Strabismus can result in amblyopia, which is a 
major cause of vision loss in the United States. The 
causes of strabismus are not well understood, but a 
defect in central nervous system control over the 
oculomotor system is thought to play a role. Mater- 
nal cigarette smoking during pregnancy as a risk 
factor for childhood strabismus was recentiy 
evaluated. A population-based, case-control study 
was conducted and evaluated all incident cases of 
strabismus diagnosed during a 21 -month period ft'om 
1985 to 1986 in nine pediatric ophthalmology centers 
in Baltimore. Cigarette smoking was associated vwth 
esotropia but not exotropia for those women who 
smoked throughout pregnancy. The association 
between maternal smoking and esotropia was only 
seen in low-birth weight infants and infants in the 
upper-half of the birth weight distribution. The 



16 



NEI Annual Report— FY 1993 Extramural Research 

authors conclude that cigarette smoking may have a 
direct toxic effect on the developing nervous system, 
which can lead to abnormalities such as strabismus. 



17 



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 Epidemiology 
(DBE) comprises 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, respectively; Dr. Roy 
Milton is the head of the Biometry Section. 

The DBE has three main functions: research, 
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 populations, 
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 education in 
biometric and epidemiologic principles and methods 
for the vision research community. This program 
consists of courses, workshops, a fellowship program 
for ophthalmologists, publications, and consultation 
and collaboration on research. 

The Division provides biometric and 
epidemiologic assistance to NEI intramural and 
extramural staffs and to vision researchers in the 
pubhc and private sectors. The assistance ranges 
from consultation to collaboration as coinvestigator. 



Research Highlights 

The Krypton-Argon Regression 
Neovascularization Study 

This randomized multicenter clinical trial was de- 
signed to compare the efficacy of red krypton with 
blue-green argon laser photocoagulation for the 
management of high-risk proliferative diabetic 
retinopathy. Scatter laser photocoagulation with 
either argon or krypton appears to be equally effec- 
tive in arresting neovascularization of the disc. 



The Linxian Cataract Studies 

The Linxian Cataract Studies were two ran- 
domized clinical trials conducted in China that 
studied the effect of vitamin and/or mineral sup- 
plements on the risk of developing age-related 
cataracts. In these studies of populations with 
chronic deficiencies of multiple nutrients, use of the 
supplements was associated with a decreased risk of 
nuclear cataract. Additional research is underway to 
determine whether these findings ^ply to less 
nutritionally deficient populations. 

The Eye Disease Case-Control Study 

In a large epidemiologic study of neovascular 
AMD, an increased risk of disease was associated 
with cigarette smoking and higher levels of serum 
cholesterol. Decreased risk was associated with 
postmenopausal use of estrogens and higher serum 
levels of carotenoids. Results from the study are 
consistent with a hypothesis linking risk factors for 
cardiovascular disease with AMD. 

The hypothesis that higher serum levels of 
micronutrients with antioxidant c^abilities may be 
associated with a decreased risk of AMD was 
evaluated in the Eye Disease Case-Control Study. 
Persons with higher levels of carotenoids and those 
with higher levels of an antioxidant index derived 
from serum measurements of vitamins C and E, 
carotenoids, and selenium also showed a decreased 
risk of macular degeneration. Results from this 
observational study are now being tested in a cUnical 
trial. 

A study was conducted to evaluate the relative 
anatomic position of the crossing vessels at the site 
of occlusion in eyes with branch retinal vein oc- 
clusion. In 99% of eyes with a branch retinal vein 
occlusion, the artery was located anterior to the vein 
at the obstructed site. At comparable nonoccluded 
crossings, the artery was located anterior to the vein 
less than 65% of the time. The finding suggests a 
possible role for mechanical obstruction in the 
pathogenesis of branch retinal vein occlusion. 



21 



Division of Biometry and Epidemiology 



ISEI Annual Report— FY 1993 



In a study designed to identify risk factors for 
idiopathic rhegmatogenous retinal detachment, only 
one clearly relevant risk factor, myopia, emerged 
from the analysis. An eye with a spherical 
equivalent refractive error of -1 to -3 diopters had a 
fourfold increased risk of retinal detachment com- 
pared with a nonmyopic eye. Data from the study 
suggest that almost 55% of nontraumatic detach- 
ments in eyes without previous surgery are at- 
tributable to myopia. Results from the study are 
consistent with a hypothesis suggesting that the 
etiology of retinal detachment is related to the 
architecture of the eye, rather than to systemic 
factors. 

A large epidemiologic study reported an increased 
risk of branch retinal vein occlusion in persons with 
a history of systemic hypertension, a history of 
cardiovascular disease, an increased body mass index 
at age 20, and a history of glaucoma. Risk of vein 
occlusion decreased with higher levels of alcohol 
consumption and high-density lipoprotein cholesterol. 
The data suggest a cardiovascular risk profile for 
patients with branch retinal vein occlusion and 
indicate that 50% of branch retinal vein occlusioas 
may be due to hypertension. 

The Sorbinil Retinopathy Trial 

This multicenter trial was designed to assess the 
ability of sorbinil, an aldose reductase inhibitor, to 
retard the development and progression of diabetic 
complications. Results for retinopathy have 
previously been published. The study now reports 
that no benefit was found from sorbinil in slowing 
the development of clinical diabetic polyneuropathy. 

The Early Treatment Diabetic Retinopathy 
Study 

This multicenter, randomized clinical trial of 
aspirin versus placebo among diabetics examined 
mortality and morbidity from all causes with special 
emphasis on cardiovascular events. There were no 
harmful effects of aspirin, and the suggestion of 
beneficial effects was similar to previous studies of 
mainly nondiabetic persons. 



Research Activities 

Clinical Trials 

The Early Treatment in Diabetic Retinopathy 
Study 

The Early Treatment in Diabetic Retinopathy 
Study (ETDRS) was designed to determine when to 
use photocoagulation for diabetic retinopathy. 
Patients with macular edema, preproliferative 
retinopathy, and mild or moderate proliferative 
retinopathy were studied. Three forms of 
photocoagulation treatment, ranging from restricted 
focal treatment to complete panretinal 
photocoagulation, were compared with no 
photocoagulation. In addition, the study evaluated 
the placebo-conttolled effects of daily administration 
of aspirin on the incidence of microvascular and 
macrovascular complications. The study also inves- 
tigated factors associated with the progression of 
disease. 

Recruitment was completed in March 1985 with 
the enrollment of 3,711 patients. In December 1985, 
the study reported that focal photocoagulation of 
clinically significant diabetic macular edema substan- 
tially reduces the risk of visual loss. It was further 
reported that focal tteatment increases the chances of 
visual improvement, decreases the frequency of 
persistent macular edema, and causes only minor 
visual field losses. 

Sixteen ETDRS reports have been published. 
Additional manuscripts are in preparation. 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 plaiming group. The ETDRS results 
of aspirin effects on mortality and morbidity in 
patients with diabetes were analyzed and published. 
Analyses in progress include the effect of aspirin on 
vitreous hemorrhage, risk factors for severe visual 
loss, and risk factors for development of high-risk 
proliferative diabetic retinopathy. In addition, 
patients with mild to proliferative retinopathy are 



22 



NEI Annual Report— FY 1993 



Division of Biometry and Epidemiology 



being followed with extensive psychophysical testing 
in the NEI Clinical Center to determine the 
mechanisms for loss of visual acuity in diabetic 
retinopathy. 

The Sorbinil Retinopathy Trial 

Dr. Daniel Seigel served as project officer for the 
Sorbinil Retinopathy Trial (SRT) until his retirement 
in November 1991. Sorbinil, a drug manufactured 
by Pfizer Laboratories, is an aldose reductase in- 
hibitor that has potential to prevent or retard diabetic 
neuropathy and retinopathy. The NEI provided 
scientific leadership for this multicenter clinical trial, 
which was funded by Pfizer. Approximately 500 
patients were randomized to treatment and follov^oip, 
which ended in mid-1988. The results for 
retinopathy were published in 1990. No large benefit 
of treatment was observed. The effect of the treat- 
ment on neuropathy was summarized in a paper that 
was published this year. 

The Krypton-Argon Regression of 
Neovascularization Study 

The Clinical Trials Branch began the Krypton- 
Argon Regression of Neovascularization Study 
(KARNS) in three pilot clinics in December 1983. 
The major objective of this randomized clinical trial 
is to compare krypton laser with argon laser pan- 
retinal photocoagulation for treating neovas- 
cularization on the optic nerve head caused by 
diabetic retinopathy. Twenty-nine new clinics were 
enrolled 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. Results 
of the KARNS were presented at the American 
Academy of Ophthalmology (AAO) in November 
1992 and will appear in Ophthalmology. 

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 



and/or 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 examinations 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 DBE and a 
Chinese ophthalmologist. Dr. Tian-Sheng Hu, from 
Beijing. Findings for cataract were published this 
year and are discussed in the research results section 
of the report of the Division of Collaborative Clinical 
Research. 

Intramural Program Clinical Trials 

Drs. Ferris and Chew are collaborating with Dr. 
Robert B. Nussenblatt on four additional randomized 
clinical trials in the NEI Intramural Program of the 
Clinical Center: (1) a trial of a sustained-release 
intraocular drug delivery system for gancyclovir 
therapy of CMV in patients with AIDS; (2) a trial to 
evaluate the efficacy of a heparin-surface modified 
intraocular lens in reducing the incidence and 
severity of postoperative inflammatory episodes 
following extracapsular surgery in uveitis patients 
with cataracts; (3) a trial of anti-inflammin, a pep- 
tide, in the treatment of anterior uveitis; and (4) a 
trial of S-antigen tablets in patients with uveitis. 

Other 

Dr. Seigel, as a special expert, continues to 
represent the NEI on the Data Monitoring Committee 
of the United Kingdom Prospective Diabetes Study, 
a clinical trial of alternative treatment regimens in 
the management of patients with diabetes. Followup 
is scheduled to continue in this study until 1994. 

Epidemiology 

The Age-Related Eye Disease Study 

The Age-Related Eye Disease Smdy (AREDS) is 
designed to collect natural history data of 4,600 
patients between the ages of 55 and 78 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 
during the 10-year AREDS study period will provide 
an opportunity to evaluate factors associated with the 



23 



Division of Biometry and Epidemiology 



NEI Annual Report— FY 1993 



development of cataracts. In addition, a clinical trial 
will be perfonned to determine whether antioxidants 
(vitamins C, E, and beta-carotene) and zinc would 
prevent the development or retard the progression of 
AMD and cataract. There are 1 1 Clinical Centers, a 
Photographic Reading Center, a Central Laboratory, 
and a Coordinating Center. Identification of study 
participants began in September 1990. In November 
1992, participants were evaluated with qualifying 
visits, and participants were randomly assigned to the 
study medications beginning in February 1993. Drs. 
Ferris (chairman), Sperduto (director of Leas 
Project), and Chew are directing the scientific aspects 
of the AREDS; Dr. Natalie Kurinij is the project 
officer. 

The Eye Disease Case-Control Study 

The Eye Disease Case-Control Study (EDCCS) is 
designed to identify risk factors for neovascular 
macular degeneration, idiopathic branch retinal vein 
occlusion, idiopathic central vein occlusion, rheg- 
matogenous retinal detachment, and idiopathic 
macular hole. Dr. Sperduto is study chairman, Ms. 
Rita Hiller is director of Data Analysis, and Dr. 
Chew is a member of the project team. All data 
have been collected. Five manuscripts were 
published this year: Risk Factors for Neovascular 
AMD, Antioxidant Status and Neovascular AMD, 
Arteriovenous Crossing Patterns in Branch Retinal 
Vein Occlusion, Risk Factors for Idiopathic Rheg- 
matogenous Retinal Detachment, and Risk Factors 
for Branch Retinal Vein Occlusion. 

The Diabetes in Early Pregnancy Study 

Dr. Emily Chew and Ms. Nancy Remaley, in col- 
laboration with Dr. James Mills of the National 
Institute of Child Health and Human Development 
(NICHD), are examining the effects of pregnancy on 
diabetic retinopathy in the Diabetes in Early Pregnan- 
cy Study (DEEPS). Data collection terminated in 
1985. A manuscript has been prepared. 

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

In Parma, Italy, the Italian-American Natural 
History Study of Age-Related Cataract will estimate 
the rates of development and progression of the 
different types of lens opacities and the associated 
risk factors. Dr. Sperduto is the project officer; Dr. 
Milton and Ms. Remaley are on the project team. 



Data collection began in May 1989 with baseline 
data from the Italian-American Case-Control Study 
of Senile Cataract and was completed in May 1993. 
Analyses of development and progression of age- 
related cataract are under way. 

The Framingham Offspring Eye Study 

Dr. Sperduto is the project officer, Dr. Milton is 
the alternate project officer, and Drs. Marvin J. 
Podgor and Valeria Freidlin and Ms. Hiller are 
members of the project team for the Framingham 
Offspring Eye Smdy (FOES). This study is designed 
to examine familial relationships for age-related 
cataract and AMD among parents examined in the 
Framingham Eye Study (1973-1975) and their 
children examined between 1989 and 1991. Dr. 
Podgor has used generalized estimating equation 
methodology in the analyses of these data. A 
manuscript describing the study's findings for 
cataract has been prepared. 

Other 

A manuscript has been submitted for publication 
on risk factors for strabismus, using data fi-om the 
NICHD Collaborative Study and in collaboration 
witii Dr. Mark Klebanoff, NICHD. The DBE project 
team includes Drs. Chew, Tamboli, Zhao, Podgor, 
and Ms. Remaley. 

Statistical Methods 

Dr. Marvin Podgor and Dr. Joseph Gastwirth 
from the George Washington University collaborated 
in the investigation of various tests for the two- 
sample problem with location and scale change 
alternatives. Dr. Podgor presented some of these 
results at the NIH Conference on Current Topics in 
Biostatistics and at the 1993 Joint Statistical 
Meetings. A paper has been accepted for 
publication. 



Professional 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 monitoring committees for clinical 
trials, training of staff fellows, invited and 



24 



NEI Annual Report— FY 1993 



Division of Biometry and Epidemiology 



contributed presentations at professional society and 
other meetings, advisory committees for grant-sup- 
ported cooperative agreements, and technical advisors 
to the World Health Organization (WHO). 



Publications 

Bouzas EA, Freidlin V, Parry DM, Eldridge R, 
Kaiser-Kupfer MI: Lens opacities in 

neurofibromatosis 2: further significant cor- 
relations. BrJOphthalmomi6y.354-351, 1993. 

ETDRS Investigators: Aspirin effects on mortality 
and morbidity in patients with diabetes mellitus. 
ETDRS report No. 14. JAMA 268(10): 1292-300, 
1992. 

Ferris HI FL: Diabetic retinopathy. Diabetes Care 
16:322-323, 1993. 

Ferris HI FL: Issues in management of diabetic 
retinopathy. Hospital Practice 28(5):7, 1993. 

Ferris DI FL, Freidlin V, Kassof A, Green SB, 
Milton RC: Relative letter and position difficulty 
on ETDRS visual acuity charts. Am J Ophthal- 
mol 116(6):735-740, 1993. 

Glenn GM, Linehans WM, Hosoe S, et al.: 
Screening for von Hippel-Lindau disease by DNA 
polymorphism analysis. JAMA 267(9): 1226- 1231, 
1992. 

Lasa MSM, Podgor MJ, Datiles MB, Caruso RC, 
Magno BV: Glare sensitivity in early cataracts. 
Br J Ophthalmol 77:489-491, 1993. 

Magno BV, Freidlin V, Datiles MB: Reproducability 
of the NEI Scheimpflug cataract imaging system. 
Invest Ophthalmol Vis Sci 35(7):3078-3084, 1994. 

Nussenblatt RB, de Smet MD, Rubin B, et al.: A 
masked, randomized, dose-response study bet- 
ween cyclosporine A and G in the treatment of 
sight-threatening uveitis of non-infectious origin. 
Am J Ophthalmol 115(5):583-591, 1993. 

Podgor MJ, Gastwirth JL: On nonparametric and 
generalized tests for the two-sample problem with 
location and scale change alternatives. Stat Med 
13(5-7):747-758, 1994. 



Prior MJ, Prout T, Miller D, Ewart R, Kumar D and 
Early Treatment Diabetic Retinopathy study 
Research Group: C-peptide and the classification 
of diabetes mellitus patients in the Early Treat- 
ment Diabetic Retinopathy Study. ETDRS report 
No. 6. Ann Epidemiol 3:9-17, 1993. 

Rodgers GP, Walker EC, Podgor MJ: Is "relative" 
hypertension a risk factor for vaso-occlusive 
complications in sickle cell disease? Am J Med 
5d 305:150-156, 1993. 

Sastry SM, Sperduto RD, Waring GO, Remaley NA, 
Lynn MJ, Blanco PE, Miller DN: Relationship of 
radial keratotomy and intraocular pressure. 
Refractive and Corneal Surgery 9(6):459^64, 
1993. 

Singerman LJ, Chew EY, Ferris HI FL, Murphy RP, 
Brucker AJ, Remaley NA, and The Krypton 
Argon Regression Neovascularization Study 
(KARNS): Randomized comparison of krypton 
vs. argon scatter photocoagulation for diabetic 
disc neovascularization. KARNS report No. 1. 
Ophthalmology 100(11): 1655-1664, 1993. 

Sorbinil Retinopathy Trial Research Group: The 
Sorbinil Retinopathy Trial: neuropathy results. 
Neurology 43:1141 -\\49, 1993. 

Sperduto RD, Hu T-S, Milton RC, et al.: The 
Linxian Cataract Studies — Two nutrition interven- 
tion trials. Arch Ophthalmol 111:1246-1253, 
1993. 

Sperduto RD: Epidemiologic aspects of age-related 
cataract. In: Tasman W, Jaeger EA (eds): 
Duane's clinical ophthalmology. Philadelphia, 
J.B. Lippincott Co, 1992, Chapter 73 A, pp 1-5. 

The Eye Disease Case-Control Study Group: Risk 
factors for neovascular age-related macular 
degeneration. Arch Ophthalmol 110:1701-1708, 
1992. 

The Eye Disease Case-Control Study Group: An- 
tioxidant status and neovascular age-related 
macular degeneration. Arch Ophthalmol 111:104- 
109, 1993. 

The Eye Disease Case-Control Study Group: Risk 
factors for idiopathic rhegmatogenous retinal 
detachment. Am J Epidemiol 137:749-757, 1993. 



25 



Division of Biometry and Epidemiology 



NEI Annual Report— FY 1993 



Eye Disease Case-Control Study Group: Risk factors 
for branch retinal vein occlusion. Am J Ophthal- 
mol 116:286-296, 1993. 

Zhao J, Sastry SM, Sperduto RD, Chew EY, 
Remaley NA, and The Eye Disease Case-Control 
Study Group: Arteriovenous crossing patterns in 
branch retinal vein occlusion. Ophthalmol 
100:423-428, 1993. 



26 



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 includes the reduction of 
the prevalence of blindness, visual impairment, 
and eye disease worldwide through basic and applied 
research and training. Although excellent ophthalmic 
procedures and eye-care delivery systems are acces- 
sible in the developed world, adequate health care is 
not readily available in all parts of the developing 
world. This widening gap in visual health between 
developed and developing nations threatens to have 
ominous consequences. If present trends continue, 
the number of blind people — ^today estimated at 24 
million — will more than quadruple during the next 
40 years. Tragically, as many as 90% of these blind 
people will live in developing countries. 

This large-scale disablement caused by blindness 
is not only a costly obstacle to economic develop- 
ment, it is a catastrophic loss of human potential in 
the very parts of the world most desperately in need 
of a healthy workforce. In addition, because more 
than 80% of all cases of blindness can be considered 
avoidable — that is, they could have been prevented 
or could be cured using available and locally ap- 
propriate technology — such deprivation is a truly 
needless denial of a basic human right for millions 
and millions of people. Therefore, the NEI under- 
takes international activities to facilitate the develop- 
ment and application of effective prevention and 
intervention programs. These efforts are coordinated 
by the Institute's Office of International Program 
Activities (OBPA), which was aeated in February 
1989. OIPA enhances NEI's international programs, 
which include: 

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

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

• Conducting controlled clinical evaluations of 
promising research findings. 



• Exchanging information on recent scientific 
advances and their appropriate application to 
visual problems. 

NfEI currently supports international research on 
several blinding diseases that have a major 
worldwide impact: cataract, onchocerciasis, ocular 
toxoplasmosis, glaucoma, diabetic retinopathy, and 
vitamin A deficiency. During the past year, the NEI 
has continued to support investigations of important 
blinding eye diseases with worldwide impact. These 
studies are implemented through bilateral agreements 
with the U.S. Government, other types of country-to- 
country programs (such as those supported by the 
U.S. Agency for International Development [U.S. 
AID]), and through collaborative activities with the 
WHO, the Pan-American Health Organization, and 
with foundations and private and voluntary or- 
ganizations such as the International Association of 
Lions Clubs. 



Research Activities 

Cataract 

Because cataract is responsible for about one-half 
of the developing world's curable blindness and is a 
major problem for the United States as well, the NEI 
has developed a collaborative research program that 
includes projects to prevent blindness from cataract 
with collaborating groups in Italy, India, and Latin 
America. Additionally, health services research 
expertise from the NEI is made available to selected 
collaborating partners through training activities and 
the conduct of joint research projects. 

An NEI-supported randomized clinical trial to 
compare intracapsular cataract surgery plus aphakic 
spectacles with extracapsular cataract extraction plus 
implantation of an intraocular lens (lOL) is being 
conducted at the Aravind Eye Hospital in Madurai, 
India. The trial's primary comparison concerns 
operative and postoperative complications. 



29 



International Program Activities 



NEI Annual Report— FY 1993 



Secondary evaluation endpoints include measurement 
of vision function assessed by interview using a 
multi-item questionnaire and appraisal of economic 
impact in terms of direct and indirect cost associated 
with blindness and cataract surgery. 

The Collaborative Italian-American Case Control 
Study of Age-Related Cataract, which was started in 
September 1986, as part of the program of 
cooperation in biomedical research between the 
United States and Italy, has now been completed. 
The objectives of the study were to: (1) identify risk 
factors for age-related cataract, (2) evaluate metiiods 
of in vivo cataract classification, and (3) compare the 
findings with those of parallel studies being con- 
ducted in the United States and India. Data were 
collected at the Institute of Ophthalmology at the 
University of Parma. The Laboratory for 
Epidemiology and Biostatistics at the Istituto 
Superiore di Sanita in Rome served as the study's 
coordinating center. Data collection ended in April 
1989, after 1,477 subjects had been entered into the 
system. Four papers describing the study's findings 
have been published. 

Ihe Collaborative Italian-American Study of the 
Natural History of Age-Related Cataract has added a 
four-year follov^p component to the recently 
completed case-control study of age-related cataract. 
Approximately 1,000 subjects with cataracts and 300 
subjects fi-ee of cataracts are being examined every 
six months for four years to collect data on the 
natural history of the various types of cataracts. 
Data collection began in May 1989. The objectives 
of the natural history study are to estimate the rates 
of development and progression of the various types 
of lens opacities, identify risk factors associated with 
the development and progression of cataracts, and 
determine whether the risk factors affecting rates of 
progression differ for the various types of leas 
opacities. 

Data collection ended in April 1993 at the 
Institute of Ophthalmology, University of Parma. 
The Laboratory for Epidemiology and Biostatistics, 
Istituto Superiore di Sanita in Rome serves as the 
Coordinating Center. Preliminary findings from the 
study were presented in Stresa, Italy, at the 1992 
International Congress of Eye Research meeting. 
Preliminary findings from the study were presented 
at the meeting of the Association for Research in 
Vision and Ophthalmology in May 1993. A paper 



describing incidence and progression rates for 
specific cataract types has been prepared for 
publicatioa 

International collaborators have been established 
by scientists in the NEI's Laboratory of Mechanisms 
of Ocular Diseases, Section on Cataract to further 
our understanding of the relationship between en- 
zyme deficiency diseases and cataract For example, 
these NEI scientists have begun a candidate gene 
study to determine whether a deficiency in sorbitol 
dehydrogenase (SDH) in a family where several 
members have congenital cataracts is due to changes 
in SDH gene structure or expression. This study is 
possible through the cooperation of the Unidad de 
Investigacion Biomedica Hospital de Pediatria, 
Instituto Mexicano del Soguro Social, Guadalajara, 
Mexico. 

Vitamin A Deficiency 

Although not a major problem in the United 
States, vitamin A deficiency worldwide affects an 
estimated 14 million children annually. It is the 
world's major cause of childhood blindness, accoun- 
ting for 250,000 to 500,000 new cases of blindness 
per year. In addition, children die at higher rates 
from common childhood infections if they are 
deficient at any level of severity. The NEI supports 
basic research on the interaction of nutrients such as 
vitamins A, C, and E on retinal and other eye tissue 
development. Such investigations can lead to clinical 
interventions that may help alleviate morbidity from 
malnutrition eye disease. In addition, the NEI has 
provided technical consultation for a study in south 
India that has shown an impressive reduction in 
childhood mortality associated with improved 
vitamin A nutritional status, and other efforts to 
transfer this technology to alleviate world blindness 
are under way. 

Particularly in Asia, vitamin A deficiency is a 
public health problem and the leading cause of 
blindness among preschool-age children. The most 
effective way of providing affordable prevention 
programs is under study by the University of 
Michigan and the Nepal Netra Jyoti Sangh, Nepal's 
national society for the prevention and control of 
blindness. OIPA is providing technical oversight for 
this three-year project for the U.S. Department of 
Health and Human Services (DHHS) Office of 
International Health and the U.S.AID East Bureau. 



30 



NEI Annual Report— FY 1993 



International Program Activities 



Glaucoma 

Open-angle glaucoma is the leading cause of 
blindness among African Americans and is a major 
cause of visual impairment and disability. The 
incidence of glaucoma has not been measured 
precisely in any population, and the risk factors 
related to its development are largely unknown. In 
the Barbados Eye Study more than 4,200 persons 
between the ages 40 and 86 years were examined 
from 1988 to 1992 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 disorders from individuals free of disease in 
the Barbados prevalence survey. Also, risk factor 
analysis will be conducted for associations with 
development of glaucoma and to characterize those 
who have progressive eye disease. 

The Early Manifest Glaucoma Trial is a ran- 
domized, controlled clinical trial designed to deter- 
mine whether and to what extent reduction of lOP 
influences the course of chronic open-angle 
glaucoma. Investigators at the University of Lund in 
Malmo, Sweden, collaborating with investigators at 
the State University of New York at Stony Brook, 
will study an estimated 300 patients with newly 
diagnosed disease. Participants will be randomized 
either to pressure-lowering treatment or to obser- 
vation without treatment Both groups will be 
followed closely 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. 

Retinal Degenerations 

In collaboration with protein biochemists at the 
Karolinska Institute in Stockholm, Sweden, NEI 
cataract researchers are investigating the evolutionary 
relationships of i^-crystallin, an enzyme/crystallin of 
certain species, with other oxido-reductases. Es- 
tablishing such relationships with enzymes of known 
function should help in identifying the physiological 
roles of ^-crystallin both in the lens and in other 
tissues where it is present at low levels. 



Diabetic Retinopathy 

The United Kingdom Prospective Diabetes Study 
is a prospective randomized study of different 
therapies to determine whether improved blood 
glucose control or improved blood pressure control 
of noninsuhn-dependent diabetes will reduce mor- 
bidity and mortality. The study began in 1977 and 
has recruited 5,102 newly diagnosed diabetic 
patients. Patients who fail to respond to diet therapy 
are randomized to diet therapy or "active 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 ACE inhibitor or beta-blocker to "less tight 
control." The development and progression of 
diabetic retinopathy in these patients is being as- 
sessed by retinal photography. The study is currently 
completing 10 years of patient followup. 

Management for Eye-Care Delivery Course 

As a WHO Collaborating Center for the Preven- 
tion of Blindness, the NEI offered a course at the 
Aravind Eye Hospital in Madurai, India, in January 
1993, on management for eye-care delivery. 

The purpose of this five-day course was to 
enhance the effectiveness of mid- and senior-level 
managers of eye-care programs and facilities. The 
course broadened the management perspective of the 
students, extended their understanding of decision- 
making, and developed their problem-solving skills. 
Emphasis was placed on demonsfrating the ap- 
plication of operations research and management 
science to "real world" problems. 

Individually and in small discussion groups, 
participants analyzed each case by identifying the 
basic problems involved, characterizing the relevant 
background setting and facts, formulating an ^- 
propriate analytical framework or model, and 
generating alternative solutions. Subsequentiy, in a 
large-group classroom setting, all participants ex- 
changed views concerning the cases and tested their 
conclusions. Group discussion forced participants to 
examine critically their own assumptions and to 
narrow their thinking to a plan of action. Members 
of the faculty guided the classroom discussion and 
ensured that all significant issues were addressed and 
that there was full participation. 



31 



International Program Activities 



NEI Annual Report— FY 1993 



Consultation to the World Bank 

The director, deputy director, and special advisor 
to the director, NEI, have participated as consultants 
to the World Bank in the development of a proposal 
by the Government of India for a major initiative in 
cataract blindness control. Technical meetings were 
held in New Delhi and Madurai to provide the 
knowledge base upon which training and surgical 
guidelines can be developed for a significant expan- 
sion of cataract surgery with explicit attention to the 
quality and extent of vision restoration. 



Activities With International and 
Multinational Organizations 

In FY 1993 NEI staff continued to provide tech- 
nical advice to Lions Clubs International in the 
development of their $100 million SightFirst 
initiative, a global sight-conservation program aimed 
at substantially reducing the prevalence and incidence 
of preventable and curable vision loss. 

Also in FY 1993, NEI continued its activities as 
a WHO Collaborating Center for the Prevention of 
Blindness. The NEI director continues to serve on 
the WHO'S Special Advisory Panel in the Prevention 
of Blindness, and the assistant director for Inter- 
national Program Activities serves on the Global 
Advisory Committee. Other NEI staff members 
have, on request, given consultations to the WHO 
program. In addition, an ophthalmologist from India 
visited the NEI, under the auspices of a WHO 
fellowship, to study cataract etiology and prevention. 



NEI continues working closely with non- 
governmental organizations in designing service and 
research programs to reduce the prevalence of 
blindness, regardless of its etiology, throughout the 
world. 

Extramural Programs 

In FY 1993, NEI granted 14 awards to foreign 
institutions in eight countries. Research and training 
projects were supported in lens and cataract, 
glaucoma, visual system development, photorecep- 
tors, phototransduction, visual cortex, visual abnor- 
malities, Leber disease, nutrition of the eye, ocular 
complications of diabetes, and the prevention of 
blindness. Awards covered both basic and clinical 
research projects. 

Intramural Programs 

NEI continues to serve as an international center 
for research and training on eye disease. In FY 
1993, 18 visiting fellows, 22 visiting associates, 13 
visiting scientists, 21 special volunteers, and eight 
guest researchers, from more than 20 countries 
conducted research in NEI's Bethesda, Maryland, 
facilities. Their work included basic laboratory 
investigations on the molecular structure and 
development of the visual system, sensory and motor 
disorders of vision, and the biochemical bases of 
retinal and corneal diseases and cataract develop- 
ment. In addition, visiting scientists collaborated 
with NEI investigators in clinical studies to define, 
treat, and prevent vision disorders such as genetic 
and developmental defects, ocular inflammatory 
disease, and ocular complications due to systemic 
conditions such as diabetes. 



32 



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 Legislation 
(OSPL) is responsible for a broad and diverse 
range of management activities that support and 
further the NEI's mission. Among these are 
providing leadership and direction for program 
planning, analysis, evaluation, and legislative 
functions, including the development and main- 
tenance of a computerized management information 
system and public information and scientific 
reporting services in support of the NEI's research 
programs. 

This year has been a period of significant change 
and yet has also been a time of significant ac- 
comphshment. Mr. Julian Morris, who had served 
the NEI for more than 20 years in positions ranging 
from information officer to associate director for 
science policy and legislation, succumbed after a 
lengthy illness. As a tribute to his numerous 
contributions to the institute, the NEI staff and the 
National Advisory Eye Council (NAEC) honored his 
memory by dedicating the most recent long-range 
plan for vision research to him. 

During this past fiscal year, the three sections that 
make up the OSPL were elevated to branch level. 
Mr. Michael P. Davis, who had served as acting 
associate director during Mr. Morris' illness, was 
selected to fill the vacancy. Subsequently, Dr. 
Carmen P. Moten, a program analyst within the 
Policy, Legislation, Planning, and Evaluation Branch 
(PLPEB), was selected as chief of that branch. 

Also of great significance was the completion of 
Vision Research— A National Plan: J 994-] 998. 
After final updating by staff, the plan was sent to 
MERIT awardees for their comments and sugges- 
tions, prior to final review and approval by the 
NAEC. Development and publication of such a 
comprehensive plan by so small an office, especially 
during a period complicated by many external 
factors, was an enormous undertaking. Without the 
hard work and extra effort by Dr. Moten and Mr. 
Whitaker of the PLPEB, Dr. McLaughlin and tiie 
program directors firom tiie extramural research 
program, and a very talented group of publication 



experts at CSR, Inc., final publication would not 
have occurred. We are greatly indebted to them for 
their assistance in bringing this excellent document 
to completion. 



Policy, Legislation, Planning, and 
Evaluation Branch 

Carmen P. Moten, Ph.D., Chief 

The PLPEB advises the NEI director on program 
plaiming, analysis, evaluation, and legislation 
and serves as the focal point within the Institute for 
these functions. In addition, PLPEB develops and 
executes a comprehensive program planning strategy 
for the Institute, including the periodic development 
of a national five-year vision research plan in con- 
junction with the NAEC; plans, coordinates, carries 
out, and/or monitors NEI program evaluations; and 
prepares recurring and ad hoc program analyses in 
response to requests fi"om the NIH, the Public Health 
Service (PHS), and the Department 

During FY 1993, the principal activities for the 
PLPEB were: 

• Preparation of material for the 1993-1994 Bien- 
nial Report of the Director, NIH, describing 
research accomplishments, outiining future oppor- 
tunities, and assessing important policy issues. 

• Preparation of briefing materials for the confir- 
mation hearing of NIH Director-Designate Dr. 
Harold Varmus. 

• Support of the NEI extramural grant information 
retrieval system by assigning scientific, biotech- 
nology, diabetes, and other codes to awarded 
grants for the purpose of analyzing and reporting 
research activity in areas of interest to the NEI, 
NIH, DHHS, Congress, or non-governmental 
organizations and individuals. 



35 



Science Policy and Legislation 



NEI Annual Report— FY 1993 



• Preparation of materials for the Congressional 
Appropriations Committee Report — Decade of the 
Brain. 

• Preparation of materials for NIH-coordinated 
activities with PHS agencies. 

• Preparation for publication by NIH The 1993- 
1994 Prevention Annual Report. 

• Preparation of materials for the NIH director on 
supported research related to FDA-approved 
drugs. 

• Preparation for publication by NIH of The 1992 
Annual Report on Rare Disease Research Ac- 
tivities. 

• Reparation of briefing materials for the General 
Accounting Office (GAO) on activities duplicated 
among PHS agencies. 

The PLPEB has also been involved in 
researching, writing, and editing a variety of reports 
requested by the NIH, PHS, DHHS, Congress, and 
non-governmental organizations and individuals that 
include the following: 

Report to Congress on sickle cell anemia research 
and the role of minority institutions in research. 

Review of the draft update of the Healthy People 
2000: Public Health Service Action Report. 

NEI submission on fibromyalgia for the Congres- 
sional Appropriations Report. 

NEI submission for the Diabetes Mellitus 
Interagency Coordinating Committee (DMICC) 
Annual Report. 

Scientific advances for the NIH FY 1994 
Congressional Justification. 

Report on breast cancer-related research during 
FY 1992, in response to an NCI request. 

Recommendations for the implementation of the 
NIH Strategic Plan, in response to an OSPL 
request. 

Review of the draft report — Public Version of the 
Strategic Plan. 

Support of the NIH Conference on Disease 
Prevention Research. 

Review of the Cross-Cutting for Healthy People 
2000 Progress Report— American Indians and 
Alaskan Natives; Adolescents and Young Adults: 
Hispanic Americans; and Women. 



Recommendations for technical changes to the 
H.R.4, the NIH Revitalization Act of 1993. 

Review of the draft report — Clinician 's Handbook 
of Preventive Services 1993 and Clinical Preven- 
tive Services: What Works and What It Costs, 
supported by the National Coordinating Com- 
mittee on Chnical Preventive Services (NCCCPS). 

Report on space medicine-related research, in 
response to a NIH director request. 

Report on research use of FDA-approved drugs, 
in response to a Congressional request. 

Report on federally funded current research, 
training, and intervention projects related to injury 
control. 

Briefings for the GAO. 

— Breast cancer 

— Immunology 

— Diabetes 

— Environmental health 

Policy briefing materials for the director, NIH. 

Report on adolescent-related research, in response 
to a NICHD Office of Demographic and 
Behavioral Sciences request 

Report on trauma-related research, in response to 
an NIH director request. 

Submission of highlights of drug-related research 
for Congressman Waxman's request for infor- 
mation concerning NIH-supported research on 
drugs. 

Final review of the draft report — Implementation 
Plan on Health and Behavior Research. 

Report to the Office of the Inspector General for 
an on site discussion of the Institute's mission 
and program activities. 

For the NEI Scientific Reporting Branch, data on 
inti-amural and extramural research in the fol- 
lowing areas: 

— Dry eye 

— AMD 

— Glaucoma 

— Cataract 

— RP 

— Sjogren syndrome 



36 



NEI Annual Report— FY 1993 



Science Policy and Legislation 



— Ocular implants 

— Corneas 

— Retinal transplants 

• For the NEI Financial Management Branch, 
information on intramural and extramural research 
costs for the following areas: 

— Biotechnology 

— Prevention 

— Immunology 

— Vaccine development 

— Decade of the Brain 

— Breast cancer 

— Tuberculosis 

— Aging 

— Nutrition 

— AIDS 

— Women's health issues 

— Diabetes 

— Cancer 

— Sexually transmitted diseases 

The PLPEB also provided editorial review of a 
variety of letters, reports, and other narrative 
materials for other offices within the NEI. 



Management Information Systems 
Branch 

David Scheira, Ph.D., Chief 



During the past fiscal year, the Management 
Information Systems Branch (MISB) has made 
significant changes to its local area network (LAN) 
configuration prompted by the move of the NEI's 
Extramural and Collaborative Program to Executive 
Plaza South (EPS) in April 1993. To accommodate 
this move, MSB configured a new LAN at EPS, 
connected to the NEI's Building 31 LAN via NIHnet 
using TCP/IP. MISB designed the star topology for 
the EPS LAN, based upon unshielded twisted pair 
UTP cabling. 



To support this distributed configuration of two 
LANs connected over NIHnet, the total number of 
network file, print, and database servers was 
increased from six to eight. The MISB procured the 
needed computer hardware, designed the network 
cabling and EPS machine room arrangement, and 
reassembled all 40 workstations at EPS after the 
move. During the fiscal year, MISB also configured 
additional Dell 486 computers to replace the last of 
the NEI's outmoded Zenith 286 PCs, yielding a total 
of 95 NEI workstations. MISB also upgraded the 
network cards in all workstations to Etherlink II or 
III for improved performance and reliability. 

During the past fiscal year, the MISB also 
upgraded all client network software to LAN 
Manager 2.1a. The network's database server was 
upgraded to a Dell 486/50 with 2.1 gigabytes of disk 
space to support greatly expanded database 
functionality. SQL Bridge was installed and con- 
figured to allow transparent access by Building 31 
NEI staff to database systems, which used the NEI's 
SQL server located at EPS. 

Remote printing capability via BARRHASP was 
added to the EPS LAN and was enhanced to allow 
more flexible routing to different network printers at 
the Building 31 site. Daily network backup 
procedures using two digital/audiotape (DAT) drives 
were enhanced to ensure more reliable operation. An 
uninterrupted power supply unit (UPS) was procured 
and installed at the NEI's EPS site, providing con- 
tinued operation in the event of a temporary power 
loss and an automatic smooth network shutdown in 
the event of a protracted power outage. 

The MISB provided extensive enhancements to its 
grants information systems during FY 93. Microsoft 
SQL Server, the database server for all systems, was 
upgraded to version 4.2 to allow enhanced 
functionality. JAM and JAM/DBI, the client tools 
for these systems, were also upgraded to allow 
additional functionality. The existing NEI snapshot, 
council letter, and grants coding systems were 
upgraded for this new environment. 

Three new systems for, respectively, user-friendly 
grants queries, CRISP queries, and pay plan 
management were designed and programmed by 
MISB staff. Automated security and login functions 
integrated with LAN login were developed by MISB 
staff. These functions provide automatic login to 
grants information systems for NEI staff and also 



37 



Science Policy and Legislation 



NEI Annual Report— FY 1993 



automatic tracking of system usage. Database 
system status information has been integrated into 
this automatic logon procedure, so that users receive 
messages concerning system shutdowns when they 
occur and estimated times of resumed operation upon 
logon to these systems. 

Automatic daily check and backup procedures 
have been implemented through custom 
programming by MISB for all active NEI databases. 
These procedures are automatically run overnight 
from an NEI LAN server with results automatically 
recorded in a log table that can be instantly checked 
by MISB staff. 

The NEI's weekly grants update batch procedures 
have been fine-tuned to provide virtually no weekday 
system downtime during FY 1993. These procedures 
continue to be nm each Sunday by MISB staff to 
allow full system availability during the work week. 

During the end of the fiscal year, these update 
procedures, now miming in Paradox, were 
completely reprogrammed by MISB staff in 
Microsoft SQL Server Transact SQL language. 
When tested and implemented in FY 1994, this 
reprogramming will allow Paradox operations to be 
fully superseded by the more reliable, state-of-the-art 
client-server architecture. This reprogramming effort 
will streamline the NEI's database configuration, 
provide enhanced reliability and maintainability, and 
establish a foundation for additional advanced system 
development for both grants and other NEI functions. 

The MISB has continued to provide custom 
information reports to NEI staff for internal use and 
public distribution, with 107 new requests logged for 
FY 1993 and rapid turnaround achieved in every 
case. Weekly and monthly reports, as well, continue 
to be provided. In addition to its own programnung 
efforts, the MISB has continued to support NEI staff 
in the use of information resources provided by the 
Division of Research Services (DRG), the National 
Library of Medicine (NLM), and other sources, 
including the DRG information system, CRISP, 
FOCUS, WYLBUR, MEDLINE, Gratefiil Med, 
Legislate, the electronic NIH library catalog. Gopher, 
and other specialized systems. 

During the past fiscal year, MISB has 
implemented upgrades to several of the software 
packages it operates while continuing to provide 
support for its full LAN software array, including 
Harvard Graphics, Quattro, Paradox, Calendar, FTP 



PC/TCP, Microsoft Mail, Virus scaimer, and other 
packages used for specialized functions. The MISB 
has continued to support all computer hardware in- 
house, with service calls made only to order parts not 
internally available or to handle unusual problems. 
This internal support has resulted in substantial 
savings to the NEI. 

The MISB has arranged for the services of a high 
school student intern during FY 1993, who assisted 
with its evaluation of a client-server gateway that the 
Division of Cancer Research and Treatment (DCRT) 
is now in the process of procuring for NIH-wide use. 
This gateway will allow NEI transparent access into 
personnel and administrative database systems whose 
data is stored in the DB2 mainframe database sys- 
tem. The MISB has continued to be a leader in the 
development of client-server database systems at the 
NIH, and MISB staff members have demonstrated 
NEI systems at various intercampus forums. 

The MISB has continued to handle a number of 
IRM functions for the NEI, including its environment 
and resources report, strategic plan, tactical plan, 
budget report, and security functions. The MISB 
staff members have continued to represent the NEI 
on a number of NIH-wide committees, including the 
Office Technical Coordinators and its network 
subcommittee, the ADP Extramural Programs Coor- 
dinating Committee and its steering committee, the 
Database Technology Task Force, the NIH lead users 
group, the Campus Users Research Exchange, and 
the Technical LAN Coordinators Committee. 



Scientific Reporting Branch 

Judith A. Stein, M.A., Chief 



This year, the Scientific Reporting Branch (SRB) 
provided numerous reporting and public infor- 
mation activities in support of NEI programs. 
Specific activities included the development of 
responses to inquiries received from the general 
public, professionals, and the media; the development 
and dissenunation of information and education 
materials; development of A Celebration of Vision 
Research, highlighting the NEI's 25th anniversary; 
planning, implementation, and coordination of the 



38 



NEI Annual Report— FY 1993 



Science Policy and Legislation 



National Eye Health Education Program (NEHEP); 
maintenance of an NEI exhibit program; and 
preparation of reports to the Congress. Specific 
accomplishments in these areas are ouflined below. 

• The SRB responded to approximately 17,000 
written, telephone, and in-person inquiries from 
the general public, patients and their families, 
students, health professionals, the media, and 
other professionals. This figure represents almost 
a threefold increase in inquiries from FY 1992. In 
addition, staff members responded to 15 pieces of 
controlled correspondence. These correspon- 
dences included responses to congressional in- 
quiries and Presidential greetings and 
proclamations. To support the public inquiry 
function of this office, the SRB staff developed 
and/or updated publications, including a new 
brochure for people at risk for AMD. 

• A new edition of the book. Clinical Trials Sup- 
ported by the National Eye Institute was 
produced. This book describes 16 nationwide 
extramural clinical trials supported by the NEI 
and, for the first time, five intramural studies 
conducted at the NIH. The book will be 
promoted to practitioners through exhibits at the 
upcoming meetings of the AAO and the 
American Academy of Optometry and through 
public service advertisements and announcements 
in various professional journals. 

• The results of the Retinitis Pigmentosa Vitamin 
Study were aimounced in May. This included the 
writing and dissemination of a press release to the 
print and electronic media A "Dear Colleague" 
letter was prepared and distributed to all members 
of the AAO and the American Optometric As- 
sociation. 

• To highlight the NEI's 25th aimiversary, A 
Celebration of Vision Research was initiated. The 
objectives of this activity are to provide the 
American public with a report on its investment 
in vision research, highlighting the achievements 
and firontiers of publicly funded vision research; 
increasing awareness of the benefits derived from 
vision research; and stimulating interest in 
biomedical research. A traveling science museum 
was developed that will present the progress and 
future of vision research by highlighting the 



anatomy and physiology of the eye and visual system 
through the use of interactive modules; artifacts firom 
the past such as eyeglasses, advertisements, equip- 
ments, etc; common eye diseases and disorders, 
focusing on both basic and clinical research; and 
predictions for the future of vision research. In 
addition, the development of a school program for 
grades four to eight was initiated. 

• The NEHEP continued to expand its efforts to 
reach people at risk for glaucoma or diabetic eye 
disease. This included the distribution of the 
three NEHEP education kits to individuals 
responsible for educating people at risk. Since 
the kits became available in the spring of 1992, 
more than 37,350 kits have been distributed. In 
addition, initial plans were developed to expand 
the diabetic eye disease program to reach Native 
Americans and Hispanics. This included con- 
ducting literature searches, identifying or- 
ganizations, and reviewing current materials 
designed for these audiences. 

The membership in the NEHEP Partnership grew 
from 43 to 50 organizations. A Partnership 
action plan was developed to monitor activities as 
well as to identify opportunities for future 
involvement in the NEHEP. Technical assistance 
was provided to many community-based or- 
ganizations to increase their participation in the 
NEHEP, particularly through the involvement of 
local Partnership members. 

Plans were made to hold the Third National Eye 
Health Education Conference in December 1993 
in Pennsylvania. The purpose of this conference 
will be to foster the development of local eye 
health education programs. The conference will 
provide the NEHEP Partnership with the opportu- 
nity to work together and to learn new skills that 
can be used to implement programs at the local 
level. 

• SRB staff members represented the NEI at the 
meetings of several professional organizations, 
including the American Diabetes Association, the 
Association of State and Territorial Directors of 
Public Health Education, the American As- 
sociation of Diabetes Educators, and the National 
Association of Area Agencies on Aging. 



39 



Office of the Scientific Director 



Report of the Scientific Director 



Robert B. Nussenblatt, M.D. 



This past year has been one of change and 
achievement in the Intramural Research Program 
of the National Eye Institute (NEI). Work has 
progressed in both clinical and basic research 
spheres. There has continued to be a heavy empha- 
sis in the area of AIDS (acquired immune deficiency 
syndrome), with an ongoing clinical trial to evaluate 
the effectiveness of a sustained-release device for 
ganciclovir placed into the eye for the treatment of 
cytomegalovirus (CMV) retinitis. A randomized 
masked study to immunomodulate uveitis has con- 
tinued. This attempt at using oral tolerization is an 
attempt to down-regulate inflammatory disease in 
patients' eyes without the use of medications. 

In the area of basic research, there have been 
developments in exciting new concepts about gene 
regulation, as well as continued advancement toward 
the ultimate goal of the use of gene therapy to treat 
eye diseases. Following are a few highlights of 
research achievements by the NEI intramural scien- 
tists during Fiscal Year 1993. 



Laboratory of Mechanisms of Ocular 
Diseases (LMOD) 

LMOD investigators have continued studies on a 
broad range of topics relating to the biology of 
various tissues, looking in depth at the molecular 
mechanisms responsible for certain ocular disorders. 
The major emphases of this group continue to be 
cataract and the ocular complications of diabetes. 
This year the group has shown, using site-directed 
mutagenesis, that the histidine at position 1 10 of the 
enzyme aldose reductase appears critical for catalytic 
activity. Studies have been instituted to evaluate a 
family with congenital cataracts whose members 
have a probable defect in the sorbitol dehydrogenase 
gene. 

On the clinical level the group also has evaluated 
the protein composition of normal human lenses and 
cataracts. Dr. Fielding Hejtmancik and his col- 
leagues have been studying the structure and function 



as well as the relationships of p-crystallins. At the 
same time, they are conducting gene mapping studies 
on a variety of genetic disorders that have ocular 
manifestations, including Usher's syndrome type I. 
These researchers have mapped two independent 
genes to chromosome 2. 

Dr. W. Gerald Robison has continued to refine 
and better characterize the rat model for diabetic 
retinopathy. Rats with this disorder will develop 
striking angiopathy in the retina. 



Laboratory of Sensorimotor Research 

(LSR) 

This Laboratory of international standing has 
continued to concentrate its research efforts on 
the brain mechanisms underlying the visual sense 
and visually controlled movements. Skilled motor 
control is one of the tasks the LSR scientists have 
concentrated on, particularly in relation to the visual 
control of eye movements. They have integrated, in 
a superb fashion, the observations made in humans, 
using an excellent animal model, the Rhesus mon- 
key. Its use permits exploration of not only the 
exact behavioral mechanisms related to visual motor 
behavior but also the underlying brain mechanisms. 
One area of particular interest has been the genera- 
tion of rapid or saccadic eye movements. 

Dr. Fred Miles and his group have attempted to 
increase understanding of the problem solving that 
goes into generating very rapid saccades. Some 
work has centered on the evaluation of what happens 
when monkeys or humans are confronted with 
different-sized images that are presented before each 
eye. Building on past observations. Dr. Miles' group 
has found that humans immediately adjust the 
amplitude of their eye movements in ways that are 
appropriate to the size of the stimulus. These LSR 
researchers have conjectured that the horizontal 
disparity in the images detected by the visual system 
controls this movement. They were able to 
reproduce this phenomenon in the monkey, thereby 



43 



Office of the Scientific Director 



NEI Annual Report— FY 1993 



permitting further evaluation of the brain mecha- 
nisms underlying this control. In parallel fashion, 
Dr. Wurtz and his group have evaluated the control 
of movement through the environment and the 
stabilization of posture. 

Dr. Lance Optican, who has an ongoing interest 
in the way neurons convey visual information, has 
demonstrated that nem"ons convey information about 
visual features by using a temporal code. This work 
may elucidate the fascinating concepts of how the 
brain processes information that ultimately leads to 
visual perception. 

Dr. Michael Goldberg and his collaborators have 
used a variety of techniques to evaluate the control 
of eye movements in the monkey. More recently 
they have applied these techniques to the human 
situation. In recent work, using PET scanning, they 
have identified in the human the approximate region 
in which frontal eye fields are located, which they 
had previously noted in the monkey. 

During this past year the whole LSR moved to 
the new Silvio O. Conte Building. This facility 
provides a state-of-the-art environment for nonhuman 
primate research. 



Laboratory of Ocular Therapeutics 
(LOT) 

This Laboratory has continued to focus on the 
development of new ophthalmic drugs — aldose 
reductase inhibitors as well as anticataract agents. 
This year more effective and less toxic aldose 
reductase inhibitors that are unrelated to previous 
aldose reductase inhibitors have been developed 
through the use of biochemical, pharmacological, and 
computer molecular design techniques. 

The Laboratory has continued long-range studies 
evaluating the effect of galactose on dogs. The 
retinal changes that have been seen are typical of 
diabetic retinopathy as it progresses to the prolifera- 
tive stage. This dog model is the first experimental 
model that demonstrates both clinical and histolog- 
ical changes found in all stages of diabetic 
retinopathy. 



Laboratory of Molecular and 
Developmental Biology (LMDB) 

LMDB investigators continue to focus on under- 
standing the fundamentals of gene expression 
and cellular differentiation in the eye. Major empha- 
sis is placed on the lens. On the basis of their earlier 
observations that lens crystallins ^pear to be multi- 
functional proteins that are expressed outside the lens 
and eye, they have broadened their research during 
the past year to include new areas of metabolism and 
gene expression in various tissues. Of interest is the 
fact that transcriptional factors involved in the 
expression of crystallin genes are present in many 
tissues and are used to control numerous biological 
processes. The implications of gene control in the 
eye go far beyond this organ. 

Of great importance this year has been the 
LMDB's identification of regulatory elements re- 
quired for expression of genes in the eye and other 
tissues. Regulatory elements may be functionally 
redundant, that is, removing a regulatory element 
will not necessarily eliminate expression of the gene. 
A great deal of emphasis has been placed on the 
expression of proto-oncogenes and cyclins, which 
have been linked to the normal processes of cellular 
differentiation in the lens, as well as to the cell cycle 
and growth control. 

The development of a transgenic facility within 
this Laboratory has expanded the NEI's ability to use 
transgenic animal models. An ongoing, fruitful 
interaction between the LMDB and the Laboratory of 
Immunology (LI) has resulted in genetic engineering 
experiments which have the potential to contribute 
animal models for research on autoimmune diseases 
of the eye. 



Ophthalmic Genetics and Clinical 
Services Branch (OGCSB) 

The OGCSB conducts clinical and laboratory 
research on gene expression and molecular 
interactions important to the eye. There is also an 



44 



NEI Annual Report— FY 1993 



Office of the Scientific Director 



application of clinically relevant research findings for 
the prevention, diagnosis, and treatment of diseases 
affecting the eye and visual system. The Branch has 
continued using different systems to develop objec- 
tive and subjective methods of monitoring and 
documenting opacities in the human lens. Among 
various objective systems that have been utilized are 
the Scheimpflug camera and the retroillumination 
camera. Other subjective systems utilized include 
the LOCS-2 grading system, as well as contrast 
sensitivity and glare testing. 

The OGCSB has continued its ongoing cataract 
research, carefiilly documenting cataracts in patients 
using a variety of techniques. Small pieces of tissue 
from cataracts extracted extracapsularly are used for 
various laboratory tests. Abnormal proteins have 
been identified by immunoblotting techniques as well 
as protein sequencing. It has been shown that in 
aging there is an acidic shift of proteins and that an 
increased number of polypeptide species exist within 
the molecular weight range of the crystallins. 

The Branch also has been a leader in the study of 
gyrate atrophy. Dietary intervention studies will 
continue in families with two affected siblings. A 
marked decrease in the retinal progression of this 
disorder now can be seen in the children who began 
the dietary intervention at an early age. This original 
work will lead to the exciting area of gene therapy. 
These studies will be performed in collaboration with 
the Laboratory of Immunology (LI). 



Laboratory of Retinal Cell and 
Molecular Biology (LRCMB) 

The research focus of the LRCMB has been to 
elucidate new genes and biochemical mecha- 
nisms that deal in particular with the retinal pigment 
epithelial (RPE) complex, in both health and disease. 
The long-term goal of this group is to establish basic 
information that will permit the Intramural Program 
to apply rational methods of gene therapy to various 
disorders. Several retina-specific genes identified by 
subtractive cloning have been located on the short 
arm of the X chromosome. 

In a similar fashion, LRCMB scientists have 
cloned RPE-specific genes that appear unique to the 
RPE. A new 65-kD protein of potential immuno- 
logic importance has been isolated from the human 



RPE and its gene has been cloned. This is the first 
RPE-specific gene to be reported and characterized. 

Work also has centered around a pigment epitheli- 
um-derived factor, which in very low concentrations 
causes the extension of elaborate neuronal processes 
from cultured retinoblastoma cells. It is hoped that 
this work may ultimately lead to understanding cone 
neuron development. 

LRCMB researchers continue to collaborate with 
LI investigators in studies of the immunopathology 
of experimental autoimmune uveitis. 



Laboratory of Immunology (LI) 

The LI is dedicated to the evaluation, diagnosis, 
and treatment of ocular inflammatory diseases. 
The research carried out by this group is both clinical 
and basic. In the clinical arena, the group continues 
to have a major conamitment to the study of the 
ocular complications of AIDS. The group has been 
interested in establishing noninvasive methods for 
diagnosing the presence of CMV retinitis in a non- 
ophthalmic setting. The use of the cell flare meter 
has helped in this regard. It shows an increase in the 
protein content in the anterior chamber that appears 
in AIDS patients who have CMV retinitis. This 
machine also is being used for a variety of thera- 
peutic studies. 

A randomized study to evaluate the usefulness of 
an intraocular slow-release device containing ganci- 
clovir that releases the drug for an 8-month period 
was in progress this past year. Patient recruitment 
continues but will end shortly. Newer medications 
such as rapamycin have been studied extensively in 
the experimental uveitis model. In addition, planning 
has begun for the use of monoclonal antibody 
therapy. The initial target is the interleukin 2 (IL-2) 
receptor. 

The Laboratory has had a long-term interest in 
toxoplasmosis. During the past year LI researchers 
have developed a reliable ocular model for toxoplas- 
mosis in which retinal as well as brain cysts of this 
origin can be demonstrated. This model has been 
used to evaluate the virulence of various strains, 
including those obtained from southern Brazil. 

RPE transplants have been studied in some detail 
by the LI. One accomplishment is the establishment 



45 



Office of the Scientific Director 



NEI Annual Report— FY 1993 



of a reliable method for studying rejection phenome- 
na of RPE cells. The group also has pursued its 
long-term interest in experimental uveitis models. 
The evaluation of various systems has resulted in 
further elucidation of the toleragenic state induced 
with the feeding of uveitogenic antigens. In parallel 
with these studies is an ongoing randomized masked 
study that will more fully evaluate the usefulness of 



S-antigen feeding in patients with intraocular inflam- 
matory disease. 

During the past year the group also has continued 
working on the development of a gene therapy 
approach for gyrate atrophy. The work to date has 
shown the feasibility of this approach; various 
studies have been designed to develop optimum ways 
by which this gene can be transfected into cells. 



46 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00065-16 OSD 



PROJECT NUMBER 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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 



SECTION 



INSTITUTE AND LOCATION 

NEI, MH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

0.2 



PROFESSIONAL: 

0.2 



OTHER: 

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 study of the physiological organization of neurons of the visual system of primates, with 
emphasis on the chromatic properties of color-opponent ganglion cells and of cells from the lateral geniculate 
nucleus and the primary visual cortex of macaques. 



47 



PHS 6040 (Rev. 5/92) 



Office of the Scientific Director 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

The purpose of this project is to study the neural 
organization underlying the processing of visual 
information at different levels of the primate visual 
system. 

Methods 

This research includes intracellular and extracellular 
recordings from single neurons, extracellular record- 
ings of mass responses, computer video stimulation, 
and tangent screen chromatic and spatial stimulation. 

Major Findings 

The studies have been resumed only recently, after 
underground construction work ended both along the 
wall separating the laboratory rooms from the street 
and in a nearby building across the street. The 
construction work made microelectrode recordings 
from single neurons almost impossible due to the 
nearly continuous shaking of the ground, which was 
transmitted to the recording system despite attempts 
at vibration isolation. In addition, street digging by 



heavy machinery resulted in damage to the computer 
hard disks. 

The studies involve an attempt of simultaneous 
recordings from both ganglion cell axons and genicu- 
late cells receiving input from such axons to examine 
spectral property transformations between these two 
levels of the visual pathway. 

Significance to Biomedical Research and the 
Program of the Institute 

Numerous behavioral, psychophysical, and electro- 
physiological studies show that the visual perfor- 
mance and characteristics of macaques and humans 
are extremely similar to one another. An understand- 
ing of nonhuman primate physiology provides a 
useful animal model for human visual function. 

Proposed Course 

These studies will be continued. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Processing — 
Visual Processing and Amblyopia (Structure and 
Function) 



48 



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



PROJECT NUMBER 

ZOl EY 00122-13 OSD 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or less. Title must fit or) or>e lir\e betweer) the borders.) 

Anatomical 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 



SECTION 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

0.8 



PROFESSIONAL: I OTHER: 

0.8 I 0.0 



CHECK APPROPRIATE BOX(ES) 

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

□ (a1) Minors 
D (a2) Interviews 



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

This project involves 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 (1) the anatomical association 
of outer-retinal cells selectively stained with tissue-reactive dyes and (2) the pattern distribution of cones in 
the retinas of human donors. 

Systematic study of the anatomical association at the light-microscope level of blue cones and horizontal and 
bipolar cells selectively stained by several tissue-reactive dyes in the macaque retina continues. The results 
have provided information on the probable retinal circuitry of the blue-sensitive cone pathway of primate 
retina. 

Retinal cone studies of eyes from human donors also continue. Evidence of a cone population with a point 
pattern resembling that of blue-sensitive cones selectively stained by tissue-reactive dyes has been obtained 
in quickly fixed, well-preserved retinas of some donors with a clinical history of diabetes. Cone density 
studies are being conducted on the retinas of macaques and human donors of various ages to resolve issues 
on the degree of primate photoreceptor losses with aging. 



49 



PHS 6040 (Rev. 5/92) 



Office of the Scientific Director 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

This project was designed to study the anatomical 
properties and neural organization of the primate 
visual system. 

Methods 

This project involves retinal histological processing, 
intravitreal injection of dyes, computer modeling and 
spatial statistical analyses of point and area patterns, 
silver staining of cells and myelin, histological 
processing of the cerebral cortex, deoxyglucose 
labeling, autoradiogr^hy, and cytochrome oxidase 
labeling. 

Major Findings 

1. Human donor retinas fixed within 3 hours or 
less of time of death show a cone population with a 
point pattern distribution resembling that of the cones 
identified as blue-sensitive ones (i.e., absence in the 
central most region of the fovea, a peak density in 
the parafovea, and a regular though slightly disor- 
dered spacing in which stained cones are separated 
by two to three unstained cones) in donors with a 
reported longstanding clinical history of diabetes. 
This finding is consistent with clinical and psycho- 
physical observations of a dysfunction of blue- 
sensitive cones in diabetic retinopathy. Fixation of 
the donor retinas in as short a time as possible after 
death continues to be a major obstacle in the obtain- 
ing of well-preserved material from a sizable number 
of cases. 

2. Thin serial sections of macaque retinas, 
stained with a tissue-reactive dye that selectively 
stains blue-sensitive and some post receptoral cells, 
are being examined systematically by light microsco- 
py to trace the anatomical relationship between 
selectively stained blue cones, HI horizontal cells, 



and blue-cone bipolar cells of the more peripheral 
macaque retina. Data obtained so far provide evi- 
dence that two or three blue cones are commonly 
contacted by blue-cone bipolar cells in peripheral 
retina, whereas a single blue cone is typically con- 
tacted by a single blue-cone bipolar cell. These 
results indicate that the blue-sensitive cone system 
behaves in a manner similar to that of the so-called 
midget cell system, with increasing retinal eccen- 
tricity. 

3. A modification of the tissue-reactive staining 
technique used to label blue-sensitive cones of 
primate retina has provided initial results of a sub- 
labeling of the "unstained" cones into two apparentiy 
distinct populations. Because these populations may 
represent the green-sensitive and red-sensitive cones, 
several j^proaches are being tried to enhance such a 
labeling, which, if successful, would pennit a direct 
observation of the point pattern of all three primate 
cone types. 

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 function- 
al prop)erties of these cones investigated in different 
basic disciplines but also to the clinical research and 
diagnosis of acquired retinal disease. The data 
obtained from the eyes of diabetic human donors are 
particularly promising in this respect. 

Proposed Course 

These studies will be continued. 

NEI Research Program 

Retinal and Choroidal Diseases — ^Fundamental 
Processes and Retinal Disorders (Retinal Organiza- 
tion, Neurotransmission, and Adaptation) 



50 



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



PROJECT NUMBER 

ZOl EY 00135-21 OSD 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Biochemistry of Retina and Pigmented 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, NET 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Office of the Scientific Director 



SECTION 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 

1.0 



PROFESSIONAL: 

1.0 



OTHER: 

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.) 

Effects of nutrition, oxidation, and other environmental factors Oight 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). 
Preoxidation in polyunsaturated fatty acids in debris led 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 1-4 footcandles (PC), and 27% of the rats 
developed mature cataracts by 5-12 months. 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. 

A study of effects of environmental lighting on incidence of bilateral mature cataracts in pink-eyed RCS rats 
fed the natural ingredient diet (NIH-07) has been completed. Incidence of bilateral cataracts was 5% in rats 
reared in 1-4 PC of cyclic light but was 25% in rats reared in 10 PC constant light, 70% in 25 PC constant 
light, and 100% in 22- to 28-day-old rats given high-intensity light (700 PC) for 48 hours. 

In RCS rats reared at 1-4 PC, a purified diet (AIN-76A) fortified with antioxidants (0.4% p-carotene + 0.01% 
BHT) prevented PSO and mature cataracts. Currently 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 (23-53 postnatal days) if NIH-07 had been fed. A study using higher concentrations of 
vitamin E has been completed, but the histopathological evidence in the retina has not been evaluated. 



51 



PHS 6040 (Rev. 5/92) 



Office of the Scientific Director 



NEI Annual Report— FY 1993 



Project Description 

Additional Personnel 

J. Samuel Zigler, Jr. Ph.D. 
Joseph J. Knapka Ph.D. 



Dennis Bernard 



M.S. 



Chief, LMOD, NEI 
Nutrition Consul- 
tant, Veterinary 
Research Program 
(VRP), National 
Center for Research 
Resources (NCRR) 
Nutritionist, VRP, 
NCRR 



Objectives 

This project is designed to study the biochemical and 
bionutritional relationships between lens, retinal 
photoreceptors, retina, retinal pigment epithelium 
(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. Diseases in which the RPE may be in- 
volved are of particular interest. 

Methods 

The Royal College of Surgeons (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 is being used as a tool to 
combat lipid peroxidation in the RCS rat retina and 
to prevent water-soluble toxic aldehyde byproducts 
from reaching and damaging the lens. The RCS 
cataract is not genetic, because the mutant gene is 
expressed 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 varying risk factors and preventive 
measures. 

Defined diets are prepared and fed to congenic 
affected and unaffected RCS rats in controlled 
experiments. The diets are fed to young breeding 
pairs prior to producing their first offspring and to 
their offspring after weaning, so that the experimen- 
tal animals will have received their diets from 
conception to date of observation. Clinical findings 
are recorded after indirect ophthalmoscopic and 



biomicroscopic slit-lamp examination. Postmortem 
examinations of the eye include dissecting microsco- 
py and light microscopy of stained specimens. At 
appropriate times, photography is used to record in 
vitro or in vivo data. Analytical methods include 
flameless atomic absorption, standard biochemical 
assays by spectrophotometry and fluorometry, and 
separation procedures. Special environmental light- 
ing conditions are employed to determine their 
histopathological effects on the retina and the lens. 

Major Findings 

1. Previous work had yielded data on the inci- 
dence of bilaterality of mature cataracts in the pink- 
eyed, tan-hooded retinal dystrophic RCS rat under 
different intensities and duration of light, except for 
the standard conditions of cyclic light at 1-4 foot- 
candles (FC) inside the cages. The omission 
occurred because the early data were obtained during 
a collaborative agreement in which any rat that 
developed a single cataract was sent for examination, 
and such rats could not be returned to observe 
bilateral occurrence. When our animal room was 
changed in location and in type of lighting (i.e., 
incandescent instead of fluorescent), it seemed wise 
to determine (1) whether incidence of cataract was 
the same and (2) the incidence of bilaterality. The 
diet in all these experiments on bilaterality was the 
standard natural ingredient diet (NIH-07). This diet 
contains all nutrients required by rats but permits the 
cataracts to occur. 

The results showed the same incidence of cataract 
in incandescent as in fluorescent lighting (27%); 5% 
of the rats had bilateral cataracts by 1 year of age. 
In the previous studies, the incidence of bilaterality 
was greater in rats exposed to constant light: (a) 10 
FC from 3 weeks to 1 year (25%), (b) 25 FC from 3 
weeks to 1 year (70%), and 700 FC in 65-day-old 
rats in a short-term experiment of 2 days (100% of 
15 rats). It seems possible that the 48-hour exposure 
at 700 FC could be reduced by using a program of 
short alternating exposures to light and darkness to 
produce the same percentage of cataracts and bilater- 
ality, since this regimen has been shown to produce 
light damage in the retina Thus, such experiments 
might be completed within the veterinary require- 
ment of holding a rat for only 24 hours after remov- 
ing it from the animal facility and also reduce the 
stress to the animal. 



52 



NEI Annual Report— FY 1993 



OfTice of the Scientific Director 



At 700 FC exposure for 48 hours, bilateral mature 
cataracts also were produced in congenic control 
RCS rats (15 of 15 rats), but the lag time before 
appearance of the cataracts was greater than in 
dystrophic rats (13-15 months, as compared to 9-12 
months). Furthermore, cataracts only occurred if the 
light exposure was preceded by a long period of dark 
adaptation (18 days). If short exposures to light and 
darkness could be substituted in these experiments, 
this possible model of age-related cataract could be 
pursued further. 

The requirement for long dark adaptation for 
production of mature cataracts in control RCS rats is 
consistent with our hypothesis that the cataracts are 
secondary to retinal light damage in both dystrophic 
and control rats. Long dark ad^tation of normal 
retina increases the content of rhodopsin by 50%, to 
parallel the situation in dystrophic rats, in which the 
retinal content of rhodopsin is 70-100% greater than 
normal because of accumulation of rod outer seg- 
ment debris from failure in phagocytic activity of the 
mutant RPE. 

In vitro, retinaldehyde has been shown to act as 
a photosensitizer to generate singlet oxygen, a highly 
energetic oxidant for polyunsaturated lipids, as well 
as proteins. In retinas with a high rhodopsin content, 
the retinaldehyde released by bleaching may exist in 
the free state long enough to act as a sensitizer to 
generate singlet oxygen. In accord with the hypothe- 
sis, lens opacities would be initiated by water-soluble 
toxic lipid peroxidation products from degenerating 
retina, carried through the vitreous to attack mem- 
branes of lens cells and fibers. The prevention of 
cataracts by antioxidants would begin in the retina, 
with quenching of singlet oxygen by vitamin E and 
beta-carotene, and continue as these and other 
antioxidants combat secondary products of peroxida- 
tion. 

2. Antioxidant diets that prevent 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 pro- 
tected. Last year we studied the AIN-76A purified 
diet, which contains twice the normal concentration 
of all the minerals in the AIN mineral mix plus 0.4% 
beta-carotene and 0.01% BHT, as well as 1,000 
mg/kg vitamin C and 150 mg/kg vitamin E. After 
the rats consumed this diet, histopathological exami- 
nation showed retarded retinal degeneration during 



the time the cataracts would have had their onset 
(23-53 postnatal days) if the NIH-07 diet had been 
fed. This year we fed that same diet containing 
increased concentrations of vitamin E to explore 
whether retinal degeneration can be delayed further. 
The eyes from rats of different ages have been fixed 
for histopathological study, but the results are not yet 
available. The dystrophic retina is extremely sensi- 
tive to light damage and to peroxidation of its lipids. 
This sensitivity, a fundamental part of the pathophys- 
iology in the RCS rat, makes it more vulnerable to 
the defect of the RPE; furthermore, it may provide a 
clue to the underlying disease. 

Significance to Biomedical Research and the 
Program of the Institute 

The program goal of preventing posterior subcapsular 
cataracts (PSC) in RCS rats has been achieved, and 
the goal of slowing the rate of retinal degeneration 
has been advanced. When the retinal degeneration is 
slowed through 55 postnatal days, the cataracts are 
prevented. These results were obtained using bio- 
nutrition with a purified diet supplemented with 
antioxidants. PSC occurs in many varieties of 
human hereditary retinal degeneration known as 
retinitis pigmentosa (RP), as well as in so-called age- 
related cataracts and in some persons treated with 
steroids or exposed to short-wave radiation. None of 
the well-known types of human RP appears to show 
the problem of RPE phagocytosis found in the RCS 
rat; however, the number of types of RP appears to 
be very great, and many have not been explored for 
this phenomenon. 

Cataract is a predominant cause of vision loss and 
blindness in the United States and in the worid. 
Effective prevention in humans has not been devel- 
oped; the only treatment is removal of the opaque 
lens. In the United States, the surgical treatment is 
safe, and intraocular lens replacement is highly 
successful. However, the annual cost of cataract 
surgery totals $2 billion and will continue to rise as 
tlie population ages. Cataract surgery, therefore, has 
been targeted for reduction as an important cost- 
saving plan, and prevention or slowing of cataract 
development has become imperative. 

Among the known risk factors for cataractogene- 
sis are ocular characteristics of the RPE and iris 
color, as well as exposure to sunlight and ultraviolet 
(UV) radiation. In humans, these and other risk 
factors are difficult to control and quantitate. In 



53 



Office of the Scientific Director 



NEI Annual Report— FY 1993 



RCS rats, however, the existence of both pink-eyed 
and black-eyed dystrophic and congenic control 
strains provides for control of ocular characteristics, 
and artificial illumination in the animal room pro- 
vides the equivalent of sunlight and known UV 
radiation. In the RCS rat, the cataracts are secondary 
to the retinal degeneration, in which peroxidation of 
polyunsaturated fatty acids leads to water-soluble 
toxic aldehydes that are carried through the vitreous 
to the back of the lens, where they can damage the 
cell membranes of lens cells and fibers. Principles 
involved in this example of cataractogenesis may 
have relevance to some of the types of human 
cataract, including factors in slowing or preventing 
cataracts and retinal degeneration. 

An initiative in the National Institutes of Health 
(NIH) Strategic Plan is the prevention or delay of 
cataract through nutrient-specific dietary intervention. 
Vitamins E and C and beta-carotene, as well as the 
antioxidant-associated trace minerals zinc and seleni- 
um, are to be tried in populations where diets are 
considered to be inadequate. 

In an animal model, there is total control over the 
content and intake of nutrients, whereas this is 
difficult or impossible in a human setting. Our diets 
are fed to the parents prior to conception, to the 
female during pregnancy and in the lactation period, 
and to the experimental offspring during its entire 
life. In RCS rats, we employed a purified ingredient 
diet supplemented with twice the usual concentra- 
tions of normal minerals (including zinc and seleni- 
um), vitamins E and C, and beta-carotene. With this 
diet, the cataracts were totally prevented, and retinal 
degeneration was delayed through the time when the 



cataract would have had an onset if a standard 
natural ingredient rat diet had been fed. With the 
natural ingredient diet, cataract incidence was 27% in 
this rat strain under standard light conditions (cyclic 
light giving 1-4 FC intensity inside the cage). The 
percentage of cataracts seen with this diet increased 
with the light intensity, whereas rearing in darkness 
prevented cataract. 

Proposed Course 

Histopathological effects of feeding a high vitamin E 
antioxidant diet to the pink-eyed dystrophic rat will 
be evaluated to determine whether the retinal degen- 
eration has been slowed fiirther, beyond 55 postnatal 
days. Histopathological material firom all previous 
rats with mature cataracts will be examined to 
compare the populations of lens epithelial cells. As 
pink-eyed, tan-hooded dystrophic breeders become 
available fi-om the NIH foundation colony, lens 
epithelial cell whole mounts will be prepared to 
compare the numbers of cells in lenses with and 
without cataract. 

Until now, studies of the mutant autosomal 
recessive rdy gene on Chromosome 3 of the RCS rat 
have not been feasible because the rat genome had 
not been well smdied. However, knowledge of the 
rat genome has been proceeding apace, and a collab- 
orative study to locate this gene, which appears to be 
involved in RPE phagocytosis, may soon be possible. 

NEI Research Program 

Cataract — Pathogenetic Mechanisms 

Retina — ^Retinitis Pigmentosa and Other Inherited 

Disorders 



54 



Laboratory of Immunology 



Report of the Chief, Laboratory of Immunology 

Robert B. Nussenblatt, M.D. 



The Laboratory of Immunology has finished its 
seventh year. Sections of the Laboratory include 
Immunology and Virology, headed by Dr. John J. 
Hooks; Experimental Immunology, headed by Dr. 
Igal Gery, who is also the Deputy Chief of the 
Laboratory; Immunoregulation, whose acting head is 
Dr. Rachel Caspi; Experimental Immunopathology, 
whose head is Dr. Chi-Chao Chan, and Clinical 
Immunology, whose acting head is Dr. Marc de 
SmeL For the recently formed Section on Molecular 
Biology, I serve as the acting head of an interdisci- 
plinary group. 



Section on Clinical Immunology 

The Section on Clinical Immunology has contin- 
ued to focus on major questions of clinical 
relevance. Studies centering on the evaluation of the 
use of the Molteno glaucoma implant and 5-fluoro- 
uracil combined with trabeculectomy continue. Final 
results still have not been obtained in this random- 
ized long-term endeavor. On the basis of our obser- 
vations, we are rather optimistic about the outcome 
for the long-term efficacy of the Molteno implant, 
although scientific evaluation of the results is still 
needed. 

The Section continued its study of patients with 
AIDS (acquired immune deficiency syndrome), in 
collaboration with the National Institute of Allergy 
and Infectious Diseases. Evaluation of the safety of 
adnunistering anticytomegalovirus (anti-CMV) 
hyperimmunoglobulin to patients at risk for CMV 
retinitis was concluded. A randomized study to 
evaluate a slow-release implant filled with gancyclo- 
vir is being actively tested in AIDS patients with 
CMV retinitis. These implants, placed directiy into 
the eye through the porous plana, are calculated to 
release small but therapeutically effective amounts of 
gancyclovir over an 8-month period. This random- 
ized study may yield information about an important 
alternative to systemic anti-CMV therapy for patients 
who cannot tolerate the intravenous infusions or who 



possibly do not have a specific indication for system- 
ic therapy. Recruitment has been brisk, and we hope 
that results will be obtained within the next calendar 
year. 

Also continuing are pediatric AIDS studies in 
which children are evaluated for the incidence of 
ocular infection. This study is done in conjunction 
with Dr. Philip Pizzo and his National Cancer 
Institute laboratory. 

The Section also has been particularly interested 
in the development of immunosuppression. A 
randomized masked study to look at the effectiveness 
of oral tolerization has been in progress this past 
year. The research group is attempting to evaluate 
the usefulness of S-antigen (S-Ag) given per os in 
the induction of tolerance in uveitis patients. Initial 
observations in a pilot study have demonstrated a 
positive therapeutic response to feeding. The aim is 
to complete this randomized study within the next 
year and to have final results shortly thereafter. 



Section on Molecular Biology 

To acquire a better understanding of the 
approaches to gene therapy, this group has 
focused on regulation of the omithine-6-aminotrans- 
ferase (OAT) gene in vivo as well as on genetic 
modification of somatic cell Unes mediated by 
recombinant retroviruses. Dr. Moncef Jendoubi 
placed human OAT cDNA under the control of the 
enhancer-promoter regulatory elements derived fi'om 
the Moloney murine leukemia virus long-terminal 
repeat, then transfected this construct into a safe 
packaging cell line (GP-i-E-86) to produce provirus 
particles. Supernatant from the ecotropic OAT 
producers of cell lines were useid to transduce mouse 
embryonal fibroblasts as well as stem cells. The 
recombinant retrovirus transferred the OAT gene to 
the recipient cells, which produced an immunore- 
active OAT. Northern blot analysis confirmed the 
presence of an OAT transcript in the transduced cell 
fines, even after a long time in vitro. 



57 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



The Human Genome Group was established, and 
several individuals from various parts of the Labora- 
tory have participated. The major goal is the devel- 
opment of a particular form of gene ther^y in the 
treatment of gyrate atrophy. This gene therapy 
would entail use of the OAT gene. The work has 
shown that this gene can be transfected and has 
potential for use in such therapy. We hope that in 
the not too distant future this therapy will become a 
reality at the National Eye Institute. 



Section on Immunoregulation 

This Section has maintained interest in the devel- 
opment and study of animal models of experi- 
mental ocular autoimmune disease. The work has 
centered particularly on the characterization of the 
murine experimental autoimmune uveitis (EAU) 
model because the mouse offers some very important 
advantages over other rodent models of uveitis. In 
addition, the group has been actively involved in the 
establishment of antigen-specific T-cell lines and 
clones, which permit investigators to identify and 
characterize cells capable of inducing ocular immu- 
nomodulation. 

This year the group has shown that the severity of 
inflammation and tissue damage in athymic rats is 
correlated with the proportion of lymphocytes in the 
intraocular infiltrate while the infiltrate in euthymic 
rats was predominantly lymphocytic with fewer 
monocytes and even fewer neutrophils. The sparse 
infiltrate in athymic rats was largely monocytic and 
had a relatively high proportion of neutrophils and 
eosinophils. It is interesting that reconstituted 
animals had an intermediate histological picture. 
These results indicate that recruited nonspecific T 
cells play a very major role in the pathogenesis of 
disease. 

Collaboration with Dr. Charles Egwuagu contin- 
ues in studies of the T-cell receptor (TCR) genes of 
cell lines and clones at the molecular level. Data 
collected to date indicate that TCR (variable region 
gene) usage in uveitis differs from that reported for 
other autoimmune diseases; it also may be more 
heterogeneous. It is interesting that recent experi- 
ments confirm our group's initial observations that 
TCR Vp8.2 may be a pathogenic clonotype in S-Ag- 



induced uveitis, whereas TCR VP8.3 may be one of 
the pathogenic clonotypes in uveitis induced by 
interphotoreceptor retinoid-binding protein (IRBP). 
These findings are exceptionally important because 
of their impact on the development of therapeutic 
strategies that would specifically target pathogenic 
cells via the T-cell receptor. 

Using a BIO. A strain of mice, the group has 
developed a pathogenic T-cell line specific to whole 
IRBP. Such cell lines are interesting for multiple 
reasons, including the fact that the TCR profile of 
the line changes with time in culture. There appears 
to be progressive enrichment in the vp8.2 and Vp8.6 
TCR-expressing cells. In the mouse this would 
suggest that VP8.2 and vp8.6 represent pathogenic 
clonotypes in IRBP-induced uveitis. 



Section on Experimental Immunology 

Continuing its long-term investigation of the 
pathogenesis of inflammatory eye diseases, this 
Section has found that the induction of EAU by 
bovine IRBP in the Lewis rat involves a unique 
immunologic relationship. Lymphocytes sensitized 
against the immunodominant and uveitogenic deter- 
minant of IRBP do not recognize the rat homolog of 
this determinant; rather, they are stimulated by other 
peptides of the rat IRBP. In this description of the 
phenomenon, a surrogate peptide determinant of an 
organ-specific antigen is used to initiate an autoim- 
mune pathogenic response. 

In addition, the group has been actively involved 
in the S-Ag feeding oral tolerance studies. They 
have overcome the inaccessibility of large amounts 
of human S-Ag by using recombinant DNA technol- 
ogy to produce human S-Ag in Escherichia coli 
bacteria. 

During the past year the group has evaluated the 
novel immunomodulator linomide, which was found 
effective in inhibiting EAU development in rats and 
mice actively immunized with S-Ag or IRBP. On 
the other hand, treatment with linomide had no effect 
on the development of EAU adoptively transferred 
by presensitized lymphocytes. These findings 
suggest that linomide would probably not be useful 
for the treatment of uveitis in humans. 



58 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Section on Experimental 
Immunopathology 



group also has demonstrated that cell adhesion 
molecules are important for both antigen sensitization 
and inflammatory cell infiltration into the eye. 



This new Section had an extremely productive 
year. Immunohistochemistry and in situ hybrid- 
ization have been used to identify and topographical- 
ly localize immunocompetent cells and analyze the 
alteration of surface markers on ocular resident cells 
and their cytokines in experimental uveitis models as 
well as in ocular tissues. The T-cell dominance of 
the response remains one of the major factors that is 
seen again and again. Moreover, the migration of 
inflammatory cells from the vessels into the target 
appears to be directed by adhesion molecules that 
can be expressed on the vascular endothelium as well 
as other resident cells in the eye. 

The group also has evaluated specimens firom 
human ocular tissues with various diseases, including 
uveitis, retinal disease(s), conjunctival and corneal 
diseases, metabolic genetic diseases, and tumors. 
The group's demonstration of the presence of a 
major lens protein, oB-crystallin, in retinoblastoma 
suggests that oB-crystallin is involved in tumor 
growth and/or is a marker for general oncogenic 
stress in retinoblastoma. 

Cell adhesion studies remain an area of great 
interest. The studies performed this year demonstrate 
that monoclonal antibodies against intercellular 
adhesion molecule 1 (ICAM-1) and its counterrecep- 
tor, lymphocyte function-associated antigen 1 (LFA- 
1), inhibit the development of EAU in mice. The 



Section on Immunology and Virology 

This Section has continued to emphasize its 
interest in the study of the retinal pigmented 
epithelial (RPE) cell. The use of monoclonal anti- 
bodies has helped in identifying a 67-kD protein that 
appears to be an RPE-specific epitope. Its sequence 
homology is similar to that of the intermediate 
filament of protein. The group also has demon- 
strated that inflanmiatory mediators such as lipopoly- 
saccharide, tumor neaosis factor (TNF-a), and 
interleukin 1 (IL-1) induce interleukin 6 (IL-6) gene 
expression and secretion by human RPE cells. This 
effect also is noted to be synergistic with interferon 
gamma (IFN-y). RPE cell transplantation continues 
to be a major area of interest for this group as well. 

During the past year the group developed a 
reproducible model for ocular toxoplasmosis. The 
induction of this model has provided the Laboratory 
with an opportunity to evaluate strain virulence. A 
strain of toxoplasmosis virulent in humans and 
isolated from Brazil was shown to be particularly 
virulent in a mouse model. Studies have begun to 
evaluate the mechanisms involved in toxoplasmosis 
cyst formation as well as attempts to counteract the 
infection. 



59 



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



PROJECT NUMBER 



ZOl EY 00279-02 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Study of Immunosuppressants for the Treatment of Uveitis in Animal Models 



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

LI, NEI 
LI, NEI 



PI: 


Francois G. Roberge 


M.D. 




Others: 


Chi-Chao Chan 


M.D. 






Marc D. de Smet 


M.D. 






Robert B. Nussenblatt 


M.D. 






Dan Martin 


M.D. 






Margaret Cheung 


M.D., 


Ph.D 




David Parks 


M.D. 





Visiting Scientist 
Head, Section on 
Immunopathology 
Visiting Scientist 
Scientific Director 
Senior Staff Fellow 
Senior Staff Fellow 
Senior Staff Fellow 



LLNEI 
NEI 
LI, NEI 
LI, NEI 
LLNEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Clinical Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.7 



PROFESSIONAL: 



0.7 



OTHER: 



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.) 

The goals of the project are to acquire a better understanding of the immunopathogenic mechanism of 
noninfectious intraocular inflanmiatory diseases (uveitis) and to develop treatment and prevent the 
complications associated with these diseases. This past year we studied two specific aspects of the application 
of the new noncytotoxic inmiunosuppressant rapamycin (RAPA) in the treatment of uveoretinitis: 

(1) evaluation of the synergistic effect of RAPA with cyclosporine A (CsA) or dexamethasone (Dex) and 

(2) evaluation of the effect of RAPA on complications of uveitis, fibrosis, and intraocular membrane 
formation. We also evaluated the role played by nitric oxide (NO) in anterior uveitis. 

We demonstrated that combining RAPA with CsA or Dex can inhibit experimental uveitis in vivo at greatly 
reduced doses of each drug, compared with the doses necessary when these three drugs are used singly. We 
showed that RAPA also inhibits proliferation of human fibroblast and retinal pigment epithelial (RPE) cells, 
indicating the advantage of using RAPA in the treatment of uveitis, in which fibrosis and membrane formation 
are common complications. We also demonstrated that NO is an important mediator of endotoxin-induced 
anterior uveitis. 



60 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Additional Personnel 

Bruce Pfeffer Ph.D. 



Senior Staff Fellow, 
LRCMB, NEI 



Clinical Protocol Numbers 

91-191 
91-187 

Objectives 

The immunosuppressive treatment of autoimmune 
disease such as uveoretinitis usually has to be sus- 
tained for a long period of time. To avoid treatment 
complications as much as possible, we try to develop 
treatment using noncytotoxic drugs. In particular, we 
seek to develop combination ther^y that minimizes 
the toxicity of each drug while maximizing the 
beneficial effects. Previously we had demonstrated 
by in vitro studies that the combinations of rapamy- 
cin (RAPA) with cyclosporine A (CsA) and RAPA 
with dexamethasone (Dex) had a synergistic effect on 
the inhibition of the proliferation of retinal antigen- 
primed lymphocytes. This year we tested these 
results in the in vivo rat model of experimental 
autoinunune uveoretinitis (EAU). We evaluated the 
effect that RAPA could have on the fibrosis and 
membrane formation in the eye following inflamma- 
tion. 

We also have studied the role of nitric oxide 
(NO) in a model of anterior uveitis induced with 
endotoxin (lipopolysaccharide). NO is the oxidation 
product of one of the guanidino nitrogens of L- 
arginine. The reaction is catalyzed by two different 
forms of the enzyme nitric oxide synthase, which 
have specific tissue distributions. NO is produced 
after stimulation with endotoxin. The synthesis of 
NO can be competidvely blocked with L-arginine 
analogues such as N°-nitro-L-arginine methyl ester 
(L-NAME). The inhibition is enantiomer restricted, 
the D form of analogues being inactive; it is also 
fully reversible with an increased concentration of L- 
arginine. We used the specificity of L-NAME 
inhibitory action to evaluate indirectly the role of NO 
in endotoxin-induced uveitis (EIU). 



nated on Day 28 after immunization. RAPA was 
delivered by continuous intravenous (i.v.) infusion by 
means of a miniosmotic pump implanted in the 
abdominal cavity. CsA or Dex was given by intra- 
muscular (im) injection once a day. The disease was 
evaluated by histopathology. 

Two human RPE lines, between passages 4 and 8, 
and human skin fibroblasts were used. Proliferation 
assays were done in quadruplicate in Dulbecco's 
modified Eagle's medium/F12 medium, in microtiter 
plates at 6 X 10^ cells per well. Proliferation was 
measured by the incorporation of ^H-thymidine, 
added after 24 hours for 18 hours of incubation. 
Stimulants were 5% fetal bovine serum; IGF-1 (50 
ng/ml); and aPGF, bFGF, EGF, and PDGF (10 ng/ml 
each). RAPA, CsA, FK 506, or solvent alone was 
added at the initiation of culture in concentiations 
ranging from 10"* to 10"'* M. Toxicity of the drugs 
was assessed by a quantitative tetrazolium-based 
colorimetric assay. 

Uveitis was induced in rats with subcutaneous 
LPS. The animals were treated with L-NAME, an L- 
arginine analog acting as a specific inhibitor of NO 
synthesis. Ocular inflammation was evaluated by 
measuring the protein concentration and leukocyte 
number in the aqueous humor of one eye and by 
histopathology of the contralateral eye. 

Major Findings 

We found that we could effectively inhibit EAU by 
using a combination of RAPA with CsA in which 
the doses were reduced by factors of 10 and 5, 
respectively, compared with the doses necessary 
when the drugs were used alone. Similarly, in the 
RAPA with Dex combination, the doses were re- 
duced by fourfold and fivefold, respectively. 

We found that RAPA could significantiy inhibit 
the proliferation of human retinal pigment epithelial 
cells as well as human fibroblasts, thus RAPA could 
be indicated for patients in whom uveitis is compli- 
cated by fibrosis and retinal membrane formation. 

We demonstrated for the first time that NO is a 
crucial mediator of EIU and that its inhibition may, 
in the future, become an avenue of therapy for 
anterior uveitis. 



Methods 

EAU was induced by immunization with S-antigen 
in Hunter's adjuvant Treatment was given for 14 
days, starting on Day 7, and the experiment termi- 



Significance to Biomedical Research and the 
Program of the Institute 

Our study on combination therapy for EAU has 
shown that the observed in vitro synergy between 



61 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



RAPA and CsA and between RAPA and Dex was 
effective in vivo. Such combination therapy, if 
applied to humans, could significantly reduce the 
toxic side effect of the treatment while allowing good 
control of the disease. In addition, RAPA could 
benefit patients for whom uveitis is complicated by 
intraocular fibrosis and membrane formation, two 
complications that are often responsible for a large 
part of vision loss. 

The newly discovered role for NO in anterior 
uveitis could lead to improved therapy by finding 
inhibitors of the production of NO in the eye. 

Proposed Course 

In the fiiture, we intend to develop a clinical trial for 
the treatment of uveitis with RAPA in humans. A 
protocol has been developed and will be submitted to 
the producer of the drug. 

The study concerning NO and uveitis will be 
developed in the direction of identifying the cells 
responsible for the effect found in the eye. In 
addition, different types of NO inhibitors will be 
tested to find the one(s) most appropriate for uveitis 
therapy. 



NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

Li Q, Lopez JS, Caspi RR, Roberge FG, Nussenblatt 
RB, Kador PF, Chan C-C: Suppression of S- 
antigen induced experimental autoimmune uveo- 
retinitis in Lewis rats by oral administration with 
COS- 13080, a thromboxane synthetase inhibitor. 
Exp Eye Res 57:601-608, 1993. 

Roberge FG, Kozhich A, Chan C-C, Martin DF, 
Nussenblatt RB, de Smet, MD: Inhibition of 
cellular transfer of experimental autoimmune 
uveoretinitis by rapamycin. Ocular Immunol 
Inflam 1:269-73, 1993. 

Roberge FG, Xu D, Chan C-C, de Smet MD, Nuss- 
enblatt RB, Chen H: Treatment of autoimmune 
uveoretinitis in the rat with rapamycin, an inhibi- 
tor of lymphocyte growth factor signal transduc- 
tion. Curr Eye Res 12:197-203, 1993. 



62 



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



PROJECT NUMBER 



ZOl EY 00262-04 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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 Ph.D., M.P.H. Senior Research Scientist LI, NEl 



Others: Igal Gery 



Robert B. Nussenblatt 
Rachel Caspi 
Rashid Mahdi 



Ph.D. 

M.D. 
Ph.D. 



Head, Section on LI, NEI 
Experimental Immunology 

Scientific Director NEI 

Visiting Associate LI, NEI 

Biologist LI, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 

SECTION 

Section on Experimental Immunology 

INSTITUTE AND LOCATION 

NEI, NTH, Bethesda, MP 20892 

TOTAL STAFF YEARS: TpROFESSIONAL: 



0.7 



0.7 



OTHER: 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 
n (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 intraocular 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). We had 
shown previously that vpS-expressing T cells accumulate in the retina during EAU. Because the 
vpS-expressing T cells comprise cells with distinct functional activities, we sought to identify the uveitogenic 
subpopulation by analyzing the T-cell antigen receptor (TCR) genes and the cytokines they secrete in the retina 
during EAU. Our results indicate that the Vp8 response is highly dependent on the antigen used for disease 
induction: Whereas only Vp8.2* T cells were found in the retinas of animals with S-Ag EAU, both vp8.2* 
and Vp8.3* T cells were present in the retinas of rats with IRBP EAU. In terms of the pattern of cytokine 
production, both Thl- and Th2-like lymphokine profiles were observed, and the cytokine detected in the retina 
was found to be dependent on the antigen used for disease induction. Our current data suggest that the T-cell 
Vp8 response in the retina during EAU is heterogeneous. Thus, despite the fact that there is a bias toward • 
use of Vp8* cells in the retina during EAU, a single anti-Vpg antibody cocktail may not confer full protection 
from uveitis because several autoantigens and an unknown number of immunopathogenic T-cell clonotypes 
may be involved in the induction of EAU. 



63 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

This project is aimed at determining the clonality of 
the T lymphocytes that mediate ocular autoimmune 
diseases. Identification of the pathogenic T-cell 
subset in experimental autoimmune uveoretinitis 
(EAU) is relevant to our goal of developing anti-T- 
cell receptor (TCR) ther^ies for the treatment of 
uveitis. Our effort during Fiscal Year 1993 focused 
on analyses of T cells present at the autoinunune site 
and the lymphokines they produce. 

Methods 

In situ, reverse-transcribed polymerase chain reaction 
(RT/PCR) was used to examine T-cell populations in 
the retinas of athymic and euthymic Lewis rats at 
different stages of EAU. We used the cDNAs 
generated to amplify transcribed genes that code for 
rat VP TCR chains, T-cell subset markers (CD4 and 
CDS), and cytokines associated with autoimmune 
pathology — ^interferon-gamma (IFN-y), tumor necro- 
sis factor (TNF-a), transforming grovith factor (TGF- 
P), interleukin 2 (IL-2), and interleukin 6 (IL-6). 
Conventional recombinant DNA techniques such as 
Southern blot hybridization, PCR, cDNA construc- 
tion, densitometry, and DNA sequencing were used 
to analyze the resultant PCR products. 

Major Findings 

Analysis of TCR Vp5 repertoire induced by S-antigen 
(S-Ag) and interphotoreceptor retinoid-binding 
protein (IRBP). — We analyzed TCR genes expressed 
in response to immunopathogenic epitopes of human 
S-Ag or bovine IRBP by the PCR assay. We found 
that expression of VP8 genes in the retinas of S-Ag- 
immunized rats was restricted to vp8.2 TCR, while 
both Vp8.2 and Vp8.3 TCR isoforms were detected 
in IRBP-immunized rats. vps.T T cells were not 
detectable in the retinas of rats with either IRBP- or 
S-Ag-induced EAU. 

Analysis of Vp5 repertoire in adoptively trans- 
ferred EAU. — In contrast to EAU induced by active 
immunization, all three vps subfamily members 
were detected in the retina in EAU induced by 
adoptive transfer of IRBP- or S-Ag-specific uveito- 
genic T cells. Skewing of the vps repertoire during 
EAU induced by active immunization points out a 
potential confounding factor in vaccination studies in 



which complete Freund's adjuvant is a component of 
the vaccine preparations. The earliest Vp8* T cells 
detected in the retina were of the VP8.2 clonotype. 
This subpopulation was followed by vps.B- and 
Vp8.1 -expressing clonotypes. Similar to the re- 
sponse to active immunization, the initial appearance 
of VP8.3* cells was subsequently followed by a rapid 
decline in the numbers of VP8.3* lymphocytes. 

Analysis of cytokine production in the retina 
during EAU. — In adoptively transferred EAU, the 
cytokines in which production in the retina could be 
correlated with EAU pathogenesis are TNF-a, EFN-y, 
and IL-6. However, the temporal appearance of 
these cytokines in the retina was dependent on the 
antigen used for eliciting the uveitogenic T cells. 

Significance to Biomedical Research and the 
Program of the Institute 

Our current data show selective accumulation of 
Vp8* cells in the retina during EAU. However, the 
T-cell response is not oligoclonal, and distinct Vp8 
subfamilies were differentially activated by the 
autoantigens S-Ag and IRBP. Furthermore, the 
patterns of lymphokine production indicate that 
distinct T-cell subsets may initiate IRBP- and S-Ag- 
induced EAU. Taken together, these findings sug- 
gest that a single anti-Vp antibody cocktail may not 
confer full protection from uveitis because several 
autoantigens and an unknown number of immuno- 
pathogenic T-cell clonotypes may be involved in the 
pathogenesis of EAU. 

Proposed Course 

Fumre analyses of uveitogenic T-cell clonotypes and 
the lymphokines they produce during EAU will be 
performed in nude rats. Because these rats do not 
normally produce T cells, we expect the intraretinal 
T-cell repertoire in these rats to be limited. This 
would allow for a more concise analysis of the 
identity of the relevant autoaggressive T cells in- 
volved in uveitis. 

NEI Research Program 

Retinal and Choroidal Diseases — ^Inflammatory 
Disorders 

Publications 

Egwuagu CE, Bahmanyar S, Mahdi R, Nussenblatt 
RB, Gery I, Caspi R: Predominant usage of 



64 



NEI Annual Report— FY 1993 Laboratory of Immunology 

Vp8.3 T cell receptor in a T cell line that induces uveoretinitis induced by two (Afferent retinal anti- 
experimental autoimmune uveoretinitis. J Clin gens. J Immunol 151:1627-1636, 1993. 
Immunol Immmopathol 65:152-160, 1992. Mahdi RM, Caspi RR, Nussenblatt RB, Gery I, 
Egwuagu CE, Caspi R, Mahdi R, Gery I, Nussenblatt Egwuagu CE: Selective accumulation of vps* T 
RB: Evidence for selective accumulation of lymphocytes in EAU. Invest Ophthalmol Vis Sci 
Vp8+ T lymphocytes in experimental autoimmune 34(4)(suppl): 1 144, 1993. 



65 



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



PROJECT NUMBER 



ZOl EY 00280-02 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 
Eaopic Expression of Inlerferon-Gamma in the Eyes of Transgenic Mice and Rats Induces Ocular Pathology and MHC Qass 11 Gene Expression 



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



Others: Robert B. Nussenblan 

Chi-Chao Chan 

Ana B. Cbepelinsky 



Jorge Sztein 
Rashid Mahdi 



M.D. 
M.D. 

Ph.D. 



D.V.M., Ph.D. 



Scientific Director 
Head, Section on 
Immunopathology 
Head, Section on 
Regulation of Gene 
Expression 
Visiting Associate 
Biologist 



NEI 
LI, NEI 

LMDB, NEI 



LI, NEI 
LI, NEI 



COOPERATING UNITS (it any) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 

Section on Experimental Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.2 



PROFESSIONAL: 



1.2 



OTHER: 



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.) 

To investigate the consequences of ectopic expression of interferon gamma (IFN-y) in the lens and how this 
lymphokine growth factor may influence the fate of cells committed to a particular differentiation state, we 
used the murine (xA-crystallin promoter (oACry) to direct the expression of IFN-y to the lens of transgenic 
mice. Two transgenic mouse lines were established using two distinct strains, Balb/c and P/B/N. In both the 
aACry-IFN-y/Balb/c and cxACry-IFN-y/FYB/N transgenic mice, the essential histopathological features 
observed were very similar at all developmental stages studied (i.e., 12- to 18-day embryos, newborns, adults), 
suggesting that maldevelopment of ocular tissues of these mice resulted from oAGry-IFN-Y expression. 
Recently we have generated transgenic rats using the oACry-IFN-y cDNA construct, making our transgenic 
rats the first transgenic rats to be generated at the National Institutes of Health. 

Constitutive expression of IFN-y in the lens induced ocular pathology and aberrant major histocompatibility 
complex (MHC) class-II protein synthesis in several ocular tissues. These transgenic mice and rats provide 
powerful models for understanding the molecular pathways governing synchronized programmed differentiation 
of ocular tissues and for studying the linkage between aberrant MHC expression and predisposition to 
autoimmune diseases. 



66 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Objectives 

This project is designed to generate transgenic 
animals (rats and mice) that selectively secrete 
interferon-gamma (IFN-y) in their eye tissues for use 
in studying the bioregulatory actions of IFN-y in the 
eye. Because aberrant expression of major histocom- 
patibility complex (MHC) molecules is an early 
event in a number of human autoimmune diseases 
and IFN-y induces high levels of MHC class II 
protein biosynthesis, these transgenic mice and rats 
are ideally suited for studying (1) the effects of IFN- 
y on the physiology of the eye and (2) the role of 
elevated MHC class D in predisposition to auto- 
immune diseases such as diabetes and uveitis. 

Methods 

Transgenic animals were generated according to 
standard transgenic mouse techniques; however, for 
the transgenic rats, adjustments in the hormone 
treatment regimen were necessary to obtain embryos 
used for microinjection of the construct. Transgenic 
and wild-type (WT) phenotypes were characterized 
by histologic examination of representative tissue 
sections. We used conventional recombinant DNA 
techniques — such as Southern blot hybridization, 
polymerase chain reaction, cDNA construction, 
densitometry, and DNA sequencing — to characterize 
DNAs and RNAs derived from WT and transgenic 
mice. 

Major Findings 

The most notable effects of IFN-y in the transgenic 
mice include cataract, microphthalmia, blepharophim- 
osis, microphakia, impairment of lens fiber forma- 
tion, arrest of retinal differentiation, serous retinal 
detachment with the presence of macrophages in the 
subretinal space, persistent hyperplastic primary 
vitreous, and corneal vascularizatioa MHC class II 
mRNA levels were significantly increased in the 
transgenic eyes, and MHC class II proteins were 
expressed in the cornea, iris, ciliary body, choroid, 
lens, and retinal pigment epithelium. 



Significance to Biomedical Research and the 
Program of the Institute 

The generation of transgenic rats is a major technical 
accomplishment that now allows us to generate rats 
containing other DNA constructs. This is particularly 
important considering that several ocular diseases, 
such as uveitis and cataract, are better suited for 
study in the rat than in the mouse. Our data suggest 
that oACry-IFN-y transgenic mouse ocular cells 
express functional IFN-y receptors and that inter- 
action of IFN-y with its receptor induced biochemical 
and morphological changes in the transgenic eyes. 
These transgenic mice and rats provide a powerful 
model for understanding the molecular pathways that 
govern synchronized, programmed differentiation of 
ocular tissues and for studying the linkage between 
aberrant MHC expression and predisposition to 
autoimmune diseases. 

Proposed Course 

We intend to further dissect the molecular basis of 
IFN-y actions in the eye, placing particular emphasis 
on the rat model. A major focus will be the study of 
transcriptional factors that mediate oACry-IFN-y 
effects. 

NEI Research Program 

Retinal and Choroidal Diseases — ^Inflammatory 
Disorders 

Publications 

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 pathology and 
MHC class n gene expression. Invest Ophthal- 
mol Vis Sci, in press. 

Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi 
R, Nussenblatt RB, Chepelinsky AB: Ganmia 
interferon gene expression in the lens of trans- 
genic mice directed by the ctA-crystallin gene 
promoter. Invest Ophthalmol Vis Sci 34(4) 
(suppl):846, 1993. 



67 



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



PROJECT NUMBER 



ZOl EY 00069-16 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Immune Responses to Ocular Antigens 



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



PI: Igal Gery 

Others: Alexander Kozhich 
Xiaowen Wu 
Nancy Miller-Rivero 
Barbara Vistica 
Norman Chanaud UI 
Robert B. Nussenblatt 



Ph.D. 


Head, Section on 
Experimental Inmiunology 


LI, NEI 


Ph.D. 


Visiting Fellow 


LLNEI 


M.D. 


Visiting Fellow 


LLNEI 


M.D. 


IRTA Fellow 


LLNEI 


B.A. 


Microbiologist 


LLNEI 


B.A. 


Special Volunteer 


LI, NEI 


M.D. 


Scientific Director 


NEI 



COOPERATING UNITS (it any) 

Biotechnology Unit, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases 
(Joseph Shiloacfa, Ph.D.); Center for Neurologic Diseases, Brigbaro and Women's Hospital, Boston, MA (David Hafler, M.D.); Department 
of Medicine, Hadassah Hospital, Jerusalem, Israel (Shimon Slavin. M.D.) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Experimental Immunology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



4.2 



PROFESSIONAL: 



3.8 



OTHER: 



0.4 



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.) 

Targeted at learning about the pathogenesis of inflammatory eye diseases grouped under the term "uveitis," 
this project continued to focus mainly on learning about ocular antigens that induce experimental autoimmune 
uveoretinitis (EAU), an animal model for uveitis in man, and on procedures that modulate this disease. Three 
major achievements of this study are as follows: (1) We found tiiat the induction of EAU by bovine 
interphotoreceptor retinoid-binding protein (IRBP) in the Lewis rat involves a unique immunologic reaction 
in which lymphocytes sensitized against the immunodominant and uveitogenic determinant of this protein do 
not recognize the rat homolog of this determinant, but, rather, are stimulated by another peptide of the rat 
IRBP. This is the first description of a phenomenon in which a "siuTogate" peptide determinant of an organ- 
specific antigen is used to initiate an autoimmime pathogenic process. (2) To overcome the inaccessibility of 
large amounts of human S-antigen (S-Ag), we have used recombinant DNA technologies to produce this 
human antigen in Escherichia coli bacteria. The recombinant human S-Ag was found to aoss-react with 
native bovine S-Ag and to be similarly capable of inducing EAU in Lewis rats. In addition, the recombinant 
human S-Ag was used to identify the immunodominant peptide determinants of this antigen, i.e., the peptides 
recognized by lymphocytes sensitized against whole human S-Ag. Three regions of the molecule were found 
to harbor immunodominant determinants, with different peptides being selected as dominant in rats of different 
inbred strains. (3) A novel immunomodulator, linomide, wqs found to be effective in inhibiting EAU 
developing in rats and mice actively immunized with S-Ag or IRBP, respectively. This drug also inhibited 
the humoral and cellular immune responses in these animals. On the other hand, treatment with linomide had 
no effect on the development of EAU adoptively transferred by presensitized lymphocytes. The latter finding 
suggests that linomide cannot be useful for the treatment of uveitis in humans, in whom immunopathogenic 
processes are preactivated. 



68 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Objectives 

Studies conducted during Fiscal Year (FY) 1993 
were aimed at the following: (1) to fiirther analyze 
the immune responses to the immunodominant and 
highly uveitogenic peptide 1181-1191 of bovine 
interphotoreceptor retinoid-binding protein (IRBP); 
(2) to prepare recombinant human S-antigen (rHumS- 
Ag) and test its immunological properties — (a) its 
immunogenicity and uveitogenicity in rats, (b) its 
cross-reactivity with bovine S-antigen (S-Ag), and 
(c) identification of the peptide determinants in its 
sequence that are immunodominant in various rat 
strains; and (3) to investigate the effect of linomide, 
a novel immunomodulator, on the development of 
experimental autoimmune uveoretinitis (EAU) in rats 
and mice. 

Methods 

Peptides were synthesized on an Applied Biosystems 
synthesizer 430A. Bovine S-Ag was extracted from 
frozen retinas by Dr. Joseprti Shiloach (National 
Institute of Diabetes and Digestive and Kidney 
Diseases). rHumS-Ag was expressed in Escherichia 
coli according to the method described by H. F. 
Oettinger et al. (7 Neuroimmunol 44:157-162, 1993) 
using a cDNA coding for human S-Ag. A large 
batch of the recombinant antigen was prepared by 
Dr. Shiloach and was extracted by 8 M urea. Con- 
ventional procedures were used for immunization of 
animals and for testing their immune responses and 
development of EAU. The affinity of peptides 
toward major histocompatibility complex (MHC) 
molecules on antigen-presenting cells (APC) was 
assessed by the capacity of the tested peptide to 
inhibit the binding of biotinylated bovine IRBP 
peptide 1181-1191 to adherent cells from syngeneic 
spleens. Linomide was provided by Dr. S. Slavin 
(Hadassah Hospital, Jerusalem). The drug was 
administered to experimental animals via the drink- 
ing water at 1 mg/ml. 

Major Findings 

Studies concerning IRBP peptide 1181-1191. — ^As 
indicated in our report for FY 1992, the rat homolog 
of bovine IRBP peptide 1181-1191 is immuno- 
logically inactive in the Lewis rat and is not recog- 
nized by lymphocytes sensitized to the bovine 



sequence. Since the bovine peptide is immunodomi- 
nant and highly uveitogenic in Lewis rats (Sanui et 
al., J Exp Med 169:1947, 1989), lymphocytes sensi- 
tized against it must recognize a determinant of the 
rat ERBP molecule to initiate the pathogenic process 
of EAU. Our studies now show that this target 
determinant is likely to be the rat IRBP peptide 273- 
283: (1) sequence 273-283 of IRBP, which is ex- 
tremely conserved, is identical in bovine and rat; 

(2) rat peptide 273-283 is recognized by lymphocytes 
sensitized against bovine peptide 1181-1191 and 
stimulates them to proliferate; and (3) moreover, rat 
273-283 is superior to bovine 1181-1191 in its 
capacity to stimulate uveitogenicity in lymphocytes 
sensitized against the bovine peptide. 

The lack of immunogenicity of the rat IRBP 
peptide 1181-1191 in Lewis rats was found to be 
related to the poor afiinity of this peptide toward the 
MHC molecules on the APC of Lewis rats. (T 
lymphocytes recognize antigenic determinant only in 
the context of the MHC molecule.) The poor affinity 
of the rat peptide 1181-1191 was shown by its 
inability to competitively inhibit the binding of the 
bovine 1181-1191 to Lewis rat spleen cells. In line 
with the assumption that the lack of immunogenicity 
of rat peptide 1181-1191 in Lewis rats is due to its 
poor affinity to the Lewis MHC, we found that this 
rat peptide is immunogenic and uveitogenic in rats of 
the Buffalo inbred strain; the Buffalo MHC class n 
is RT1^ while the Lewis MHC molecule is RTl'. 
Moreover, bovine 1181-1191 was found to be 
nonuveitogenic and nonimmunogenic in the Buffalo 
rat. 

Studies with rHwnS-Ag. — ^Partially purified 
rHuraS-Ag, expressed in E. coli, was examined for 
its immunological activities in rats. Our findings 
were as follows: (1) rHumS-Ag resembled bovine 
S-Ag (extracted from retinas) in its capacity to 
induce EAU in Lewis rats; the minimal dose of both 
preparations was 2.5 pg per rat (when injected in 
complete Freund's adjuvant, along with Bordetella 
pertussis). (2) There were moderate levels of cross- 
reactivity between the two preparations when tested 
by either antibody or cellular immune responses. 

(3) Lymphocytes from rats immunized with rHumS- 
Ag were used to identify the immunodominant 
peptide determinants of human S-Ag, namely the 
determinants against which the immune response is 
targeted in animals immunized with the whole 
antigen. When tested against 40 overlapping syn- 



69 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



thetic peptides that extend the whole antigen se- 
quence, sensitized lymphocytes from Lewis rats 
immunized with rHumS-Ag responded against 
peptides derived from three sequence regions: 61-80, 
171-190, and 281-310. The highest lymphocyte 
response was observed against peptide 281-300, 
which is also uveitogenic in Lewis rats. On the 
other hand, we observed low proliferative responses 
with peptides 341-360 and 351-370, which are highly 
uveitogenic in the Lewis rat. 

Testing the responses of rHumS-Ag-sensitized 
lymphocytes from rats of inbred strains other than 
Lewis revealed that the same three regions of human 
S-Ag provide the inamunodominant peptides for the 
response of all tested strains, but we saw remarkable 
differences among the strains with regard to their 
response to individual peptides. Thus, the highest 
response of Buffalo rats was aimed at peptides 61-80 
and 71-90; ACI rats responded mainly to peptides 
71-90 and 181-200, while BN rats responded at the 
highest levels against peptides 71-90 and 291-310. 

The effects of linomide in the EAU system. — 
Linomide (quinoline-3-carboxamide) is a unique 
ixnmunomodulator that both enhances natural killer 
cell activity and suppresses autoimmune processes. 
Treatment with linomide inhibited the development 
of actively induced EAU: In mice, 1 1 of 15 controls 
had EAU versus none of the 15 in the treated group; 
in rats, 11 of 12 controls developed EAU versus 5 of 
12 in the treated group. The disease developed later 
and was less severe in treated rats than in control 
rats. Linomide treatment also significantly sup- 
pressed humoral and cellular immune responses in 
both mice and rats. In contrast, however, treatment 
with linomide had no effect on the development of 
adoptively transferred EAU or on the immune 
responses in the recipient animals. 

Significance to Biomedical Research and the 
Program of the Institute 

1. Our findings with IRBP peptide 1181-1191 
reveal for the first time a unique immunological 
system in which lymphocytes sensitized against an 
immunodominant peptide of a xenogeneic organ- 
specific protein do not recognize the autologous 
homolog but, instead, initiate the pathogenic auto- 
immune process by recognizing a "surrogate" peptide 
epitope of the autologous molecule. This unique 
phenomenon is made possible by the unusual four- 



fold structure of the IRBP molecule, allowing cross- 
reactivity between "repeats" on different regions of 
the molecule. In addition, our observations with this 
immunological system provide new evidence of the 
pivotal role of the affinity of a peptide toward the 
MHC in determining the immunogenicity of the 
peptide in animals using that MHC molecule. 

2. The limited supply of human retinas has 
restricted in the past usage of the human S-Ag in the 
various immunological studies concerning the in- 
volvement of this retinal antigen in human diseases. 
Consequently the large majority of studies have been 
carried out with the bovine protein. The successful 
expression of immunologically active rHumS-Ag 
thus provides us with an essentially limitless supply 
of this antigen. It is expected that rHumS-Ag will 
become a useful tool for analyzing autoimmunity in 
uveitic patients. Moreover, rHumS-Ag may be the 
antigen of choice in clinical studies in which oral 
tolerance with retinal antigens will be used as a 
modality to modulate the pathogenic process of 
autoimmune-mediated uveitis. 

3. Our study with linomide provides the first 
data concerning the effect of this immunomodulator 
on an autoimmune ocular disease. Moreover, oiu: 
data indicate that, unlike its effectiveness in inhibit- 
ing inunune responses of the afferent type, tinomide 
has no effect on the efferent limb of the immune re- 
sponse. This new observation underscores the 
uniqueness of the mode of action of this compound, 
but it casts doubt on the future usefulness of lino- 
mide in chnical conditions. 

Proposed Course 

Our future efforts will focus on the following issues: 
(1) the relationship between the affinity of IRBP 
peptides to MHC molecules and the immunogenicity 
and uveitogenicity of these peptides in rats of various 
inbred strains and (2) analysis of the system in which 
feeding with uveitogenic antigens protects animals 
against the development of EAU and immune re- 
sponse toward these antigens. In particular, we will 
examine the capacity of rHumS-Ag to induce oral 
tolerance. We will probe the possibility of using this 
antigen in the treatment of patients with uveitis. 

NEI Research Program 

Retina] and Choroidal Diseases — Inflammatory 
Disorders 



70 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Publications 

Casper- Velu LE, Verougstraete C, Gery I, Nussen- 
blatt RB: Ultrastructural changes of retinal 
vascular endothelial cells at the onset of experi- 
mental autoimmune uveitis, in Dernouchamps JP, 
Verougstraete C, Caspers-Velu L, Tassignon MJ 
(eds): Recent Advances in Uveitis. Amsterdam, 
Kugler Publications, 1993, pp 103-110. 

Egwuagu CE, Bahmanyar S, Mahdi RM, Nussenblatt 
RB, Gery I, Caspi RR: Predominant usage of 
VP8.3 T cell receptor in a T cell line that induces 
experimental autoimmune uveoretinitis (EAU). 
Clin Immunol Immunopathol 65:152-160, 1992. 

Egwuagu CE, Mahdi RM, Nussenblatt RB, Gery I, 
Caspi RR: Evidence for selective accumulation 
of Vp8+ T lymphocytes in experimental auto- 
immune uveoretinitis induced with two different 
retinal antigens. J Immunol 151:1627-1636, 
1993. 

Fujino Y, Li Q, Chung H, Hikita N, Nussenblatt RB, 
Gery I, Chan C-C: Immunopathology of experi- 
mental autoimmune uveoretinitis in primates. 
Autoimmunity 13:303-309, 1992. 



Gery I, Nussenblatt RB: Immunologic basis of 
uveitis, in Pepose JS, Holland GN, Wilhelmus 
KR (eds): Ocular Infection and Immunity. 
Philadelphia, Mosby-Year Book, Inc, in press. 

Kasner L, Chan C-C, Cordella-Miele E, Gery I: The 
effect of chlorpromazine on endotoxin-induced 
uveitis in the Lewis rat. Curr Eye Res 11:843- 
848, 1992. 

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. 

Oppenheim JJ, Gery I: From lymphodrek to inter- 
leukin 1 (IL-1). Immunol Today 14:232-234, 
1993. 

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

Suh EDW, Vistica BP, Chan CC, Raber JM, Gery I, 
Nussenblatt RB: Splenectomy abrogates the 
induction of oral tolerance in experimental auto- 
immune uveoretinitis. Curr Eye Res 12:833-839, 
1993. 



71 



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



PROJECT NUMBER 



ZOl EY 00288-01 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Gene Therapy for 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 
Robert B. Nussenblatt 



M.D., 


Ph.D. 


Visiting Associate 


LI, NEI 


Ph.D. 




Visiting Fellow 


LI, NEI 


Ph.D. 




Visiting Fellow 


LLNEI 


M.D. 




Scientific Director 


NEI 



COOPERATING UNITS (it any) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Genetics and Molecular Immunology 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



3.9 



PROFESSIONAL: 



3.9 



OTHER: 



0.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
□ (a1) Minors 
n (a2) Interviews 



□ (b) Human tissues [x] (c) Neither 



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

The goals of our main project are to get a better understanding of the physiological regulation of the ornithine 
5-aminotransferase (OAT) and its regulation in vivo. In humans, the mutation of OAT leads to the 
degeneration of the choroid and retina, causing a gyrate atrophy disease. There is no treatment for this human 
genetic disease; the only feasible approach would be to submit the patient to gene therapy via modified 
somatic cell lines. 

To accomplish this task, we are focusing on (1) the regulation of OAT gene in vivo and (2) the genetic 
modification of somatic cell lines mediated by recombinant retroviruses. With the idea of applying gene 
therapy, Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed. The 
human OAT cDNA was placed under the control of the enhancer-promoter regulatory elements derived from 
the Moloney murine leukemia virus long-terminal repeat. The construct was transfected into a safe packaging 
cell line, GP-hE-86, to produce provirus particles. Supernatant from these ecotropic OAT producer cell lines 
was used to transduce mouse C57B1/6 embryonal fibroblasts and embryonic stem cells. We showed that the 
recombinant retrovirus transfers the OAT gene to the recipient cells, which produce an immunoreactive OAT. 
Northern blot analysis confirmed the presence of an OAT transcript in the transduced cell lines, even after a 
long period of time in vitro. 



72 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Objectives 

Ornithine 5-aminotransferase (OAT, L-omithine:2- 
oxoacid aminotransferase, EC 2.6.1.13) is a nuclear- 
encoded mitochondrial matrix housekeeping gene 
that catalyzes the reversible transamination of orni- 
thine to glutamate semialdehyde. Biochemical 
analyses have shown that OAT is synthesized as 49- 
kD precursor monomer cleaved to a 45-kD protein 
on entry into the mitochondria, where assembly into 
the active homohexameric form of the enzyme 
occurs. This enzyme is expressed constitutively at 
low levels in the liver and kidney and at much 
higher levels in the retina, where OAT function 
seems to be critical for vision. 

Methods 

In humans, a genetic deficiency of OAT results in 
gyrate atrophy (GA) of the choroid and retina — a 
rare, blinding chorioretinal degeneration with associ- 
ated cataract, inherited as an autosomal recessive 
trait. Such OAT deficiency disrupts ornithine and 
arginine catabolism and results in a 10- to 15-fold 
accumulation of ornithine in all body fluids. In 
addition to their visual problems, some GA patients 
exhibit muscular dystrophy. Using a variety of 
techniques, including RNase A protection, single- 
strand conformational polymorphism analysis, and 
polymerase chain reaction, recent studies have shown 
that the unique OAT gene in GA patients contains 
firameshift, nonsense, and missense mutations that 
lead to the inactivation of the gene. 

A diet low in arginine has been shown to possibly 
delay the onset of this disease, but this approach is 
difficult to follow and may not be applicable to all 
patients. Therefore, since no therapy has been shown 
to be particularly effective, gene therapy in somatic 
cells (i.e., insertion of a functional OAT gene into 
patients' cells) could be a reasonable therapeutic 
alternative for patients suffering from GA. 

Vector construction. — ^The 1.4-kilobase (kb) 
EcoRI/HindHI fragment containing the entire human 
OAT cDNA was inserted into EcoRI/XhoI linearized 
retroviral vector LXSN to generate the recombinant 
vector pLXSN/OAT. All plasmids were grown in 
Escherichia coli strains HB 101 and DH 5 Alfa. 

Cell lines. — The murine retroviral packaging cell 
line GP+E-86 was grown in Dulbecco's modified 



Eagle's medium containing 10% (v/v) calf serum. 
We seeded ecotropic cells at 2 x 10^ cells per 10 cm 
dish and transfected with 10 ng of undigested 
pLXSN/OAT plasmid DNA, using the calcium- 
phosphate method. Twenty-four hours later cells 
were washed twice with phosphate-buffered saline 
(PBS) and grown in the presence of 1 mg/ml of 
geneticin (G418) for 1 week in 24- well plates. 
Supematants from different wells were harvested, 
filtered through 0.2 \xn\ filters, and tested for viral 
titer on 14-day-old C57BI/6 murine embryonal 
fibroblasts. After 1 day, the culture medium was 
replaced by G418 culture selection medium. Resis- 
tant clones were stained with methylene blue and 
counted. 

OAT immunodetection. — Subconfluent 10-cm 
dishes of transduced and nontransduced murine 
embryonal fibroblast cells were grown for 16 hours 
in the culture conditions described above, then 
harvested and lysed in 500 mM sodium chloride 
(NaCl), 50 mM Tris (pH 7.5), 1% NP40. Protein 
extracts from the same number of transduced and 
nontransduced cells were subjected to preparative 
sodium dodecyl sulfate/polyacrylamide gel electro- 
phoresis (SDS/PAGE) and transferred to nylon filters 
(Schleicher and Schuell). After saturation in PBS 
solution containing 5% nonfat dry milk, the strips 
were incubated with anti-OAT or with preimmune 
serum at the same dilution — 1/100 — ^for 1 hour at 
room temperature, then washed three times at 20°C 
for 20 minutes in 1% NP-40, 150 mM NaCl, and 50 
mM Tris/HCl (pH 7.5). The strips were incubated 
with goal anti-IgG antibodies conjugated with peroxi- 
dase. Anti-antibodies were visualized by visiblof™ 
AP. 

Major Findings 

We have achieved the construction of Moloney 
murine leukemia virus (Mo-MuLV)-based recombi- 
nant retrovirus vectors expressing a human OAT 
cDNA under the control of a Mo-MuLV long-termi- 
nal repeat. Neomycin phosphotransferase was 
included as a selectable marker in these recombinant 
retroviruses. Murine embryonal primary fibroblasts, 
as well as embryonal stem cells, have been trans- 
duced with helper virus-free recombinant refrovirus 
particles generated from a GP-^E-86 packaging cell 
line previously ttansfected with the described con- 
struct. We have successfully transduced several cell 
lines, as shown by Northern hybridization analysis 



73 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



and immunodetection via rabbit polyclonal antibodies 
raised against human OAT. 

Significance to Biomedical Research and the 
Program of the Institute 

The improvement in visual function on reduction of 
ornithine accumulation suggests that the physio- 
pathology of GA may involve (1) a direct toxic 
effect of ornithine; (2) a deleterious effect of meta- 
bolic alterations, occurring as a result of hyperomi- 
thinemia; or (3) both. However, despite many 
efforts, no therapy has been totally successful in 
treating this genetic disease. 

It is possible to correct a genetic disease by 
directing the treatment to the site of the defect itself 
(i.e., "the mutated gene"), rather than against second- 
ary or pleiotropic effects due to the mutant gene. A 
retrovirus-based delivery vehicle is likely to be the 
best choice for introducing a functional gene into 
somatic cells, thus achieving gene therapy of heredi- 
tary genetic diseases. Since the first successful 
retrovirus-mediated transfer of the adenosine deamin- 
ase gene into lymphocytes of patients suffering from 
a lethal immune deficiency, gene ther^y has been 
viewed as a reasonable, feasible approach to treating 
human genetic diseases. Since then, work has 
focused on several other genes, such as those encod- 
ing low-density lipoprotein, factor IX, and the cystic 
fibrosis transmembrane conductance-regulator gene. 
Further research is under way, with the aim of 
clinical trial. 

As shown by several groups, retroviral vectors 
can stably introduce genes into a variety of cultured 
cells. Defective retroviruses have been proposed as 
carriers to transfer functional genes to patients 
suffering from human genetic diseases. 

To attempt gene transfer via somatic cell lines to 
patients suffering from genetic deficiency of OAT, 
we designed and made a Mo-MuLV-based retroviral 
vector carrying a functional human OAT gene. We 
analyzed its stable integration and expression in 
murine embryonal fibroblasts and embryonic stem 
cells. To test the production of OAT transcript from 
the recombinant retroviral virus present in the frans- 
duced cell lines, we performed Northern blot analy- 
sis, using a total RNA preparation from cell lines 



both transduced and nontransduced by OAT refro- 
virus. The results obtained show the production of 
significant amounts of mRNA transcript exclusively 
in the transduced cell lines that hybridize with an 
OAT cDNA probe. No reaction was detected in 
wild-type fibroblasts. 

The absence of cross-reaction with murine OAT 
may be due to the high stringency during Northern 
blot washes or to the fact that mouse embryonal 
fibroblasts do not produce OAT enzyme. Moreover, 
when Western blot analysis of proteins extracted 
from the various cell lines used (including transduced 
mouse embryonal fibroblasts) was performed with 
rabbit polyclonal antibodies raised against two 
peptides of human OAT, we observed a specific 
reaction in only those extracts prepared from die cell 
lines transduced by the OAT provirus. The same 
polyclonal antibodies tested in Western blot against 
protein exfracts from human retinal pigmented 
epithelium — fibroblasts and HeLa cell lines — showed 
a similar reaction on just one polypeptide of the 
same apparent molecular weight as that detected in 
the transduced fibroblasts. Taken together, these 
results reveal in transduced cells the expression of a 
single OAT transcript and a single OAT polypeptide. 

We show here the ability to produce a retrovirus 
vector carrying and expressing a functional human 
cDNA coding for OAT, opening up the possibility of 
considering replacement gene ther^y for OAT- 
deficient patients who suffer from GA disease. 

Proposed Course 

Our future efforts will focus on the following issues: 

(1) the enzymatic activity of OAT produced by 
genetically modified somatic cell lines, which we 
will further investigate via recombinant refrovirus; 

(2) assessment of the target cell types for gene 
product delivery; (3) investigation of the effect of 
overexpression of OAT in transgenic mice that 
express human OAT; and (4) characterization of the 
murine OAT genomic gene, prior to engineering the 
appropriate homologous recombination vector. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and Other 
Inherited Disorders 



74 



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



PROJECT NUMBER 

ZOl EY 00232-08 LI 



PERIOD COVERED 

October 1, 1992 to September 30. 1993 



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

Interferon System in Cellular Function and Disease 



PRINCI PAL 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 M.S. Biologist LI, NEI 

Chandrasekharam Nagineni Ph.D. Visiting Scientist 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 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: PROFESSIONAL: OTHER: 

0.5 0.2 0.3 



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.) 

Cytokines, such as interferon-gamma (IFN-y) and interleukin 2 (IL-2), are a group of specialized hormone-like 
proteins which 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 miCToenvironment. 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 epithelium (RPE) cells. Furthermore, IFN-y- 
activated RPE cells can process and present antigeas to helper T lymphocytes. 

Experimentally we demonstrated that isolated human RPE cells can be induced to produce another 
lymphokine, IL-6, following incubation with IFN-y. IL-6 is a potent inflammatory cytokine capable of 
enhancing antibody production and cytotoxic T-cell activities. 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. 

Retinoblastoma cells are an important model for exploring human malignancy and differentiation. Using these 
cells we showed that EFN-y can regulate MHC cla.ss 1 genes at both transcriptional and posttranscriptional 
levels. In addition, this modulation is not associated with downregulation of N-myc oncogene expression. 

These observations indicate that IFN-y-induced MHC class 1 and class II antigen expression 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. 



75 



PHS 6040 {Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

This project is designed to determine the bioregula- 
tory actions of interferon (IFN) and other cytokines 
and to evaluate their regulatory actions in the patho- 
genesis of disease. 

Methods 

We assayed human IFN, using inhibition of vesicular 
stomatitis virus plaque formation in human amnion 
(WISH) cells. IFNs were characterized by neutral- 
ization of antiviral activity with monoclonal anti-IFN 
immunoglobulin. Interleukin 2 (IL-2) biological 
activity was assayed by induction of proliferation of 
CTL cells. Interleukin 6 (IL-6) activity was assayed 
by an enzyme-linked immunosorbent assay, immuno- 
blot assays, and Northern blots. Analytical flow 
cytometry was used to quantitate retinal proteins. 
Gene transcription techniques, such as Northern blot 
analysis and nuclear runoff transcription assays, are 
being used to evaluate IFN-y modulation of retinal 
proteins. 

Major Findings 

IFN activation of retinal pigment epithelial (RPE) 
cells. — Numerous studies indicate that a variety of 
autoinunune diseases are associated with the DFN-y- 
induced tissue-specific expression of major histocom- 
patibility complex (MHC) class 11 molecules. During 
the past 5 years, we have identified various steps that 
may be involved in ocular immunopathologic mecha- 
nisms. In these studies of retinal degenerations and 
autoimmune diseases, we showed that a critical 
regulatory cell in the retina, the RPE cell, is capable 
of expressing MHC class II determinants. We also 
can detect IFN-y in situ and in retinas from patients 
with inflammatory eye diseases, as well as in MHC 
class Il-positive RPE cells. In addition, freshly 
isolated hiunan RPE cells can express these determi- 
nants following treatment with IFN-y. 

In animal model systems, we found that inocula- 
tion of recombinant IFN-y induces la expression of 
ocular cells, and treatment with anti-la antibodies can 
eliminate or inhibit experimental autoimmune uveitis. 
Most 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. 



During the past year we have provided the most 
recent piece of experimental evidence implicating a 
role for cytokine-activated RPE cells in autoimmune 
phenomena by showing that the RPE cell is capable 
of producing the cytokine IL-6. RPE cell cultures 
were established from human donor eyes. These 
isolated RPE cells do not produce IL-6 alone; IFN-y 
induces these cells to produce IL-6 in a dose-depen- 
dent manner. Moreover, EFN-y can synergize with 
tumor necrosis factor (TNF) to produce IL-6 in 
human RPE cells. IL-6 is a potent cytokine which 
can act on B lymphocytes to induce growth and 
antibody production. It can also act on T lympho- 
cytes to induce IL-2 production, IL-2 receptor 
expression, and cell proliferation. These studies 
further substantiate the concept that cytokine-medi- 
ated activation of RPE cells may be a basic compo- 
nent of ocular immunity and may have major inmiu- 
nological consequences for RPE cell transplantation 
studies. 

Cytokine-induced modulation of cellular proteins 
in the retina and retinoblastoma. — Retinoblastoma 
cells are an important model for exploring human 
malignancy and differentiation. These multipotent 
embryonic cells are capable of differentiating into 
neuronal, glial-like, and RPE-like elements. We 
have shown that flow cytometric analysis can be 
used to measure the expression of both cytoplasmic 
and cell surface proteins in retinoblastoma cells. We 
used this technique to monitor changes in the expres- 
sion of selected cellular proteins after exposure to 
specific cytokines and found that MHC class I 
molecules were augmented by IFN-a and IFN-y but 
not by TNF. However, the MHC class 11 molecules 
were augmented by IFN-y but not by BFN-a or TNF. 
The neuronal markers IRBP and PR-6, the glial-like 
marker GFAP, and the RPE cell markers RPE-9 and 
RPE- 15 were not altered by any of the cytokines 
tested. 

The mechanism of induction of MHC class I and 
II antigens by IFN in retinoblastomas is not known. 
We therefore have initiated studies to compare 
IFN-a, BFN-P, and IFN-y in their ability to induce 
the expression of class I antigens and to investigate 
the role of transcriptional and posttranscriptional 
mechanisms in this induction. We found that IFN-y 
increased HLA class I antigen expression and in- 
duced a fivefold increase in its transcription rate. 
Posttranscriptionally IFN-P and IFN-y increased 



76 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Steady-State mRNA for the HLA-B7 gene. These 
effects were not associated with down-regulation of 
N-myc oncogene nuclear transcription. Moreover, 
dexamethasone did not affect the IFN-y-induced 
expression of HLA class I molecules. These studies 
implicated both transcriptional and posttranscriptional 
mechanisms in the modulation of class I molecule 
expression by IFNs. 

Significance to Biomedical Research and the 
Program of the Institute 

These studies highlight the fact that the release of 
cytokines, such as IFN-7, within the ocular microen- 
vironment and the subsequent induction of cytokines 
and MHC class I and II antigen expression on 
resident and infiltrating cells may be critical elements 
in a cascade leading to ocular cell destruction. The 
retinal cell that may play a critical role in auto- 
immune uveitis is the RPE cell. IFN-y-induced 
activation of RPE cells may participate in auto- 
immune disease in the ocular microenvironment. 

Cytokines produced and localized in the eye may 
play critical roles in normal physiology, pathogenic 
mechanisms, and therapeutic approaches. Since 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 in transplantation. 



Proposed Course 

We plan to continue our evaluation of the role of 
cytokines in autoinununity and inflammation. We 
are now developing systems in rat models to monitor 
directly the effects of altering cytokine production on 
inflammatory eye diseases. Moreover, we will 
continue to characterize the antigen-presenting ability 
of the RPE cell in a variety of antigens and viruses. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

Barez S, Boumpas D, Percopo C, Anastassiou ED, 
Hooks JJ, Detrick B: Modulation of major 
histocompatibility complex (MHC) class I genes 
in human retinoblastoma cells by interferons: 
Evidence for both transcriptional and post-tran- 
scriptional regulation. Invest Ophthalmol Vis Set 
34:2613-2621, 1993. 

Detrick B, Hooks JJ: Cytokines and effector mole- 
cules in human immunology, in Leffell MS, Bias 
WB, Donnenberg AD, Rose MR (eds): CRC 
Handbook of Human Immunology. Boca Raton, 
CRC Press, in press. 



77 



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



PROJECT NUMBER 



ZOl EY 00233-08 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (BO characters or less. Title must lit on or\e Ime between the borders.) 

Studies on the Bioregulatory Aspects of the Retinal 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 
Caroline Percopo 
T. Michael Redmond 
David Parks 
Robert B. Nussenblatt 



Ph.D. 


Visiting Scientist 


LLNEI 




M.S. 


Biologist 


LI, NEI 




Ph.D. 


Research Biologist 


LRCMB, 


NEI 


M.D. 




LI, NEI 




M.D. 


Scientific Director 


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 Campocbiaro, 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, Bethesda, MP 20892 



TOTAL STAFF YEARS; 



0.9 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



0.5 



OTHER: 



0.4 



[x] (b) Human tissues □ (c) Neither 



SUMMARY OF VlfORK (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. We are using these techniques and reagents to 
evaluate molecular, biochemical, and biological properties of the RPE cells. 

Since the monoclonal antibodies detect epitopes present solely on RPE cells, they provide a unique opportunity 
to evaluate a variety of aspects of RPE cell development and function. Studies on RPE cell development 
indicate that the epitopes appear only after the cells have begun terminal differentiation. Moreover, studies 
on RPE migration demonstrate the value of these antibodies in evaluating epiretinal membrane formation. The 
RPE epitope is a 67-kD protein that is closely associated with the microsomal membrane. A cDNA clone that 
has been isolated codes for a protein which does not match any other sequences in the data bases. Studies 
are in progress to propagate and transplant RPE cells in various animals. We have propagated human RPE 
cells in vitro and evaluated their ability to respond to cytokine activation. 

RPE cells respond to retinal aberrations by dying, proliferating, migrating, losing phagocytic function, 
expressing major histocompatibility complex (MHC) class II antigens, and presenting antigens to T 
lymphocytes. The techniques and reagents obtained in these studies allow us to evaluate the mechanisms 
involved in aberrant responses of RPE cells. Moreover, they provide a framework for evaluating RPE cell 
transplantation. 



78 



PHS 6040 {Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Objectives 

The aim of this project is to evaluate the molecular, 
biochemical, and various biologic properties of 
retinal pigment epithelial (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 are generated in mice by 
fusing mouse spleen cells with myeloma cells. 
Antibodies to the RPE cells are evaluated by immu- 
noperoxidase and Western blot assays. The effects 
of drugs and cytokines are evaluated by cell viabiUty 
and proliferation assays and by nuclear transcription 
runoff assays. 

Major Findings 

Evaluation of epitopes identified by monoclonal 
antibodies. — ^We have identified two mouse IgG3 
monoclonal antibodies that react with RPE cells from 
a variety of species, ranging from man to frog. 
Since these antibodies detect epitopes present solely 
on RPE cells, they provide us with the unique 
opportunity to evaluate a variety of aspects of RPE 
cell development and function. 

Electron microscopic immunocytochemistry 
revealed labeling patterns for the two RPE antibodies 
that are very similar. In human eyes, staining was 
localized to surface and intracellular membranes and 
the cytoplasm. Staining occurred predominantly on 
the j^ical surface of the RPE cells. The RPE pro- 
tein, a 65-kD protein, was isolated by polyacrylamide 
gel electrophoresis and fransferred to nitrocellulose 
blot, and the sequence of its amino acid residues was 
determined. The amino acid sequence was used to 
design a synthetic cDNA probe. A bovine cDNA 
library was screened, and cDNA clones were isolated 
and characterized. The cDNA insert is 3,1 15 base 
pairs; the open reading frame encodes a protein of 
533 amino acids. The RPE protein does not match 
any other sequence in the data bases. The protein 
expressed in Escherichia coli has a molecular weight 
similar to that of the native protein. In addition, we 
used Northern blotting with the cDNA to detect 
protein mRNA in RPE cells. 



In studies of the developmental expression of 
RPE and photoreceptor determinants in the rat retina, 
we had previously shown that the expression of these 
determinants in rats is absent the day of birth and 
detectable at postnatal Day 6. Recent studies show 
that RPE cells express their determinants shortly 
before the first outer segments are detected and that 
the posterior-anterior progression of outer segment 
formation matches a similar progression of the 
expression of the RPE determinants. These data 
indicate that the RPE resumes its maturation during 
the first postnatal week and that RPE maturation and 
outer segment growth can be correlated. 

RPE cell transplantation. — ^Recent studies indicate 
that RPE cell transplantation may be beneficial in 
restoration of the retinal architecture in selected 
retinal degenerations. It is essential to develop 
methods for large-scale preparations of RPE cell 
cultures for somatic cell genetic engineering manipu- 
lation. We are in the process of evaluating various 
parameters for human and rat RPE cell culture and 
transplantation. Preliminary smdies show that we 
can successfiiUy inoculate human RPE cells into the 
rat refina Evaluation of the immunologic parameters 
of this transplantation process is under way. 

Significance to Biomedical Research and the 
Program of the Institute 

The monoclonal antibodies developed in this smdy 
are the first directed solely at the RPE cell. These 
antibodies are potentially useful in identifying RPE 
cells in situ and in vitro. These antibodies, which 
can be used to monitor RPE cellular fiinctions, may 
provide a better understanding of the role of RPE 
cells in retinal degenerative disorders. Identification 
of the cDNA and proteins detected by the mono- 
clonal antibodies may provide the framework on 
which to evaluate specific RPE cell fiinctions. RPE 
cell transplantation to correct retinal degenerative 
processes is being actively investigated in a number 
of laboratories. The studies reported here provide 
the basis for evaluation of RPE cell transplantation. 

Proposed Course 

1. We will continue to characterize these anti- 
bodies and their effects on cell function in vivo and 
in vitro. 

2. We will isolate, propagate, and characterize 
RPE cells for transplantation studies in animals and 



79 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



humans. We will design effective ways to maintain 
the cell in culture and to measure and monitor cell 
function. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

Hamel CP, Tsilou E, Harris E, Pfeffer BA, Hooks JJ, 
Detrick B, Redmond TM: A developmentally 
regulated microsomal protein specific for the 
pigment epithelium of the vertebrate retina. J 
Neurosci Res 34:414-425, 1993. 



Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick 
B, Redmond TM: Molecular cloning and expres- 
sion of RPE65, a novel retinal pigment epithe- 
lium-specific microsomal protein that is post- 
transcriptionally regulated in vitro. J Biol Chem 
268:15751-15757, 1993. 

Vinores SA, Orman W, Hooks JJ, Detrick B, Camp- 
ochiaro PA: Ultrastructural localization of RPE- 
associated epitopes recognized by monoclonal 
antibodies in human RPE and their induction in 
human fibroblasts by vitreous. Graefe's Arch 
Clin Exp Ophthalmol 231:395-400, 1993. 



80 



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



PROJECT NUMBER 



ZOl EY 00240-07 LI 



PERIOD COVERED 

October 1, 1992 to September 30. 1993 



TITLE OF PROJECT (BO characters or less. Title must lit on arte 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 Burnier 
Ingeborg Kirch 
Yusuke Komuraski 



M.S. 
M.D. 
M.D. 
M.D. 
M.D. 



Biologist 
Guest Worker 
Visiting Scientist 
Guest Worker 
Guest Worker 



LI, NfEI 
LI, NEI 
LI, NEI 
LLNEI 
LI, NEI 



COOPERATING UNITS (if any) 

DepartmeDi of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.); Department of Pathology, 
Uniformed Services University for 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. MP (William Bums, M.D.) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL: 



OTHER: 



1.0 



0.8 



0.2 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[xl (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) antiviral therapeutic actions of cytokines and drugs, and (4) molecular diagnosis and 
pathogenesis of cytomegalovirus (CMV) infections in man. We have estabhshed that murine coronavirus 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 the virus and progressive retinal degeneration, even months after the 
virus is gone. The virus replicates predominantly in MuUer cells and also can be detected in retinal pigment epithelium 
(RPE) and photoreceptor cells. 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, i.e.. some 
strains of mice are resistant to virus-induced retinal degenerative changes. The pathologic changes also are closely related 
to the development of antiretinal 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 hiunans. 

The need for effective drug treatment and prevention of herpes virus and other viral diseases has assumed growing 
importance. We found that leukoregulin, a naturally occurring immunologic cytokine, not only increases the antiviral 
actions of the drug acyclovir but also directiy inhibits herpes simplex virus replication, demonstrating that combination 
immunotherqjy and chemotherapy can produce substantial inhibition of herpes virus replication and providing a rationale 
for applying this approach to treating virus infections. 

Studies initiated this past year indicate that CMV is capable of replicating within human RPE cells in vitro; however, 
replication is limited at the level of immediate early protein production. The low frequency of expression of immediate 
early viral proteins in RPE cells and the subsequent slow replication of CMV may be critical variables in terms of their 
relationship to viral persistence and activation within the retina. 

81 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

This project was designed to determine the various 
effects of virus infections on the ocular microenvi- 
ronment and to study modes of antiviral therapy. 

Methods 

This study involves the propagation and quantitation 
of viruses, such as herpes simplex virus type 1 
(HSV-1), coronaviruses, and cytomegalovirus 
(CMV), both in vitro and in vivo. It also includes 
immunocytochemical analysis of infected cells and 
tissues. Techniques used in characterization of virus 
infection include flow cytometric analysis, Western 
blot analysis, Northern blot analysis of HSV thymi- 
dine kinase, in situ hybridization, and amplification 
of viral genes by polymerase chain reaction (PCR). 
Techniques used in characterization of antivirus 
antibodies include enzyme-linked immunosorbent 
assay and neutralization assays. 

Major Findings 

Coronavirus infection in the eye. — ^The murine 
coronavinis, mouse hepatitis virus (MHV), JHM 
strain, induces a retinal degenerative disease in adult 
Balb/c mice. Tlie disease consists of an acute phase 
lasting from 2 to 8 days, during which virus is 
detected within the retina and initial pathology is 
noted in the retinal pigment epithelium (RPE) and 
photoreceptor layers. This is followed by a late 
phase lasting from 1 to 14 weeks, during which virus 
is not detected but retinal degenerative changes 
continue, with reduction of the photoreceptor layer, 
loss of interphotoreceptor retinoid-binding protein, 
and retinal detachments. This model provides 
evidence that viruses can trigger retinal degenerative 
processes and may offer insight into pathogenic 
mechanisms in retinal degenerative diseases of 
humans. During the past year we have evaluated 
three aspects of this disease process: (1) immuno- 
logic aspects of the disease, (2) genetic predisposi- 
tion to the disease, and (3) use of electroretinography 
(ERG) to monitor the disease process. 

In the coronavirus-induced retinopathy, the late 
phase of the JHM-induced disease was associated 
with the lack of direct evidence for viral replication 
within the retina. This observation suggested that the 
continued degenerative process may be associated 



with alterations directly induced by virus replication 
during the first few days after infection, or it may be 
associated with additional factors. Because viruses 
are known to trigger an autoimmune phenomenon 
and some human retinopathies may be associated 
with autoantibody formation, we studied the possible 
production of antiretinal autoantibodies. We found 
that the retinal degenerative process is associated 
with the presence of antiretinal autoantibodies. In 
total, 22 of 23 sera samples collected from 10 to 70 
days after JHM virus inoculation of Balb/c mice 
contained antiretinal autoantibodies. These autoanti- 
bodies were not found in sera from normal or mock- 
injected mice. 

Antibodies to retinal tissue were identified by two 
distinct patterns of immunoperoxidase staining on 
frozen sections of normal rat eyes — ^retinal autoanti- 
bodies and RPE autoantibodies. The antiretinal 
autoantibodies first appeared as IgM class antibodies 
which shifted to IgG class autoantibodies. The 
anti-RPE cell autoantibodies were predominantly of 
the IgG class. Sera positive for these autoantibodies 
did not stain with liver or kidney sections, but two of 
three did react with rat brain sections. 

We also evaluated a second mouse strain, CD-I, 
because these animals respond to JHM virus inocula- 
tion by developing only the early phase of this 
disease, i.e., vasculitis. On Day 10 postinoculation 
(pi) the retinal architecture had a normal ^pearance. 
In these mice, which are free of a retinal degenera- 
tion, antiretinal autoantibodies are not produced. 
However, as noted in the Balb/c mice, antivirus 
neutralizing antibodies were produced in the infected 
CD-I mice. These findings suggest a role for 
autoimmunity in the pathogenesis of murine corona- 
virus-induced retinal degeneration. 

Since the genetic composition of the host and the 
virus can determine the response to infection and the 
resulting pathology, we evaluated the effect of MHV 
infections on different strains of mice. The JHM 
and A59 strains of MHV were propagated in rat L2 
cells. Balb/c, CD-I, and A/J mice were inoculated 
by the intravitreal route with 10"^ TCID50/O.5 pi of 
virus or with uninfected tissue culture preparations 
(mock injection). At various times after infection, 
the eyes were removed and evaluated histologically, 
and sera were assayed for the presence of virus- 
neutralizing antibody. Both JHM and A59 strains of 
MHV induced similar retinal diseases. 



82 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



We observed two distinct phases of coronavirus- 
induced retinopathy, retinal vasculitis (Days 3-6) and 
retinal degeneration (Days 10-20), in Balb/c mice. In 
contrast, we saw only the early stage of disease in 
CD-I mice. We observed typical retinal vasculitis at 
3-6 days pi. However, by Day 10 pi the retinal 
architecture had returned to a normal appearance. 
No retinal degeneration was observed. The third 
strain, A/J mice, displayed a biphasic disease but 
with a mild degenerative component. All strains of 
mice responded to the retinopathy by developing 
antivirus-neutralizing antibody at similar levels. 
These studies demonstrate that the pathologic mani- 
festations of a virus infection in the retina can be 
influenced by the genetics of the host. 

The third phase of these studies incorporated ERG 
evaluation of the development of the virus-induced 
retinopathy. At various times after inoculation, 
animals were dark-adapted for 60 minutes and 
anesthetized, after which ERGs were recorded 
between a wick electrode touching the cornea and a 
needle electrode placed subcutaneously at the fore- 
head. Light stimuli were provided by a xenon arc 
light source and focused onto the eye via a fiberoptic 
bundle. We varied the light intensity via neutral - 
density filters. Five responses were averaged for 
each light intensity. 

On Day 3 pi all virus-infected mice had slightly 
depressed ERGs and retinal vasculitis was seen. In 
contrast, on Days 8 and 20 pi all these mice had 
subnormal or abolished responses, and retinal degen- 
erative changes were clearly apparent. On Day 10 pi 
57% of the mice (4/7) had abolished responses and 
43% (3/7) had subnormal responses. On Day 22 pi 
50% (4/8) had abolished responses, while the remain- 
ing 50% had subnormal ERGs. Mice inoculated with 
virus-free tissue culture preparations had minor 
alterations in their ERG patterns. However, at Day 
20 pi these mock-injected mice displayed ERG 
patterns similar to those of normal, uninfected mice. 
ERG studies in this murine model provide an indirect 
but objective means of measuring visual function, 
serving as the basis for future studies of treatment 
effects. 

In summary, this model is characterized by the 
replication of JHM virus in the retina, producing an 
acute necrotizing disease of the sensory retina that 
results in only a mild inflammatory response and a 
long-lasting disease (>14 weeks). In these studies 
we identified a progressive degenerative disease in 



the retina that may be initiated by an acute virus 
infection in the absence of major inflammatory 
response. During the past year these studies have 
clearly indicated that this retinal degenerative process 
has viral, immune, and genetic components. How the 
genetic and immunologic factors interact to influence 
the development of retinal degenerations is the 
intriguing aspect of this model. 

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 in order 
to use seroepidemiologic approaches for the detection 
of virus and viral antigens via immunocytochemical 
staining, in situ hybridization, and PCR assays. 

Antiviral therapeutic actions of cytokines and 
drugs. — The need for effective treatment and preven- 
tion of herpesvirus and other viral diseases has 
assumed growing importance during the past 10 
years. The development of targeted antiviral agents 
through combination ther^y is becoming an impor- 
tant strategy. One strategy consists of the develop- 
ment of cytokines or lymphokines in combination 
with chemother^y to treat malignancy and infec- 
tions. Using this approach, we recently showed that 
the cytokine leukoregulin could enhance the anti- 
HSV actions of acyclovir (ACV). 

Cytokines are a group of specialized hormone-like 
proteins that can exert profound influences on 
cellular development and a variety of cellular func- 
tions. As a lymphokine that performs unique regula- 
tory activities in transformed cells, the leukoregulin 
molecule, which is produced by a variety of lym- 
phoid cells, is a multifunctional cytokine. It can 
prevent chemical carcinogen transformation, inhibit 
neoplastic cell proliferation, and augment target cell 
sensitivity to natural killer cell cytotoxicity. Further- 
more, this cytokine has been shown to increase 
membrane permeability of tumor cells and to in- 
crease drug uptake in these cells. We recently 
showed that leukoregulin can selectively increase 
membrane permeability in HSV-1 -infected cells but 
not in normal (i.e., uninfected) cells. 

In addition, we have recently shown that leuko- 
regulin enhances the anti-HSV actions of ACV. The 
cells were exposed to the cytokine and/or ACV for 
only 3 hours early in the replication cycle. Because 
the continued presence of ACV greatly enhances 
antiviral activity, we evaluated the effect of the 



83 



Laboratory of Immunology 



NEI Annua] Report— FY 1993 



continuous presence of leukoregulin on HSV replica- 
tion. Human amnion epithelial (WISH) cells were 
infected with HSV-1 (Wendy and F strains) and 
vesicular stomatitis virus (VSV). Following a 90- 
minute incubation period, we washed the cells and 
treated them with media, leukoregulin, ACV, or 
leukoregulin plus ACV. We evaluated virus replica- 
tion by plaque assays while testing virus and cellular 
protein expression by immunoblotting. The continu- 
ous presence of leukoregulin (0.1 unit) inhibited 
HSV-1 plaque formation by 50-80% in the Wendy 
and F strains, respectively. In contrast, leukoregulin 
did not affect VSV replication. Immunoblot analysis 
revealed that the expression of the 89-kD HSV-1 
protein was inhibited by 50%, whereas the cellular 
protein, actin, was not affected by leukoregulin 
treatment Moreover, leukoregulin treatment did not 
alter the ability of the cells to incorporate tritiated 
thymidine. 

Initial evaluations of the effect of leukoregulin on 
HSV transcription indicated that the cytokine did not 
alter the level of expression of HSV tk mRNA. 
These studies show that leukoregulin not only 
enhances the antiviral actions of ACV but also can 
act to inhibit HSV-1 replication directly. These 
findings, which demonstrate that combination immu- 
notherapy (cytokines) and chemotherapy can substan- 
tially inhibit herpesvirus replication, provide rationale 
for the application of this approach to the interven- 
tion of virus infections. 

CMV replication within the retina. — CMV infec- 
tions are frequent complications in kidney and bone 
marrow transplant patients and HIV (human inmiu- 
nodeficiency virus) patients. Because the mecha- 
nisms by which CMV is activated and replicates 
within the retina are not known, we evaluated the 
ability of human CMV to initiate replication in 
human RPE cells and compared the results with 
finding in studies of human fibroblasts (HEL) and 
WISH cells. Human RPE cells obtained from donor 
eyes were propagated in vitro and infected at an 
input multiplicity of 1. CMV replication was evalu- 
ated in three ways: (1) detection of viral antigen by 
immunofluorescence and flow cytometry, (2) detec- 
tion of virus-induced cpe, and (3) assaying for 
infectious virus. 

We found no evidence of viral replication in the 
WISH cells. In contrast, CMV replication was 
detected in both RPE and HEL cells. In HEL cells, 
IE, E, and L proteins were detected at 5, 48, and 48 



hours, respectively. In RPE cells these proteins were 
detected somewhat later — at 24, 48, and 72 hours. 
We noted a striking difference in the percentage of 
cells expressing IE protein: After 72 hours, 100% of 
the HEL cells expressed IE protein, whereas after 7 
days, less than 1 % of the RPE cells expressed this 
protein. Analysis of the production of infectious 
virus revealed that viral infectivity and cpe were 
maximal at Day 5 in HEL cells and at Day 30 in 
RPE cells. 

This study demonstrates that, although all of the 
RPE cells were capable of becoming infected with 
CMV, less than 1% of the cells expressed IE viral 
proteins early in the infection cycle. The low fre- 
quency of expression of IE viral protein in RPE cells 
and the subsequent slow replication of CMV may be 
critical variables in terms of their relationship to viral 
persistence and activation within the retina. 

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 animals. This model has many unique features. 
It is capable of inducing acute infection in the 
presence of mild retinal vascular inflanmiation. The 
initial retinal damage is followed by clearance of the 
virus and progressive retinal destruction, even 
months after the virus is gone. Moreover, develop- 
ment of the retinal degenerative process is deter- 
mined by the genetics of the host; it involves the 
development of antiretinal autoantibodies. This 
model should assist us in understanding the patho- 
genesis of selected human diseases of unknown 
etiology. 

We have identified a cytokine, leukoregulin, that 
selectively increases membrane permeability in virus- 
infected cells. We also have shown that combined 
cytokine and drug therapy can produce substantial 
inhibition of HSV replication. Moreover, the contin- 
uous presence of the cytokine can directly inhibit 
virus replication. The data from these studies pro- 
vide rationale for the application of this approach to 
the interventive treatment of virus infections. 

We have shown that CMV replicates within RPE 
cells in a slow, limited manner. Evaluation of the 
molecular aspects of this defect may provide critical 
clues in terms of the virus' ability to establish 



84 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



persistent infections and the factors initiating viral 
activation within the retina. 

Proposed Course 

1. We will continue to evaluate coronavirus 
infections of the eye. The role(s) of genetic factors 
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 will initiate studies to determine whether 
certain viruses can replicate in retinal tissues and 
cells. Infected cells will be evaluated for the release 
or expression of uveitogenic proteins. 

3. We will continue to collect samples and 
initiate studies to detect the involvement of viruses in 
human eye diseases. 

4. We will continue to evaluate combinations of 
leukoregulin and chemotherapeutic agents for the 
management of virus infections. 

5. We will evaluate the molecular diagnosis and 
pathogenesis of CMV infections in the eye. 



NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

Bumier M, Wang Y, Detrick B, Hooks JJ: Retinal 
manifestations of a murine coronavirus infection: 
A histopathological and ultrastructural study. Exp 
Pathol, in press. 

Hooks JJ: Ocular virology, in Tabbara K (ed): 
Infections of the Eye. Boston, Little, Brown & 
Co, in press. 

Hooks JJ, Percopo C, Wang Y, Detrick B: Retina 
and retinal pigment epithelial cell autoantibodies 
are produced during murine coronavirus retinop- 
athy. J Immunol 151:3381-3389, 1993. 

Wang Y, Detrick B, Hooks JJ: Coronavirus replica- 
tion within the retina: Analysis of cell tropism in 
mouse retinal cell cultures. Virology 193:124- 
137, 1993. 



85 



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



PROJECT NUMBER 



ZOl EY 00287-01 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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 Burnier 
Robert B. Nussenblatt 



M.D. 


Guest Worker 


LI, NEI 


Ph.D. 


Visiting Scientist 


LLNEI 


M.D. 


Visiting Scientist 


LI, NEI 


M.D. 


Scientific Director 


NEI 



COOPERATING UNITS (if any) 

National Institute of Allergy and Infectious Diseases (R. Gazzinelli, M.D.) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Immunology and Virology 



INSTITUTE AND LOCATION 

NEI, NTH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.5 



PROFESSIONAL: 



0.5 



OTHER; 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (at) Minors 

□ (a2) Interviews 



□ (b) Human tissues [x| (c) Neither 



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 or acquired infections and as a manifestation of immunosuppression, particularly 
as a result of transplantation or AIDS (acquired immune deficiency syndrome). Due to the recent resurgence 
of acquired ocular toxoplasmosis in Brazil and the worldwide complications of toxoplasmosis in HIV (human 
immunodeficiency virus) infections, we initiated smdies to develop a model of acquired toxoplasmosis to 
evaluate the molecular mechanisms of pathogenesis and therapeutic strategies. 

We have developed an animal (murine) model of ocular toxoplasmosis that is characterized by retinal 
inflammation, chorioretinal scarring, 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. 



86 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunoiogy 



Project Description 

Objectives 

This project was designed to develop an animal 
model of acquired ocular toxoplasmosis, which 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 strains in vitro and in vivo, 
as well as immunocytochemical analysis of infected 
cells and tissues. Techniques used in characteriza- 
tion of T. gondii infections include histopathology, 
immunocytochemistry, in situ hybridization, and 
Western blot analysis. Techniques used in character- 
ization of antitoxoplasmosis antibodies include 
enzyme-linked immimosorbent assay. 

Major Findings 

Adult Swiss Webster, C57BL6, and Balb/c mice 
were inoculated by the subcutaneous route or intra- 
peritoneal route with 10 T. gondii cysts (S2C9 or 
ME49 strains) in a 1 -ml volume. At various times 
after inoculation (i.e.. Days 7, 14, 21, 28, and 42), 
we sacrificed the mice and removed and fixed the 
eyes and brains in 10% buffered formalin. Fifteen 
hematoxylin and eosin-stained sections of brain and 
eye were evaluated for the presence of 7. gondii 
cysts. 

By Day 14, 100% of the mice had developed 
cysts in the brain. Retinal inflammation also was 



noted in 100% of the animals by Day 14, and 
chorioretinal scars were observed in mice inoculated 
with both strains of T. gondii. Retinal cysts were 
found in mice 28 and 42 days after inoculation with 
the MB49 strain and 14 and 42 days after inoculation 
with the S2C9 strain in Swiss Webster mice. T. 
gondii cysts in the retina were detected in C57BL6 
mice at 14, 21, 28, and 42 days after inoculation 
with the S2C9 strain. This smdy has identified an 
animal model of ocular toxoplasmosis characterized 
by retinal inflammation, chorioretinal scarring, retinal 
disorganization, and cyst formation. 

Significance to Biomedical Research and the 
Program of the Institute 

This is the first model of acquired toxoplasmosis that 
consists of retinal inflammation, degeneration, and 
parasitic cyst formation. It will allow us to evaluate 
the efficacy of new antiparasitic drugs in controlling 
the development of retinal cyst formation and retinal 
inflanmiation and scarring. 

Proposed Course 

We will evaluate drugs and cytokines in the control 
of the ocular manifestations of acquired toxoplasmo- 
sis infections. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 



87 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00277-02 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Role of Retinal Pigment Epithelium in Retinal Disorders 



PRINCIPAL INVESTIGATOR (List other 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 (il 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, NTH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.0 



PROFESSIONAL: 



1.0 



OTHER: 



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 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 Unes 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 exposed to bacterial lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-a), and interleukins 
1 alpha and 1 beta (IL-loc, IL-lp) secreted large amounts of interleukin 6 (IL-6). Immunoblot and Northern blot analysis 
confirmed the presence of posttranslationally modified IL-6 protein and mRNA levels, respectively. Interferon-gamma 
(IFN-y) acted synergistically with other mediators to stimulate 3- to 5-fold increases in IL-6 secretion. We extended our 
studies to examine the expression and secretion of intercellular adhesion molecule- 1 (ICAM-1), a cell surface Ugand for 
lymphocyte function-associated antigen- 1 (LFA-1) expressed during inflammatory reactions, by human RPE. Wi^-y, 
TNF-oc, and IL-1 increased significantly both cell surface expression (detected by unmunofluorescence staining) and 
secretion into the medium (detected by ELISA). Secretion (shedding) of ICAM-1 by RPE cells in the presence of a 
mixture of EFN-y (100 u/ml), TNF-a (1 ng/ml), and IL-1 (I ng/ml) was cumulative, suggesting that combining these 
cytokines results in potent inflammatory reactions. The response of RPE cells to inflammatory mediators was rapid and 
sustained in the presence of stimulants but reversed to control levels quickly upon withdrawal, suggesting the reversibiUty 
of the responses of RPE to inflammatory signals. The effects of transforming growth factor beta (TGF-p) on RPE 
functions at cellular and molecular levels also are being studied. TGF-p increased the expression of heme oxygenase- 1, 
an enzyme reaction that generates the antioxidant bihrubin, which helps in cellular defense mechanisms against oxidative 
stfess. 

The results clearly show that human RPE cells respond to specific inflammatory signals or infections by increased cellular 
expression and secretion of IL-6 and lCAM-1, which may in tum perpetuate immune reactions in the pathogenesis and/or 
prevention of retinal and choroidal diseases. 

88 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Objectives 

Human retinal pigment epithelial (RPE) cell cultures 
have been established from human donor eyes. 
Primary cell lines of RPE are used as an in vitro 
model to study the effects of growth factors, inflam- 
matory mediators, and toxic compounds on biochem- 
ical, cellular, and molecular aspects of RPE structure 
and function. The usefulness of RPE cultures also 
are being evaluated for transplantation to restore 
retinal function in hereditary and age-related disor- 
ders. 

Methods 

Primary cell cultures are prepared by initial seeding 
of either freshly isolated RPE cells or RPE-choroid 
explants. Cells are grown in minimum essential 
medium supplemented with 10% fetal calf serum, 
nonessential amino acids, and antibiotics. We are 
attempting to develop serum-free hormonally defined 
medium to render cultured cells more suitable for 
fransplantation therapy with minimal immune-related 
complications and consequent graft rejection. 

Techniques required for cell culture, immunofluo- 
rescence, cytokine, and ICAM-1 assays by enzyme- 
linked immunosorbent assay (ELISA), gel elecfro- 
phoresis, and Western and Northern blotting for pro- 
teins and RNA are developed and standardized in our 
laboratory to carry out these studies. 

Major Findings 

In the past, the age of the donor was considered very 
critical in preparing human RPE cultures because 
eyes from donors over 50 years did not yield fruitful 
cell lines, probably due to senescence-associated loss 
of viability. In these experiments, RPE cells were 
first dissociated from the eye cups by digestion with 
proteolytic enzymes, freatment that might have 
caused initial contamination with nonepitheUal cells, 
from which it is impossible to purify epithelial cells. 
Therefore, we modified this method, using RPE-cho- 
roid explants, native and without harsh enzyme 
freatment, to initiate cell growth. Then, by careful 
monitoring of clusters of cells growing around the 
explants, we were able (on the basis of morphology 
combined with experience) to select purely epithelial 
cells and discard nonepitheUal cells at the primary 
culture stage. 



Using this technique, we established primary cell 
lines of human RPE from eyes obtained from 81- 
and 87-year-old donors. The epitheUal nature of 
these cell lines was confirmed by inmiunochemical 
staining with monoclonal antibodies to cytokeratin. 
All of the cells expressed cytokeratin at different 
passages (3 to 10). Immunoblotting analysis of 
cellular proteins indicated cytokeratin 18 as the 
predominant cytokeratin in these cells. Because RPE 
is the only epithelial cell in the posterior segment 
(choroid-RPE-retina), these results establish without 
doubt that the cell lines developed are, in fact, RPE. 
The feasibility of using donor eyes from a population 
over 70 years of age for preparing RPE cultures is 
demonsfrated. 

Human RPE cultures secrete large quantities of 
inflammatory cytokine, interleukin 6 (IL-6), when 
exposed to inflammatory mediators — ^lipopolysacca- 
ride (LPS), tumor necrosis factor alpha (TNF-a), 
IL-la, and IL-ip. Western blot analysis revealed 
posttranscriptionally processed forms of IL-6 in the 
secreted proteins. Although interferon-y (IFN-y) 
induced the lowest levels of IL-6 by itself, it acted 
synergistically with other cytokines to stimulate 
threefold to fivefold increases in IL-6 secretion by 
RPE. Analysis of IL-6 secretion by ELISA and the 
expression of IL-6 mRNA by Northern blotting 
indicated rj^id, sustained RPE responses to inflam- 
matory mediators that can be reversed quickly upon 
withdrawal of the stimulus. Cell surface expression 
and secretion (shedding) of intercellular adhesion 
molecule 1 (ICAM-1), a cell surface glycoprotein 
ligand for lymphocytes, by RPE was significantiy 
stimulated by inflammatory cytokines — IFN-y, 
TNF-a, and IL-1. The presence of all these cyto- 
kines together appears to induce more potent secre- 
tion of ICAM-1. Regulation of the expression of 
ICAM-1 in RPE is under investigation through the 
use of monoclonal antibodies and cDNA probes. 

Our observations suggest that, in response to the 
presence of the inflammatory cytokines produced by 
macrophages and lymphocytes that, for example, 
infiltrate the eye during inflammation caused by 
infection or autoimmune disease, RPE can locally 
produce IL-6 and ICAM-1. In turn, IL-6 aids in the 
proliferation and differentiation of lymphoid cells to 
regulate immunological phenomena. Secreted or cell 
surface ICAM-1 expressed on RPE helps in homing 
and concentration of lymphocytes near the sites of 
inflammation for immunoregulation. 



89 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Elevated levels of intravitreal IL-6, IL-1, TNF-a, 
and IFN-y were reported in proliferative and other 
noncomplicated retinal detachments. Moreover, 
intravitreal injection of IL-1, TNfF-a, or IL-6 induced 
uveitis in experimental animal models. These studies 
implicate local but not systemic increase in these 
cytokines as initiators of uveitis. Increases in the 
circulating ICAM-1 levels in the serum of uveitis 
patients and expression of ICAM-1 in epiretinal 
membranes in proliferative vitreoretinopathy, prolif- 
erative diabetic retinopathy, and macular pucker were 
reported, suggesting involvement of ICAM-1 in 
various diseases. Our studies indicate that RPE, 
possibly in association with other resident cells, 
reacts to inflammatory stimuli and participates in the 
immunopathologic mechanisms by secreting IL-6 and 
ICAM-1. 

Basic fibroblastic growth factor (bFGF) and 
transforming growth factor beta (TGF-|3) secreted by 
RPE are known to have both autocrine and paracrine 
actions on retina and choroid. bFGF and TGF-P are 
involved in various biological processes, such as cell 
proliferation, differentiation, wound healing, immu- 
nosuppression, and apoptosis. The roles of bFGF 
and TGF-P in RPE functions and the regulation of 
secretion of these growth factors by RPE are being 
investigated. We have studied the expression of 
heme oxygenase- 1 (HO-1), an enzyme known to 
respond to oxidative stress, heat shock, heavy metals, 
and inflammatory agents, that offer protection against 
oxidative damage. Among ocular tissues, RPE has 
the highest activity of HO-1. Using specific poly- 
clonal antibodies and PCR-generated probes, we have 
demonstrated that TGF-P increases HO-1 levels by 
fourfold to fivefold in human RPE cells within 4 
hours. Toxic compounds — cadmium, lead, mercury, 
arsenite, and iodoacetate — are the most potent 
inducers of HO-1 in RPE. HO-1 catalyzes the 
oxidation of heme into biliverdin and carbon mon- 
oxide. Biliverdin is converted by nonlimiting enzy- 
matic reaction into bilirubin, an antioxidant that 
offers cells protection against heat and oxidative 
stress. 

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 RPE functions and 
for further elucidation of the mechanisms of RPE 
involvement in the pathogenesis 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 pigmen- 
tation upon serial passaging of cells and (2) lack of 
clear intercellular junctions and in vivo-like morpho- 
logical appearance. These changes may be due to 
cytoskeletal reorganization and partial dedifferentia- 
tion. Our immediate goal is to examine the mecha- 
nisms by which RPE cultures can be induced to 
resume in vivo characteristics. This will be achieved 
by selecting specific media composition, the addition 
of growth and differentiating factors, and/or culturing 
on suitable extracellular matrix. Development of a 
fully differentiated RPE cell line is crucial, not only 
for understanding cellular functions but also for 
cellular transplant therapy. 

Continuing to evaluate the effects of inflammatory 
cytokines and bacterial endotoxins on RPE cell 
cultures, we will address three areas: (1) influences 
of these factors on cellular cytoskeletal organization, 
intercellular junctions, and adhesion properties; 

(2) effects on cell functions (e.g., membrane perme- 
ability and solute transport and phagocytosis); and 

(3) characterization of proteins such as growth 
factors, cytokines, and proteolytic enzymes secreted 
by RPE in response to various stimuli. These studies 
are likely to shed light on the role of RPE in the 
pathophysiology of the retina and choroid, tissues 
that are in close proximity to and directly influenced 
by RPE. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases, Macular 
Degeneration, Photoreceptors, and Retinal Pigment 
Epithelium 

Publications 

Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader 
GJ, Wiggert B: Transforming growth factor-p 
increases the expression of heme oxygenase 1 in 
human retinal pigment epithelial cells. Invest 
Ophthalmol Vis Sci 34(4)(suppl):1451, 1993. 

Nagineni CN, Detrick B, Hooks JJ: Interferon-y acts 
synergistically with inflammatory mediators to 
induce expression of interleukin 6 by human 
retinal pigment epithelial cells. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1020, 1993. 



90 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl EY 00222-08 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Immunopathology in Eyes With Experimental and Clinical Ocular Diseases 



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

Kourosh Dastgheib M.D. IRTA FeUow LI, NEI 

Deborah Luyo Technician LI, NEI 

Scon M. Whitcup M.D. Medical Officer LI, NEI 

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

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

Igal Gery Ph.D. Deputy Chief LI, NEI 

Robert B. Nussenblan M.D. Scientific Director NEI 



COOPERATING UNITS (if any) 

Department of Ophthalmology, Kunime University, Kurume, Japan (Manabu Mochizuki, M.D.) 



LAB/BRANCH 



Laboratory of Inmiunology 



SECTION 



Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 

4.4 



PROFESSIONAL: 

3.4 



OTHER: 

1.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects □ (b) Human tissues [xj (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, the alteration of surface markers on 
ocular resident cells, and their cytokines in animals with experimental autoimmune uveoretinitis or endotoxin- 
induced uveitis (EAU, EIU) and in human ocular tissues with various diseases were analyzed by 
immunohistochemical smdies and in situ hybridization. T lymphocytes were the predominant infiltrating cells 
in EAU, yet both maaophages and polymorphonuclear neutrophils (PMNs) were the predominant infiltrating 
cells in EIU. Migration of the inflammatory cells from tlie vessels into the target site is directed by adhesion 
molecules, which can be expressed on vascular endothelium and other resident cells in the eye. Mast cells 
appear to participate in the immunopathogenesis of EAU and EIU. T-lymphocyte specificity is directed to 
small fragments of antigen bound to cell surface major histocompatibility complex (MHC) molecules, which 
are presented on the surface of specialized antigen-presenting cells. The expression of MHC class 11 antigens 
was observed on ocular resident cells such as retinal pigment epithelium (RPE), retinal endothelium, 
keratocytes, fibroblasts, and ciliary epithelium in rodents. Both the infiltrating cell subpopulation and the 
expression of class II antigens and adhesion molecules on ocular resident cells can be altered by various 
immunomodulating agents and cytokines. 

Specimens from human ocular tissues with various diseases — such as uveitis, retinal disease, conjunctival and 
corneal diseases, metabolic genetic diseases, and tumors — are studied using immunohistochemical and in situ 
hybridization techniques as well as light and electron microscopic evaluation. In uveitis, immunocompetent 
cells and lymphokines are valuable adjuncts to clinical diagnosis, and they are determinants of disease course 
and prognosis. In nonuveitic conditions, alteration of cellular membrane surface markers and intracytoskeleton 
of the ocular resident cells may imply 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. 

91 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

This program is designed to evaluate the clinical 
manifestation, histopathology, and immunopathology 
of the ocular tissue when experimental autoimmune 
uveoretinitis (EAU) and endotoxin-induced uveitis 
(EIU) are induced and/or modulated by various 
immunosuppressive agents in various animal species. 
Ocular tissues obtained from patients with various 
diseases, including inflammatory and noninflamma- 
tory disorders, also are studied. The infiltrating 
inflammatory cells, ocular resident cells and their 
products, and various lymphokines and cytokines are 
examined. The findings will help us understand 
ocular inflammation and the pathogenesis of each 
disease examined in humans. 

Methods 

Clinical examinations include flashlight and slit-lamp 
examinations as well as examination of the fundus of 
animals and patients under the dissecting microscope. 
Pathological examinations include routine histologic 
techniques for light and electron microscopy, immu- 
nofluorescence, avidin-biotin-peroxidase complex 
methods, and in situ hybridization techniques. 

Major Findings 

We continued to study the immunopathology of 
various inflammatory cells and ocular resident cells 
in different experimental models of uveitis. We have 
observed that, prior to the infiltration of inflamma- 
tory cells into the eye, the number of mast cells in 
the anterior uvea decreases and is consistent with 
mast cell degranulation in EAU and EIU. This 
observation suggests that anterior uveal mast cells 
participate in uveitis by releasing vasoactive amines 
to alter the integrity of the blood-ocular barrier and 
amplify the inflammatory process. 

Toward understanding the immunopathological 
process in various experimental uveitides, we have 
examined the efficacy of different anti-inflammatory 
mediators and immunomodulating agents in these 
animal models. For example, we have found that 
feeding animals rat chow mixed with CGS- 13080, a 
thromboxane synthetase inhibitor, suppresses the 
development of clinical and histopathological EAU. 
Inhibition of this enzyme results in a reduction of 
thromboxane B^ and an increase of prostaglandin E 



in the serum, thus altering the inflammatory mediator 
and suppressing EAU. 

Antibodies against adhesion molecules are able to 
abrogate EAU and EIU because expression of 
adhesion molecules precedes the flux of inflamma- 
tory cells into the eye. The cytokine cascade in the 
inflammatory process is complicated. Tumor necro- 
sis factor a has a paradoxical role in EIU. Immuno- 
suppressive agents — ^in particular the inhibitors of T- 
cell function, such as cyclosporine A, FK 506, and 
rapamycin, which interfere with the release of 
lymphokines — are potent and effective medications 
to treat EAU, a T-cell-mediated autoimmune uveo- 
retinitis. 

Using immimopathological techniques, we exam- 
ine ocular tissues obtained from patients with various 
ocular diseases to help visualize the pathology and 
the kinetics of the specific disease process. The 
findings provide useful information for understanding 
the pathological mechanisms of the disease, deter- 
mining the diagnosis, and guiding the subsequent 
management of patients. 

We found collagen dysgenesis in Reis-Buckler 
corneal dystrophy. The presence of immature 
collagen type III and poorly developed collagen type 
I may contribute to the pathogenesis of Reis-Buckler 
dystrophy. In Cogan-Reese syndrome, we found that 
the iris nevi are cells that originate in the neural crest 
and have numerous melanosomes, junctional com- 
plexes, and basement membrane. We demonstrated 
the presence of oB-crystallin, a major lens protein in 
retinoblastoma, suggesting that oB-crystallin is 
involved in tumor growth and/or is a marker for 
general oncogenic "stress" in retinoblastoma We 
also have shown the presence of tachyzoites and the 
role of T lymphocyte in congenital toxoplasmosis. 

Using in situ hybridization, we detected the RNAs 
of botii interleukins 2 and 4 in the conjunctiva of 
ocular onchocercal patients, suggesting that Th2 cells 
and their lymphokines are important for localized 
host responsiveness to ocular onchocerciasis. 

In practice, correct handling and processing of 
surgical specimens obtained from vitrectomy and/or 
chorioretinal biopsy can yield important information, 
in particular, the diagnosis of intraocular large B-cell 
lymphoma (central nervous system lymphoma) and 
progressive chorioretinal lesions of unknown etiol- 
ogy. Once the diagnosis is made, the appropriate 
treatment can be offered. 



92 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



We are investigating other experimental models, 
e.g., allergic conjunctivitis, melanin-protein-induced 
uveitis (EMIU), and acquired toxoplasmosis, and 
their resemblance to other ocular inflammatory 
diseases in humans. We hope to better understand 
the mechanisms of ocular inflammation and evaluate 
the effects of different therapeutic approaches in 
these different new models. 

Significance to Biomedical Research and the 
Program of the Institute 

Immunopathological findings on experimental uve- 
itides have provided information on various inflam- 
matory cells and ocular resident cells during the 
process of ocular inflammation. This information 
helps us to choose and evaluate novel pharmacologic 
agents and provide better therapeutic intervention of 
uveitis in humans. Studies of ocular tissues obtained 
from patients with various disorders have enabled us 
to gain information on the mechanism, diagnosis, and 
management of these ocular diseases. This informa- 
tion is useful in treating patients not only with 
uveitis but also with ocular tumors and congenital 
disorders. 

Proposed Course 

Various experimental models, including EAU, EIU, 
EMIU, allergic conjunctivitis, and acquired toxoplas- 
mosis, will be studied clinically, histopathologically, 
and immunopathologically in different species and 
strains. Various pharmacological agents and the role 
of cytokines, lymphokines, enzymes, and cellular 
surface markers will be evaluated in these models. 
Also, we propose continuation of analysis of human 
specimens in the study of their immunopathogenesis. 

NEI Research Program 

Retinal and Choroidal Disease — Inflammatory 
Disorders 

Publications 

Brezin AP, Kasner L, Thulliez P, Li Q, Daffos F, 
Nussenblatt RB, Chan C-C: Ocular toxoplasmo- 
sis in the fetus: Immunohistochemistry and DNA 
amplification. Invest Ophthalmol Vis Sci 
34(4)(suppl):1001, 1993. 

Brezin AP, Kasner L, Thulliez P, Li Q, Daffos F, 
Nussenblatt RB, Chan C-C: Ocular toxoplasmo- 



sis in the fetus: Immunohistochemistry and 
DNA amplification. Retina, in press. 

Bucci FA Jr, Li Q, Luyo D, Tanner J, Chan C-C: 
Detection of T lymphocytes in patients with 
allergic conjunctivitis. Invest Ophthalmol Vis Sci 
34(4)(suppl):853, 1993. 

Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover 
S, Hansen CT, Wilder RL: Recruitment of 
antigen-nonspecific cells play a pivotal role in the 
pathogenesis of a T-cell-mediated organ-specific 
autoimmune disease, experimental autoimmune 
uveoretinitis. J Neuroimmunol 41:171-1^^, 1993. 

Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover 
S, Hansen CT, Wilder RL: Recruitment of naive 
T cells plays a pivotal role in the pathogenesis of 
experimental autoimmune uveoretinitis. Invest 
Ophthalmol Vis Sci 34(4)(suppl):902, 1993. 

Chan C-C, Cogan DG, Bucci FS, Barsky D, Li Q, 
Crawford MA: An anterior corneal dystrophy 
with dyscollagenosis (Reis-Buckers type?). 
Cornea 12:451-460, 1993. 

Chan C-C, Hikita N, Dastgheib K, Whitcup SM, 
Gery I, Nussenblatt RB: Immunopathology of 
experimental autoimmune anterior uveitis 
(EAAU). Invest Ophthalmol Vis Sci 34(4) 
(suppl):999, 1993. 

Chan C-C, Li Q, Brezin AP, Whitcup SM, Egwuagu 
C, Otteson EA, Nussenblatt RB: Immunopathol- 
ogy of ocular onchocerciasis. 3. Th-2 helper T 
cells in the conjunctiva. Ocular Immunol Inflam 
1:71-77, 1993. 

Chepelinsky AB, Overbeek PA, Chan C-C, Jamieson 
S, Dickson C, Parker DM, Robinson W: Int2 
ectopic expression induces differentiation of 
secretory epithelia in the eyes of transgenic mice. 
Invest Ophthalmol Vis Sci 34(4)(suppl):1222, 
1993. 

Dastgheib K, Hikita N, Sredni B, Albeck M, Nussen- 
blatt RB, Chan C-C: Ocular inflammation stimu- 
lated by the immunomodulator ASIOI. Invest 
Ophthalmol Vis Sci 34(4)(suppl):1483, 1993. 

Egwuagu CE, Sztein J, Chan C-C, Reid W, Mahdi 
R, Nussenblatt RB, Chepelinsky AB: Gamma- 
interferon expression in the eyes of transgenic 
mice disrupts differentiation of the lens and 
retina. Invest Ophthalmol Vis Sci 34(4)(suppl): 
1455, 1993. 



93 



Laboratory or Immunology 



NEI Annual Report— FY 1993 



Fujino Y, Li Q, Chung H, Hikita N, Nussenblatt RB, 
Chan C-C: Immunopathology of experimental 
autoimmune uveoretinitis in primates. Autoimmu- 
nity 13:303-309, 1992. 

Kara Y, Caspi RR, Wiggert B, Chan C-C, Streilein 
JW: Use of ACAID to suppress interphotorecep- 
tor retinoid-binding protein-induced experimental 
autoimmune uveitis. Curr Eye Res 1 l(suppl):97- 
100, 1992. 

Hikita N, Chan C-C, Mochizuki M, Maturi R, Nus- 
senblatt RB, Whitcup SM: Topical FK 506 
inhibits endotoxin-induced uveitis (EIU). Invest 
Ophthalmol Vis Sci 34(4)(suppl):1480, 1993. 

Holland EJ, Chan C-C, Bergstrom L, Palestine AG, 
Nussenblatt RB: Kinetics of corneal transplant 
rejection in the rat penetrating keratoplasty model. 
Cornea, in press. 

Holland EJ, Hardten DR, Murali S, Lushine K, 
DeMartelaere S, Olevsky OM, Mindrup EA, 
Karlstad C, Chan C-C: Effect of topically admin- 
istered platelet-derived growth factor on corneal 
wound strength in penetrating keratoplasty. 
Invest Ophthalmol Vis Sci 34(4) (suppl):1375, 
1993. 

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. 

Kasner L, Chan C-C, Whitcup SM, Gery I: The 
paradoxical role of tumor necrosis factor-alpha in 
endotoxin-induced uveitis. Invest Ophthalmol Vis 
Sci 34(4)(suppl):1480, 1993. 

Kupfer C, Chan C-C, Bumier M Jr, Kaiser-Kupfer 
MI: Histopathologyofthe ICE syndrome. Trans 
Am Ophthalmol Soc 90:149-160, 1992. 

Lai JC, Chan C-C, Li Q, Whitcup SM: Treatment 
with corticosteroids and cyclosporine A inhibits 
the expression of cell adhesion molecules in 
experimental autoimmune uveitis (EAU). Invest 
Ophthalmol Vis Sci 34(4)(suppl):1206, 1993. 

Li Q, Fujino Y, Caspi RR, Najafian F, Nussenblatt 
RB, Chan C-C: Association between mast cells 
and the development of experimental autoimmune 
uveitis in different rat strains. Clin Immunol 
Immunopathol 65:294-299, 1992. 

Li Q, Hikita N, Whitcup SM, Nussenblatt RB, Chan 
C-C: Allergic conjunctivitis induced by com- 



pound 48/80 in C57BL/6NCR mice. Invest 
Ophthalmol Vis Sci 34(4)(suppl):857, 1993. 

Li Q, Lopez JS, Caspi RR, Roberge FG, Nussenblatt 
RB, Kador P, Chan C-C: Suppression of S- 
antigen-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. 

Li Q, Whitcup SM, Fujino Y, Nussenblatt RB, Chan 
C-C: The role of mast cells in endotoxin induced 
uveitis. Invest Ophthalmol Vis Sci 34:256-259, 
1993. 

Martin DF, DeBarge LR, Nussenblatt RB, Chan C-C, 
Roberge FG: Synergistic effect of rapamycin and 
cyclosporine A on the inhibition of experimental 
autoimmune uveoretinitis. Invest Ophthalmol Vis 
Sci 34(4)(suppl):1476, 1993. 

Martin DF, Chan C-C, de Smet MD, Palestine AG, 
Davis JL, Whitcup SM, Burnier MN Jr, Nussen- 
blatt RB: The role of chorioretinal biopsy in the 
management of posterior uveitis. Ophthalmology 
100:705-714, 1993. 

Parks DJ, Hikita N, Nagineni C, Hooks JJ, Chan 
C-C, Nussenblatt RB, de Smet MD: Immunohis- 
tochemistry of xenogeneic RPE transplants in the 
rat: A model for graft rejection. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1095, 1993. 

Pineda R U, Chan C-C, Ni M, Hayden BJ, Johnson 
MA, Chader GJ: Human retinoblastoma cells 
express oB-crystallin in vivo and in vitro. Curr 
Eye Res 12:239-245, 1993. 

Rizzo LV, Silver PB, Hakim F, Chan C-C, Wiggert 
B, Caspi RR: Establishment and characterization 
of an IRBP-specific T-cell line that induces EAU 
in BIO. A mice. Invest Ophthalmol Vis Sci 34(4) 
(suppl):1143, 1993. 

Roberge FG, Kozhich A, Chan C-C, Martin DF, 
Nussenblatt RB, de Smet MD: Inhibition of 
cellular transfer of experimental autoimmune 
uveoretinitis by rapamycin. Ocular Immunol 
Inflam 1:269-273, 1993. 

Roberge FG, Xu D, Chan C-C, de Smet MD, Nus- 
senblatt RB, Chen H: Treatment of autoimmune 
uveoretinitis in the rat with rapamycin, an inhibi- 
tor of lymphocyte growth factor signal transduc- 
tion. Curr Eye Res \2:\91-lQ'i,\99l. 

Shah DN, Piacentini MA, Bumier MN Jr, McLean 
IW, Nussenblatt RB, Chan C-C: Inflammatory 



94 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



cellular kinetics in sympathetic ophthalmia. A 
study of 29 traumatized (exciting) eyes. Ocular 
Immunol Inflam 1:255-262, 1993. 

Silver PB, Rizzo LV, Chan C-C, Donoso LA, Wig- 
gert B, Caspi RR: Identification of a putative 
epitope in the IRBP molecule that is uveitogenic 
for mice of the H-2b h^lotype. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1482, 1993. 

Vistica B, Gery I, Chan C-C, Nussenblatt RB, Whit- 
cup SM: Anti-ICAM-1 and anti-LFA-1 mono- 
clonal antibodies (mAbs) inhibit in vitro prolifera- 
tion of a uveitogenic cell line. Invest Ophthalmol 
Vis Sci 34(4)(suppl):1144, 1993. 

Westeren AC, Chan C-C, de Smet MD, Nussenblan 
RB, Roberge FR: Evaluation of the immunosup- 
pressant SK&F 106610 in the treatment of experi- 
mental autoimmune uveoretinitis. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1477, 1993. 

Whitcup SM, DeBarge LR, Caspi RR, Haming R, 
Nussenblatt RB, Chan C-C: Monoclonal anti- 
bodies against ICAM-1 (CD54) and LFA-1 



(CD 11 a/CD 18) inhibit experimental autoimmune 
uveitis. Clin Immunol Immunopathol 67: 143-150, 
1993. 

Whitcup SM, DeBarge LR, Rosen H, Nussenblatt 
RB, Chan C-C: Monoclonal antibody against 
CSl lb/CD 18 iniiibits endotoxin-induced uveitis. 
Invest Ophthalmol Vis Sci 34:673-681, 1993. 

Whitcup SM, de Smet MD, Rubin BI, Palestine AG, 
Martin DF, Burnier M Jr, Chan C-C, Nussenblatt 
RB: Intraocular lymphoma: Clinical and histo- 
pathologic diagnosis. Ophthalmology 100:1399- 
1406, 1993. 

Whitcup SM, Hikita N, Shirao M, Mochizuki M, 
Nussenblatt RB, Chan C-C: Effect of monoclonal 
antibodies against ICAM-1 (CD54) and LFA-1 
alpha (CDl la) in the prevention and treatment of 
endotoxin-induced uveitis (EIU). Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1143, 1993. 

Whitcup SM, Nussenblatt RB; Price FW Jr, Chan 
C-C: Expression of cell adhesion molecules in 
corneal graft failure. Cornea 12:475-480, 1993. 



95 



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, 1992 to September 30, 1993 



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

Immunopathology of Ocular Diseases in Humans 



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



PI: Chi-Cbao Chan 

Others: Robert B. Nussenblatt 

Qiao Li 

Marc D. de Smet 
Raymond DeBarge 
Scott M. Wfaitcup 
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 
Inununopathology 
Scientific Director 
Visiting Fellow 
Visiting Scientist 
Senior Staff Fellow 
Staff Medical Officer 
Visiting Associate 
Visiting Scientist 
Staff Fellow 
Visiting Associate 



LI, NEI 

NEI 
LI, NEI 
LI, NEI 
LLNEI 
LLNEI 
LI, NEI 
LI, NEI 
LLNEI 
LINE] 



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'Hdpital de la Piti6, Paris, France 
(Phuc LeHoang, M.D.) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.0 



PROFESSIONAL: 



OTHER: 



0.0 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



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

This project has been terminated and combined with Project No. ZOl EY 00222-08 LI. 



96 



PHS 6040 (Rev. 5/92) 



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 September 30, 1993 



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

Cytokines and Ocular Antigens in the Eye 



PRINCIPAL INVESTIGATOR (List other profossional 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. 
Ph.D. 

M.D. 
M.D. 



Scientific Director LI, NEI 

Head, Section on LI, NEI 

Experimental Immunology 
Visiting Fellow LI, NEI 

Fellow LI, NEI 



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 



OTHER: 



0.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[xl (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. 



97 



PHS 6040 (Rev. 5/92) 



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



PROJECT NUMBER 



ZOl EY 00268-03 LI 



PERIOD COVERED 



October 1, 1992 to September 30. 1993 



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

The Diagnosis and Treatment of Human Uveitis 



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

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



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



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



Scientific Director 
Visiting Scientist 
Medical Officer 



NEI 
LI, NEI 
LI, NEI 



COOPERATING UNITS (if any) 

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



LAB/BRANCH 

Laboratory of Immunology 



SECTION 



Section on Inmiunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



1.0 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



PROFESSIONAL; 



1.0 



OTHER: 



0.0 



□ (b) Human tissues □ (c) Neither 



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

The goal of this project is to develop improved methods for diagnosing and treating human uveitis. Three studies of 
diagnosis are as follows: (1) We examine biopsy and pathology specimens from patients with uveitis and AIDS to 
develop improved diagnostic tests and to understand better the pathophysiology of inflammatory eye disease. Ongoing 
studies of intraocular lymphoma show that multiple vitrectomies or lumbar punctures are required to diagnose about one- 
third of the patients. Appropriate, prompt handling of pathology specimens by an experienced cytopathologist remains 
critical to making the correct diagnosis. (2) To improve methods for diagnosing ocular sarcoidosis, we test lacrimal gland 
and conjunctival biopsies for the presence of interferon-gamma; Kveim antigen; interleukins 2, 3, 4, 6, and 8; and T-c^U 
receptors believed to be specific for this disease. This year we retrospectively reviewed 46 patients with biopsy-proven 
sarcoidosis and 21 with uveitis. In patients with ocular involvement, the most sensitive diagnostic test was the pulmonary 
diffusing capacity (DLCO), which diminished in 78% of patients tested. Corticotropin-releasing hormone tests are 
performed on patients with uveitis to determine whether a defective hypothalamic-pituitary-adrenal axis is associated with 
increased risk for autoimmune ocular inflammatory disease. (3) Our study of animals with endotoxin-induced uveitis 
(EIU) showed that tumor necrosis factor alpha causes a paradoxical exacerbation of ocular disease. 

In the area of treatment, we have three projects: (1) We are continuing a masked, randomized crossover study to 
compare acetazolamide with placebo for the treatment of uveitic cystoid macular edema; to date 31 patients have been 
recruited. (2) Topically applied FK 506 was used to treat EIU in the rat. Ocular inflammation was reduced significandy 
in animals treated with topical FK 506 (0.3% and 0.05%) when compared with control animals, a finding that may be 
useful in the treatment of acute ocular inflammation in humans. (3) The optimal therapy for intraocular lymphoma 
remains unclear; however, previous studies suggest that untreated patients die within 1 year of diagnosis. Retrospective 
review of 11 patients with intraocular lymphoma treated with radiation, chemotherapy, or both showed substantial 
treatment-related mortality. In a joint protocol with the National Cancer Institute, we now are investigating alternative 
treatment regimens for central nervous system lymphoma. 



98 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Additional Personnel 

Emily Chew M.D. Visiting Scientist, 

BEP, NEI 

Frederick Ferris, III M.D. Chief, Clinical Trials 

Branch, BEP, NEI 

George P. Chrousos M.D. Diabetes Epidemiology 

Branch (DEB), National 
Institute of Child 
Health and Disease 
(NICHD) 

Daniel Martin M.D. Senior Staff Fellow, 

LI, NEI 

George Mastorakos M.D. Visiting Scientist, 

DEB, NICHD 

Igal Gery Ph.D. Deputy Chief, LI, NEI 

Clinical Protocol Numbers 

90-EI-132 
91-EI-30 
91 -EI- 139 
92-EI-0070 

Objectives 

The goal of this study is to develop better methods 
for the diagnosis and treatment of human uveitis. 
We also are interested in defining the pathophysiol- 
ogy of inflammatory eye diseases by analyzing 
human tissue and animal models of uveitis. 

Methods 

Diagnosis of Uveitis 

1. To improve the diagnostic yield of conjuncti- 
val and lacrimal gland biopsies for sarcoidosis, we 
are examining tissue specimens using immunohisto- 
chemical staining. Conjunctival and lacrimal gland 
biopsies will be performed on 10 patients with 
known sarcoidosis and snap frozen in O.C.T.® 
Immunohistochemical staining will be performed 
using an avidin-biotin-peroxidase complex. Primary 
monoclonal antibodies against T-cell markers, T-cell 
receptors, Kveim antigen, and various interieukins 
will be applied. The results will be compared with 
those of biopsies from patients with other uveitic 
conditions, such as Behcet's disease, to determine the 
specificity of these results. We also have reQ-ospec- 
tively reviewed the records of patients with biopsy- 



proven sarcoidosis to determine the sensitivity of 
current tests obtained to diagnose sarcoidosis. 

2. Intraocular lymphoma often masquerades as 
an idiopathic uveitis, which delays the stan of appro- 
priate therapy. We continue to collect data on 
patients diagnosed with intraocular lymphoma. 

3. We are performing corticotropin-releasing 
hormone tests to access the hypothalamic-pituitary- 
adrenal axis in patients with autoimmune uveitis. 
Subnormal Cortisol production in response to this 
hormone may predispose patients to the development 
of autoimmune disease. 

4. The pathophysiology of endotoxin-induced 
uveitis (EIU) is being studied using immunohisto- 
chemistry, histology, and monoclonal antibodies 
against various cytokines. 

Treatment of Uveitis 

1. The efficacy of acetazolamide for the treat- 
ment of uveitis-associated macular edema is being 
evaluated in a masked, crossover study comparing 
acetazolamide with placebo. Visual acuity and the 
height of the macular edema measured by fluorescein 
angiography are the primary endpoints. 

2. We are testing the efficacy of topically 
applied FK 506, a new immunosuppressive agent, for 
the treatment of acute anterior uveitis, using the 
animal model of EIU in the rat Histologic evidence 
of intraocular inflammation and aqueous humor 
protein concentrations are compared between treated 
and control animals. 

3. In an investigation of treatment for patients 
with intraocular lymphoma, we are reviewing both 
morbidity and mortality. In addition, we are partici- 
pating in a joint protocol with the National Cancer 
Institute to study chemotherapy on non-Hodgkin's 
lymphoma arising in the central nervous system 
(CNS) or the eye. 

4. We are comparing frabeculectomy combined 
witii subconjunctival 5-fluorouracil to the Molteno 
implant for the treatment of glaucoma secondary to 
uveitis. 

Major Findings 

1. We retrospectively reviewed 46 patients with 
biopsy-proven sarcoidosis, 21 with uveitis. In 
patients with ocular involvement, only 61% had 
abnormal chest x-rays; 36% had an elevated angio- 
tensin-converting enzyme. The most sensitive 



99 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



diagnostic test was the pulmonary diffusing capacity, 
which was diminished in 78% of the patients tested. 
There was no statistically significant difference 
between the test results of sarcoidosis patients with 
and without uveitis. Among 21 uveitis patients, 14 
(67%) had visual acuity of 20/40 or worse in at least 
one eye. Poor visual acuity (20/200 or worse) was 
predominantly caused by secondary glaucoma. 

2. Examination of the use of topical FK 506 for 
the treatment of EIU showed that the mean anterior 
chamber cell count per microliter and the median 
histologic grade of ocular inflammation (scale of to 
4) were significantly decreased in rats treated with 
topical FK 506 0.05% and FK 506 0.3% when 
compared with those of placebo-treated rats. The 
blood levels of FK 506 in rats treated with topical 
0.05% and 0.3% FK 506 were 1.2 and 2.9 mg/ml, 
respectively— more than tenfold lower than levels 
obtained with systemic therapy at a dose of 1 mg/kg. 

3. Retrospective review of 11 patients with 
intraocular lymphoma treated with radiation, chemo- 
therapy, or both showed that 5 patients died a mean 
of 21 months after diagnosis while 6 have survived 
a mean of 33 months. We performed autopsies on 
three of the five patients who died. Interestingly, no 
residual lymphoma was found in any of the three, 
whose deaths were felt to result from treatment- 
related complications, predominantly severe leuko- 
encephalopathy. This substantial treatment-related 
mortality suggests that improved ther^)eutic regi- 
mens are needed. We are currently involved in a 
joint protocol with the National Cancer Institute, 
investigating alternative treatment regimens for CNS 
lymphoma. 

Significance to Biomedical Research and the 
Program of the Institute 

Uveitis accounts for about 10% of visual impairment 
in the United States. A major goal of the ^fEI is to 
improve the methods for diagnosing and treating 
uveitis in an attempt to preserve useful vision in 
patients with inflammatory eye disease. 

Proposed Course 

We will continue patient recruitment for the clinical 
trials of cystoid macular edema, corticotropin-releas- 
ing hormone, sarcoidosis, uveitic glaucoma, and 
intraocular lymphoma. We have completed our 
initial studies, showing the effectiveness of topically 
applied FK 506 for the treatment of EIU, and we are 
planning to investigate the use of Uposome-bound 



FK 506 to improve ocular penetration of topically 
applied compounds. In addition, studies on the 
effect of cytokines on ocular inflammatory disease 
will continue. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 

Publications 

Chan C-C, Li Q, Brezin AP, Whitcup SM, Egwuagu 
C, Otteson EA, Nussenblatt RB: Immunopathol- 
ogy of ocular onchocerciasis. 3. Th-2 helper T 
cells in the conjunctiva Ocular Immunol Inflam 
\:1\-11, 1993. 

Fenton RM, Rubin BI, de Smet MD, Whitcup SM, 
Nussenblatt RB: A prospective study of 5-FU 
trabeculectomy vs. single plate Molteno implant 
in patients with panuveitis complicated by glauco- 
ma refractory to prior therapy. Invest Ophthalmol 
Vis Sci 34(4)(suppl):897, 1993. 

Hikita N, Chan C-C, Mochizuki M, Maturi R, Nus- 
senblatt RB, Whitcup SM: Topical FK 506 
inhibits endotoxin-induced uveitis (EIU). Invest 
Ophthalmol Vis Sci 34(4)(suppl):1480, 1993. 

Kasner L, Chan C-C, Whitcup SM, Gery I: The 
paradoxical role of tumor necrosis factor-alpha in 
endotoxin-induced uveitis. Invest Ophthalmol Vis 
Sci 34(4)(suppl):1480, 1993. 

LI Q, Hikita N, Whitcup SM, Nussenblatt RB, Chan 
C-C: Allergic conjunctivitis induced by com- 
pound 48/80 in C57BL/6NCR mice. Invest 
Ophthalmol Vis Sci 34(4)(suppl):857, 1993. 

Li 0. Whitcup SM, Fujino Y, Nussenblatt RB, Chan 
C-C: The role of mast cells in endotoxin-induced 
uveitis. Invest Ophthalmol Vis Sci 34:256-259, 
1993. 

Martin DF, Chan C-C, de Smet MD, Palestine AG, 
Davis JL, Whitcup SM, Burnier M Jr, Nussenblatt 
RB: The role of chorioretinal biopsy in the 
management of posterior uveitis. Ophthalmology 
100:705-714, 1993. 

Whitcup SM, de Smet MD, Rubin BI, Palestine AG, 
Martin DF, Burnier M Jr, Chan C-C, Nussenblatt 
RB: Intraocular lymphoma: Clinical and histo- 
pathologic diagnosis. Ophthalmology, 100:1399- 
1406, 1993. 

Whitcup SM, Fenton RM, Pluda JM, de Smet MD, 
Nussenblatt RB, Chan C-C: Pneumocystis carinii 
and Mycobacterium avium-intracellulare infection 
of the choroid. Retina 12:331-335, 1992. 



100 



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



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



PROJECT NUMBER 



ZOl EY 00269-03 LI 



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

Ocular Toxicity of 2^3^-DideoxyinosiDe (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 LI, NEI 



Others: Robert B. Nussenblatt 
Marc D. de Smet 
Rafael Caruso 



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



Scientific Director 
Visiting Scientist 
Visiting Scientist 



NEI 
LI, NEI 
OGCSB, 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.) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 

Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.4 



PROFESSIONAL; 



0.4 



OTHER: 



0.0 



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.) 

2',3'-Dideoxyinosine (ddl), a purine analog with antiretroviral activity currentiy used to treat patients with 
AIDS (acquired immune' deficiency syndrome), is being used to treat both adults and children in clinical 
protocols at the National Institutes of Health. The purpose of this study is to follow prospectively patients 
treated with ddl for the development of ocular complications secondary to drug toxicity. Ninety-five children 
with symptomatic (CDC class P-2) HIV (human immunodeficiency virus) infection were enrolled in a phase 
I/II study to assess the safety and antiretroviral activity of ddl. Five children developed peripheral atrophy 
of the retinal pigment epithelium during ddl therapy. The two children with the most severe retinal atrophy 
were enrolled in the smdy at the highest dose level studied (540 mg/m^/day). Electro-oculograms were 
abnormal in one of three patients with retinal toxicity who could be tested. A group of 75 adults treated with 
ddl are being followed with periodic fundus examinations and electro-oculograms. During the past year 
similar retinal lesions were found in one adult patient treated with ddl. 



101 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 



Additional Personnel 



Daniel Martin 

Margaret Cheung 
David Parks 
John J. Hooks 



Caroline Percopo 



M.D. Senior Staff Fellow, 

LI, NEI 

M.D. Senior Staff Fellow 

M.D. Senior Staff Fellow 

Ph.D. Head, Section on 

Immunology and 

Virology, LI, NEI 

M.S. Biologist, LI, NEI 



Objectives 

The goal of this study is to monitor patients treated 
with 2',3'-dideoxyinosine (ddl) for the development 
of ocular complications. 

Methods 

Every 3-4 months patients treated with ddl are given 
complete eye examinations, including dilated oph- 
thalmoscopy and fundus photography of any abnor- 
mal retinal findings. Patients treated with the higher 
dosages of ddl also receive periodic electro-oculo- 
grams to assess the electrophysiologic function of the 
retinal pigment epithelium (RPE). 

Major Findings 

1. Five children have now developed peripheral 
atrophy of the RPE during ddl therapy. The lesions 
are scalloped areas of RPE atrophy with hyperpig- 
mented borders. They 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 has remained 
unaffected. During the past year no discrete retinal 
lesions have developed in any other children treated 
with ddl. 



2. One adult patient treated with ddl developed 
progressive, well-circumscribed atrophic retinal 
lesions of the peripheral RPE, similar to those in the 
children. The lesions appeared after 32 months of 
ddl treatment; the total dosage received was 264 g 
(approximately 3.3 g/kg). Adjacent areas of RPE 
mottling also were seen. Visual acuity and electro- 
oculography were normal in this patient. 

Significance to Biomedical Research and the 
Program of the Institute 

ddl is a drug with in vitro and in vivo activity 
against HIV (human immunodeficiency virus) infec- 
tion. One mission of the NEI is to monitor patients 
for the development of ocular toxicity and to assess 
the effect such toxicity has on vision. 

Proposed Course 

We will continue to follow all patients treated with 
ddl at the NIH for signs of ocular manifestations of 
ddl toxicity or HIV infection. We are performing 
serial electro-oculograms in adults treated with ddl. 

NEI Research Program 

Retinal Diseases — ^Photoreceptors and Retinal Pig- 
ment Epithelium 

Publications 

Nguyen B-Y, Shay LE, Wyvill KM, Pluda JM, 
Brawley O, Cohen RB, Whitcup SM, Venzon DJ, 
Broder S, Yarchoan R: A pilot study of sequen- 
tial therapy with zidovudine (AZT) plus acyclo- 
vir,dideoxyinosine, dideoxcytidine in patients with 
severe human immunodeficiency virus infection. 
J Infect Dis 168:810-817, 1993. 



102 



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



PROJECT NUMBER 



ZOl EY 00270-03 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Cell Adhesion Molecules in Ocular Inflammation 



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

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



Others: Chi-Chao Chan 



Robert B. Nussenblatt 



M.D. 



M.D. 



Head, Section on 
Inmiunopathology 
Scientific Director 



LI, NEI 
NEI 



COOPERATING UNITS (if any) 

Biochemical and Molecular Pathology, Merck Sharp & Dohme Research Laboratories (Hugh Rosen, M.D.); 
Immunology Section, Roberts Pharmaceutical Corporation (Ron Haming, Ph.D.); Department of 
Ophthalmology, Kurume University School of Medicine, Fukuoka, Japan (Manabu Mochizuki, M.D.) 

LAB/BRANCH 

Laboratory of Immunology 



SECTION 



Section on Immunopathology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.4 



PROFESSIONAL: 



0.4 



OTHER: 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



[x] (b) Human tissues □ (c) Neitiier 



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

Cell adhesion molecules are surface proteins important for antigen sensitization and the migration of leukocytes to sites 
of inflammation. We are studying the expression of cell adhesion molecules in ocular inflammation, investigating the 
blocking of cell adhesion molecules as a treatment for uveitis and other ocular inflammatory diseases, and examining the 
effect of immunosuppressive agents on cell adhesion molecule expression in eyes with experimental autoimmune uveitis 
(EAU). 

We previously showed that intercellular adhesion molecule 1 (lCAM-1) is expressed in eyes with EAU before the 
infiltration of inflammatory cells. Further experiments showed that monoclonal antibodies against lCAM-1 and its 
counter-receptor lymphocyte function-associated antigen 1 (LFA-1) inhibit EAU development in mice. 

We demonstrated that cell adhesion molecules are important for both antigen sensitization and inflammatory cell 
infiltration into the eye, with additional sets of experiments showing the following: (1) that monoclonal antibodies against 
both lCAM-1 and LFA-1 will prevent inflammatory cell infilti-ation of the eye induced by endotoxin, and importanUy 
that these antibodies can inhibit ocular inflammation even when administered after signs of inflammation have been noted; 
and (2) that monoclonal antibodies against lCAM-1 and LFA-1 can inhibit in vitro proliferation of a uveitogenic cell line 
by interfering with the interaction between lymphocytes and antigen-presenting cells. These results suggest that cell 
adhesion molecules play an important role in the development of uveitis and that blockage of cell adhesion molecules 
may provide a new therapeutic approach for patients with inflammatory eye disease. 

Finally, we examined the effect of inununosuppressive agents on the expression of cell adhesion molecules in animals 
with EAU. Ocular expression of cell adhesion molecules was delayed and downregulated in animals treated with 
corticosteroids and cyclosporine following immunization with retinal S-antigen. Downregulation of cell adhesion 
molecule expression may be one of the mechanisms by which immunosuppressive agents inhibit ocular inflammation. 



103 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEl Annual Report— FY 1993 



Project Description 



Additional Personnel 




Rachel Caspi 


Ph.D. 
NEI 


Visiting Associate, LI, 


Igai Gery 


Ph.D. 


Deputy Chief, LI, NEI 


Qian Li 


M.D. 
NEI 


Visiting Fellow, LI, 



Objectives 

The goal of this project is to examine the role of cell 
adhesion molecules in ocular inflammation. We are 
studying the expression of cell adhesion molecules in 
eyes with uveitis and examining the effect of block- 
ing these adhesion molecules on the development of 
ocular inflammatory disease. We also are investigat- 
ing the role of ceU adhesion molecules in antigen 
sensitization. By blocking cell adhesion molecules 
or preventing the expression of these surface pro- 
teins, we hope to be better able to treat patients with 
ocular inflammatory disease. 

Methods 

Animal models of ocular inflammation. — ^Endotoxin- 
induced uveitis (EIU) is induced by injecting 100 ^g 
of Salmonella typhimurium endotoxin into one 
footpad of a Lewis rat or 200 |ig into one footpad of 
a C3H-hen mouse. Experimental autoimmune uveitis 
(EAU) in mice is induced by immunizing BIO.A 
mice with 50 \ig of interphotoreceptor retinoid- 
binding protein (IRBP) in complete Freund's adju- 
vant, with pertussis toxin injected intraperitoneally. 

Histology and immunohistochemistry of ocular 
inflammation. — ^Enucleated animal eyes and human 
ocular tissue are immediately snap frozen and em- 
bedded in O.C.T.® The expression of cell adhesion 
molecules and the presence of cytokines are then 
assessed by immunohistochemical staining with 
avidin-biotin-peroxidase complex (ABC) on frozen 
sections of ocular tissue. Eyes also are embedded in 
methyl methacrylate, and 4-|jm sections are examined 
for histologic evidence of inflammation. 

Treatment of ocular inflammation by blocking cell 
adhesion molecules. — In an attempt to inhibit the 
development of ocular inflammation, we treated 
animals with infraperitoneal injections of monoclonal 
antibodies against ICAM-1 or LFA-1 before the 
induction of either EIU and EAU. 



Effect of monoclonal antibodies against ICAM-1 
or LFA-1 on cell proliferation of a uveitogenic cell 
line. — Mouse anti-rat ICAM-1 (CD54) monoclonal 
antibody (mAb), designated 1A29, and mouse anti-rat 
LFA-1 (CDl la) mAb, designated WT.l, were kindly 
provided by Dr. Miyasaka (Tokyo, Japan). mAbs 
were incubated with irradiated Lewis rat thymocytes 
(antigen-presenting cells [ APCs]) at concentrations of 
10, 1, 0.1, and ^g/ml for 2 hours. These cells were 
then added to lymphocyte cultures comprised of 
CD4-(- T cells of a highly uveitogenic cell line, 
sensitized against IRBP-derived peptide R15 (se- 
quence 1181-1191), and stimulated with peptide R15 
(1 or 0.01 )iM) or with concanavalin A (con A). The 
cultures, which consisted of 2 x 10^ lymphocytes and 
1 X 10^ or 5 X 10^ APCs in 0.2 ml of medium, were 
processed as previously detailed (Cell Immunol 
122:251, 1989). 

Effect of corticosteroids and cyclosporine A (CsA) 
on the expression of cell adhesion molecules in eyes 
with EAU. — ^EAU was induced in 36 female Lewis 
rats by injecting into one hind footpad 30 MS of 
retinal S-antigen in complete Freund's adjuvant 
containing 0.25 mg of Mycobacterium tuberculosis. 
Rats were then treated with daily intramuscular 
injections of 0.2 mg/kg methylprednisolone (MP), 3 
mg/kg CsA, or olive oil as a confrol. Rats were 
sacrificed 0, 7, 10, 14, 21, and 28 days after immuni- 
zation. Each right eye was processed for routine 
histology, and each left eye was immediately sn£^ 
frozen for immunohistochemical staining, using an 
avidin-biotin-peroxidase technique and primary 
antibodies against ICAM-1 (CD54), LFA-1 alpha 
(CDl la), E-selectin, major histocompatibility com- 
plex (MHC) class n antigens (RTIB and RTID), 
CD4+ T cells (W3/25), and CD8+ T cells (0X8). 
Slides were then graded by two masked observers. 

Major Findings 

1. When treatment was given at the time of 
endotoxin injection, the mean number of inflamma- 
tory cells infiltrating the eye on histologic sections 
was 469.2 ±51.9 (standard error of the mean [SEM]) 
for controls, 13.8 ± 2.6 for rats receiving anti-ICAM- 
1 mAb (p < 0.001), and 195.8 ± 48.8 for rats receiv- 
ing anti-LFA-1 mAb (p < 0.001). When treated after 
the start of inflammatory disease, the mean number 
of infiltrating inflammatory cells ± SEM was 273.0 
± 30.7 for controls, 6.4 ±1.7 for rats receiving anti- 



104 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



ICAM-1 mAb (p < 0.001), and 54.2 ± 7.6 for rats 
receiving anti-LFA-1 mAb (p < 0.001). The mean 
numbers of cells per microliter of aqueous humor 
were 1867.6 ± 321.8 for controls, 21.7 ± 5.3 for rats 
receiving anti-ICAM-1 mAb (p < 0.001), and 295.1 
± 71.2 for rats receiving anti-LFA-1 mAb (p < 
0.001). Treatment with mAbs against ICAM-1 and 
LFA-1 significantly inhibited the development of 
EIU and was effective in treating clinically evident 
ocular inflammatory disease. 

2. In mice with EAU, ocular inflammation, 
graded clinically by examination of the fundus 14 
and 21 days after immunization, was significantly 
decreased in animals treated with anti-ICAM-1 (p < 
0.01 at days 14 and 21) and with anti-LFA-1 anti- 
body (p < 0.01 at days 14 and 21). 

3. In cultures containing 5 x 10^ APCs, anti- 
LFA-1 mAb (10 ng/ml) inhibited the lymphocyte 
response to 1 and 0.01 \M of R15 by 58% and 74%, 
respectively. mAbs against ICAM-1 were less 
inhibitory, reducing the responses to R15 by 23% 
and 30% for doses of anti-LFA and R15 mAb, 
respectively. Decreasing the APC concentration had 
little effect on the antibody activity, while decreasing 
the mAb concentration to <1 jig/ml almost complete- 
ly eliminated their inhibitory cs^acity. mAbs against 
LFA-1 and ICAM-1 inhibited the interaction between 
APCs and lymphocytes of a uveitogenic cell line. 
We propose that this activity plays a major role in 
the inhibition of EAU in animals treated with mAbs 
against LFA-1 and ICAM-1. 

4. By 14 days after inununization, ICAM-1, E- 
selecfin, and MHC class II antigens were strongly 
expressed on the vascular endothelium of the iris, 
ciliary body, choroid, and retina of control rats; 
infiltrating lymphocytes expressing LFA-1 also were 
noted in these eyes. In contrast, 28 days after 
immunization, rats treated with MP and CsA still had 
only mild expression of ICAM-1, E-selectin, and 
MHC class II antigens, and few infiltrating lympho- 
cytes were noted on histologic sections. Ocular 
expression of cell adhesion molecules was delayed 
and down-regulated in animals treated with MP and 
CsA following immunization with S-antigen. Ex- 
pression of class II antigens and infiltration with 
inflammatory cells also were diminished in eyes with 
decreased expression of cell adhesion molecules. 
Down-regulation of cell adhesion molecule expres- 
sion may be one of the mechanisms by which 
corticosteroids and CsA inhibit ocular inflammation. 



Significance to Biomedical Research and the 
Program of the Institute 

One major mission of the NEI is to understand the 
mechanisms of sight-threatening eye diseases so that 
new and effective therapies can be developed. The 
expression of cell adhesion molecules appears to be 
a fundamental mechanism in the development of 
intraocular inflammation. With this understanding, 
we hope to develop new anti-inflammatory therapy 
for ocular inflammation, which accounts for approxi- 
mately 10% of the visual impairment in the United 
States. 

Proposed Course 

We plan to continue our experiments on the expres- 
sion of cell adhesion molecules in eyes with ocular 
inflammatory diseases, including uveitis, corneal 
disease, and uveitic glaucoma. We are examining 
the roles of additional cell adhesion molecules such 
as VCAM-1 and VLA-4 in ocular inflammation. In 
addition, we plan to study the pharmacokinetics of 
antibodies against cell adhesion molecules, adminis- 
tered topically or intraocularly, to determine the 
feasibility of local therapy. 

NEI Research Program 

Retinal Diseases — Inflammatory Diseases 

Publications 

Lai JC, Chan C-C, LI Q, Whitcup SM: Treatinent 
with corticosteroids and cyclosporine A inhibits 
the expression of cell adhesion molecules in 
experimental autoimmune uveitis (EAU). Invest 
Ophthalmol Vis Sci 34(4)(suppl):1206, 1993. 

Vistica B, Gery I, Chan C-C, Nussenblatt RB, Whit- 
cup SM: Anti-ICAM-1 and anti-LFA-1 mono- 
clonal antibodies (mAbs) inhibit in vitro prolifera- 
tion of a uveitogenic cell line. Invest Ophthalmol 
Vis Sci 34(4)(suppl):1144, 1993. 

Whitcup SM, DeBarge LR, Caspi RR, Haming R, 
Nussenblatt RB, Chan C-C: Monoclonal anti- 
bodies against ICAM-1 (CD54) and LFA-1 
(CDlla/CD18) inhibit experimental autoimmune 
uveitis. Clin Immunol Immunopathol 67: 143-150, 
1993. 

Whitcup SM, DeBarge LR, Rosen H, Nussenblatt 
RB, Chan C-C: Monoclonal antibody against 
CDllb/CD18 inhibits endotoxin-induced uveitis. 
Invest Ophthalmol Vis Sci 34:673-681, 1993. 



105 



Laboratory of Immunology NEI Annual Report — FY 1993 

Whitcup SM, Hikita N, Shirao M, Mochizuki M, Whitcup SM, Nussenblatt RB, Price FW Jr, Chan 

NussenblattRB, Chan C-C: Effect of monoclonal C-C: Expression of cell adhesion molecules in 

antibodies against ICAM-1 (CD54) and LFA-1 corneal graft failure. Cornea, 12:475-480, 1993. 
alpha (CDl la) in the prevention and treatment of 
endotoxin-induced uveitis (EIU). Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1143, 1993. 



106 



DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00184-11 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Cellular and Immunogenetic Mechanisms in Uveitis 



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

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

Acting Head, Section on 
' Immunoregulation 

Others: Phyllis Silver 
Luiz Rizzo 
Chj-Chao Chan 



B.S. 


Biologist 


LLNEI 


M.D. 


Visiting Associate 


LI, NEI 


M.D. 


Head, Section on 
Immunopathology 


LI, NEI 



COOPERATING UNITS (If any) 

Laboratory of Immunobiology, Rega Instituut, Katholjeke Universiteit, Leuwen, Belgium (A. Billiau, M.D.; H- Heremans, Ph.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, WiUs Eye Hospital, 
Philadelphia, PA (Larry A. Donoso, M.D., Ph.D.) - 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 

Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



2.6 



PROFESSIONAL: 



2.6 



OTHER: 



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.) 

Cellular mechanisms in ocular immunologically mediated disease are being studied in animal models of 
experimental autoimmune uveoretinitis (EAU). Rats and mice are immunized with retinal -derived antigens, 
or synthetic peptides representing fragments of these antigens, to induce EAU. Susceptibility to disease is 
being evaluated in various strains of known genetic makeup in the hope of delineating the hereditary 
mechanisms that predispose to uveitis. EAU in rats and mice serves as a template for the evaluation of new 
drugs and compoimds as well as for the study and characterization of the participating cells and their factors. 
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 
studied to develop strategies for in vivo targeting of the autoimmune cells. The goal of these studies is 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. 



107 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 

Additional Personnel 

Robert B. Nussenblatt M.D. Chief, LI, NEI 
Charles E. Egwuagu Ph.D. StaffFellow,LI,NEI 
Igal Gery Ph.D. Head, Section on 

ExperimentaJ 
Immunology, LI, 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 
involved in ocular autoimmunity in a general sense. 
Experimental autoimmune 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 separate and selectively 
grow various T-cell subsets. The goals are (1) to 
continue to establish and characterize the murine 
EAU model because the mouse offers some impor- 
tant advantages over other rodents as a model of 
EAU; (2) to use the EAU model in rodents for the 
study of cellular mechanisms in ocular autoimmuni- 
ty; this is done in large part by establishing and 
using retinal antigen-specific T-cell lines and clones, 
permitting us to identify and characterize cells 
capable of ocular immunomodulation, learn about 
migration and localization of autoimmune lympho- 
cytes, and study their interactions with other lym- 
phoid and nonlymphoid cells in eliciting effector 
mechanisms; (3) to use tiie EAU model as a template 
for the development of immunotherapeutic jqjproach- 
es designed to target autoimmune lymphocytes 
directly or to disrupt specific stages in the autoim- 
mune inflammatory cascade; and (4) to use the 
murine EAU model for the study of various genetic 
mechanisms controlling susceptibility to ocular 
autoimmune disease. The study and understanding 
of these parameters will help not only in the devel- 
opment of new therapies but possibly in the preven- 
tion of ocular disease. 

Methods 

Rats and mice of various strains are inmiunized with 
purified S-antigen (S-Ag) or interphotoreceptor 
retinoid-binding protein (IRBP) in complete Freund's 



adjuvant or with various pathogenic peptides derived 
from these proteins. After disease development, eyes 
are processed for histopathology and examined for 
disease, and lymphoid cells are taken from the blood, 
lymph nodes, or eyes. Cells thus obtained are placed 
in culture either with mitogen or with the retinal 
antigen with which the donor animal was immunized. 
Responses of the immune cells are studied. 

Cells also are expanded in culture and used in 
attempts to transfer EAU to nonimmune animals in 
order to find out tiie cell population responsible for 
disease induction. Long-term cell lines are devel- 
oped and in some cases are cloned by either the soft 
agar bilayer or tiie limiting dilution technique. These 
lines or clones are tiien tested for functional charac- 
teristics such as the ability to induce ocular disease, 
production of soluble mediators, expression of 
various cell surface molecules, response to therapeu- 
tic agents, and interactions with other cells in culture. 

Major Findings 

We previously had studied the importance of "non- 
specific" T-cell recriiitment in the immunopathogene- 
sis of uveitis by using congenitally athymic Lewis 
rats (LEW.mu/mu), which are deficient in functional 
endogenous T cells but are otherwise syngeneic with 
the euthymic Lewis rats that develop characteristical- 
ly severe EAU. The uveitogenic stimulus was 
delivered in the form of phenotypically and function- 
ally homogeneous pathogenic T-cell lines specific to 
the major pathogenic epitope of either the intracellu- 
lar photoreceptor protein, S-Ag, or the extracellular 
photoreceptor matiix protein, IRBP. Previous data 
indicated that, depending on the T-cell line used, 
EAU in athymic rats was either drastically reduced 
in severity or absent Susceptibility was restored 
when the athymic animals were reconstituted with 
immunocompetent T cells from syngeneic euthymic 
donors. 

We now have shown that the severity of inflam- 
mation and tissue damage are correlated with the 
proportion of lymphocytes in the intraocular infil- 
ti^ate: The infiltrate in euthymic rats was predomi- 
nantly lymphocytic, with smaller numbers of mono- 
cyte-macrophages and even fewer neuti-ophils, 
whereas the sparse infiltrate in athymics was largely 
monocytic and had a relatively high proportion of 
neutrophils and eosinophils. Reconstituted animals 
had an intermediate histological picture with respect 



108 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



to the infiltrating cell types and disease severity. 
Our results indicate that recruited nonspecific T cells 
play a major role in the pathogenesis of disease. 

Furthermore, the data suggest that the extent of 
dependence on recruitment may be influenced by the 
antigenic specificity of the T-cell line and could be 
connected to the "accessibility" of the target antigen 
in vivo. This is the first direct demonstration that 
recruitable "nonspecific" T lymphocytes are neces- 
sary for the expression of the disease itself. 

In collaboration with Dr. Charles Egwuagu, we 
are continuing to study the T-cell receptor (TCR) 
genes of these lines and clones on the molecular 
level. The data indicate that TCR variable-region 
gene usage in uveitis differs from that reported for 
some other autoimmune diseases and may be more 
heterogeneous. We currently are studying TCR 
expression in the inflamed eyes of Lewis rats immu- 
nized with various retinal antigens or adoptively 
transferred with T-cell lines of the appropriate 
specificity. 

Because our previous findings with athymic rats 
suggest that the majority of lymphocytes infiltrating 
the eye are likely to be recruited cells, we are look- 
ing at the earliest stages of disease induction as well 
as at cells that infiltrate the eye in athymic nude rats 
injected with pathogenic T cells. The results appear 
to confirm our previous findings: TCR Vp8.2 may be 
a pathogenic clonotype inS-Ag-EAU, whereas TCR 
vp8.3 may be one of the pathogenic clonotypes in 
IRBP-EAU. The results also indicate tiiat additional 
clonotypes such as Vpl4 may be involved. These 
findings could impact on the development of thera- 
peutic strategies designed to specifically target the 
pathogenic cells through their T-cell receptors. 

In the mouse model of EAU, we have developed 
a pathogenic T-cell line specific to the whole IRBP 
molecule in the BIO.A strain of mice (I-A"). The line 
was developed from draining lymph nodes of IRBP- 
immunized mice using a protocol similar to that used 
for the derivation of uveitogenic T-cell lines in the 
rat (alternating cycles of stimulation with antigen and 
expansion in interleukin 2). After the fourth weekly 
stimulation with IRBP, we tested the line for patho- 
genicity and found that it induced uveitis at 5 x 10* 
cells per mouse. The line elaborated an unrestricted 
lymphokine profile, suggesting that both Thl-type 
and Th2-type cells were present 



With continued stimulations in culture, the cell 
line became progressively more pathogenic. After 16 
cycles the cell line was pathogenic at cell numbers as 
low as 10^ cells per mouse. The TCR profile of the 
line also changed wdth time in culture, with progres- 
sive enrichment in Vp8.2 and VP6 TCR -expressing 
cells (64% and 16%, respectively), suggesting that 
vp8.2 and Vp6 may represent pathogenic clonotypes 
in IRBP-EAU in the BIO.A mouse. The line current- 
ly is being cloned to test this hypothesis and to 
further characterize the pathogenic cells with respect 
to their Thl-like or Th2-like identity. Another 
IRBP-specific pathogenic T-cell line was developed, 
using a similar cultiu-e protocol, from eyes of uveitic 
BIO.A mice. The line was pathogenic at 5 x 10* 
cells per mouse. These results suggest that the 
pathogenic cells infiltrate and are physically present 
in the eye during uveitis. 

In all animal species, as well as in humans, the 
genetic makeup of an individual determines the 
regions of the uveitogenic protein molecule that 
evoke an autoimmune response. It is important to 
study the nature of these epitopes and their relation 
to different major histocompatibility complex types 
because the findings could potentially be extri^lated 
to the situation in humans. In collaboration with Dr. 
Larry Donoso (Wills Eye Hospital, Philadelphia), 
who synthesized overlapping peptides representing 
the entire sequence of the human IRBP molecule, we 
are engaged in an ongoing effort to identify epitopes 
that are pathogenic in the three previously identified 
susceptible mouse H-2 haplotypes, namely, H-2'', 
H-2', and H-2\ The peptides are being systematical- 
ly screened by immunizing animals from strains 
representing the three susceptible haplotypes. Pep- 
tides that cause EAU are being studied further with 
respect to the identity of the minimal pathogenic 
sequence, immunodominance, and the fine specificity 
of the response. Pathogenic T-cell lines are raised to 
these peptides, and their TCR usage is studied. 

Peptide LRHNPGGPSS AVPLLLSYFQ, represent- 
ing a highly conserved sequence in the IRBP mole- 
cule and sparming amino acids 461^80, was found 
to be pathogenic in C57BL/10, but not in the other 
mouse strains. The disease scores obtained with the 
peptide (50-250 |ig) were lower than those obtained 
with tiie whole IRBP molecule (50-100 ^ig). A 
lymphocyte line specific to the peptide was able to 



109 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



adoptively transfer low-grade uveitis to syngeneic 
recipients. Mice immunized with the peptide, or 
with whole IRBP, had positive DTH to the immuniz- 
ing, but not to the reciprocal, antigen. Lymphocytes 
of IRBP-immunized mice did not proliferate in vitro 
in response to the peptide; however, positive lympho- 
cyte responses to IRBP could sometimes be detected 
in peptide-immunized mice and the peptide-specific 
lymphocyte line proliferated in vitro to IRBP. Thus, 
peptide 461-480 appears to contain an epitope 
pathogenic to mice of the H-2'', but not H-2'' or H-2', 
haplotypes. The low pathogenicity of peptide 461- 
480 in comparison to that of whole IRBP, as well as 
its lack of immunological recognition by IRBP- 
immunized mice, in vivo or in vitro, suggests that it 
may be a minor, immunologically nondominant 
epitope. This is the first uveitogenic epitope de- 
scribed for the mouse EAU model. Several addition- 
al sites, pathogenic for the other haplotypes, have 
been tentatively identified and are being studied. 

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 identificaUon and character- 
ization of the cells involved in ocular autoimmunity, 
and of their functions, will provide new understand- 
ing of inflammatory ocular diseases. Successful 
immunomodulation of EAU in animal models usually 
has served as a good predictor of the clinical success 
of a given therapeutic modality. Continued study of 
basic mechanisms involved in the immunopathogene- 
sis of uveitis in animal models will aid in the devel- 
opment of novel immunotherapeutic approaches for 
the control of uveitis in humans. 

Proposed Course 

This project will continue so that more information 
about the basic mechanisms in experimental uveitis 
may be obtained. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 



Publications 

Caspi RR. Experimental autoimmune uveoretinitis in 
rats and mice, in Cohen I, Miller A (eds): Guide- 
hook to Animal Models for Autoimmune Diseases. 
Academic Press, in press. 

Caspi RR: Immunogenetic aspects of cUnical and 
experimental uveitis. Reg Immunol 4:321-330, 
1992. 

Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover 
S, Hansen CT, Wilder RL: Recruitment of naive 
T cells plays a pivotal role in the pathogenesis of 
experimental autoimmune uveoretinitis. Invest 
Ophthalmol Vis Sci 34(4)(suppl):902, 1993. 

Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover 
S, Hansen CB, Wilder RL: Recruitment of 
antigen-nonspecific cells plays a pivotal role in 
the pathogenesis of a T-cell-mediated organ- 
specific autoimmune disease, experimental 
autoimmune uveoretinitis. J Neuroimmunol 
47:177-188, 1993. 

Caspi R, Nussenblatt R: Natural and therapeutic 
control of ocular autoimmunity — rodent and man, 
in Kazatchkine M, Roitt I (eds): Autoimmunity. 
Wiley and Sons, Inc, in press. 

Egwoiagu CE, Mahdi RM, Gery I, Nussenblatt RB, 
Caspi RR: Evidence for selective accumulation 
of VP8+ T lymphocytes in experimental auto- 
immune uveoretinitis induced by two different 
retinal antigens. J Immunol 151:1627-1636, 
1993. 

Mahdi RM, Caspi RR, Nussenblatt RB, Gery I, 
Egwuagu CE: Selective accumulation of Vp8-i- T 
lymphocytes in EAU. Invest Ophthalmol Vis Sci 
34(4)(suppl):1144, 1993. 

Rizzo LV, Silver PB, Hakim F, Chan C-C, Wiggert 
B, Caspi RR: Establishment and characterization 
of an IRBP-specific T-cell line that induces EAU 
in BIO. A mice. Invest Ophthalmol Vis Sci 34(4) 
(suppl):1143, 1993. 

Silver PB, Rizzo LV, Chan C-C, Donoso LA, Wig- 
gert. B, Caspi RR: Identification of a putative 
epitope in the IRBP molecule that is uveitogenic 
for mice of the H-2'' haplotype. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1482, 1993. 



110 



NEI Annual Report-FY 1993 Laboratory of Immunology 

Whitcup SM, DeBarge LR, Caspi RR, Haming R, 
Nussenblatt RB, Chan C-C: Monoclonal anti- 
bodies against ICAM-1 (CD54) and LFA-1 
(CD 11 a/CD 18) inhibit experimental autoimmune 
uveitis. Clin Immunol Immunopathol 67:143-150, 
1993. 



Ill 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00218-08 LI 



PERIOD COVERED 

October 1. 1992 to September 30, 1993 



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

Ocular Manifestations of the Acquired Immune Deficiency Syndrome 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator. J (Name, title, laboratory, and institute affiliation) 
PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI 

Others: 



Roben B. Nussenblan 


M.D. 


Scott Whitcup 


M.D. 


Margaret Cheung 


M.D. 


David Parks 


M.D. 


Dan Martin 


M.D. 


Francois Roberge 


M.D. 


Chi-Chao Chan 


M.D. 



Scientific Director 


NEI 


Senior Staff Fellow 


LI. NEI 


Senior Staff Fellow 


LI. NEI 


Senior Staff Fellow 


LI, NEI 


Senior Staff FeUow 


LI, NEI 


Visiting Scientist 


LI. NEI 


Head, Section on 


LI, NEI 


Immunopatbology 





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 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS; 



2.0 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 
[x] (a1) Minors 
□ (a2) Interviews 



PROFESSIONAL: 



2.0 



OTHER: 



0.0 



[x] (b) Human tissues □ (c) Neither 



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

Patients suffering from AIDS (acquired immune deficiency syndrome) are at risk of developing significant 
ocular problems, either as a result of HIV (human immunodeficiency 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 1993 the two major 
emphases have been (1) CMV retinitis detection and therapy and (2) pediatric AIDS. All currently available 
drugs are virostatic. Early detection is the most effective means of ensuring that pafients will preserve long- 
term vision. We have continued to evaluate the useftilness of a laser photometric device to help screen patients 
for the presence of ocular inflammation and/or CMV retinitis. We also have looked at the use of alternative 
methods of followup using tangent saeens and Amsler grids to help determine early recurrences. We have 
initiated a study using an implantable slow-release device for ganciclovir. This study is still in its early stages. 
Due to the sustained nature of the release, it is possible that this approach will lead to prolonged remissions, 
as compared to standard therapy. 

In FY 1993 we continued to evaluate the incidence of ocular infection in about 220 children with AIDS. The 
incidence of complications is rare, which has prompted us to reduce the frequency of foUowups to every 6 
months instead of every 4 months. However, it is still important that parents or guardians monitor their 
AIDS-affected children for symptoms and signs of visual loss. 



112 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Additional Personnel 

Susan Mellow R.N. 

Clinical Protocol Number 

90-EI-208 

Objectives 

This project's primary objective is to develop meth- 
ods of identifying and treating known ocular compli- 
cations of HIV (human immunodeficiency virus) 
infection in such a way as to prevent significant loss 
of vision. The second, equally important objective 
is the identification of new manifestations of ocular 
involvement from the AIDS (acquired immune 
deficiency syndrome) virus itself and related oppor- 
tunistic ocular infections. The third objective is to 
recognize complications related to the therapeutic 
agents used or the mode of administration. 

Methods 

This project entails the clinical evaluation, diagnosis, 
and treatment of retinitis in AIDS patients. It also 
involves the development of novel methods of 
ther^y for the various forms of retinitis observed. 
Study of pathologic 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 
evaluation and treatment of cytomegalovirus (CMV) 
retinitis. CMV is a major vision-threatening infec- 
tion found in patients with AIDS. Preservation of 
useful vision for a prolonged period of time requires 
early detection. In the present context, this can only 
be done by careful funduscopic examination by a 
trained eye care professional. In asymptomatic 
individuals, such exams are usually normal. Exami- 
nations in these patients are both time consuming 
and costiy, with very few having any ocular lesions. 
The development of a screening device that could 
help to detect, within the eye, conditions predispos- 
ing to CMV retinitis and other disorders is highly 
desirable. 

A device capable of detecting even minute fluctu- 
ations in the anterior chamber flare has been evalu- 
ated over the past 2 years. It consists of a low- 



intensity laser beam focused on the anterior chamber 
of the eye. Under normal circumstances, none of the 
incident light is reflected, as there is neither protein 
nor cells in the anterior chamber. In the presence of 
inflammation, part of the laser beam is reflected back 
through the cornea to a detector that measures the 
intensity of the reflected light in photons per milli- 
second. The intensity of this reflected light corre- 
lates directiy with the intensity of inflammation in an 
affected eye. On average the test itself takes only 15 
minutes for both eyes, and it is simple enough that it 
can be performed in a nonophthalmic cUnic. We 
recentiy have evaluated data from 80 patients with 
and without CMV retinitis who were subjected to 
this test We found that the technique was 100% 
sensitive in detecting patients with CMV retinitis 
when the cutoff count was 8.0 photons per milli- 
second; the corresponding specificity was 77%. 

Early detection can lead to preservation of vision, 
but it also commits the patient to life-long intra- 
venous (IV) therapy with anti-CMV drugs. A device 
recentiy has been developed that slowly releases 
ganciclovir directiy into the eye. This alternative to 
systemic therj^jy may be particularly useful in 
patients who cannot tolerate IV infusions of antiviral 
drugs. We have developed a protocol to evaluate the 
safety and efficacy of the device in patients who 
have not been treated previously with an anti-CMV 
agent. The protocol is designed to compare the rates 
of progression between patients in whom ther^y is 
delayed with the rates in patients implanted with a 
device that releases the drug over 8 months. Only 
patients with peripheral non-sight-threatening disease 
are ehgible for the study. One important concern has 
been the lack of systemic coverage and its possible 
effects on patient survival. While the study is not 
designed to answer this question, patients will be 
followed carefully to determine whether this form of 
therapy has any adverse effect on their survival. 
Lack of systemic side effects from anti-CMV therapy 
and the patient's ability to stay on anti-HIV agents 
may in fact be of greater benefit to survival. So far, 
a dozen patients have been recruited out of a total of 
35. 

We have continued to follow about 200 children 
who developed AIDS by various means. We have 
been particularly struck by the lower incidence of 
CMV in this population, where the overall incidence 
is about 1.6%. However, ia children who have low 
total T-cell counts (below 100/mm^), the risk in- 



113 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



creases to 16%. By following children every 6 
months, we have been able to detect all cases of 
ocular involvement, provided that the children's 
parents or guardians are periodically screened for the 
presence of vision loss. 

Significance to Biomedical Research and the 
Program of the Institute 

The AIDS epidemic is a major public health concern. 
CMV retinitis remains the number one cause of 
blindness among patients infected with the AIDS 
virus. Early diagnosis is important because all drugs 
currently available are only virostatic and not viro- 
cidal; thus, some progression of the lesion is seen in 
more than 50% of patients, despite anti-CMV ther- 
apy. Inasmuch as most patients present late with 
well-established disease, a device able to screen and 
identify patients with early lesions is highly desir- 
able. New therapeutic modalities that are cost- 
effective and reduce the incidence of progression or 
the development of resistant strains are necessary. 
Reports of strains resistant to gancyclovir are increas- 
ing in number. 

The number of children infected with AIDS is on 
the rise. A good understanding of the epidemiology 
of AIDS in terms of ocular disease is highly desir- 
able. We are therefore continuing to follow these 
children prospectively to identify the frequency and 
type(s) of ocular complications they are likely to 
develop. 

Proposed Course 

In the coming fiscal year we plan to evaluate further 
the laser photometer to determine possible ways of 
increasing the specificity of the device in detecting 
CMV retinitis. We will continue to evaluate the 
slow-release device in patients with newly diagnosed 
CMV retinitis. We also are plarming to use new 
therapeutic agents for the treatment of CMV retinitis. 



NEI Research Program 

Retina] and Choroidal Diseases — Inflanmiatory 
Disorders 

Publications 

de Smet MD: Ocular consequences of human inmiu- 
nodeficiency virus infection. Ophthalmol Clin 
North Am 6:117-126, 1993. 

de Smet MD, Butler KM, Rubm BI, Whitcup SM, 
DeBarge LR, Martin DF, Pizzo PA, Nussenblatt 
RB: The ocular complications of HIV in the 
pediatric population, in Demouchamps JP, 
Verougstraete C, Capsers-Velu L, Tassignon MJ 
(eds): Recent Advances in Uveitis, Proceedings 
of the Third International Symposium on Uveitis. 
New York, Kugler Publications, 1993, pp 
315-319. 

Muccioli M, Belfort R, Podgor M, Sampaio P, 
Hayashi S, Neves R, Lottemberg C, Kim MK, de 
Smet M, Nussenblatt RB: The diagnosis of 
intraocular inflammation and CMV retinitis in 
HIV infected patients by laser flare photometry. 
Invest Ophthalmol Vis Sci 34(4)(suppl):1110, 
1993. 

Polls MA, de Smet MD, Baird BF, Mellow S, 
Falloon J, Davey RT, Kovacs JA, Palestine AG, 
Nussenblatt RB, Masur H, Lane HC: Increased 
survival of a cohort of patients with acquired 
immimodeficiency syndrome and cytomegalovirus 
retinitis who received sodium phosphonoformate 
(foscamet). Am J Med 94:115-1^0, 1993. 

Whitcup SM, Fenton RM, Pluda JM, de Smet MD, 
Nussenblatt RB, Chan C-C: Pneumocystis carinii 
and Mycobacterium avium-intracellulare infection 
of the choroid. Retina 12:331-335, 1992. 



114 



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



PROJECT NUMBER 



ZOl EY 00266-04 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Characterization of Immune Responses to S- Antigen 



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 
Margaret Cheung 
Fran90is Roberge 



Ph.D. 

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



Head, Section on LI, NEI 
Experimental Immunology 

Scientific Director NEI 

Senior Staff Fellow LI, NEI 

Visiting Scientist LI, NEI 



COOPERATING UNITS (it any) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI, NTH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.6 



PROFESSIONAL: 



0.6 



OTHER: 



0.0 



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.) 

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 ATCs, results 
in the production of small polypeptide subunits. These small polypeptides must then be protected from further 
degra£lation and transported to the cell surface where the interaction with the T cell takes place. 

In FY 1993 we identified an intracellular protein that is capable of binding a major epitope of IRBP. It was 
identified first in rat B cells, which are good antigen-presenting cells. This binding protein qjpears to belong 
to the heat shock family of proteins, and its production appears to be upregulated under conditions of cellular 
stress. These stresses can be exogenous, such as heat, or they can result from more physiologic stresses, such 
as stimulation by lectins and bacterial cell wall products. This protein appears to be present not only in animal 
cells but also can be detected in human B cells. 



115 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 



Additional Personnel 

Sumeet Mainigi 

Kalpana Rengerajan Ph.D. 

Gerald J. Chader Ph.D. 

Barbara Wiggert Ph.D. 



Biologist, LI, NEI 

Biologist, LRCMB. 

NEI 

Chief, LRCMB, NEI 

Head, Section on 

Biochemistry, 

LRCMB, NEI 



Clinical Protocol Numbers 

84-EI-214 
79-EI-49 

Objectives 

In Fiscal Year (FY) 1993 the study has concentrated 
mainly on identification and isolation of the intracel- 
lular binding proteins involved in preventing degra- 
dation of key immunopathogenic epitopes of inter- 
photoreceptor retinoid-binding protein (IRBP) and S- 
antigen (S-Ag) within antigen-presenting cells of 
Lewis rats and humans. 

Methods 

Using B-cell lysate, we isolated the intracellular 
binding protein for R-15 (1169-1191), a major 
immunopathogenic epitope of IRBP, on a cyanogen 
bromide Sepharose 4b column. Because this particu- 
lar protein is produced in very small amounts within 
cells, large numbers of B cells were needed. In rats, 
these were first obtained by panning, but to increase 
the yield and the purity of the cell population, we 
used a magnetic separator with paramagnetic beads. 
The efficiency of the separation was greatly in- 
creased by this approach, and it required much less 
time. This approach also ensured a highly viable 
population of cells, which appeared to be much more 
responsive to physiologic stressors than those ob- 
tained by paiming. 

R-15 also has been shown to cause an immune 
resptonse in peripheral blood lymphocytes of some 
patients with uveitis; thus, an attempt was made to 
identify and isolate a similar intracellular binding 
protein in human antigen-presenting cells — namely, 
the B cell. To produce large quantities of B cells, 
we used the Epstein-Barr virus (EBV) to transform 



peripheral blood lymphocytes from patients and 
normal controls. EBV causes B cells to proliferate 
indefinitely. Although these cells are infected with 
a virus and are perpetually in a blastic phase, their 
antigen-presenting capabilities are not affected. Once 
transformed, these cells can be grown in very high 
numbers in a variety of cell growth systems. A 
combination of stirrer flasks, tissue culture flasks, 
and cell factories was found to be the most efficient 
way of growing these cells in sufficient numbers. 
Once the appropriate cell number was obtained, we 
subjected the cells to specific physiologic stressors to 
increase the production of the cellular binding 
protein. 

Major Findings 

Our previous studies in the Lewis rat have shown 
that several fi-agments of S-Ag are able to induce a 
strong immune response when tested in vitro. These 
fragments are normally produced by endocytic 
enzyme degradation of a parent protein such as S-Ag 
or IRBP. Partially degraded fi-agments must then be 
protected firom further degradation and transported to 
the cell surface, where they can associate with class 
II antigens. Recently it has been suggested that 
proteins belonging to the heat shock family of 
proteins might play a role in preventing enzymatic 
degradation and in carrying antigens to the cell 
surface. In FY 1993 we showed that antigen-pre- 
senting cells contain a protein that is able to bind to 
the immunodominant determinant of IRBP (sequence 
1169-1191). This pepfide-bihding protein has a 
molecular weight similar to that of other heat shock 
proteins (HSP). By Western blot, it stains positively 
to monoclonal antibodies directed against the consti- 
tutive and inducible forms of HSP70. Binding does 
not appear to occur to all peptide firagments of IRBP, 
as shown in some preliminary experiments using 
Sepharose columns activated with different epitopes 
of IRBP. We also have isolated a similar protein 
from transformed human B cells. This protein has 
the same molecular weight and staining characteris- 
tics as the protein isolated from the rat cells. In both 
cell types, the protein is produced in larger amounts 
when the cell is activated by exogenous stress (heat) 
or by agents such as lipopolysaccharide. In addition 
to the 70-kD protein, there appears to be a secondary 
peak at 40-kD that is nearly always present. Its 
exact nature and role remain to be determined. 



116 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Significance to Biomedical Research and the 
Program of the Institute 

Intracellular processing is a crucial step in the 
generation of an immune response. These studies 
suggest that certain intracellular proteins may play a 
determining role in the selection of the peptidic 
determinants that are ultimately presented at the cell 
surface. In addition, exogenous and endogenous 
factors appear to regulate the synthesis of these 
proteins. Identification of these intracellular proteins 
and the mechanisms that regulate their synthesis may 
give us further insights on the mechanisms of antigen 
presentation and possibly a means of regulating 
aberrant antigen presentation. 

Proposed Course 

In the coming year the main emphasis will be on 
further characterization of the intracellular binding 
protein. We will attempt to determine the binding 
characteristics of the protein and to determine the 
factors that can enhance its synthesis. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

de Kozak Y, Mirshahi M, Stiemer R, de Smet M, 
Frank R, Faure JP: Modulation of S-antigen 



induced EAU by neonatal injection of peptides 
from S-Ag or TNF-a or by anti-idiotypic anti- 
body. Exp Eye Res 55(suppl 1):S84, 1992. 

de Kozak Y, Stiemer RH, Mirshahi M, Frank RW, 
de Smet M, Faure JP: Humoral immune response 
against S-antigen/TNF-alpha common epitope in 
rat EAU suppressed by the monoclonal antibody 
S2D2. CurrEyeRes ll(suppl):119-127, 1992. 

de Smet MD, Mainigi S, Nussenblatt RB: Immuno- 
genicity and immunopathogenicity of peptide 
determinants of human S-Ag in various rat 
strains. Invest Ophthalmol Vis Sci 34(4)(suppl): 
1143, 1993. 

Rengarajan K, de Smet MD, Chader GJ, Wiggert B: 
B cells in Behcet patients contain a heat shock 
protein that binds to a fragment of IRBP. Clini- 
cal Immunology Society, Denver, CO, 1993, 
p72A. 

Rengarajan K, de Smet MD, Chader GJ, Wiggert B: 
Identification of a heat shock protein that binds to 
a peptide causing autoimmune uveitis. Keystone 
Meeting on Molecular Chaperones: Function in 
Protein Folding and Cellular Metabolism. Key- 
stone, CO, October 1992. 

Rengarajan K, de Smet MD, Chader GJ, Wiggert B: 
Identification of a heat shock protein that binds to 
peptide 1169-1191 of IRBP causing autoimmune 
uveitis. Invest Ophthalmol Vis Sci 34(4)(suppl): 
1482, 1993. 



117 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00276-02 LI 



PERIOD COVERED 



October 1, 1992 to September 30, 1993 



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

Surgical Management of Uveitis 



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



PI: 
Others: 



Marc D. de Smet 

Francois Roberge 
Margaret Cheung 
Scott Whitcup 
David Parks 
Dan Martin 
David Callanan 
Naofurm Hikita 
Ray DeBarge 
Richard Feoton 



M.D. 


Visiting Scientist 


LI, ^fEI 


M.D. 


Visiting Scientist 


LI, NEI 


M.D. 


Senior Staff Fellow 


LI, NEI 


M.D. 


Senior Staff Fellow 


LI, NEI 


M.D. 


Senior Staff Fellow 


LI, NEI 


M.D. 


Senior Staff Fellow 


LLNEI 


M.D. 


Senior Staff Fellow 


LI, NEI 


M.D. 


Visiting Associate 


LLNEI 


M.D. 


Senior Staff Fellow 


LI. NEI 


M.D. 


Senior Staff Fellow 


LLNEI 



COOPERATING UNITS (It any) 

Clinical Oncology Program, Medicine Branch, National Cancer Institute (Robert Wittes, M.D.) 



LAB/BRANCH 

Laboratory of Immunology 



SECTION 

Section on Inamunoregulation 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.75 



PROFESSIONAL: 



1.75 



OTHER: 



0.0 



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.) 

Patients with uveitis often develop ocular complications that require surgery to prevent permanent loss of 
vision. Surgery in these patients has been particularly challenging because the surgery itself can induce severe 
inflammation. The exact timing of the surgery and the choice of postoperative immunosuppressive therapy 
given to the patient often determine the outcome. Proper handling of the specimen is essential, particularly 
in cases of intraocular lymphoma in which the lymphoma cells are particularly fragile. In patients with 
recurrent disease, doing an air-fluid exchange can provide the necessary cells to make a diagnosis. Glaucoma 
remains an important complication in patients with uveitis. In all cases, standard trabeculectomies stop 
functioning after a few months. We are continuing to compare the use of 5-FU and. Molteno implants in 
patients with uveitis and glaucoma who require surgery. We have enrolled 12 patients in the smdy. 

The use of intraocular lenses following cataract extraction in patients with uveitis is being addressed in a 
randomized double-masked study to compare modified intraocular lenses with standard lenses in patients with 
uveitis that has been under control for at least 3 months. So far three patients have been enrolled in the study, 
and no significant complications have been seen. 

In experimental models, we evaluated the effect of different methods of inmiunomodulation on graft rejection. 
In a corneal graft rejection model in rats, we evaluated the kinetics of inflammatory cell infiltration into the 
graft with and without FK 506 treatement. We also began to evaluate the effect of feeding class I and class 
II antigens on the rejection rate of corneal grafts. We also have initiated a study of retinal pigment epithelial 
cell transplantation in the Lewis rat. The immunohistochemical characteristics of the graft were studied for 
several weeks. Results thus far indicate that rejection occurs at the same rate as in any other tissue. 



118 



PHS 6040 (Rev, 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 



Additional Personnel 

Igal Gery 



Chi-Chao Chan 



Susan Mellow 
Susan Whitcher 



Ph.D. Head, Section on 

Experimental 

Immunology, LI, NEI 
M.D. Head, Section on 

Immunopathology, 

LI, NEI 
R.N. Nurse, LI, NEI 

Psychologist, LI, NEI 



Clinical Protocol Numbers 

79-EI-49 

87-EI-104 

92-EI-157 

Objectives 

The objectives of this project are as follows: (1) to 
develop rational surgical approaches for patients with 
intraocular inflammation, because appropriate surgi- 
cal modalities are needed to properly manage the 
complications that arise with chronic intraocular 
inflammation; (2) to devise rational methods for 
sampling intraocular tissues and to develop the 
methodology needed to obtain clinically useful 
information from limited tissue samples; (3) to test 
new methods of suppressing graft rejection in animal 
models; and (4) to determine the immunology of 
graft rejection in the subretinal space. 

Methods 

Patients who have developed ocular complications as 
a result of ocular inflammation and who require 
surgery and patients for whom an appropriate diag- 
nosis can only be made with surgery are eligible for 
one of the patient protocols described above. Pa- 
tients with vitritis and retinitis of unknown etiology 
in whom a nonspecific trial of immunosuppression is 
contraindicated may, according to the protocol, 
undergo vitrectomy and chorioretinal or endoretinal 
biopsy to obtain a diagnosis. The tissue specimen is 
partitioned for microbiology, electron microscopy, 
immunohistochemistry, and polymerase chain reac- 
tion. 

Patients with a suspected intraocular lymphoma 
undergo an intraocular lymphoma workup with 
appropriate computed tomography or magnetic 
resonance imaging scans and lumbar punctures. If 



these are negative, a vitrectomy is performed and the 
cells are studied by immunohistochemistry. Patients 
who have intraocular lymphoma are entered in a 
central nervous system (CNS) lymphoma protocol 
and followed prospectively. 

Patients with glaucoma and uveitis are entered in 
a double-masked trial of either trabeculectomy with 
5-fluorouracil or Molteno implant. They are then 
followed prospectively to determine the degree of 
postoperative inflammation and to determine how 
effective the procedure is over time. 

For patients with cataracts and uveitis under good 
control and minimal intraocular inflammation, the 
protocol calls for a cataract extraction and random- 
ization to a standard intraocular lens or a modified 
lens with a heparin coating. Patients are then moni- 
tored postoperatively for the appearance of inflamma- 
tion via laser cell flare meter. They also are moni- 
tored for the appearance of cellular deposits on the 
lens surface. 

In animals, we test the efficacy of oral feeding of 
class I and class II antigens in preventing corneal 
graft rejection. We also evaluate the kinetics of 
inflammatory cell infiltration in corneal grafts that 
are treated with a placebo or FK 506. This model 
uses heterotopic grafts taken from Fisher rats and 
sewn into Lewis rats. To evaluate the rejection 
characteristics of retinal pigment epithelial (RPE) 
cells under conditions that would favor rejection, 
human RPE cells are implanted into the subretinal 
space of Lewis rats using a fransscleral approach. 
Animals are serially sacrificed at preset times to 
determine the severity of rejection and the type of 
cell infiltration occurring within the grafted tissue. 
Standard immunohistochemistry for avidin-biotin- 
peroxidase reactions is used in these experiments. 
Finally, using the endotoxin-induced uveitis model, 
we are testing a method of measuring, in a noninvas- 
ive way, the inflammation present in the anterior 
chamber of rats. The device used is a laser photom- 
eter that already is being used in patients to monitor 
anterior chamber inflammation. Measurements of 
anterior chamber inflammation made with the device 
are correlated with measurements of the protein in 
the anterior chamber. 

Major Findings 

In Fiscal Year 1993 we performed several diagnostic 
vitrectomies for intraocular lymphoma. This particu- 
lar lymphoma, a subtype of CNS lymphomas, is on 



119 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



the rise. There are three times more CNS lym- 
phomas being diagnosed today than 10 years ago. 
Prior to performing a vitrectomy, we perform a 
complete workup, including an MRI brain scan and 
lumbar puncture on each patient Several patients 
were referred to us after unsuccessful attempts to 
diagnose the tumors elsewhere. Invariably, after 
reviewing the charts, we found that the specimens 
had been poorly processed or had been allowed to sit 
for too long. We have found that it is imperative to 
bring a specimen to the pathology laboratory while 
the vitrectomy is under way to ensure adequate cell 
viability. In a patient who has had a previous 
vitrectomy and in whom there are still cells floating 
in the vitreal cavity, a simple air/fluid exchange may 
be all that is necessary to make the diagnosis. 

We have found that pretreatment with pulse 
methylprednisolone is an effective way of decreasing 
preoperative inflammation. This has been particular- 
ly useful in cases in which it has been necessary to 
perform a vitrectomy while there was still evidence 
of inflammation. In patients undergoing the Molteno 
implant interim analysis seems to suggest that 
Molteno implants maintain a lower pressure for a 
longer period of time. Most trabeculectomies fail by 
about 1 8 months, while Moltenos are still functioning 
after 24 months. We also have found that in all 
postoperative cases, the use of topical nonsteroidal 
agents such as diclofinac significantly reduce the 
amount of inflammation. This is particularly visible 
in glaucoma patients. No data analysis is yet avail- 
able on the intraocular lens trial, which has just 
begun. 

In the animal studies, we were able to demon- 
strate that topical drops of FK 506 are an effective 
means of stopping corneal graft rejection. FK 506 
has a predominant effect on T cells, inhibiting both 
the activation of these cells and their recruitment into 
the transplanted tissue. It appears to down-regulate 
the expression of both class I and class II antigens as 
well as adhesion molecules in the transplanted 
cornea Using these drops, we are now looking at 
the influx of cells into the graft tissue to determine 
the early events involved in graft rejection. Prelimi- 
nary studies of feeding lymphocytes of donor ani- 
mals to recipients prior to corneal grafting are 
showing promising results. There appears to be a 
delay in corneal graft rejection, but the effect is not 
yet statistically significant 



Studies on the kinetics of RPE rejection in the 
subretinal space of Lewis rats reveal that, when 
human RPE cells are used, graft rejection occurs 
within 14 days. This is the same rate of rejection 
observed for other tissue grafts; The infiltrating cell 
population is mixed, containing both T and B cells. 
This is the first good demonstration of graft rejection 
in RPE transplantation. Prior to these experiments, 
several claims had been made that graft rejection did 
not occur. Studies using the laser photometer have 
shown that it is possible to take measurements of 
anterior chamber flare from rat eyes. The major 
advantages of this technique are that it can be 
performed on live, anesthetized animals; measure- 
ments can be repeated over time; and the measure- 
ments are given as numerical values, making data 
analysis much easier. 

Significance to Biomedical Research and the 
Program of the Institute 

Uveitis is the cause of 10% of visual impairment in 
the United States. Ocular complications that require 
surgery for correction are common in these patients, 
despite adequate inmiunosuppression. Developing 
appropriate surgical modalities of treatment is thus an 
important endeavor. Similarly, conditions exist in 
which appropriate therapy can only be given once 
the proper diagnosis has been made from intraocular 
tissue. Developing the means of obtaining a minimal 
amount of tissue and properly processing it is thus of 
major significance. 

Proposed Course 

This study will continue to investigate methods of 
surgically managing patients with uveitis. Patient 
enrollment continues. In the animal models, we will 
continue to study new methods of modulating graft 
rejection. We also will proceed with our study of 
the immunology of RPE transplantation and factors 
that may influence rejection, such as the method of 
graft insertion. A transvitreal approach may prove to 
be less inflammatory. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

DeBarge LR, Fenton R, Nussenblatt RB, de Smet 
MD: Quantitative determination of the flare in 



120 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



the anterior chamber of the rat by a noninvasive 
method. Invest Ophthalmol Vis Sci 34(4)(suppl): 
1481, 1993. 

Fenton RM, Rubin BI, de Smet MD, Whitcup SW, 
Nussenblatt RB: A prospective study of 5-FU 
trabeculectomy vs. single plate Molteno implant 
in patients with panuveitis complicated by glauco- 
ma refractory to prior therapy. Invest Ophthalmol 
Vis Sci 34{4)(suppl):897, 1993. 

Hikita N, Lopez JS, Chan C-C, Mochizuki M, 
Nussenblatt RB, de Smet MD: Effect of topical 
FK 506 on the rejection of corneal allograft in the 
Lewis rat. 2nd International Symposium on 



Ocular Inflammation, Jerusalem, Israel, Aug 30- 
Sep 3, 1992. 

Martin DP, Chan C-C, de Smet MD, Palestine AG, 
Davis JL, Whitcup SW, Bumier MN, Nussenblatt 
RB: The role of chorioretinal biopsy in the 
management of posterior uveitis. Ophthalmology 
100:705-714, 1993. 

Parks DJ, Hikita N, Nagineni C, Hooks JJ, Chan 
C-C, Nussenblatt RB, de Smet MD: Immuno- 
histochemistry of xenogeneic RPE transplants in 
the rat: A model for graft rejection. Invest Oph- 
thalmol Vis Sci 34(4)(suppl):1095, 1993. 



121 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00115-15 LI 



PERIOD COVERED 

October 1, 1992 to September 30. 1993 



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

Cyclosporine Therapy in Uveitis 



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

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



Others: Marc D. de Smet 
Scott Whitcup 
Chi-Chao Chan 

Richard Fenton 
Dan Martin 



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

M.D. 
M.D. 



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



LI, NEI 
LI, NEI 
LI, NEI 

LI, NEI 
LI, NEI 



COOPERATING UNITS (il any) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 



Section on Immunoregulation 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.75 



PROFESSIONAL: 



0.75 



OTHER: 



0.0 



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.) 

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



122 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Immunology 



Project Description 

Additional Personnel 

Bany Grubbs 

Clinical Protocol Number 

81-EI-33 



Biologist, LI, NEI 



Objectives 

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

Methods 

Patients 18 years or older, of either sex (females not 
pregnant) who have not done well on more conven- 
tional medical ther^y have been admitted to this 
study. All patients must have bilateral sight-threaten- 
ing uveitis of noninfectious etiology that was not 
satisfactorily 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-Ag, to assess evidence of cellular immune 
memory, which is considered to be the in vitro 
equivalent of the ananmestic 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' clinical and immunologic courses are 
closely monitored. Specific attention is given to 
renal function change, a frequent side effect. Pa- 
tients who need to continue CsA for over 1 year 
because of their ocular disease may be asked to 
undergo renal biopsy for evaluation of the reversible 
and irreversible components to CsA renal toxicity. 
Some patients entered on previous CsA studies still 
followed in tiie eye clinic will continue to be moni- 
tored for their renal function to determine how and 
when cyclosporine dosage can safely be tapered. 



Major Findings 

CsA has been effective in the treatment of some 
cases of posterior uveitis. Decreased inflammatory 
activity and improved visual acuity was seen in most 
patients Created to date. The particular responsive- 
ness to this agent by patients with the ocular mani- 
festations of Behget's disease has been corroborated 
by a masked randomized trial performed in Japan. 
The improvement in the chnical condition was 
supported by a concomitant improvement in elecfro- 
physiologic test results, particularly in contrast 
sensitivity. 

Patients freated with CsA had no abnormalities of 
natural killer cell activity before the initiation of 
tiierapy, nor was any noted afterward. CsA signifi- 
cantiy decreased skin test responsiveness but did not 
alter lymphocyte proliferation or antibody production 
in patients. 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 3 months approximately 78% of the 
patients entering this open study were considered 
therapeutic successes, while 62% were considered 
successes at 1 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 compared these biopsies to 
those from age-matched conttols. An irreversible 
component of CsA toxicity could be identified: in the 
main, renal tubular atrophy accompanied by intersti- 
tial fibrosis. The majority of the individuals' biop- 
sies had normal serum creatinine values, but a 
correlation could be made between the alterations 
noted and previous serum creatinine elevations for 
some period of time. The cyclosporine A/G trial has 
shown that the two cyclosporines have overall equal 
value in treating uveitis. However, CsG was more 
effective than CsA in reducing cystoid macular 
edema, particularly at lower dosages. 

Significance to Biomedical Research and the 
Program of the Institute 

Uveitis is one of the most frustrating problems in all 
of ophthalmology. Present modes of therapy for 
patients with severe ocular inflammatory disease are 
inadequate and nonspecific. CsA appears effective 
in treating posterior uveitis of noninfectious etiology. 
This is tiie first new agent in decades to be found 



123 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



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 cyclosporine combi- 
nations will continue. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 



Clinical use of 
Int Ophthalmol 



Publications 

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

Nussenblatt RB, de Smet MD, Rubin B, Freidlin V, 
Whitcup SM, Davis J, et al: A masked random- 
ized, dose-response study between cyclosporine A 
and G in the treatment of sight-threatening uveitis 
of noninfectious origin. Am J Ophthalmol 
115:583-591, 1993. 



124 



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



PROJECT NUMBER 



ZOl EY 00278-02 LI 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Oral Admimstration of Antigen and the Ocular Immune Response 



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

PI: Robert B. Nussenblatt M.D. Scientific Director LI, 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) 



LAB/BRANCH 



Laboratory of Immunology 



SECTION 

Section on Immunoregulation 

INSTITUTE AND LOCATION 

NEI, NTH, Bethesda, MP 20892 

TOTAL STAFF YEARS 



0.8 



PROFESSIONAL: 



OTHER: 



0.3 



0.5 



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.) 

The effect of oral administration of various antigens on the ocular immune response has been tested in the 
animal model for a 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 S-Ag. A putative suppressor 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 has been put together. A pilot study performed in two patients showed the induction of such 
tolerance. 



125 



PHS 6040 (Rev. 5/92) 



Laboratory of Immunology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

Exploring means of immunomodulation has been a 
major role in this laboratory. While extensive 
experimentation has used various immunosuppressive 
agents, there also has been a major thrust in attempts 
to use other modes of immunosuppression. The goal 
of this series of experiments, both in animals and in 
humans, is to test the efficacy of oral tolerance with 
uveitogenic antigens in the treatment of animals 
induced with experimental autoimmune uveitis and 
in patients with bilateral sight-threatening posterior 
and intermediate uveitis of an autoimmune nature. 

Methods 

Six- to 10- week-old Lewis rats of either sex are used 
for these experiments. Animals are fed various 
antigens both before and after the induction of 
experimental uveioretinitis. The antigens include 
whole molecules such as the retinal S-antigen (S-Ag) 
and interphotoreceptor retinoid-binding protein 
(IRBP), as well as their fragments. In a subset of 
experiments, some animals also undergo splenectomy 
before the initiation of the exjjeriments; other ani- 
mals receive sham procedures. We are attempting to 
evaluate the clinical course of the disease and corrob- 
orate the clinical observations with histopathology at 
various points after 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 toleragenic state. 

In the studies performed with patients, individuals 
who have bilateral uveitis of a noninfectious cause 
and are 18 years or older (either sex) are considered 
for the study. In addition, their lymphocytes must 
demonstrate an in vitro proliferative response to the 
retinal S-Ag. The patients also need to be on sys- 
temic immunosuppressive ther^y, whether it be 
corticosteroids, cytotoxic agents, or cyclosporine. 
The goal of this study is to determine whetlier the 
addition of oral feeding of retinal antigeas will 
induce a toleragenic state in individuals who need 
high amounts of immunosuppressive therapy to 
control their disease. 

This study is performed in a randomized, double- 
masked fashion in which some patients receive S-Ag, 
other patients receive a retinal mixture containing 



several antigens, and still other patients receive 
placebo. The intent is to reduce the amount of 
immunosuppressive therapy that the patients are 
taking. We hope that a toleragenic state can be 
induced by feeding these antigens. 

Major Findings 

In the animal study, the spleen appears to play an 
important role in the induction of oral tolerance of 
S-Ag. In addition, the spleen is essential for adop- 
tive transfer of tolerance by splenocytes from donors 
fed S-Ag. Thus, it would be logical to assume that 
the spleen acts as a site for induction and/or amplifi- 
cation of cells with suppressive activity. 

The pilot study demonstrated that, at least in two 
patients, a toleragenic state can be induced by 
feeding antigen at the dosages planned for this study. 
One patient with par planitis and one with Behcet's 
disease have been able either to come off medication 
completely or to be reduced to exceptionally low 
dosages. 

Significance to Biomedical Research and the 
Program of the Institute 

Uveitis is one of the most frustrating problems in all 
of ophthalmology. The present modes of ther^y for 
patients with severe ocular inflammatory disease all 
have limitations, in particular because of their sec- 
ondary effects. By identifying patients with an 
immune response to the retinal S-Ag, we will be able 
to induce an immunosuppressive state without the 
use of pharmacologic agents. Furthermore, the 
induced tolerance would be antigen specific. 

Proposed Course 

The randomized study will begin shortly. 

NEI Research Program 

Retinal and Choroidal Diseases — Inflammatory 
Disorders 

Publications 

Nussenblatt RB, de Smet MD, Weiner HL, Gery I: 
The treatment of the ocular complicafions of 
Behcet's disease with oral tolerizafion, in Wechs- 
ler B, Godeau P (eds): Sixth International Con- 
ference on Behcet's Disease. New York: Ex- 
cerpta Medica, 1993. 



126 



NEI Annual Report— FY 1993 Laboratory of Immunology 



Weiner HL, Miller A, Khoury SJ, Zhang ZJ, AL- 
Sabbagh A, Brod SA, Lider O, Higgins P, Sobel 
R, Matsui M, Sayegh M, Carpenter C, Eisenbarth 
G, Nussenblatt RB, Hafler DA: Suppression of 
organ-specific autoimmune diseases by oral 
administration of autoantigens. Progress in 
Immunology VII. Eight International Congress of 
Immunology, Budapest, 1992. 



127 



Laboratory of Mechanisms of Ocular Diseases 



Report of the Acting Chief, Laboratory of Mechanisms of Ocular 
Diseases 



J. Samuel Zigler, Jr., Ph.D. 



Investigators in the Laboratory of Mechanisms of 
Ocular Diseases (LMOD) have continued to 
conduct studies on a broad range of topics relating to 
the biology of various tissues in the normal eye and 
the molecular mechanisms responsible for certain 
ocular diseases. The major emphasis has been on 
cataract and the various ocular compUcations of 
diabetes. 



Section on Cataract 

Dr. Deborah Carper and her colleagues have 
concentrated their efforts on the role of aldose 
reductase, which produces polyols, in causing diabet- 
ic complications and on the possible effect of sorbi- 
tol dehydrogenase, which metabolizes polyols, in 
protecting against such pathologies. Specifically, 
site-directed mutagenesis studies have demonstrated 
that the histidine at position 110 of aldose reductase 
is critical for catalytic activity. Also, a study has 
been instituted on a family with congenital cataracts, 
whose members have a probable genetic defect in the 
sorbitol dehydrogenase gene. 

Dr. Donita Garland's group has made major 
advances in its collaborative study on the protein 
composition of normal human lens and cataracts. 
The addition of a scanner capable of quantifying the 
complex images obtained by two-dimensional elec- 
trophoresis and software with which to compare and 
analyze the data provides the tools necessary to 
address the important questions raised by this investi- 
gation. In addition, this group is investigating the 
effects of metals, including copper, iron, and zinc, on 
the lens crystallins and has found that both oxidation 
and aggregation are induced by such exposure in 
vitro. 

Dr. Fielding Hejtmancik and his group are study- 
ing structure/function relationships of P-crystallins 
and doing gene-mapping studies on a variety of 
genetic diseases with ocular findings. One such 
disease is Usher's syndrome type I, for which two 



independent genes have been m^ped on chromo- 
some 2. One of these genes, which causes Usher's 
syndrome in the Acadian population, has been 
localized within a 6cM portion of the chromosome. 

Dr. Paul Russell's group has concentrated its 
efforts on the biology of the lens epithelium and on 
development of lens organ culture techniques. 
Smdies on lens epithelium have included analysis of 
protective mechanisms induced by various types of 
stress and analysis of the process whereby lens 
epithelial cells differentiate into fibers. These studies 
include tissue culture approaches as well as analyses 
of the epithelial layer from intact lenses. A novel 
method has been developed to assess the integrity of 
lenses in organ culture. This technique provides 
quality control information which allows the re- 
searcher to reject imperfect lenses before committing 
them into experiments. 

Dr. Samuel Zigler' s group also has been working 
with the lens organ culture system, using it as a 
means of screening potential anticataract drugs. This 
group also is investigating the functions of lens 
crystallins, in particular the role of a-crystallin as a 
molecular chaperone. Definitive proof of noncova- 
lent complex formation between a-crystallin and the 
early non-nafive forms of denaturing proteins has 
been obtained, as has evidence for marked differ- 
ences in the protection of apo- and holo- forms of 
some enzymes. 



Section on Pathophysiology 

Dr. W. Gerald Robison, Jr., has continued to 
refine and better characterize the rat model for 
diabetic retinopathy. Multiple angiopathies were 
present in the retinas of these rats following 24 
months of galactose feeding. In contrast, galactose- 
fed animals given an aldose reductase inhibitor did 
not develop such pathologies. The data support the 
hypothesis that aldose reductase is the primary player 
in the formation of retinopathy in this model. 



131 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00201-09 LMOD 



PERIOD COVERED 

October 1. 1992 to September 30, 1993 



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

Structure and Expression of Polyol Pathway Enzymes 



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



PI: Deborah Carper 

Others: Susan Old 

Takeshi Iwata 



Ph.D. 

Ph.D. 
Ph.D. 



Biologist 

Staff Fellow 
Visiting Associate 



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: 



3.0 



PROFESSIONAL: 



3.0 



OTHER: 



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.) 

In diabetes, the accumulation of sorbitol is believed to be a Icey factor in initiating cataract, retinopathy, and 
neuropathy. We are interested in controlling the accumulation of sorbitol by regulating the action of the two 
enzymes of the sorbitol pathway: (1) aldose reductase (AR), which reduces glucose to sorbitol, and 
(2) sorbitol dehydrogenase (SDH), which oxidizes sorbitol to fructose. Our aim is to design innovative 
methods to inhibit the action of AR or increase SDH, with the purpose of reducing sorbitol accumulation in 
diabetic tissues. 

Site-directed mutagenesis of AR has been a major priority of our laboratory. We have made amino acid 
substitutions in the rat and human AR and determined that some of these changes affect the kinetics of the 
protein with its substrate. For example, when histidine at position 1 10 was changed to glutamine, the activity 
of AR was reduced dramatically. TTie HI lOQ mutant protein showed very little activity with glyceraldehyde 
(1% of normal) and no activity with ;7-nitrobenzaldehyde. Other histidine substitutions did not acutely alter 
the kinetics of AR, supporting the finding that HI 10 plays an essential role in catalysis. These structure/ 
function studies should help define the active site and locate the target areas of the current AR inhibitors. 

Another strategy to control sorbitol accumulation is to regulate SDH. We have determined the primary 
sequence of human SDH and characterized part of the SDH gene. Molecular genetic studies also are under 
way to test for an SDH genetic defect in a family presenting with congenital cataracts and lowered SDH 
enzyme activity. By evaluating the expression of SDH at the gene level, we may be able to evaluate its role 
in sorbitol accumulation in diabetes and other genetic diseases. 



132 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The objective of this project is to study regulation of 
the enzymes of the polyol pathway. 

Methods 

The methods employed include molecular biology, 
protein chemistry, and cell biology techniques. 

Major Findings 

Structure/function studies. — Mutant forms of the 
aldose reductase (AR) protein were synthesized using 
the polymerase chain reaction. Sequencing verified 
the amino acid substitution. The mutant proteins 
were expressed in bacteria, purified on three columns 
using different biochemical characteristics of the 
protein, then tested for enzyme activity. Of the six 
histidines we mutated, histidine at position 110 was 
discovered to play an essential role in enzyme 
catalysis. When histidine 110 was changed to 
glutamine, AR activity was reduced to only 1% of 
the normal. The K^ for glyceraldehyde changed 
from 0.12 mM for the normal to 12.5 mM for the 
HI 10 mutant. No reductase activity was observed 
for HI lOQ using p-nitrobenzaldehyde as a substrate. 
Ultraviolet circular dichoism and NADPH fluores- 
cence quenching indicated that HllOQ was not 
substantially altered in structure or in its ability to 
bind NADPH. Because of its location in the active 
site pocket, histidine 110 has been proposed to be a 
hydrogen donor in the catalytic mechanism of AR. 
From these findings, using site-directed mutagenesis, 
we conclude that HI 10 plays a critical role in the 
catalytic mechanism of AR, although further studies 
will be needed to determine the exact nature of its 
action. 

Expression of human AR in transgenic mice. — A 
cDNA that encodes human AR was ligated with a ^- 
crystaUin promoter. The construct was injected into 
mice. Several mice were found to carry the trans- 
gene and were bred to produce separate Fj genera- 
tions. Evaluation of the presence of the enzyme and 
its polyol product are now under way. Expression of 
human AR in transgenic mice will facilitate in vivo 
drug design studies. 

Characterization of sorbitol dehydrogenase (SDH) 
in a family with congenital cataracts. — We have 
obtained and sequenced over 50% of the gene for 



human SDH. Previously we determined the 
complete coding sequence of the protein. With this 
information we have begun studies to determine a 
possible genetic defect in a family reported to have 
reduced levels of SDH and congenital cataracts. Our 
preliminary nucleotide-sequencing data have indi- 
cated a difference between this family and normal 
controls. 

Significance to Biomedical Research and the 
Program of the Institute 

AR has been implicated in diabetic cataracts, retinop- 
athy, and neuropathy. Side effects and lack of 
efficacy of AR inhibitors in diabetic clinical trials 
have emphasized the need for innovative approaches 
to AR inhibition. Our research is a rational approach 
to designing new types of inhibitors by characteriz- 
ing the structure/function aspects of the protein and 
evaluating the signals that regulate this enzyme. In 
addition, we feel that by understanding the regulation 
of SDH — ^the other enzyme of the polyol pathway — 
we may be able to modulate more fully the accumu- 
lation of sorbitol in diabetes. 

Proposed Course 

The project will continue via site-directed mutagene- 
sis of AR protein to localize the critical amino acid 
residues in the active and inhibitor binding sites. We 
will complete the structure of SDH and analyze the 
gene in a family with congenital cataracts. 

NEI Research Program 

Cataract — Molecular Genetics 

Publications 

Bateman JB, Kojis T, Diep A, Klisak I, Heinzmaim 
BS, Carper D, Nishimura C, Mohandas T, 
Sparkes RS: Mapping of aldose reductase gene 
sequences to human chromosomes 1, 3, 7, 9, 11, 
14 and 18. Genomics, in press. 

Lin L-R, Carper D, Yokoyama T, Reddy V: Effect 
of hypertonicity on aldose reductase, alphaB- 
crystallin and organic osmolytes in retinal pig- 
ment epithelium. Invest Ophthalmol Vis Sci 
34:2352-2359, 1993. 



133 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



HHUJbUI INUMOtM 



ZOl EY 00189-10 LMOD 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



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

Oxidation of Proteins in Cataractogenesis 



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

PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI 



Others: Jose Jimenez 

Lorenzo Merola 
Kenichi Matsuno 



Ph.D. 

M.S. 

Ph.D. 



Visiting Fellow 

Chemist 

IRTA 



LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (it any) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI. NTH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



4.0 



PROFESSIONAL: 



OTHER: 



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.) 

The proteins of the normal human lens and cataracts of various etiologies are being characterized. These 
studies include identifying the major protein species and the modified forms of these proteins, mapping the 
protein composition tiiroughout the lens, and quantitadng changes in the levels of these proteins in 
cataracts. 

An enzyme that protects proteins against thiol-dependent oxidative inactivation has been identified in bovine, 
rat, and primate lens. The enzyme has been purified and identified by sequence analysis. Seventy percent 
of the amino acid sequence has been obtained. 

The interaction of copper, iron, and zinc with a number of proteins, including lens crystallins, has been 
studied. All three metals alter the solubility of the lens crystallins and many of the other proteins studied. 
Copper and iron are metals generally thought to be involved in the metal-catalyzed oxidation of proteins. In 
addition, these studies show them capable of inducing aggregate formation. 



134 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The immediate objectives of tiiis project are (1) to. 
identify and characterize the types of protein modifi- 
cations found in cataracts of various etiologies, (2) to 
investigate the role of oxidation in the formation of 
these modifications, (3) to study the interaction 
between metals and crystallins and the effects of 
these interactions on crystallin solubility and aggre- 
gate formation, and (4) to characterize one of the 
enzymes that protect lens proteins against thiol- 
dependent metal-catalyzed oxidation. 

Methods 

Bovine, rat, and human tissues were used for these 
studies. After human lens material was obtained 
from donors' eyes via cataract surgery, we employed 
classical methods to purify bovine and rat lens 
proteins. Other methods used were standard proce- 
dures for studying proteins, including two-dimension- 
al gel electrophoresis, high-pressure liquid chroma- 
tography, ultraviolet/visible spectroscopy, fluores- 
cence, circular dichroism, electron spin resonance, 
amino acid analysis, and inmiunotechniques. 

Major Findings 

1. Copper, zinc, and iron, but not calcium, 
induced aggregate formation in bovine lens extracts 
and solutions of the crystallins. Aggregation, mea- 
sured by light scattering, was time dependent, occur- 
ring at metal-to-protein ratios greater than 1.0 and 
varying, depending on the metal and protein. Zinc 
induced the aggregation of P- and a- but not y- 
crystallin. Copper and zinc induced aggregation of 
a number of other proteins, but they had no effect on 
lysozyme and papain. One explanation for the lack 
of effect on these two proteins is that they are basic 
proteins. However, copper induced aggregation of y- 
crystallin is also a basic protein. 

The affinity of copper and zinc for these proteins 
is relatively low (K^ max is about 10"^ M). The 
addition of EDTA, DETAPAC, L-histidine, or L- 
cysteine prevented zinc- and copper-induced protein 
aggregation and caused complete disaggregation. 

These studies suggest that histidine is the amino 
acid involved in the aggregation induced by zinc and 
possibly copper. The treatment of a- and ^-crystal- 
lin and trypsin inhibitor with diethylpyrocarbonate 



prevented aggregation. Increasing the pH to 8.0 
substantially increased the zinc-induced aggregation 
of a-crystallin but not that of P-crystallin. No pH- 
induced change in conformation of either protein was 
observed by fluorescence studies, suggesting the 
effect may have been on the pK of an amino acid. 
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 circular 
dichroism spectroscopy. The mechanism of metal- 
induced aggregation is not clear, but it is likely that 
it primarily involves cross-linking rather than confor- 
mation-induced protein-protein interactioa 

The presence of small amounts of zinc in the 
buffer reduced the thermal stability of a-crystallin 
and hemoglobin. 

Zinc concentrations greater than 20 ^iM induced 
cell membrane damage to rat lenses in culture, as 
measured by choline and rubidium uptake. 

Atomic absorption analysis of bovine crystallins 
indicate the presence of zinc associated with a- and 
P-crystallin. 

These studies clearly demonstrate that metals are 
known to be involved in oxidative damage and 
protection against oxidative damage bind crystallins. 
This interaction induces aggregate formation, a 
phenomenon that has been linked to cataractogenesis. 

2. A protein that appears to function in detoxifi- 
cation of the products of thiol-dependent oxidation 
has been demonstrated in cow, monkey, and rat lens 
and human trabecular meshwork cells. The protein 
has been purified to ^parent homogeneity, and about 
70% of the amino acid sequence has been obtained. 
The enzyme has been identified from the protein 
sequence as one of the detoxificafion enzymes found 
in most cells. The absence of secondary sequences 
and the copurification of the antioxidant activity and 
the detoxification enzyme during two separate 
schemes su-ongly indicate that the antioxidant activity 
is associated with this detoxification enzyme, not 
with a contaminant in the preparation. There are no 
previous reports of the antioxidant activity of tiiis 
enzyme. 

3. Analysis of the proteins in human cataract 
specimens by two-dimensional gel electrophoresis 
has continued and the techniques have been opti- 
mized. Preparation procedures to facilitate analysis 
of aspirated lens material (primarily outer cortex 



135 



Laboratory of Mechanisms of Ocular Diseases 



NEI Annual Report— FY 1993 



obtained during extracapsular cataract surgery) have 
been established. These procedures allow us to 
determine the relative amount of the major proteins 
present and the oxidation state of these proteins in 
the cataract. 

We have identified alterations in protein patterns 
and are doing quantitative analyses of the changes. 
Correlations between the altered patterns and cataract 
etiologies are being sought 

4. We have developed capillary gel chromatogra- 
phy procedures that allow the separation and accurate 
quantitation of sorbitol and galactitol. 

Significance to Biomedical Research and the 
Program of the Institute 

Oxidative processes have long been considered a 
major contributing factor in senile cataracts. Metal- 
catalyzed oxidation of the crystallins leads to protein 
modifications that mimic those seen in aging and 
senile cataracts and in brunescent lenses. These 
studies continue to demonstrate the potential for 
metal involvement in cataract formation. Not onJy 
do these metals facilitate oxidative modification of 
the proteins, they also can induce protein aggrega- 
tion. 

Understanding the lens' mechanisms of protecting 
itself against oxidative damage is important for 
developing interventions. These studies demonstrate 
the presence in the lens of an enzyme that protects 
against thiol-dependent oxidation. This activity is 
associated with a detoxification enzyme present in 
most cells and is induced imder oxidative stress. 



The importance of characterizing the proteins in 
the human lens — normal and cataractous — ^is obvious. 
It will give us a wealth of information on aging 
processes, mechanisms involved in cataractogenesis, 
and metabolic processes in this unique tissue. 

Proposed Course 

We will focus our studies for Fiscal Year 1994 on 
the following: (1) continuing the investigation of 
metal-catalyzed oxidation of lens proteins, (2) de- 
tailed characterization of the interaction of metals 
witii crystalUns and the effects on solubility, (3) anal- 
ysis of human lens proteins in cataracts and the 
normal lens, and (4) molecular biology and immuno- 
logical characterization of the enzyme that protects 
against thiol-dependent oxidation reactions. 

NEI Research Program 

Cataract — ^Lens Development and Aging 

Publications 

Bettelheim FA, Reid MB, Garland D: Hydration of 
gamma crystallins. Exp Eye Res, in press. 

Giannessi M, Del Corso A, Cappiello M, Vatarelli 
M, Marini I, Barsacchi D, Garland D, Camici M, 
Mura U: Thiol-dependent metal catalyzed 
oxidation of bovine lens aldose reductase: I. 
Studies on the modification process. Arch 
Biochem Biophys 300:423^29, 1992. 



136 



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



PROJECT NUMBER 



ZOl EY 00272-03 LMOD 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or less. Title must fit on arte lirte between trie 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 
Anthony Lloyd 
T. Padma 



Ph.D. 

Ph.D. 

M.S. 

M.D. 

Ph.D. 



Senior Fellow 
Special Volunteer 
Chemist 
IRTA Fellow 
Visiting Scientist 



LMOD, NfEI 
LMOD, NEI 
LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (if any) 

Baylor College of Medicine (J. Towbin, B. Periyman, T. Ashizawa, P. Overbeek); Univ. of Iowa (R. Smith); Univ. of Texas-Houston 
(S. Daiger); Ocular Genetics and Clinical Services Branch, NEI, NIH (M. Kaiser- Kupfer); Washington Univ. at St. Louis (M. Petrash, 
R. Hayes); Massachusetts Institute of Technology (G. Benedek, J. Pande); Osmania Univ., Hyderabad, India (J.S. Murty) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL; 



5.15 



5.15 



OTHER: 



0.0 



CHECK APPROPRIATE BOX(ES) 

[xj (a) Human subjects 
Ix] (a1) Minors 
□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



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

The 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 comprise over 90% of the soluble protein of the lens and are heavily modified in most 
cataracts. The effects which specific modifications of P- and y-crystallin structure produce on crystallin 
functions, such as stability and formation of macromolecular aggregates, are being studied in tissue culture 
cells transformed with normal and modified pA3/Al-crystalHn genes. Regions of the P-crystallin molecule 
of special interest include the amino terminal arm and tlie Greek key motifs of the core domains. The effects 
which these modifications have on lens transparency also are being studied in a transgenic mouse system in 
which a modified PA3/A1 gene is driven by an aA-crystalUn promoter. 

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, myotonic dystrophy, Duchenne muscular 
dystrophy. Long QT syndrome, cataracts, and a variety of X-linked syndromes. We currently are collecting 
families with autosomal recessive retinitis pigmentosa in preparation for study of this important group of 
diseases. 



137 



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Laboratory of Mechanisms of Ocular Diseases 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

The long-range objectives of this project include 
increasing the understanding of inherited 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 pathophysi- 
ology. These long-range objectives will be 
approached by pursuing the specific aims of identify- 
ing genes involved in inherited visual diseases and 
elucidating the mechanisms by which mutations in 
these genes cause disease. 

Methods 

Conventional cloning technology is utilized in 
preparing sequences for gene expression studies. 
These include ligation with T4 DNA ligase, screen- 
ing by NaOH miniprep methodology, and ^^P-labeled 
DNA probes, as well as allele-specific oligonucleo- 
tide hybridization to screen for specific single-base 
settings. Sequence changes are introduced by site- 
specific mutagenesis via standard methodology. 
Gene expression in Chinese hamster ovary cells (RJK 
88) and in insect cells (SF9) is enabled by the 
baculovirus expression system. Protein expression is 
monitored by standard two-dimensional gel electro- 
phoresis followed by immunoblotting. Association 
behavior is assessed by elation volume on sieve 
FPLC. 

Crystallin and other cDNAs and genomic frag- 
ments are isolated by library screening with cloned 
genes or oligonucleotides using routine metiiods. 
Sequencing is performed by cycling or using auto- 
mated fluorescent technology (ABI). 

Until recentiy, linkage analysis has involved 
conventional Southern blotting. Cell lines from NIH 
Eye Chnic patients and their family members are 
immortalized by Epstein Barr virus ti-ansformation. 
DNA is isolated by standard methodology and 
digested by restriction endonucleases. After agarose 
gel electrophoresis and Southern transfer, the result- 
ing blot is probed with isolated DNA fi-agmenLs 
labeled with ^^P by oligonucleotide labeling. Recent- 
ly, short tandem repeat (microsatellite) markers have 
been analyzed by polymerase chain reaction per- 
formed in the presence of labeled oligonucleotides 
and analyzed on sequencing gels. Linkage data 



recorded on computerized spreadsheets are subject to 
both two-point and multipoint analysis with the 
LINKAGE program package. 

Major Findings 

1. The P-crystallins, their structure, and the 
mechanisms by which heterogeneity arises among 
this family of proteins are being investigated. The 
|3A3-crystallin 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. The PA3/A1 coding sequences were 
ligated behind the RSV promoter, and RJK 88 
fibroblast cells were stably transfected with this 
construction. In addition, the PA3/A1 coding se- 
quences were inserted into the Bluebac expression 
vector (Stratagene) and expressed in SF9 cells. A 
single 26-kD protein, the predicted size of pAl- 
crystallin, was detected on Western blots of soluble 
extracts of stable clones using antibodies raised to 
crystallin peptides. However, the RJK 88 ceUs, 
transformed with the same cDNA except with codons 
gln7 and leulO mutated in vitro to stop codons, 
express only a 24-kD protein, the predicted size of 
the |3A1 -crystallin. Thus, it appears that the up- 
su-eam (PA3) start codon is preferentially used in cell 
lines, although the downstream (pAl) start codon can 
be used. 

In SF9 cells, 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 pA3-crystallin band, suggesting 
that the smaller band is created by processing or 
degradation of the larger in this system. In addition, 
clones for the mouse PA2-, PB1-, pB2-, and PB3- 
crystallin have been isolated and sequenced in 
preparation for characterization of their roles in P- 
crystallin aggregation. 

2. We have constructed an additional crystallin in 
wliich the amino-terminal arm was deleted and 
replaced by a glycine residue, an extension identical 
to that found in 72-crystalIin. This new crystallin has 
been expressed in RJK 88 and SF9 cells (Bluebac 
vector) and has an appropriate migration on Laemmli 
gels, CD-spectrum, and amino acid sequence. The 
activity of this P-crystallin in association with the 
typical 200- to 250-kD aggregates has been tested by 
FPLC on superdex 75 and superose columns. The 
nonnal pA3 polypeptide readily associates into 



138 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



homodimers, whereas the truncated |3A3 associates 
minimally if at all. SF9 cells expressing the recom- 
binant crystallins were grown in ^^S-containing 
medium, then purified and re-associated with an 
excess of lens extract containing normal crystallins 
(unlabeled) using limited urea denaturation followed 
by dialysis. Aggregation to form P-crystallin was 
assessed by FPLC on sizing columns. The recombi- 
nant full-length p-crystallin peptide aggregates into 
both dimers and tetramers, with the dimer peak 
migrating slightly before the p-light peak; however, 
the truncated pA3-crystallin migrates slightly behind 
the P-light peak and does not form obvious tetra- 
mers. These data strongly suggest that the amino- 
terminal arm of P-crystallins assists in the incorpora- 
tion of p-crystallins into higher order aggregates. 

3. We have constructed a pA3-crystallin in which 
the entire connecting peptide from the first to the 
second domain has been replaced with the corre- 
sponding sequence from 72-crystalfin. This construc- 
tion should test the hypothesis that the connecting 
peptide, which crystallographic data show is extend- 
ed in the P-crystallins and curved back on itself in 
the y-crystallins, is responsible in this fashion for the 
P-crystallins' tendency to dimerize. The P-crystallin 
with the modified connecting peptide was subjected 
to the same tests of aggregation described in Section 
2 above; it behaved essentially as the normal (un- 
modified) PA3-crystallin. The secondary structure of 
the modified P-crystallin currentiy is being confirmed 
with CD analysis. 

4. Studies of phase transition properties of the y- 
crystallin gene family have begun in collaboration 
with Drs. Mark Petrash (Washington University, St. 
Louis) and George Benedek (MIT, Boston). The 
bovine yB-crystallin has been modified at two of the 
four residues proposed to be critical for phase 
fransition behavior. Phase transition analysis of the 
expressed unmodified yZ-crystallin has begun at MIT. 

5. We also studied human genetic diseases that 
involve the eye. In addition to elucidating the 
pathogenesis of visual symptoms in inherited 
diseases, our efforts have provided reagents and 
information applicable to genomic analysis in 
general. Genetic markers in the myotonic dystrophy 
region have been used to confirm the diagnostic 
usefulness of bilateral lens opacities in the diagnosis 
of myotonic dystrophy; the data were confirmed by 
examining the trinucleotide repeat shown to be 
expanded in persons affected by myotonic dystrophy. 



The phenomenon of anticipation, long controversial 
in myotonic dystrophy, was shown to occur with 
statistical significance in the families enrolled in our 
study. In addition, earlier age of onset through 
anticipation was correlated with expansion of the 
trinucleotide repeat, although the correlation was not 
perfect We have isolated a cDNA clone correspond- 
ing to the dystrophin gene product from a mouse 
lens library and are characterizing it. 

6. Ophthalmologic diseases in humans have been 
studied by linkage analysis of RFLP markers. 
Diseases we have mapped within the past year 
include Long QT syndrome, X-linked agammaglobu- 
linemia, and Usher's syndrome type I. In addition, 
clinical and genetic heterogeneity of Usher's syn- 
drome within the Acadian population of Louisiana 
has been explored in detail. Genetic analysis con- 
firms the clinical impression that both type I and n 
of Usher's syndrome are found in the Acadian 
population, even within a single extended pedigree. 
The heterogeneity analysis described above implies 
this is due to segregation of two different, unlinked 
genes within this population. 

Two genes causing Usher's syndrome type I have 
been m^ped. In Acadians, the genetic locus is on 
chromosome 1 Ip, while in the British families in our 
study, the gene is on chromosome llq. When 
subjected to the most stringent heterogeneity analyses 
(both the H0M0G2 program and M test), these 
findings are significant at p < 0.01. This surprising 
finding implies that multiple genes can cause the 
rather specific clinical findings in Usher's syndrome. 
In detailed study of the Usher's syndrome gene on 
chromosome lip, we have used fine linkage map- 
ping and haplotype analysis to localize it to a 6-cM 
interval between tiie markers Dl 1S861 and Dl 1S928. 

Several large families with autosomal dominant 
and recessive cataracts have been ascertained, and 
samples have been collected. Genotyping of micro- 
satellite markers has begun for four of these families 
and will initially be concentrated in regions around 
candidate genes. 

Significance to Biomedical Research and the 
Program of the Institute 

Elucidation of the genetic defects causing visual 
disability will have implications far beyond the 
patient population suffering from the specific syn- 
dromes under study. Inherited diseases provide a 
means by which the molecular pathophysiology of 



139 



Laboratory of Mechanisms of Ocular Diseases 



NEI Annual Report— FY 1993 



the visual system may be understood. This knowl- 
edge can then be applied to a broad spectrum of 
diseases. This rationale also applies to the study of 
inherited diseases of which visual defects are only a 
small part. Thus, while our studies of myotonic 
dystrophy already have resulted in improved diagnos- 
tic abilities, the mechanism by which cataracts occur 
in this disease will provide insight into cataracto- 
genesis in other hereditary syndromes as well as in 
age-related and nonspecific cataracts. 

Proposed Course 

1. We will continue studies on the structure- 
function relationships of lens crystallins, concentrat- 
ing on how modifications of the terminal arms and 
possibly the interconnecting peptide between the two 
domains affect aggregation of P-crystallins. We also 
will continue to explore the effects that modificatioas 
of the Greek key motifs have on crystallin stability 
and, when applicable, lens transparency. In addition, 
we will explore the effects of modifications of y- 
crystallin sequences on the protein phase transitions 
and its relationship to cold cataract. 

2. Sample collection and linkage analysis of a 
variety of human diseases will continue. The main 
emphasis will be on inherited visual diseases, espe- 
cially Usher's syndrome type II. We are initiating a 
linkage study of autosomal dominant cataracts in 
families ascertained in collaboration with Dr. Muriel 
Kaiser (Ophthalmic Genetics and Clinical Services 
Branch) and of autosomal recessive cataracts ascer- 
tained in collaboration with Dr. J.S. Murty (Osmania 
University, India). This study will be coordinated 
with a new project to categorize and map expressed 
sequences of the human lens and the ongoing mecha- 
nistic studies on lens crystallins described above. 
Together these projects should provide a coordinated 
effort to elucidate the mechanisms of cataractogene- 
sis in the human lens. 

NEI Research Program 

Cataract — Molecular Genetics 

Publications 

Ashizawa T, Dubel JR, Dunne PW, Dunne CJ, Fu 
Y-H, Pizzuti A, Caskey CT, Boerwinkle E, 
Perryman MB, Epstein HF, Hejtmancik JF: 
Anticipation in myotonic dystrophy: Complex- 



relationships between clinical findings and struc- 
ture of the GCT repeat. Neurology 42: 1877-1893, 
1992. 

Ashizawa T, Dunne CJ, Dubel JR, Perryman MB, 
Epstein HF, Boerwinkle E, Hejtmancik JF: 
Anticipation in myotonic dystrophy: Statistical 
verification based on clinical and haplotype 
findings. Newro/o^ 42:1871-1877, 1992. 

Ashizawa T, Hejtmancik JF, Liu J, Perryman MB, 
Epstein HF, Koch DD: Diagnostic value of 
ophthalmologic findings in myotonic dystrophy: 
Comparison with risks calculated by haplotype 
analysis of closely linked restriction length poly- 
morphisms. Am J Med Genet 42:55-60, 1992. 

Ayyagari R, Smith RJH, Lee EC, Kimberling WJ, 
Jay M, Bird A, Hejtmancik JF: Heterogeneity of 
Usher syndrome type I, in Anderson RE, Holly- 
field JG, Lavail MM (eds): Degenerative Retinal 
Disorders: Clinical and Laboratory Investiga- 
tions. New York, Alan R. Liss Inc., 1992. 

Hejtmancik JF: Neurology of the visual system, in 
Conn PM (ed): Neurology, 1992. 

Hejtmancik JF, Black S, Harris S, Ward PA, Calla- 
way C, Ledbetter D, Morris J, Leech SH, Pollack 
MS: Congenital 21 -hydroxylase deficiency as a 
new deletion mutation. Detection in a proband 
during subsequent prenatal diagnosis by HLA 
typing and DNA analysis. Hum Immunol 35:246- 
252, 1992. 

Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky J: 
Inherited disorders of the eye lens, in: The 
Metabolic Basis of Inherited Disease. New York, 
McGraw Hill, 1992. 

Hejtmancik JF, Piadgorsky J: Molecular biology of 
the eye lens, in Raviola E, Dowling J (eds): 
Principles and Practice of Ophthalmology. 
Philadelphia, WB Saunders, 1992. 

Hejtmancik JF, Roberts R: Molecular genetics and 
the application of linkage analysis, in Roberts R 
(ed): Molecular Basis of Cardiology. London, 
Blackwell Scientific Publications, 1993, pp 355- 
381.' 

Keats BJ, Todorov AA, Pelias MZ, Hejtmancik JF, 
Kimberling WJ, Leppert M, Lewis RA, Smith RJ: 
Linkage studies of Usher syndrome type I: 
Exclusion results from the Usher syndrome 
consortium. Genomics 14:707-714, 1992. 



140 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



Muller B, Dechant C, Meng G, Liechti-Gallati S, 
Doherty RA, Hejtmancik JF, Bakker E, Read AP, 
Jeanpierre M, Fischbeck KH: Estimation of the 
male and female mutation rates in Duchenne 
muscular dystrophy (DMD). Hum Genet 89:204- 
206, 1992. 

Nickerson JM, Hejtmancik JF: Molecular biology 
and genetics of the retina, in Tasman W, Jaeger 
E (eds): Biomedical Foundations of Clinical 
Ophthalmology. Philadelphia: Lippincott, 1992. 

Parolini O, Hejtmancik JF, Allen RC, Belmont JW, 
Lassiter GL, Henry MJ, Barker DF, Conley ME: 
Linkage analysis and physical mapping near the 
gene for X-linked agammaglobulinemia at Xq22. 
Genomics 15:342-349, 1993. 

Smith RJH, Berlin C, Hejtmancik JF, Laties A, 
Lewis RA, Keats B, Kimberling WJ, Moller CG, 
Pelias MA, Tranebjaerg L: Clinical diagnosis of 
the Usher syndromes. Am J Med Genet, 1992. 



Smith RJH, Lee EC, Kimberling WJ, Daiger SP, 
Pelias MZ, Keats BJB, Jay M, Bird A, Reardon 
W, Guest M, Ayyagari R, Hejtmancik JF: Local- 
ization of two genes for Usher syndrome type I to 
chromosome 11. Genomics 14:995-1002, 1992. 

Smith RJH, Pelias MZ, Daiger SP, Keats B, Kimber- 
ling W, Hejtmancik JF: Clinical variability and 
genetic heterogeneity within the Acadian Usher 
population. Am J Med Genet 43:964-969, 1992. 

Towbin J A, Hejtmancik JF, Brink P, Gelb B, Zhu 
XM, Chamberlain JS, McCabe ER, Swift M: X- 
linked dilated cardiomyopathy. Molecular genetic 
evidence of linkage to the Duchenne muscular 
dystrophy (dystrophin) gene at the Xp21 locus. 
Circulation 87:1854-1865, 1993. 



141 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



PROJECT NUMBER 



ZOl EY 00237-08 LMOD 



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

Characterization of the Lens 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Nante. 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 (il any) 



LAB/BRANCH 



Laboratory of Mechanisms of Ocu lar Diseases 



SECTION 

Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH. Bethesda, MP 20892 



TOTAL STAFF YEARS: 



4.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
□ (a2) Interviews 



PROFESSIONAL: 



4.0 



OTHER: 



0.0 



B (b) Human tissues □ (c) Neither 



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

We are conunuing to develop an in vitro model to check anticataract agents. The organ culture system utilizes lenses 
from rats and inonkeys. We have developed a method to screen the lenses to detennine which might have been damaged 
m dissecuon. This is the first method to adequately predict the integrity of the lens at a very early stage in culmrerfens 

always been correct. Many lenses can mamtain clarity but are not able to transport ions and amino acids normally By 
Ifr? "^''^''^l^^^^' ^^ ^so l^ve been able to show that the main protein in the organ culmre medium is 
albumin most probably as a result of residual protein sticking to the lens during dissection. The albumin can be taken 

chilC.nf.H^fhTH""'^ ""'"^ ^ 'i^'"' '""'' ""^ '"^' P''^'"^ ^"^ ^^^ «"' «f ^^ l«"s- Cunously, when the lens is 
■of hvfh r 'ah7'" Pf™'''' "" ' '""'"^ '°^ ^'^'^^^^ ^^^^^' ^^ P«-2 ^'^^ i^ o°e of the principal proteins 
ixnreLTii'l .1 ''h " t"^' organ-culnired lenses have begun analysis of the types of messenger RNA 

Zsag^ " ' ^' "'""^ """"^'^ '^"''' '° '*'''™^' '^^ sequences of these stress-related 

lTi,r°" '^h ^T ^^'"^fr ^"^ """^"^ "^ ''"'^y '^^ 'P"'^^'" ""^^"O"^- ^^ '^on'^en's the protective mechanisms 
present ui the lens epithelium to prevent damage from oxidative sQ-ess. Work with the lens epitheUum cell lines has 
shown that the major oxidoreductase activated in an oxidative sffess system appears to be D-T diaphorase. C-crystallin 
also an oxidoreductase. is responsive to the oxidative stress and increases in the lens cells. The increa^in the 
^-crystalhn cannot by itself account for the large increase in oxidoreductase found in the stressed cells The second 
quesuon concerns cellular differentiation into fiber cells. The region of the lens where this process occurs tends to be 
wni^r^'' f^ ^^^^ ""?' '''"' conditions. We have separated certain steps in the differentiation process and 
will be better able to explore the stages at which cataracts might develop in the equatorial region of the lens. 

Work continues on the human PB-2 crystallin, which now has been successfully cloned and sequenced. The deduced 

2TZZ T J rr '""^"""'^ ^^ '°°'*''' '^^- ^'""^ ^'^ P^*'^" '^ developmentally regulated, investigation into 
the promoter activity of this gene is continuing. ^ & . eauu.. muj 

142 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The purposes of this project are (1) to understand the 
basic biological processes of the human lens and how 
they are altered in cataract formation, (2) to develop 
model systems with which to mimic these processes, 
and (3) to use these model systems to develop 
methods to test anticataract agents. 

Methods 

Among numerous biochemical and molecular biolog- 
ical methods used in this research are Northern, 
Southern, and Western blotting of mRNA, DNA, and 
proteins. In addition, various methods for quantita- 
tion of these components, such as slot-blotting, are 
done. The polymerase chain reaction is used, as is 
nucleic acid sequencing. 

Major Findings 

1. An organ culture model system to test anticat- 
aract agents has been standardized. Part of the 
model involves screening the proteins in the culture 
medium to determine lens integrity. 

2. With lenses from young animals, glutathione 
is rapidly lost in the organ culture system; however, 
this might not be the case with an older animal. 

3. One of the major proteins that leak from the 
lens under conditions of stress is PB2-crystallin. 

4. The PB2-crysta]lin has been cloned and 
sequenced from human lens. It has about 907c 
similarity with the mouse pB2-crystaUin that we had 
sequenced previously. Our deduced sequence for the 
human crystallin has been confirmed by another 
group. 

5. ^-crystallin, an oxidoreductase found in guinea 
pigs and camels, has been found in the mouse lens. 
Lens cells from transgenic animals also have i^- 
crystallin. 

6. Lens cells under oxidative stress react to the 
stress by increasing oxidoreductase activity. The 
activity that appears to be most responsible for the 
amelioration of the effects of oxidative radicals is D- 
T diaphorase, although i^-crystallin also is activated 
under oxidative stress conditions. 

7. In tissue cultures, lens cells form so called 
"lentoid bodies." We have shown that lentoid body 



formation is an early step in the maturation process 
of the lens cell. The lentoid body can form without 
the activation of certain lens-specific proteins. 

Lentoid body formation is similar to the elonga- 
tion process that occurs in the equator of the lens. In 
this equatorial area, many cataracts originate. Thus, 
understanding the steps in the differentiation proce- 
dure is necessary to understand cataract formation. 

8. Some of the proteins present in the aqueous 
humor of the eye have been identified. The aqueous 
humor is the fluid that nourishes the lens in vivo. 
The eight proteins that have been confirmed to be in 
the aqueous of the monkey are albumin, transferrin, 
ceruloplasmin, plasminogen, fibrinogen, a-1 antitryp- 
sin, HDL, and cystatin. 

Significance to Biomedical Research and the 
Program of the Institute 

The development of systems to study the lens is vital 
to understanding the mechanisms involved in cataract 
formation. The new methods and protocols that we 
have developed for organ-cultured lenses have 
enabled us to standardize this useful technique for 
the study of cataract development. Working under 
definable, reproducible conditions is an advantage in 
formulating model systems to study conditions that 
lead to loss of cell function. These studies will 
enable us to devise systems to study anticataract 
agents. 

Publications 

Chambers C, Russell P: Sequence of the human lens 
betaB2-crystallin encoding cDNA. Gene 
133:295-296, 1993. 

Du X-Y, Russell P, Zigler JS Jr: Potentiation of 
protein oxidation in cultured lenses by 2-deoxy- 
glucose and BCNU. Curr Eye Res 11:475^78, 
1992. 

Kidd GL, Reddan Jr, Russell P: Differentiation and 
angiogenic potential in two mammalian lens 
epithelial cell lines. Differentiation, in press. 

Qin C, Rao PJ, Tumminia SJ, Zigler JS Jr, Russell P: 
Loss of glutathione in the organ cultured rat lens. 
Invest Ophthalmol Vw5d34(4)(suppl):758, 1993. 

Russell P, Epstein DL: Protein analysis of monkey 
aqueous humor. Curr Eye Res 11:1239-1243, 
1992. 



143 



Laboratory of Mechanisms of Ocular Diseases 



NEI Annual Report— FY 1993 



Russell P, Epstein DL: Protein analysis of the 
rhesus monkey aqueous humor. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1280, 1993. 

Russell P, Koretz J, Epstein DL: Is primary open- 
angle glaucoma caused by small proteins? Med 
Hypothesis, in press. 

Russell P, Zigler JS Jr: Analysis of the effects of 
eye bank storage conditions on primate lens 
epithelium. Exp Eye Res 54:153-155, 1992. 



Tumminia SJ, Qin C, Zigler JS Jr, Russell P: Asses- 
sibility of the viability and integrity of mammali- 
an lenses in organ culture. Invest Ophthalmol 
Vis Sci 34(4)(suppl):756, 1993. 

Tumminia SJ, Rao PV, Zigler JS Jr, Russell P: 
Xenobiotic induction of quinone oxidoreductase 
activity in lens epithelial cells. Biochim Biophys 
Acta 8:251-259, 1993. 



144 



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



PROJECT NUMBER 



ZOl EY 00252-05 LMOD 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Cataract in the Philly Mouse Strain 



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 



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: 



0.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



PROFESSIONAL: 



0.0 



OTHER: 



0.0 



□ (b) Human tissues 



[x] (c) Neither 



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

This project has been terminated. 



145 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00105-14 LMOD 



PERIOD COVERED 

October 1. 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or less T1II9 must lit on one line between the borders.) 

Structure and Composition of Lens Crystallins with Respect to Cataractogenesis 



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

PI: J. Samuel Zigler, Jr. Ph.D. Research Biologist LMOD, NEI 



Others: Vasantha Rao 
Pedro Gonzalez 
Chuan Qin 



Ph.D. 
Ph.D. 
M.D. 



Visiting Associate 
Visiting Fellow 
Visiting Fellow 



LMOD, NEI 
LMOD, NEI 
LMOD, NEI 



COOPERATING UNITS (il any) 

Jules Stein Eye Institute, UCLA (J. Horowitz, B. Bateman); Laboratory of Molecular and Developmental 
Biology, NEI (G. Wistow, D. Lee); National Cancer Institute (M. Krishna); Centre for Cellular and Molecular 
Biology, Hyderabad, India (D. Balasubramanian; M. Rao) 



LAB/BRANCH 



Laboratory of Mechanisms of Ocular Diseases 



SECTION 



Section on Cataracts 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



4.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



PROFESSIONAL: 



4.0 



OTHER: 



0.0 



[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, places 
special emphasis on the role of the structure and function of the lens crystallins. Until recently, the crystallins 
were thought to be simply structural elements of the lens protein matrix, without any specific quantifiable 
biological function. Two recent discoveries have provided new insights and approaches to the physiological 
roles of the crystallin: (1) the crystallins either are 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. 

Our group is studying the chaperone function of a-cTystallin, with the goal of establishing its significance in 
the intact lens. Using the isolated crystallin, we have shown that it forms stable complexes with target 
proteins, specifically interacting with denaturing proteins at the earliest stage of denaturation. The specificity 
of this interaction has been demonstrated by the fact that in some instances it is strongly dependent on the 
availability of obligate cofactors of the target proteins. Studies on "enzyme/crystallins" focus on i;-crystallin, 
a major protein in the lenses of certain mammals (e.g., guinea pigs, camelids). In guinea pigs a mutation in 
the ^-crystallin gene causes hereditary nuclear cataracts, and our goal is to understand how the mutation affects 
the lenticular function(s) of the protein, leading to cataract. The ^-crystallin system also is being used to 
investigate the mechanisms of lens-specific expression of crystallin genes. 

Lens organ culture is being used as both a means of testing potential anticataract agents and a system for 
analyzing the responses of intact lenses to various cataractogenic stresses. The changes in gene expression 
induced in primate lenses by stress are being studied to identify specific proteins and processes important in 
combating stress. This information will facilitate more rational design of strategies to accomplish our ultimate 
goal: the prevention or delay of cataractogenesis. 



146 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



Project Description 

Objectives 

The primary objectives of this project are (1) to 
elucidate at the molecular level processes responsible 
for cataract development, (2) to investigate the 
structures and functions of the lens crystallins, and 
(3) to develop and use model systems for screening 
potential anticataract agents. 

Methods 

Conventional protein chemical techniques employed 
are chromatography, electrophoresis, and isoelectro- 
focusing. Immunological studies of lens proteins use 
specific antisera. Physicochemical analyses on the 
proteins are performed using high-pressure liquid 
chromatogrj^hy, fluorescence, and circular dichroism 
techniques. In lens organ culture experiments 
involving rat or monkey lenses, we 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 acids 
include RNA and DNA isolation, cDNA and gene 
cloning, DNA sequencing, various electrophoretic 
methods, and the polymerase chain reaction. 

Major Findings 

1. a-crystallin acts as a molecular ch^erone, 
forming stable complexes with various other proteins 
undergoing denaturation and preventing their aggre- 
gation. Once fully denatured, the target proteins do 
not associate with a-crystallin. Thus, like other 
chaperone proteins, a-crystallin specifically recog- 
nizes and binds proteins only in the very early stage 
of denaturation. 

2. Further evidence for the specificity of this 
reaction is provided by the finding that, with certain 
proteins, protection from aggregation by a-crystallin 
is dependent on the presence of cofactors. For 
example, ^-crystallin/quinone reductase, an NADPH- 
requiring enzyme, is efficientiy protected only in the 
presence of NADPH. 

3. The ^-crystallin cDNA from the lens of the 
llama has been sequenced and found to be highly 
similar to that of other mammals analyzed. The 
llama is a camelid and therefore of particular interest 
because, like guinea pigs, these animals have very 
high levels of lenticular ^-crystallia 



4. Our analysis of the llama ^-crystallin gene has 
revealed two promoters, one of which regulates 
normal low level expression in many tissues. This 
promoter exists in all ^-crystallin genes examined. 
A second lens-specific promoter is found only in 
species in which the protein is also a major lens 
protein (e.g., guinea pig and llama). Interestingly, 
the promoter in the llama gene is unrelated to the 
lens-specific promoter previously characterized in the 
guinea pig, suggesting that ^-crystallin/quinone 
reductase was recruited as a lens protein at least two 
different times during evolution. 

5. The human ^-crystaUin gene has been local- 
ized to chromosome lp22-p3P, and six restriction 
fragment-length polymorphisms (RFLPs) have been 
identified within the gene. 

6. Analysis of the sequences of all known lens 
crystallins reveals that, in general, they are not 
designed for high intracellular (metabolic) stability. 
Therefore, the extremely long half-lives of crystallins 
must result largely from the environment within the 
lens rather than from intrinsic properties of the 
proteins themselves. 

7. The organ-cultured rat lens loses 40% of its 
glutathione (GSH) during the first 24 hours of 
culture and more than 60% by 72 hours. This loss 
occurs even in the absence of Oj and, thus, is not the 
result of oxidative stress in the culture system. 
Interestingly, monkey lenses cultured for up to 48 
hours showed no decrease in GSH. 

8. Huorescence spectra of intact human lenses 
over a wide age range demonstrate different amounts 
and numbers of fluorophors in lenses from the 
United States relative to lenses collected and ana- 
lyzed in India. It is hoped that these analyses will 
give clues to the molecular mechanisms underlying 
the increased pigmentation and earlier onset of 
cataract in India. 

Significance to Biomedical Research and the 
Program of the Institute 

Cataract is a major public 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 smdies are 
aimed primarily at elucidating the role of the lens 
crystallins, the primary structural elements of the 
normally transparent lens matrix, in the processes 



147 



Laboratory of Mechanisms of Ocular Diseases 



NEI Annual Report — ^FY 1993 



leading to opacification. Such knowledge should 
contribute to the development of means of interven- 
tion that can prevent or delay the process of cataract 
development. 

Proposed Course 

We will continue to (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) complete 
analysis of the molecular basis underiying the high 
lens-specific expression of an enzyme/crystallin C^- 
crystallin), (3) further investigate the chaperone-like 
function of a-crystallin and determine its physiologi- 
cal significance in the normal lens and in cataract, 
and (4) evaluate gene expression in lenses under 
stress to seek proteins critical in the response to 
stress. 

NEI Research Program 

Cataract — Pathogenetic Mechanisms 

Publications 

Gonzalez P, Rao PV, Zigler JS Jr: Molecular clon- 
ing and sequencing of zeta-crystallin/quinone 
reductase cDNA from human liver. Biochem 
Biophys Res Commim 191:902-907, 1993. 

Jomvall H, Persson B, Du Bois GC, Lavers GC, 
Chen JH, Gonzalez P, Rao PV, Zigler JSJr: 



Zeta-crystallin versus other members of the 
alcohol dehydrogenase superfamily: Variability 
as a functional characteristic. FEBS Lett 322:240- 
244, 1993. 

Lee DC, Gonzalez P, Rao V, Zigler JS Jr, Wislow 
GJ: Carbonyl-metabolizing enzymes and their 
relatives recruited as structural proteins in the eye 
lens. Adv Exp Med Biol '\:\59-\()^, 1993. 

Rao CM, Zigler JS Jr: Are crystallins designed for 
high intracellular stability? Exp Eye Res 56:615- 
619, 1993. 

Rao PV, Horwitz J, Zigler JS Jr: Alpha-crystallin, a 
molecular chaperone, forms a stable complex with 
carbonic anhydrase upon heat denaturation. 
Biochem Biophys Res Commun 190:786-793, 
1993. 

Rao PV, Zigler JS Jr: Mutant zeta-crystallin from 
guinea pig hereditary cataracts has altered struc- 
tural and enzymatic properties. Exp Eye Res 
54:627-630, 1992. 

Tumminia SJ, Rao Pv, Zigler JS Jr, Russell P: 
Xenobiotic induction of quinone oxidoreductase 
activity in lens epithelial cells. Biochim Biophys 
Acta, 1203:251-253, 1993. 

Zigler JS Jr: Lens proteins, in Albert DM, Jakobiec 
F (eds): Principles and Practice of Ophthalmol- 
ogy. Basic Sciences Philadelphia, JB Saunders 
Co, 1994, pp 97-113. 



I 



148 



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



PROJECT NUMBER 



ZOl EY 00149-20 LMOD 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Ultrastructure and Function of the Cells and Tissues 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. Head, Section on LMOD, NEI 

Pathophysiology 



Others: 



Nora Laver 
Anne Groome 
Joe Hackett 
Evita By nam 
Joel Glover 



M.D. 


Special Volunteer 


LMOD, NEI 


B.S. 


Histology Technician 


LMOD, NEI 


B.S. 


Biologist 


LMOD, NEI 


B.S. 


Microbiologist 


LMOD, NEI 


B.S. 


Biologist 


LMOD, NEI 



COOPERATING UNITS (if any) 

Alcon Laboratories, Inc. (Billie M. York, Jr., Ph.D.) 



LAB/BRANCH 

Laboratory of Mechanisms of Ocular Diseases 



SECTION 

Section on Pathophysiology 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



4.75 



PROFESSIONAL: 



1.0 



OTHER: 



3.75 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 
O (a1) Minors 
□ (a2) interviews 



[xl (b) Human tissues □ (c) Neither 



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

Using the galactose-fed rat model for diabetic retinopathy, which was first developed in this laboratory, we 
designed intervention studies to test the possibility of delaying, halting, or reversing retinopathy soon after the 
earliest capillary lesions could be documented. Weanling male SD rats were divided into five groups, three 
of which received either normal lab chow or a 50% galactose diet with or without an aldose reductase inhibitor 
(ARI:ca. 11 mg/kg/day AL-3152) and two of which received 50% galactose for 6 months and then 
intervention, by either addition of inhibitor or removal of galactose. From each rat killed at 6, 18, and 24 
months, one retina was prepared for obtaining electron miaographs of capillary transections and the other was 
used for whole mounts of isolated retinal vessels. We captured images of whole and transected capillaries and 
analyzed them using computer hardware and programs specially designed for 1,024 x 1,024 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 1 to 10. 
At 6 months, when intervention began, untreated galactose-fed rats exhibited a 30%, 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 rats receiving intervention with either AL-3152-enriched or 
galactose -firee diets exhibited only grade-2 retinopathy, and rats fed control diet or galactose plus AL-3152 
throughout 18 months showed none. At 24 months, untreated rats had grade- 10 retinopathy, and both 
intervention groups had grade-8 retinopathy. Thus, 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 culture, we will investigate possible mechanisms of endothelial cell 
proliferation and subsequent pathologies. 



149 



PHS 6040 (Rev. 5/92) 



Laboratory of Mechanisms of Ocular Diseases 



NEI Annual Report — FY 1993 



Project Description 

Objectives 

This project is designed to use special diets in vivo 
and controlled media in cell cultures of ocular tissues 
to mimic the diabetic state in order to determine 
whether diabetic-like tissue changes can be prevented 
by aldose reductase inhibitors (ARIs). 

Methods 

Weanling male Sprague-Dawley rats were divided 
into five groups, three of which received either 
normal lab chow or a 50% galactose diet, with or 
without an ARJ (ca 11 mg/kg/day AL-3152), and 
two groups which received 50% galactose for 6 
months and then intervention by either the addition 
of an inhibitor or the removal of galactose Rats 
were killed at 6, 18, and 24 months. A new enzyme 
digestion procedure (elastase method) developed in 
this lab was used on the retina of one eye of each rat 
to remove all retinal tissues except the vessels. This 
provided a whole mount of the retinal vasculature 
and permitted the recognition of degenerated peri- 
cytes ("ghosts") and all of the more advanced angi- 
opathies by light microscopy. The retina of the other 
eye of each pair was sectioned and examined by 
electron microscopy. Images of whole and tran- 
sected capillaries were captured and analyzed by 
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 1 to 10. Tissue cultures of human, bovine 
and canine retinal capillary pericytes and leas' 
epithehal cells were used to investigate the 
mechamsm(s) underlying the diabetic angiopathies. 

Major Findings 

Vascular whole mounts of capillaries of rats fed 
galactose for 24 months, prepared by our new 
enzyme digestion procedure, exhibited multiple 
reunal angiopathies identical to those typical of 
human background diabetic retinopathy. These 
angiopathies did not occur in the retinas of rats fed 
a galactose diet with an ARI. The presence of 
aldose reductase was demonstrated in cultured retinal 
pericytes (1) by immunohistochemistry, shown by the 



antibody against human placental aldose reductase; 
(2) by its activity, shown by measurements of xylitol 
production in cells grown in a medium supplemented 
with xylose; and (3) by the detection of messenger 
RNA for aldose reductase. There was a compro- 
mi.sed proliferation rate in pericytes, compared with 
endothelial cells incubated in high (30 mM) sugar 
concentrations, suggesting toxicity of polyol at the 
cellular level. Aldose reductase appears to be 
involved in all the retinal complications of diabetes, 
from pericyte degeneration to microaneurysms. 

Significance to Biomedical Research and the 
Program of the Institute 

Diabetic retinopathy is mainly a disease of retinal 
capillaries. Recently potentially beneficial treatments 
and animal models have become available. How- 
ever, 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 
reanopathy have been limited by the lack of reliable 
and convenient animal models. Now, besides the 
alloxan diabetic dog and the galactosemic dog, there 
IS a galactosemic rat model. All this has been 
possible because aldose reductase is involved in 
diabetic retinopathy. Aldose reductase, which has 
been implicated in sugar cataracts, certain corneal 
healing defects, and peripheral neuropathy of diabetic 
and galactosemic animals, now appears to be in- 
volved in all lesions of background diabetic retinopa- 
thy. While the normal physiological role of this 
enzyme in most tissues remains unknown, under the 
conditions of high plasma sugar concentrations 
encountered in diabetes and galactosemia, aldose 
reductase converts these sugars to their respective 
sugar alcohols (polyols). Tliese polyols are not 
readily metabolized, nor do they penetrate cell 
membranes easily. Thus, once formed at significant 
rates, they may accumulate to very high levels in 
cells, leading to hypenonicity, alteration of ion 
permeabiUty, and eventual cell death, with conse- 
quent tissue changes such as cataract formation. 
Treatment of diabetic or galactosemic rats with 
potent ARIs, such as sorbinil or tolrestat, decreases 
tlie 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 capillaries, and decreased 
conduction velocity in nerves. 



150 



NEI Annual Report— FY 1993 



Laboratory of Mechanisms of Ocular Diseases 



By using a novel vessel preparation method, 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. 
Pericyte loss, endothehal cell proliferation, micro- 
aneurysms, shunts, occlusions, dilations, and all the 
other microangiopathies that we found in the galac- 
tose-fed rat are identical to the histopathologies 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-like retinopathy 
in galactosemic rats can be prevented with an ARI. 

Proposed Course 

The following studies are proposed for Fiscal Year 
1993. We will extend the intervention studies to 
determine how late one can interrupt the disease 
process and still obtain beneficial results by treatment 
with various ARIs. We also will examine the early 
formation of intracellular vacuoles, cell transport 
systems, the mechanism of basement membrane 
synthesis, and the relationships of these changes to 
aldose reductase in isolated retinal cells grown under 
diabetic conditions. We will manipulate the rat diets 
to shorten the time when diabetic-like retinal angiop- 
athies appear, thus improving the rat as a model for 
diabetic retinopathy. 

NEI Research Program 

Retinal Diseases — Diabetic Retinopathy 



Publications 

Laver NM, Robison WG Jr: Proliferative retinopa- 
thy stage in long-term galactose fed rats. Invest 
Ophthalmol Vis Sci 34(4)(suppl):713, 1993. 

Laver N, Robison WG Jr, Calvin HI, Fu S-CJ: Early 
epithelial lesions in cataracts of GSH-depleted 
mouse pups. Exp Eye Res 57:493-498, 1993. 

Laver NM, Robison WG Jr, Hansen BC: Demon- 
stration of retinal histopathologies in spontaneous- 
ly diabetic monkeys. Am J Clin Pathol 99(3): 
349, 1993. 

Laver NM, Robison WG Jr, Pfeffer BA: Novel 
procedures for isolating intact retinal vascular 
beds from diabetic humans and animal models. 
Invest Ophthalmol Vis Sci 34:2097-2104, 1993. 

Matthews GP, Laver N, Robison WG Jr: Electro- 
physiological and histological evaluation of inner 
retina in galactosemic rats. Invest Ophthalmol Vis 
Sci 34(4)(suppl):720, 1993. 

Robison WG Jr, Laver N: Ocular lesions in animal 
models of human diabetes, in Shafirir E (ed): 
Frontiers in Diabetes Research, Lessons from 
Animal Diabetes IV. London, Smith-Gordon and 
Company Limited, 1993, pp 145-163. 

Robison WG Jr, Laver NM, York BM, Chandler 
ML, Lou MF: ARI intervention studies of galac- 
tose induced retinopathy by computer analysis of 
retinal vessel images. Invest Ophthalmol Vis Sci 
34(4)(suppl):718, 1993. 



151 



Laboratory of Molecular and Developmental Biology 



Report of the Chief, Laboratory of Molecular and Developmental 
Biology 



Joram Piatigorsky, Ph.D. 



In its 12th year, the Laboratory of Molecular and 
Developmental Biology (LMDB) has been ex- 
panded by two sections — the Section on Regulation 
of Gene Expression, headed by Dr. Ana B. Chepelin- 
sky, and the Section on Transgenic Animals and 
Genomic Manipulation, headed by Dr. Eric Wawrou- 
sek. Dr. Chepelinsky, a valued member of the 
LMDB since its beginning, has augmented her 
research area, the crystallins, to include membrane 
proteins and their genes, as well as the effects of 
growth factors on eye development. Dr. Wawrousek, 
a former postdoctoral fellow at the LMDB, has 
returned to the National Eye Institute (NEI) after a 
brief sojourn in industry. His Section wears two 
hats. The first provides a service for the NEI — ^the 
creation of transgenic mice; the second performs 
research involving site-specific gene recombination. 
The addition of these two sections has increased the 
expertise of the LMDB and extended our usefulness 
to the NEI. The other sections of the LMDB include 
the Section on Molecular Genetics, headed by Dr. 
Joram Piatigorsky; the Section on Cellular Differenti- 
ation, headed by Dr. Peggy S. Zelenka; and the 
Section on Molecular Structure and Function, headed 
by Dr. Graeme J. Wistow. 

Not all developments are happy ones. Sadly, 
Ms. Dawn Chicchirichi, the LMDB secretary since 
the Laboratory's beginning, retired due to illness. 
Ms. Chicchirichi gave 11 years of devoted and 
excellent assistance to the LMDB and will be greatly 
missed. She has been replaced by Ms. Linda Willett. 
Ms. Willett already has become an invaluable mem- 
ber of the LMDB, and we are extremely lucky to 
have her with us. I also take this opportunity to 
thank the many NEI staff members who gave us 
great support and help during the difficult months of 
Ms. Chicchirichi 's illness so that we could keep 
functioning during the transition period. 

The LMDB's primary goal is to perform basic 
research on the molecular biology of the eye. 
Although particular attention is directed to the lens, 
the cornea and retina have not escaped our efforts; 



research projects also have focused on the role of 
growth factors on eye development. In addition, 
because lens crystalUns are multifunctional proteins 
that are expressed outside the lens and eye, the scope 
of our research has increased during the last few 
years to include new areas of metabolism and gene 
expression in various tissues. Moreover, many of the 
transcription factors involved in the expression of 
crystallin genes are present in many tissues and are 
used to control numerous biological processes. 
Consequently, our studies on eye genes have implica- 
tions for many areas of molecular, cellular, and 
evolutionary biology. This is reflected in the pleth- 
ora of general journals in which we publish our 
scientific discoveries and the fact that we often 
attend meetings that focus on broad issues of genet- 
ics, development, evolution, and molecular biology. 
Thus, the original twin purposes of using the visual 
system as a model for the structure, expression, and 
evolution of genes and incorporating general princi- 
ples of molecular biology to understanding the visual 
system continue as the core of our thinking. 

There have been many individual research accom- 
plishments by LMDB staff this year. These accom- 
plishments are detailed in the specific annual reports. 
In general, much attention has been given to the 
identification of regulatory elements required for 
expression of genes in the eye and other tissues. 
Many of these regulatory elements are commonly 
found in genes; however, each has its own special 
properties. The diversity of elements used for 
expression of eye genes is impressive, ensuring that 
we will be busy for many years sorting them all out. 
To complicate things even more, we have shown that 
the regulatory elements may be functionally redun- 
dant, i.e., removing one does not necessarily 
eliminate the expression of the gene. There are also 
many different nuclear proteins that bind to the DNA 
regulatory elements, and this year we have cloned a 
number of them. One of our biggest challenges is to 
determine which binding proteins are actually in- 
volved in regulating the genes in the animal. 



155 



Laboratory of Molecular and Developmental Biolog> 



NEI Annual Report — FY 1993 



Our studies on the expression of proto-oncogenes 
and cyclins have linked the normal process of 
cellular differentiation in the lens with the cell cycle 
and growth control, providing another example of the 
broad relevance of our research. In addition, the use 
of crystalhn promoters for directing various growth 
factors to the lens have extended cellular studies to 
consideration of growth of the entire eye. These 
genetic engineering experiments have opened the 



opportunity to develop animal models for auto- 
immune diseases of the eye, fostering communication 
between the LMDB and the NEI Laboratory of 
Immunology. The addition of the transgenic facility 
has had a major impact in increasing the dialog 
between the LMDB and other NEI laboratories. We 
look forward to additional cross-fertilization of ideas 
in the years to come. 



156 



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



PROJECT NUMBER 



ZOl EY 00238-08 LMDB 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



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

Proto-Oncogene Expression During Lens Differentiation and Development 



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 CeUular LMDB, NEI 



Others: Jo Ann Rinaudo 

Chun Yun Gao 
Emmanuel Vacchiano 
Anoradba Rampalli 
Jaspreet Arora 
Graeme Wistow 





Differentiation 


Ph.D. 


Staff Fellow 


M.D., Ph.D. 


Staff Fellow 


Ph.D. 


Staff 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, 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: 



5.2 



PROFESSIONAL 



5.2 



OTHER: 



0.0 



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.) 

This project to investigate the expression of proto-oncogenes and other cell cycle regulatory genes in the 
embryonic chicken lens seeks to determine their relationship to cell growth, quiescence, and differentiation. 
The normal developmental profiles of five nuclear proto-oncogene mRNAs (c-myc, N-myc, c-fos, c-jun, and 
p53) and the cell cycle regulatory protein, cyclin B, have been completed, and the profile of retinoblastoma 
(Rb) expression is in progress. The finding that cyclin B is present in lens-fiber cells suggests that lens-fiber 
cell differentiation may represent an aberrant form of the cell cycle. In addition to cyclin B, a number of other 
proteins normally associated with proliferating cells are expressed in postmitotic, differentiating lens cells. 
These include cyclin A, c-myc, c-fos, c-jun, and p53. Moreover, preliminary studies using explanted 
embryonic chicken lens epithelia indicate that the order of expression of these genes during differentiation is 
the same as during proliferation, further strengthening the link between differentiation and the cell cycle. The 
functional role of each of these genes during leas differentiation now is being explored through the use of 
retroviral vectors, transfection of exogenous DNA, and the production of transgenic mice. In addition, 
regulatory mechanisms governing the changes in proto-oncogene expression that accompany differentiation 
are being explored. 



157 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biology 



^fEI Annual Report— FY 1993 



Project Description 

Additional Personnel 

Milton Berger B.A. 

Tania Tolstoshev 



Graeme Wistow 



Ph.D. 



Special Volunteer, 

LMDB, NfEI 

H.S. Student, Special 

Volunteer, LMDB, 

NEI 

Head, Section on 

Molecular Structure 

and Function, LMDB, 

NEI 



Objectives 

In this project we seek to determine whether the 
expression of specific proto-oncogenes is altered 
during lens cell differentiation and, if so, to deter- 
mine the mechanism of gene regulation and the 
function of the corresponding proto-oncogene prod- 
ucts in the developing lens. The objective is to 
develop a greater understanding of the mechanisms 
underlying lens cell growth and differentiation. 

Methods 

Techniques of molecular biology are used in con- 
junction with traditional cell biology techniques. 
Conventional methods are employed for protein and 
nucleic acid analysis, including polyacrylamide gel 
electrophoresis, RNA and DNA isolation, polymerase 
chain reaction (PCR), reverse transcription PCR (RT/ 
PCR), nucleic acid hybridization, in vitro transfec- 
tion, in situ hybridization, immunocytochemistry, and 
immunoblotting. DNA/protein interactions are 
studied using DNAse I footprinting, electrophoretic 
mobility shift assays, and ultraviolet (UV) cross- 
linking. 

Studies employ 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 manmialian 
cell lines. In addition, transgenic mice are produced 
to test the function of proto-oncogenes and cell cycle 
regulatory proteins in the lens in vivo. 

Major Findings 

In the past year major progress has been made on 
studies of the cell cycle regulatory protein, cyclin B. 
Expression of this protein is known to be cell-cycle 
dependent in proliferating cells, appearing in the S 



and G2 phases of the cell cycle. Work done by Dr. 
Chun Gao has demonstrated that cyclin B is ex- 
pressed in differentiating lens fiber cells. The 
presence of cyclin B mRNA was shown by RT/PCR, 
followed by sequencing of the PCR product. 
Immunoblotting with an antibody specific for cyclin 
B following two-dimensional gel electrophoresis 
confirmed that the protein is also present in lens fiber 
cells. 

In situ hybridization of sections of 14-day embry- 
onic lenses with riboprobes for cyclin B mRNA 
showed that the mRNA is abundant in the differenti- 
ating cells at the lens equator and in the nucleated 
fiber cells, but it cannot be detected in the enucleated 
fiber cells. Cyclin B from lens fiber cells was 
affinity-purified by chromatography on pi 3™" 
Sepharose. Because the pl3 protein binds to p34'=*^ 
kinase, purificadon of cyclin B by this process 
provides evidence that it is complexed with the 
p34""^ protein. The kinase activity of this complex 
was demonstrated by using histone HI as a substrate. 
Kinase activity could be increased about twofold by 
phosphatase treatment. 

These results indicate that cyclin B, a protein 
normally expressed in the G2 phase of the cell cycle, 
is present in differentiating lens fiber cells. Since the 
cyclin B/p34"''=^ complex is known to be responsible 
for chromosomal condensation and nuclear envelope 
breakdown in mitotic cells, finding this complex in 
lens fibers and in the enzymatically active, dephos- 
phorylated form provides evidence that this same 
biochemical mechanism may be responsible for 
chromosomal condensation and nuclear envelope 
breakdown during fiber cell differentiation. 

Because cyclin B normally is expressed only in 
the G2 phase of the cell cycle, its presence in differ- 
entiating lens fiber cells suggested that the differenti- 
ation process itself may be an aberrant form of the 
cell cycle. To test this possibility, Dr. Anuradha 
Rampalli has initiated experiments to examine the 
order of expression of a number of cell-cycle mark- 
ers in differentiating explants of 6-day-old embryonic 
chick lens epithelia. Preliminary results show a 
strong, early induction of c-fos, c-jun, c-myc, and N- 
myc, followed after a lag of 5-7 hours by induction 
of p53 and after a lag of 18-24 hours by induction of 
the heat shock protein HSP70. Since c-fos, c-jun, 
and c-myc are normally expressed in early Gl, p53 
in late Gl, and HSP70 during the S and G2 phases 
in proliferating cells, the order of induction seen 



158 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



during differentiation parallels that of the nonnal cell 
cycle. Additional S-phase markers under investiga- 
tion include PCNA (the 5 subunit of DNA polymer- 
ase) and thymidine kinase. Cyclins A and B will be 
examined as markers for the G2 phase. The induc- 
tion of N-myc is not typical of proliferating cells 
and, thus, marks a significant difference between the 
two processes. 

The tumor supressor genes Rb and p53 seem to 
play key roles in preventing Gl cells from entering 
the S phase, making their role in lens differentiation 
particularly interesting. Studies by other investiga- 
tors have shown that inactivation of these gene 
products by SV40 T antigen prevents terminal 
differentiation of lens fiber cells. Dr. Rampalli has 
completed a developmental study of p53 expression 
in the embryonic chick lens, and a companion study 
of Rb expression is in progress. Her results clearly 
show that p53 mRNA and protein are expressed in 
differentiating cells at the lens equator and in the 
newly formed fiber cells, consistent with the data 
from differentiating explants and with the idea that 
differentiation and cell-cycle progression share 
important features. 

A major focus of this project continues to be the 
biological function of the proto-oncogenes expressed 
in the lens. Preliminary evidence, reported last year, 
that c-myc regulates expression of the x-crystallin/a- 
enolase gene has now been extended to demonstrate 
that the c-myc protein itself is involved in binding to 
the T-crystallin promoter. Interestingly, a x-crystal- 
lin/chloramphenicol acetyltransferase (CAT) coastruct 
with a mutation in the potential c-myc binding site 
was shown to be expressed at somewhat higher 
levels in cultured lens cells than was the wild-type 
construction, although cotransfection of a c-myc 
expression vector was no longer required to stimulate 
this expression. This observation raises the possibil- 
ity that c-myc and a negative regulatory protein may 
compete for binding to the same or overlapping sites. 
The possibility that N-myc may be such a negative 
factor is under investigation. 

The function of c-jun in the embryonic chicken 
lens has been explored using wild-type and mutated 
cDNAs for chicken c-jun cloned into the avian 
retroviral vector RCAS. Use of this vector permits 
fransfer of the c-jun constructs to cultured cells with 
efficiencies approaching 100%, making it possible to 
test for the effects of c-jun on DNA synthesis, 
differentiation, and expression of endogenous genes. 



Results obtained by Drs. Jo Aim Rinaudo and 
Emmanuel Vacchiano indicate that a negative domi- 
nant mutation of c-jun increases the levels of endoge- 
nous oA-crystallin mRNA twofold above the already 
high levels present in the control cells. The levels of 
PA3/A1 mRNA were not affected in the same cells, 
suggesting that multiple, independent pathways may 
operate in differentiating lens cells, only some of 
which are affected by c-jun. 

Overexpression of wild-type c-jun in chicken lens 
epithelial cells by means of the RCAS vector greatiy 
enhances cell proliferation and may immortalize the 
cells. Cells infected with a retrovirus bearing the c- 
jun cDNA have now undergone nine passages, with 
no apparent decrease in proliferative capacity. 
Furthermore, the cells seem to have retained their 
ability to differentiate to lens fibers; lentoid bodies 
form in the cultures when they are permitted to 
become confluent. If these cells are immortalized, 
they will be extremely useful for future studies of 
gene expression and differentiation. 

Noting the similarities between lens fiber cell 
differentiation and a^ptosis. Dr. Vacchiano has 
employed the c-jun rettoviral vector to investigate 
the role of c-jun in proliferation, differentiation, and 
apoptosis in chicken embryo lens epithelial cells. 
His results indicate that c-jun overexpression stimu- 
lates proliferation in the presence of serum, but it 
does not prevent differentiation once confluence is 
attained. In the absence of adequate levels of serum 
or growth factors, however, overexpression of c-jun 
increases the rate at which the cells enter apoptosis. 

Dr. Vacchiano also is investigating the possible 
role of bcl-2 expression in regulating lens cell 
growth and differentiation. This proto-oncogene has 
been shown in other cell types to block apoptosis 
induced by overexpression of e-myc or p53. Using 
RT/PCR, he has demonstrated that bcl-2 is expressed 
in the embryonic chicken lens. He is now preparing 
constructs that will permit overexpression of this 
gene in both cultured chicken lens epithelial cells and 
transgenic mice to determine its effect on apoptosis 
and differentiation of lens cells. Inhibition of differ- 
entiation by bcl-2 would indicate an important 
biochemical link between fiber cell formation and 
apoptosis. 

Our ongoing collaboration with Dr. Thomas Lysz 
(University of Medicine and Dentistry of New 
Jersey) has now demonstrated that endogenous 
production of 12-hydroxyeicosatetraenoic acid (12- 



159 



Laboratory of Molecular and Developmental Biology 



NEI Annual Report — F\' 1993 



HETE), a lipoxygenase pathway metabolite of 
arachidonic acid, is a required step in DNA synthesis 
stimulated by epithelial growth factor in the neonatal 
rat lens. Dr. Jaspreet Arora has used RT/PCR to 
demonstrate that 12-HETE synthesis is required for 
expression of two proto-oncogenes, c-fos and c-myc, 
whereas expression of c-jun seems to be independent 
of this pathway. Because inhibition of either c-fos or 
c-myc is sufficient to cause cell-cycle arrest, these 
findings indicate that 12-HETE production is a key 
control point in the lens epithelial cell cycle. 

Significance to Biomedical Research and the 
Program of the Institute 

The proto-oncogenes are normal cellular homologs of 
retroviral oncogenes. Since retroviral transformation 
disrupts cell growth and differentiation, it is likely 
that the proto-oncogenes are involved in the normal 
regulation of these processes. A study of proto- 
oncogene expression during lens cell differentiation 
may elucidate basic regulatory processes underlying 
lens cell growth and differentiation. Many types of 
cataract are associated with abnormal lens epithelial 
cell growth and inhibition of lens fiber cell differen- 
tiation. In addition, a number of other eye diseases 
involve loss of normal controls on cell proliferation. 
An understanding of the basic controls of cell growth 
and differentiation would further our understanding 
of these disease states. 

Proposed Course 

The following studies are in progress or are proposed 
for Fiscal Year 1994: 

1. We will test the hypothesis that the cyclin B/ 
p34'=^'=^ kinase is responsible for nuclear loss in 
differentiating lens cells by producing transgenic 
mice that express the Weel* kinase in lens fiber 
cells. This kinase inactivates the cyclin B/p34"''=^ 
kinase and would be expected to delay or prevent 
nuclear loss if cyclin B/p34"''^ is required. 

2. Due to the unexpected finding of the cyclin B/ 
p34«)c2 jynase in differentiating lens fibers and the 
noted similarities between lens differentiation and 
apoptosis, we will examine whether cyclin B is 
expressed in apoptotic cells. We will test a variety 
of apoptotic cells of divergent origins for the pres- 
ence of cyclin B and cyclin B/p34"''^ kinase activity. 



3. We will continue study of the time course of 
the expression of cell-cycle-dependent genes in 
differentiating explants of lens epithelia, with the 
addition of S and G2 phase markers to determine the 
extent to which differentiation resembles cell-cycle 
progression. 

4. We will examine ftirther the effect of c-myc 
on transcription of the x-crystallin/a-enolase gene by 
transfection studies in cells that do not express N- 
myc. We also will examine the effect on the endog- 
enous duck gene by transfection into duck fibroblasts 
and lens epithelial cells. 

5. We will examine the effect of the N-myc 
proto-oncogene on transcription directed by the x- 
crystallin/a-enolase gene using the techniques previ- 
ously used to smdy the role of c-myc. 

6. We will extend the collaborative effort with 
Dr. Lysz to other growth factors, as well as examine 
the mechanism by which 12-HETE affects expression 
of c-fos and c-myc. We will experiment to deter- 
mine whether human lenses possess the 12-lipoxy- 
genase pathway. 

7. We will explore the possible role of post- 
translational modifications of Rb and p53 proteins in 
lens cell differentiation. 

NEI Research Program 

Cataract — The Normal Lens 

Publications 

Dash A, Chung S, Zelenka PS: Expression of 
HSP70 mRNA in the embryonic chicken lens: 
Association with differentiation. Exp Eye Res, in 
press. 

Piatigorsky J, Zelenka PS: Transcriptional regulation 
of crystallin genes: cis elements, trans-factors, 
and signal transduction systems in die lens. Adv 
Develop Biochem 1:211-256, 1992. 

Wistow GJ, Shaughnessy MP, Lee DC, Hodin J, 
Zelenka PS: A macrophage migration inhibitory 
factor is expressed in the differentiating cells of 
the eye lens. Proc Natl Acad Sci USA 90:1272- 
1275, 1993. 



160 



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



PROJECT NUMBER 



ZOl EY 00126-12 LMDB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Crystallin Genes: Structure, Organization, Expression, and Evolution 



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



PI: Joram Piatigorsky 

Others: James B. Brady 

Sambath Chung 
Ales Cvekl 
Melioda Duncaii 
Peter Frederikse 
Rashmi Gopal-Srivastava 
John Haynes 
John G. Dagao 



Ph.D. Chief LMDB, NEI 

Ph.D. IRTA LMDB, NEI 

B.A. Technician LMDB, NEI 

Ph.D. Visiting Fellow LMDB, NEI 

Ph.D. IRTA LMDB, NEI 

Ph.D. Senior Staff Fellow LMDB, NEI 

Ph.D. Staff FeUow LMDB, NEI 

Ph.D. IRTA LMDB, NEI 

Howard Hughes Medical Institute/ LMDB, NEI 

NIH FeUow 
(Additional personnel listed under P rogram Description.) 



COOPERATING UNITS (if any) 

Jules Stein Eye Instimte, UCLA (J. Horwitz, Ph.D.); National Institute of Child Health and Human Development, NIH (K. Becker, Ph.D.; 
K. Ozato, Ph.D.); University of Southern California (V.M. Weis. Ph.D.; M. McFall-Ngai, Ph.D.); Medical College of Virginia (D.M. 
Stover, Ph.D.; Z.E. Zehner, Ph.D.); N.K. Koltzov Developmental Biology Institute, Russian Academy of Sciences, Moscow (R.D. 
Zinovieva, Ph.D.); Uniformed Services University of the Health Sciences (S. Bassnet, 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: 



14.0 



PROFESSIONAL 



10.8 



OTHER: 



3.2 



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.) 

The structure, expression, and evolution of crystallin genes of vertebrates and invertebrates are being studied. 
The four functional promoter elements described in the aA-crystallin gene of the mouse (DEI, oA-CRYBPl, 
PEl, and PE2) and five in that of the chicken (DE3, DE2A, DE2B, DEIA, and DEIB) are surprisingly 
different, considering the gene is orthologous in these species with the same high-level expression in the lens. 
We have cloned putative trans-acting factors which bind to the mouse oA-CRYBPl and chicken DE2A sites. 
The cxA-CRYBPl gene has been cloned and characterized; cDNA analyses indicate that it produces 
alternatively spliced mRNAs. The oA-CRYBPl protein also spears to be cleaved in a tissue-specific fashion. 
The mouse DEI site appears to bind a member of the CREB/ATF family. Four functional elements (oBE-l, 
aBE-2, (xBE-3, and MRF) have been identified in the mouse oB-crystallin enhancer; oBE-l, aBE-2, and 
txBE-3 are used in muscle and lens, while MRF binds myoD and myogenin and is muscle specific. We have 
identified in the chicken PA3/A1 -crystallin gene an enhancer containing an AP-1 consensus-binding sequence 
which increases lens transcription but is not necessary for lens specificity. Transfection and gel mobility shift 
experiments indicate that the PL-1 and PL-2 functional elements of the chicken pBl -crystallin promoter and 
the AP-l/ARE sequence of two squid crystallin promoters are necessary for activity in transfected chicken lens 
cells and bind similar nuclear proteins of the chicken lens. Six chicken nuclear proteins that bind to the PL-1 
sequence have been cloned. Transgenic mouse experiments indicate that the 52-crystallin enhancer works 
efficiently in the lens and also has modest activity in the cornea, brain, and retina. Squid glutathione 
S-transferase (GST) and two squid S-crystallin cDNAs have been expressed; GST is very active while the 
S-crystallins show little if any activity. Cephalopod cDNAs for Q-crystallin/ALDH, intermediate filament 
protein, and a- and P-tubuIin have been cloned; the genes for all but a-tubulin were lens specific. Cloned 
cubomedusan jellyfish J3-crystallin has been shown to be a novel protein. 



161 



PHS 6040 (Rev. 5/92) 



Laboraton of Molecular and Developmental Biolog> 



NEI Annual Report— FY 1993 



Project Description 

Additional Personnel 
Cynthia Jaworski Ph.D. 

Marc Kantorow Ph.D. 

Xuan Li Ph.D. 

Joan B. McDermon M.S. 

Barbara Norman 
Christina M. Sax Ph.D. 

Stanisiav 1. Tomarev Ph.D. 



Staff Fellow, 
LMDB. NEI 
IRTA. LMDB, NEI 
Visiting Associate, 
LMDB. NEI 
Biologist, 
LMDB, NEI 
Chemist, LMDB, NEI 
Senior Staff Fellow, 
LMDB. NEI 
Visiting Scientist, 
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. Particular attention is given to the 
regulation of crystallin gene expression in the devel- 
oping lens and. in the case of multifunctional crystal- 
lins and enzyme-crystallins, in nonlens tissues. 

Methods 

Conventional methods used for analysis of proteias 
and nucleic acids include polyacrylamide and agarose 
gel electrophoresis, RNA and DNA isolation, molec- 
ular hybridization (Southern and Northern blots), 
cDNA and gene cloning, DNA sequencing, recombi- 
nant DNA construction and mutagenesis, in situ 
hybridization, expression of recombinant DNAs in 
transfected cells and transgenic mice, polymerase 
chain reactions, primer extension and SI protection 
experiments, in vitro and in vivo footprinting, gel 
mobility shift analysis, chromatographic purification 
of proteins, and Western immunoblotting. 

Major Findings 

a-crystallin. — There are two a-crystallin genes, aA 
and otB. Although both are expressed principally in 
the lens, the oB gene is expressed constitutive! y in 
many other tissues and is inducible by stress. By 
contrast, there is very little expression of aA in other 
tissues (although there is some), and it is not induc- 
ible by stress. We have been continuing our studies 
on the molecular basis for expression of the mouse 
and chicken otA and the mouse oB-crystallin genes. 



At least four separate control sequences have been 
identified for the mouse ocA-crystallin gene: DEI 
(-111 to -97), oA-CRYBPl region (-75 to -48), 
TATA/PEl (-35 to -19), and PE2 (-h24 to -^43). 
Our current evidence suggests that the DEI site is a 
cyclic AMP-responsive element (CRE) that binds a 
member of the CREB/ATF family of transcription 
factors. PE2 contains both an API and a glucocorti- 
coid-responsive element. The oA-CRYBPl site 
binds a ubiquitous protein that we have cloned and 
called oA-CRYBPl. However, this site also con- 
tains a consensus sequence for the transcription 
factor, NF-kB. 

Immunoblotting experiments indicate that the 
oA-CRYBPl site binds various tissue-specific forms 
of the ocA-CRYBPl protein. Although there is no 
evidence indicating that NF-kB is used as a tran- 
scription factor for the expression of the mouse 
otA-crystallin gene, this cannot be ruled out at the 
present time. The otA-CRYBPl gene has been 
cloned and shown to consist of at least seven exons. 
Its entire cDNA sequence is almost completed, 
except for a small stretch in the middle. The en- 
coded protein contains at least four zinc fingers and 
is approximately 3(X),000 Daltons (D). It is homolo- 
gous to the human PRDII-BFl/MBP transcription 
factor. Since oA-CRYBPl is smaller than 300,000 
D on Western blots, it appears as if the protein has 
been cleaved before use. 

Sequencing multiple cDNAs has indicated that the 
primary transcript of oA-CRYBPl is alternafively 
spliced, providing another possible basis for hetero- 
geneity of this putative transcription factor. A series 
of mutated constructs inserted into transgenic mice 
have shown that the DEI and oA-CRYBPl sites are 
functionally redundant. It appears necessary for at 
least one of these control sites to interact with PEl 
and/or PE2 for lens-specific expression to occur. 

Work over the past 5 years has shown, surprising- 
ly, that different control elements are used in expres- 
sion of the orthologous mouse and chicken oA-crys- 
talUn genes. We have identified the following 
control sequences for the chicken gene: DE3 (-153 
to -140), DE2A (-144 to -134; this overiaps with 
DE3), DE2B (-128 to -118), DEI A (-114 to -104), 
and DEIB (-100 to -93). The (xA-CRYBPl se- 
quence in the chicken gene differs from that in the 
mouse gene by one nucleotide, and although it 
footprints with nuclear proteins from the chicken lens 
in DNAse I protection experiments, it does not 



162 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



appear to be functional, as judged by mutagenesis 
and transfection tests. Three cDNAs encoding 
proteins that bind to the DE2A sequence have been 
cloned from an embryonic heart and are currently 
under investigation. In contrast to the DEI region of 
the mouse oA-crystallin gene, the DEI A and DEIB 
sequences of the chicken aA gene do not appear to 
bind a member of the CREB/ATF family of tran- 
scription factors. 

In 1991 we reported the presence of an enhancer 
at positions -426 to -259 of the oB-crystallin gene 
of the mouse. This sequence behaves as a strong 
enhancer for expression in cultured muscle cells and 
as a weak enhancer in cultured lens cells. We have 
now established by mutagenesis and footprinting 
experiments the presence of at least four functional 
elements witliin this enhancer: oBE-l (-420 to 
-397), aBE-2 (-360 to -327), aBE-3 (-319 to 
-303), and the muscle regulatory factor (MRF) 
binding site (-300 to -280). The MRF contains an 
E box and is activated by the binding of either 
MyoD or myogenin. 

Last year we indicated by DNAse I footprinting 
experiments that the -148/-118 sequence was 
necessary for the specific expression of the oB gene 
in the lens. This year transgenic mouse experiments 
have shown that this sequence is necessary for 
lens-specific expression but the enhancer is not. The 
-426/-259 enhancer sequence is necessary for 
expression of the ccB gene in skeletal muscle and 
heart, and it quantitatively boosts expression in the 
lens. Thus, cxBE-1, otBE-2, and aBE-3 are used for 
expression of the oB-crystallin gene in both lens and 
muscle cells, but the MRF element is used only in 
the muscle cells. This is the first example of a 
muscle-specific control element in a crystallin gene. 

^-crystallin. — ^We have been investigating the 
|3B1 and PA3/A1 -crystallin genes in the chicken for 
several years. This year we have added the PBl- 
crystallin gene of the mouse to our studies. Previous 
experiments have established the importance of the 
PL-1 and PL-2 control elements for activity of the 
chicken PBl promoter in transfection experiments. 
The PL-1 and PL-2 sequences are present between 
position -122 and the TATA box; they resemble 
AP-1 binding consensus sequences and compete with 
AP-1 sites for binding nuclear proteins. 

This year we have selected six different cDNAs 
from an embryonic chicken heart library on the basis 
of binding to multimerized PL-1 sequences. They 



are currently under investigation. The -434/+30 
sequence of the chicken PBl -crystallin gene has been 
shown to contain the information for lens specificity 
in transgenic mouse experiments. Deletion experi- 
ments are in progress to determine whether the PL-1 
and PL-2 elements are necessary for lens specificity. 
Finally, we have cloned and are characterizing an 
approximately 18-kbp fragment that contains the 
mouse PBl -crystallin gene. 

We are continuing our studies on the chicken 
pA3/Al -crystallin gene. The -287/-254 sequence 
has been shown to possess enhancer activity for 
expression in transfected embryonic chicken lens 
epithelial cells. It contains an AP-1 consensus 
sequence and binds multiple nuclear proteins in gel 
mobility shift experiments. A T-rich tract located 
between positions -218 and -168 appears to sup- 
press activity of the -287/-254 enhancer in transfec- 
tion experiments. A series of transgenic mice have 
been produced using the reporter gene chlorampheni- 
col acetyltransferase (CAT). Fusion genes containing 
the -382/+22 or -143/+22 fragments of the pA3/Al 
gene directed lens-specific expression, indicating that 
neither the -2877-254 enhancer nor the -218/-168 
T tract is necessary for lens specificity. 

d-crystallin. — There are two linked 5-crystallin 
genes in the chicken. The 5' gene is specialized for 
lens expression, while the 3' gene encodes arginino- 
succinate lyase (ASL), making this an enzyme-crys- 
talhn. Although only the 82 gene encodes a protein 
with ASL activity, both genes are expressed in a 
developmentally regulated fashion in the lens, cor- 
nea, retina, heart, and brain of the chicken embryo. 

This year we generated transgenic mice carrying 
fusion genes comprising various combinations of the 
5-crystalUn promoters and enhancers (located in the 
third intron of the natural genes) attached to the 
bacteria] CAT reporter gene. The enhancers of both 
6-crystallin genes caused extremely high expression 
of the CAT gene in the lens of the transgenic mice, 
suggesting that a silencer for lens expression of the 
52-crystallin gene is operative in the chicken. 
Evidence also was obtained indicating that the 
5-crystallin enhancers influence expression of the 
transgenes in the transgenic mouse cornea, retina, 
and brain in a way that is consistent with the expres- 
sion of the natural genes in these tissues in the 
embryonic chicken. 

The 6-crystallin locus has been cloned from the 
duck genome to compare the regulatory sequences 



163 



Laboratory of Molecular and Developmental BiologA' 



NEI Annual Report— FY 1993 



for these two genes in the duck and the chicken. We 
have undertaken this project because, in contrast to 
the chicken 52 gene, the duck 82-crystallin gene is 
very active in the lens. It should be able to help us 
understand the mechanism of suppression of the 52 
enhancer in the chicken lens. So far, we have 
established that the duck 5-crystallin genes are linked 
as in the chicken; further characterization is in 
progress. 

S-crysiallin. — Although much research had been 
performed on the lens crystallins of venebrates, very 
little information was available concerning the major 
lens proteins of the complex eyes of invertebrates. 
Thus, several years ago we began studying inverte- 
brate crystallins. Particular attention has been given 
to the crystallins of cephalopods (squids and octopi) 
because these species have the prototypical cellular 
invenebrate lenses that have evolved independent of 
those of vertebrates. We showed earlier that S-crys- 
tallins are encoded by a family of at least 10 genes 
that are expressed specifically in the lens and are 
related to the glutathione S-transferase (GST) genes 
of vertebrates. 

Last year we cloned the squid gene encoding 
GST. In contrast to the S-crystallin genes, the GST 
gene is expressed principally in the digestive gland 
(analogous to the venebrate liver) and very little in 
the lens or other tissues of the squid. This year we 
have expressed the cDNAs for the GST gene and for 
a minor (SLl 1) and major (SL20-1) S-crystallin gene 
of the squid in a bacterial extract and assayed for 
GST activity. The results showed that squid GST 
has more enzymatic activity than any other inverte- 
brate or venebrate GST ever reported. The major 
SL20-1 crystallin had essentially no GST activity, 
and the minor SLll crystallin had slight GST activ- 
ity. Thus, the S-crystallins generally lost GST 
activity as they specialized for expression in the lens. 
Interestingly, the loss of GST activity of SL20-1 is 
associated with the insertion of a novel peptide in the 
center of the protein. There is no insertion in the 
SLl 1 protein, which has some GST activity. Muta- 
genesis experiments are in progress to identify the 
active sites for substrate binding and for enzymatic 
function. 

Transfection experiments using the CAT reponer 
gene and embryonic chicken lens epithelial cells have 
been conducted in order to identify putative control 
elements of the S-crystallin genes. An overlapping 
AP-1/antioxidant responsive element (ARE), present 



just upstream of the TATA box of the SL20-1 and 
SLll crystallin genes of the squid, is required for 
promoter activity in the transfected chicken lens 
cells. A similar sequence is present in the PL-1 and 
PL-2 functional elements of the chicken PBl -crystal- 
lin gene. Gel mobility shift and competition experi- 
ments have provided evidence that the chicken PL-1 
and PL-2 and squid AP-l/ARE regulatory sequences 
bind similar nuclear proteins of the chicken lens. 
These data raise the possibility that entirely different, 
nonhomologous crystallin genes of the chicken and 
squid have convergently evolved a similar c«-acting 
regulatory element for high expression in the lens. 
It is especially interesting that this element is a 
stress-responsive gene regulatory element, providing 
a further link between crystallins and stress proteins. 

^.-crystallin. — In addition to the major S-crystal- 
lins of cephalopods, a minor crystallin, called 
r2-crystallin, is related to aldehyde dehydrogenase 
(ALDH). This is the only known invertebrate 
crystallin that has a vertebrate counterpan (i.e., 
r| -crystallin/ ALDH found in the elephant shrew). 
Last year we cloned i2-crystallin cDNA. This year 
we finished characterizing the cDNA and the expres- 
sion pattern of the Q-crystallin gene. Sequence 
comparisons have suggested that vertebrate ALDHl/ 
ALDH2 gene duplication occurred after the diver- 
gence of cephalopods from the line giving rise to 
vertebrates but before the separation of squid and 
octopus. 

Southern blots are consistent with the presence of 
few, possibly only one, gene for ii-crystallin in 
octopus and squid, and Northern blots indicate that 
this gene is expressed specifically in the lens. 
However, it is of particular interest that Q-crystallin 
is the dominant protein in the muscle-derived, 
cellular lens of the ventral light organ in one squid 
(i.e., Euprymna scolopes). This indicates that the 
same gene has been recruited to be a crystallin in 
two entirely different lenses developing from differ- 
ent tissues. No ALDH activity has been found for 
i2-crystallin. These results are consistent with the 
idea that, like the S-crystallins, fl-crystallin evolved 
by duplication of an ancestral gene encoding ALDH 
and subsequently specialized for refraction in the 
transparent lens while losing enzymatic activity and 
expression in other tissues. 

J-crystallin. — In addition to the cephalopod 
crystallins, we have been studying the crystallins of 
cubomedusan jellyfish, which also have complex 



164 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



eyes with cellular lenses. We discovered several 
years ago that these lenses contain three apparently 
unrelated crystallins (Jl, J2, and J3). Last year we 
cloned three genes encoding Jl-crystallin polypep- 
tides and showed that they lack introns and encode 
novel proteins. This year we have initiated studies 
on the J2- and J3-crystallins. Sequences of tryptic 
peptides have indicated that the J2 and J3 polypep- 
tides are different proteins, despite their similarity in 
molecular mass (20 and 19 kD, respectively). A 
J3-crystallin cDNA has been cloned and partially 
sequenced. Like Jl, J3-crystallin appears to be a 
novel protein; no homolog is reported in the data 
base. Interestingly, although the 19-kD J3 polypep- 
tide is considerably smaller than the 35-kD Jl 
polypeptides. Northern blots indicate that the J3 
mRNA is approximately 1.4 kb in length rather than 
the 1 kb size of the J 1 mRNAs. We are now cloning 
the J3 gene(s). 

Cytoskeletal proteins. — ^This year we completed 
analysis, initiated last year, of the intermediate 
filament (IF) protein and tubulin cDNAs of cephalo- 
pod lenses. Northern blots show that a-tubulin 
mRNA is present in all tissues examined, while the 
P-tubulin and EF mRNAs are lens specific. The 
proteins encoded in the tubulin cDNA sequences are 
very similar (87-93% identical) to the corresponding 
tubulins of insects and vertebrates, as expected for 
the high degree of conservation among these cyto- 
skeletal proteins. In the IF protein, the central rod 
region is more highly conserved than the head and 
tail regions, yet even the rod region shows at most 
39% identity with any other known rod region of an 
IF protein, namely with that of the squid neuronal IF 
protein. The rod regions of the squid lens IF protein 
contained the six heptads characteristic of nuclear 
lamins, consistent with an evolutionary relationship 
between IF proteins and lamins. 

We had previously investigated the regulatory 
elements of the chicken vimentin gene because it is 
expressed relatively highly in the lens. Earlier 
transfection experiments revealed the presence of 
both positive- and negative-acting sequence elements 
within the first 767 nucleotides of the 5'-flanking 
region of the gene. This year we identified a silen- 
cer between positions -1360 and -1156 and an 
activator between positions -1612 and -1360 of the 
chicken vimentin gene. These regions, which con- 
tain numerous consensus sequences for the binding 



of transcription factors implicated in the expression 
of different crystallin genes, deserve further smdy. 

Significance to Biomedical Research and the 
Program of the Institute 

The crystallins comprise a diverse family of differ- 
entially expressed proteins that are required for the 
optical properties of the transparent lens. Under- 
standing the structure, function, and evolution of 
these protein families and their genes contributes to 
our knowledge of embryonic development, eukary- 
otic gene expression, cell differentiation, molecular 
evolution, the visual system, and cataract. That 
crystallins are multifunctional proteins expressed in 
lens and nonlens tissues adds another dimension of 
interest and has implications for metabolism, cell 
biology, and drug and gene therapy. 

Proposed Course 

The following studies are proposed for Fiscal Year 
1994: 

1. We will continue identifying ds-acting ele- 
ments in crystallin genes by mutagenesis and expres- 
sion studies. 

2. We will investigate the interaction of cis ele- 
ments of the crystallin genes by footprinting and 
function studies. 

3. We will continue cloning and characterizing 
putative transcription factors for crystallin genes by 
binding studies. 

4. We will complete the sequences of the cxA- 
CRYBPl cDNA and gene in the mouse. 

5. We will continue mutagenesis studies of the 
squid GST and S-crystallin cDNA to relate structur- 
ally the enzymatic and refractive functions of these 
proteins. 

6. We will continue cloning and characterizing 
the jellyfish crystallin genes. 

7. We will investigate the natiu-e of the noncrys- 
tallin functions of the a-crystallin polypeptides. 

8. We will continue investigations on the similar- 
ities and differences of the IF proteins of squid and 
vertebrates by conducting structural and function 
studies. 

NEI Research Program 

Lens and Cataract — Molecular Biology 



165 



Laboraton of Molecular and Developmental Biolog> 



NEI Annual Report— FY 1993 



Publications 

Brady JP, Piatigorsky J: Cloning and characteriza- 
tion of a novel zinc-finger protein-encoding 
cDNA from the mouse eye lens. Gene 
124:207-214. 1993. 

Hejtmancik JF, Kaiser MI, Piatigorsky J: Molecular 
biology of inherited disorders of the eye lens, in 
Scriver CR, Beaudet AL, Sly WS, Valle D (eds): 
The Metabolic Basis of Inherited Disease, ed 7. 
New York, McGraw-Hill P*ublishing Co, in press. 

Hejtmancik JF, Piatigorsky J: Molecular biology of 
the eye lens, in Alben DM, Jakobiec FA (eds): 
Principles and Practice of Ophthalmology: Vie 
Harvard System. Philadelphia, WB Saunders Co, 
1993. pp 168-181. 

Kantorow M, Becker K, Sax CM, Ozato K, Piatigor- 
sky J: Binding of tissue-specific forms of oA- 
CRYBPl to their regulatory sequence in the 
mouse OLA-crystallin-encoding gene: Double-label 
immunoblotting of UV-crossIinked complexes. 
Gene 131:159-165, 1993. 

Kantorow M, Cvekl A, Sax CM, Piatigorsky J: 
Protein-DNA interactions of the mouse aA-crys- 
tallin control regions. Differences between 
expressing and non-expressing cells. J Mol Biol 
230:425-435, 1993. 

Klement JF, Cvekl A, Piatigorsky J: Functional 
elements DE2A, DE2B, and DEI A and the 
TATA box are required for activity of the chicken 
oA-crystallin gene in transfected lens epithelial 
cells. J Biol Chem It'i-.fnil-bliA, 1993. 

Li X, Zelenka PS, Piatigorsky J: Differential expres- 
sion of the two 5-crystallin genes in lens and 
non-lens tissues: Shift favoring 62 expres,sion 
from embryonic to adult chickens. Dev Dynamics 
196:114-123, 1993. 

Piatigorsky J: Gene sharing in the visual system. 
Trans Am Ophthalmol Soc, in press. 



Piatigorsky J: Puzzle of crystallin diversity in eye 
lenses. Dev Dynamics 196:267-272, 1993. 

Piatigorsky J, Horwitz J, Norman BL: J 1 -crystal lins 
of the cubomedusan jellyfish lens constitute a 
novel family encoded in at least three intronless 
genes. J Biol Chem 268:11894-11901, 1993. 

Sax CM, Klement JF, Piatigorsky J: Role of the 
otA-CRYBPl site in lens-specific expression of 
the aA-crystallin gene, in Loveh PS, Mongkolsuk 
S, Trempy JS (eds): Biotechnology and Environ- 
mental Science. New York, Plenum Press, 1992, 
pp 27-33. 

Sax CM, Ilagan JG, Piatigorsky J: Functional 
redundancy of the DE-1 and oA-CRYBPl regula- 
tory sites of the mouse ocA-crystallin promoter. 
Nucleic Acid Res 21:2633-2640, 1993. 

Sax CM, Piatigorsky J: Expression of the a-crystal- 
lin/small heat shock protein/molecular chaperone 
genes in the lens and other tissues. Adv Enzymol, 
in press. 

Sax CM, Stover DM, Hagan JG, Zehner ZE, Piati- 
gorsky J: Functional analysis of chicken vimentin 
distal promoter regions in cultured lens cells. 
Gene 130:266-281, 1993. 

Tomarev SI, Zinovieva RD, Piatigorsky J: Primary 
structure and lens-specific expression of genes for 
an intermediate filament protein and a P-tubulin 
in cephalopods. Biochim Biophys Acta, in press. 

Tomarev SI, Zinovieva RD, Weis VM, Chepelinsky 
AB, Piatigorsky J, McFall-Ngai MJ: Abundant 
mRNAs in the squid light organ encode proteins 
with a high similarity to mammalian peroxidases. 
Gene 132:219-226, 1993. 

Zinovieva RD, Tomarev SI, Piatigorsky J: Aldehyde 
dehydrogenase-derived fl-crystallins of squid and 
octopus. Specialization for lens expression. J 
Biol Chem 268:11449-11455, 1993. 



166 



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



PROJECT NUMBER 



ZOl EY 00259-04 LMDB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Molecular Biology of the 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 Kayes 



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: 



0.6 



PROFESSIONAL: 



0.6 



OTHER: 



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 the past 2 years we have cloned a number of abundant cDNAs from the corneal epithelial cells of the 
mouse. Some of these encode metabolic enzymes, suggesting that enzymes may act as crystallins in the 
transparent cornea as they do in the lens. Indeed we have found that different species accumulate different 
enzymes in their corneal epithelial cells, reminiscent of the taxon specificity observed for enzyme-crystallins 
of the lens. Aldehyde dehydrogenase (ALDH) class III is the major protein in the corneal epithelial cells of 
mammals. We have employed the RACE technique to clone the 5' end of the ALDH class 3 corneal mRNA, 
identified the sequences that should constitute the 5' exon of the gene, and cloned the ALDH class 3 gene in 
an 18 kbp genomic fragment This firagment is undergoing analysis. 



167 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biology 



NEI Annual Report — FY 1993 



Project Description 

Objectives 

The project's objectives are to identify and character- 
ize the genes that are preferentially expressed in the 
epithelium and endothelium of the cornea and to 
study the molecular basis for their expression in this 
transparent structure. 

Methods 

Conventional molecular biology methods of cloning, 
sequencing, recombinant DNA construction, transfec- 
tion, and transgenic mouse production are used. 

Major Findings 

Aldehyde dehydrogenase (ALDH) class 3 comprises 
approximately 40% of the soluble protein of the 
corneal epithelial cells of the mouse and other 
mammals. Such abundance for a metabolic enzyme 
suggests that this protein is acting like an enzyme- 
crystallin and has a refractive function in the cornea 
as crystallins do in the lens. Last year we reported 
the cloning of the mouse ALDH gene. We thought 
at that time that we had approximately 2 kbp of the 
5'-flanking region as well as the structural gene. 
Thus, we constructed a P-galactosidase fusion gene 
with what we believed was 1.1 kbp of the ALDH 5'- 
flanking sequence and used this construct as a 
transgene in transgenic mice. 

Analysis of the progeny of these mice during this 
fiscal year showed that the transgene had been 
incorporated but that it was not expressed in any 
tissue, not even the cornea. We thus extracted more 
corneal RNA from mice and employed the RACE 
technique to clone the 5' end of the ALDH cDNA. 
The sequence of the resulting clones showed that 
there is an additional exon 5' beyond what we had 
cloned, implying that our P-galactosidase fusion gene 



lacked the ALDH promoter and other regulatory 
sequences for expression. Consequently the ALDH 
gene was cloned from a mouse genomic library. The 
18-kbp, cloned DNA is now undergoing analysis. 

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; consequently, it 
should be investigated. The cornea is a transparent, 
ectodermaily derived tissue like the lens; thus com- 
parative studies between it and the lens are of special 
interest with respect to transparency. Moreover, 
because of our finding that corneal epithelial cells 
show taxon-specific gene sharing of metabolic en- 
zymes as does the lens, our major tissue of research, 
comparative studies on the cornea and lens, is of 
obvious importance from developmental and evolu- 
tionary viewpoints. Finally, the cornea is particularly 
accessible for gene therapy on account of its expo- 
sure to the siuface and its association with numerous 
hereditary diseases. 

Proposed Course 

In Fiscal Year 1994 we will (1) analyze and se- 
quence the mouse ALDH class 3 gene expressed in 
the cornea, (2) identify the promoter and functional 
regulatory elements for the ALDH class 3 gene by 
transfection experiments, and (3) create and analyze 
transgenic mice containing various truncated 5'- 
flanking sequences of the ALDH class 3 gene to 
identify the sequences responsible for high expres- 
sion in the corneal epithelial cells. 

NEI Research Program 

Lens and Cataract — Molecular Biology 



168 



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



PROJECT NUMBER 



ZOl EY 00255-05 LMDB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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: Graenje J. Wistow Ph.D. Chief, Section on Molecular LMDB, NEI 

Stnicture and Function 



Others: 



Vishwas Paralkar 
Caroline Graham 
Lorenzo Segovia 
Jill Richardson 
Cynthia Jaworski 

Peggy Zelenka 



Ph.D. 

B.S. 

Ph.D. 

Ph.D. 

Pb.D. 

Ph.D. 



Visiting Associate 
Biologist 
Visiting Fellow 
Visiting Fellow 
Chemist, Section on 
Molecular Genetics 
Chief, Section on Cellular 
Differentiation 



LMDB. NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 
LMDB, NEI 

LMDB, NEI 



COOPERATING UNITS (if any) 

DNX, Inc., Princeton, NJ 



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: 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ {a2) Interviews 



PROFESSIONAL; 



4.8 



OTHER: 



1.0 



□ (b) Human tissues [x] (c) Neither 



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

Crystallins are stress-related proteins or enzymes that often maintain dual roles without gene duplication. We 
have shown that the putative lens promoter of NADPH: quinone oxidoreductase/^-crystallin is highly lens 
specific and by itself is responsible for the recruitment of this gene as a crystallin. An important functional 
element (ZPE) that binds different complexes in lens and nonlens-cell extracts has been identified. The full 
sequence of Ti-crystallin, another mammalian enzyme crystallin, has been obtained and shows identity with 
the enzyme ALDHl, another example of gene recruitmenL n-Crystallin, originally discovered in marsupial 
lenses, is a novel enzyme with NADPH-binding activity. Immunohistochemistry suggests it is preferentially 
expressed in vertebrate photoreceptors. The gene for ^ has been cloned. 

Not all important lens proteins are crystallins. We have shown that MIF, a lymphokine, is expressed in 
differentiating lens cells. The gene for human MIF has been cloned, and expression studies are under way. 
Lens P2 protein, another potential marker for the differentiation process and a possible intermediary messenger 
has been cloned and shown to belong to a lipid/retinoid-binding family. Members of the C/EBP family, 
transcription factors with important roles in differentiation and candidates for promoter binding in some 
crystallin genes, have been detected in the lens, where tlieir pattern of expression is developmentally regulated. 



169 



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Laboratorj' of Molecular and Developmental Biology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

We are investigating the molecular basis for normal 
lens structure and function, including the character- 
ization of crystallins and the mechanisms of their 
normal expression. This also has led to the identifi- 
cation of molecules, such as transcription and growth 
factors, involved in lens cell differentiation. The 
interplay of such factors is an essential pan of 
normal lens development and function. 

Methods 

A wide range of molecular biology techniques are 
used, including RNA analysis, gene and cDNA 
cloning and sequencing, functional gene promoter 
analysis in cultured cells and in transgenic mice, and 
polymerase chain reaction (PCR). We perform some 
protein analysis and make use of commercial facil- 
ities for protein sequencing. 

Major Findings 

Gene recruitment: Enzyme crystallins. — A novel 
enzyme with quinone reductase activity has under- 
gone gene recruitment in certain mammals, acquiring 
a second function as the lens structural protein ^- 
crystallin. In work by Drs. Douglas Lee and Jill 
Richardson, we have shown that recruitment of this 
taxon-specific crystallin can be explained by the leas 
specificity of an alternative promoter. By itself this 
promoter confers strongly lens-preferred expression 
in both cultured cells and transgenic niice. 

While proximal regions of the promoter have 
some activity in the transgenic brain, this is abol- 
ished by the addition of more distal regions. The 
minimal active promoter contains a region, ZPE C^- 
protected element), including a consensus C/EBP 
binding site, which is strongly and identically foot- 
printed by nuclear extracts from both mouse and 
rabbit cultured lens cells in which the promoter is 
active. In fibroblast nuclear extract, the ZPE's more 
restricted footprint is flanked by other protected 
regions that are absent or only weakly footprinted in 
lens cell extracts. In gel shift assays, the ZPE 
sequence forms specific complexes with lens-cell 
extracts but not with fibroblast extracts. Additional 
transgenic mice have been generated and show that 
sequences between positions -385 and -533 are 



required for suppression of promoter expjression in 
the brain. 

)j-Crystallin is the major component of the eye 
lens in several Australian marsupials. It also is a 
novel enzyme in other manunals, including humans, 
where it has nonlens expression in neural tissue 
(including retina), muscle, and kidney. It has strik- 
ing sequence similarity with bacterial ornithine 
cyclodeaminases, suggesting an unusual role in 
ornithine metabolism. Dr. Lorenzo Segovia has 
shown that p-crystallin preferentially binds NADPH, 
consistent with its role as a reductase. Immunohisto- 
chemistry of the developing rat shows a remarkable, 
intense reaction with retinal photoreceptors; in fact, 
our anti-p antiserum detects the earliest marker 
known for photoreceptor cells. Since the retina is 
susceptible to ornithine toxicity in gyrate atrophy, the 
expression of a novel ornithine-metaboUzing enzyme 
in photoreceptors could be significant. The kangaroo 
gene for this enzyme crystallin has been cloned and 
is being analyzed. 

Another major enzyme crystallin (up to 25% of 
total protein) in mammals is Ti-crystallin found in 
elephant shrews. Ms. Caroline Graham has cloned 
the complete cDNA sequence for this protein from 
two different species. As predicted, sequence data 
confirm that Ti-crystallin is ALDHl and that it is the 
product of a single recruited gene. cDNA clones 
will be expressed in baaerial hosts to characterize 
enzyme activity. 

Dr. Cynthia Jaworski has completed the cDNA 
sequence of human oA-crystallin and has thereby 
corrected sequence errors in older protein data ocA- 
crystallin is the single major component of the 
human lens and is related to small heat shock pro- 
teins (shsp). The quaternary structure of a-crystal- 
lins and shsps is both controversial and of great 
interest. New model structures were predicted on the 
basis of existing biochemical data. Briefly, these 
correspond to cubic and dodecahedral structures with 
coaserved intermolecular contacts. These predictions 
are stimulating biophysical investigations in other 
laboratories. 

oB-crystallin is multifunctional, serving as both 
a major structural protein in the lens and as an shsp 
in other tissues in mammals. By cloning and North- 
em analysis. Dr. Lee showed similarly that aB- 
crystallin mRNA is present in all the mamre tissues 
examined in a bird {Anas platyrhynchos), although 



170 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



there are some differences in the pattern of tran- 
scripts seen. Interestingly, sequence analysis not 
only shows that duck otB-crystallin is a member of 
the shsp family, as expected, but that this family 
shares more distant similarity with another heat 
shock protein (hsp) family, the highly conserved 
HSP70s of both eukaryotes and prokaryotes. This 
raises the interesting possibility that large and small 
hsps may share structural and perhaps functional 
features. 

Molecular markers of differentiation. — Macro- 
phage migration inhibitory factor (MIF) was original- 
ly identified as a lymphokine. However, recent work 
strongly suggests a role for MEF beyond the immune 
system. Expressed specifically in the differentiating 
cells of the immunologically privileged eye lens and 
brain, it is a delayed early response gene in fibro- 
blasts but is expressed in many tissues. In contrast 
to previous reports, we have found evidence for a 
single gene for MEF in the human genome. 
Dr. Vishwas Paralkar has shown that this small gene 
has three exons separated by introns of only 189 and 
95 bp and covers less than 1 kb. The cloned se- 
quence also includes 1 kb of 5'-flanking region 
covering the putative promoter. 

Primer extension and 5' RACE PCR of human 
brain RNA both indicate the presence of a single 
transcription start site in a TATA-less promoter. 
Northern blot analysis shows a single-size MIF 
mRNA in all human tissues examined. MIF mRNA 
is particularly abundant in the kidney and is ex- 
pressed at high levels in many other tissues but is at 
low levels in muscle and the pancreas. The relative- 
ly abundant expression of MIF in lens may have 
clinical significance, with the possibility of involve- 
ment in lens-induced endophthalmitis and uveitis. 

Another potential differentiation marker also was 
discovered by protein microsequencing of a 14-kD 
band in calf lens extract. Dr. Jaworski has now 
obtained the complete cDNA sequence for this 
protein by PCR methods. The protein is a member 
of the lipid/retinoic acid-binding family of P2 pro- 
teins. Since retinoic acid has now been implicated in 
■y-crystallin gene expression, this protein could have 
a direct role in mediating lens gene expression. 

We are interested in connections between differ- 
entiation in the well-studied adipocyte system and in 
the eye lens. In both systems there is a switch in c- 
myc expression during differentiation: Stem cells are 
marked by expression of a-enolase while P2-like 



proteins also may be associated with differentiation. 
Furthermore, C/EBP-like proteins are candidates for 
binding to an essential, tissue-specific region of the 
^-crystallin gene lens promoter. Dr. Richardson is 
examining whether C/EBP-like proteins are expressed 
in the lens. She has results from immunohistochem- 
istry associating expression of C/EBP p and 5 with 
rat lens epithelia, the relatively undifferentiated "stem 
cell" population. C/EBP 5 is most abundant in the 
central, quiescent cells, while (3 comes on down- 
stream in regions where cells are migrating and 
dividing. Both are undetectable in the terminally 
differentiated lens fiber cells. 

The human Marner cataract maps to chromosome 
16 near the locus of several cadherins, which are 
important cell adhesion molecules. The cataract 
manifests as opacities at the Y-sutures, suggesting 
that a cell-cell contact or adhesion process could be 
defective. We examined whether a cadherin could 
be involved in this cataract. Immunochemical 
methods suggest that N-cadherin is expressed in the 
eye lens, but having no sequence data, this could be 
a cross-reaction with a related, perhaps lens-specific, 
variant. 

Ms. Graham used PCR to amplify cadherin- 
related sequences in human fetal lens and sequenced 
multiple clones. All were identical to known human 
N-cadherin. We then checked the chromosomal 
location of N-cadherin by PCR, using chromosome- 
specific template DNA, confirming its published 
position on chromosome 18. It is the only major 
cadherin not on chromosome 16. 

These results suggest that the principal cadherin 
expressed in the eye lens is identical to N-cadherin 
and that there is no lens-specific variant of this 
family. The only remaining possibilities of a con- 
nection between cadherins and the Marner cataract 
are that another cadherin is expressed at low levels 
in the lens or with a particular development pattern, 
or that the mutation causes inappropriate expression 
in the lens of another cadherin. 

Significance to Biomedical Research and the 
Program of the Institute 

The discovery of fundamental mechanisms in the 
differentiation and evolution of complex tissues has 
had important results in our understanding of impor- 
tant processes in evolution and in tissue-specific 
expression. In the process, we have discovered a 
novel enzyme that has possible significance in the 



171 



Laboratory of Molecular and Developmental Biology 



NEI Annual Report — FY 1993 



human retina. We also have discovered important 
markers for cellular differentiation, including proteias 
with inflammation-related lymphokine activity. 

Proposed Course 

1. We will continue examining the molecular 
mechanisms for lens-preferred expression and for 
gene recruitment in ^, Ti, n, and x-crystallins. 

2. We will characterize the function and nonlens 
role of |i-crystallin. 

3. We will explore the molecular biology and 
fimction of MIF expressed in the lens and its pos- 
sible role in eye inflammation. 

4. We will determine the function and pattern of 
expression of the lens P2 protein, a possible second 
messenger in signal transduction. 

NEI Research Program 

Cataract — Molecular Genetics 

Publications 

Chen H, Phillips HA, Callen DF, Kim RY, Wistow 
GJ, Antonarakis SE: Localization of the human 
gene for mu-crystallin to chromosome 16p. 
Genomics 14:1115-1116, 1992. 

de Jong WW, Leunissen JAM, Wistow GJ: Eye lens 
crystallins and the phylogeny of placental orders: 
Evidence for a macroscelid-paenungulate clade? 
in Szalay FS, Novacek MJ, McKenna MC (eds): 
Mammal Phylogeny: Placentals. New York, 
Springer Verlag, 1992, pp 5-12. 

Hodin J, Wistow G: 5'-RACE PCR of mRNA for 
three taxon-specific crystallins: For each gene 
one promoter controls both lens and non-lens 
expression. Biochem Biophys Res Commun 
190:391-396, 1993. 

Kim RY, Gasser R, Wistow GJ: Mu-crystallin is a 
mammalian homologue of Agrobacterium orni- 
thine cyclodeaminase and is expressed in human 
retina. Proc Natl Acad Sci USA 899292-9296 
1992. 



Kim RY, Wistow GJ: The cDNA RPEl and mono- 
clonal antibody HMB-50 define gene products 
preferentially expressed in retinal pigment epithe- 
lium. Exp Eye Res 55:657-662, 1992. 

Kim RY, Wistow GJ: Expression of the duck 
a-enolase/T-crystallin gene in transgenic mice. 
FASEB J 7:464-469, 1993. 

Lee DC, Gonzalez P, Rao PV, Zigler JS Jr, Wistow 
GJ: Carbonyl -metabolizing enzymes and their 
relatives recruited as structural proteins in the eye 
lens, in Weiner H (ed): Advances in Experimen- 
tal Medicine and Biology, 284: Enzymology and 
Molecular Biology of Carbonyl Metabolism. New 
York, Plenum Press, 1993, vol 4, p 159-168. 

Lee DC, Kim RY, Wistow GJ: An avian oB-crystal- 
lin. Non-lens expression and sequence similar- 
ities with both small (HSP27) and large (HSP70) 
heat shock proteins. J Mol Biol 232:1221-1226, 
1993. 

Wistow G: Lens crystallins: A model system for 
gene recruitment, in Zimmer EA, White TJ, Cann 
RL, Wilson AC (eds): Methods in Enzymology: 
Molecular Evolution Producing the Biochemical 
Data. San Diego, Academic Press, 1993, vol 224, 
pp 563-575. 

Wistow G: Lens crystallins: Gene recruitment and 
evolutionary dynamism. Trends Biochem Sci 
18:301-306, 1993. 

Wistow G: Possible tetramer-based quaternary 
structures for a-crystallins and small heat-shock 
proteins. Exp Eye Res 56:729-732, 1993. 

Wistow G: I^otein structure and introns. Nature 
346:107-108, 1993. 

Wistow GJ, Shaughnessy MP, Lee DC, Hodin J, 
Zelenka PS: A macrophage migration inhibitory 
factor is expressed in the differentiating cells of 
the eye lens. Proc Natl Acad Sci USA 
90:1272-1275, 1993. 



172 



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



PROJECT NUMBER 



ZOl EY 00251-06 LMDB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (BO characters or less. Title must tit on one line between ti)e borders.) 

Genetically Engineering the Eye with the ccA-Crystallin Promoter 



PRINCIPAL INVESTIGATOR (List other protessional 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: Devonne M. Parker 



B.S. 



Biologist 



LMDB, 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 



Laboratory of Molecular and Developmental Biology 



SECTION 



Section on Regulation of Gene Expression 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.9 



PROFESSIONAL: 



0.4 



OTHER: 



0.5 



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.) 

Gamma interferon (IFN-y) expression was directed to the eyes of transgenic mice to investigate the possible 
role of this lymphokine in ocular pathogenesis. The most notable effects of IFN-y in these transgenic mice 
include microphthalmia, blepharophimosis, microphakia, impairment of lens fiber formation, arrest of retinal 
differentiation, retinal detachment, and persistent hyperplastic primary vitreous. Major histocompatibility 
complex (MHC) class n mRNA levels were significantly increased in tiie transgenic eyes, and MHC class II 
proteins were expressed in the cornea, iris, ciliary body, choroid, lens, and retinal pigment epithelium. 

Int-2/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 ectopic expression 
of int-2 during the embryonic development of the lens affected the differentiation of the entire eye, highlighted 
by the appearance of intraocular secretory glandular epithelium, similar to dermoid-like pathology. 



173 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biolo^ 



NEI Annual Report— FY 1993 



Project Description 

Additional Personnel 

Charles Egwuagu Ph.D. Scientist, Public 

Health Service, LI, 
NEI 

Chi-Chao Chan M.D. Chief, Section on 

Immunopathology, 
LI, NEI 

Robert B. Nussenblatt M.D. Clinical 

Director, LI, NEI 

Jorge Sztein D.V.M. Visiting Associate, 

Veterinary 
Research 
Resources Service, 
NEI 

Objectives 

The objective of this project is to understand how 
aberrant genetic expression of interferon gamma 
(EFN-y), int-2, or acidic fibroblast growth factor 
(aFGF), under the control of the oA-crystallin 
promoter, perturbs normal eye development in 
transgenic mice. 

Methods 

Recombinant DNA techniques used in this study 
include plasmid construction, oligonucleotide se- 
quencing, Southern and Northern hybridizations, 
DNA sequencing, primer extension, polymerase chain 
reaction (PCR), reversed transcription PCR, immuno- 
histochemistry, and the production and analysis of 
transgenic mice. 

Major Findings 

IFN-y. — This project is conducted in collaboration 
with Drs. Charles Egwuagu, Jorge Sztein, Chi-Chao 
Chan, and Roben B. Nussenblatt from the NEI 
Laboratory of Immunology. The aberrant expression 
of IFN-y in the lens of transgenic mice allowed us to 
study the effect of IFN-y on the normal development 
of the eye and the regulation of major histocompati- 
bility complex (MHC) class II gene expression by 
IFN-y in a nonlymphoid tissue such as the lens. 

We generated transgenic mice containing as a 
transgene the murine aA-crystallin promoter (-366/ 
+46) fused to the murine IFN-y coding sequence. 
The ectopic expression of IFN-y in the lens of the 
transgenic mouse affected the growth of the whole 
eye, resulting in microphthalmia and microphakia. 
The lens fiber cells were replaced by balloon cells, 



differentiation of the neuroretina into iimer and outer 
neuroblastic layers was arrested at the embryonic 
stage, and retinal detachment and the presence of 
macrophages in the subretinal space were observed J 
in the adult mouse eye. 1 

Constitutive expression of EFN-y in the lens 
induced aberrant MHC class n protein synthesis in 
several ocular tissues. This transgenic mouse serves 
as an animal model to (1) facilitate understanding of 
the molecular pathways governing synchronized 
programmed differentiation of ocular tissues and 
(2) enable study of the linkage between aberrant 
MHC class II expression and predisposition to 
autoimmune diseases. 

int-2. — This project is conducted in collaboration 
with Drs. Paul Overbeek and Michael Robinson 
(Baylor College of Medicine) and Dr. Clive Dickson 
(Imperial Cancer Research Fund). To assess whether 
ectopic expression of the proto-pncogene int-2/FGF-3 
would perturb normal eye development, we directed 
its expression to the eyes of transgenic mice with the 
miuine oA-crystallin promoter. We obtained three 
lines of transgenic mice expressing the oA-crystal- 
lin/int-2 transgene. These adult transgenic mice 
presented microphthalmia characterized by intraocu- 
lar hyperplastic glandular structures replacing the 
normal iris, ciliary body, and lens; the retinas 
showed various degrees of dysplasia. 

The intraocular glandular structures of these mice 
stained positively for int-2 and Muc-1, a marker for 
secretory epithelia. We also observed a marked 
increase in Muc-1 mRNA levels and a drastic de- 
crease in MIP (major intrinsic protein) mRNA levels, 
a marker of lens fibers, in the eyes of the adult trans- 
genic mice. Proptosis of the transgenic eye, ob- 
served as early as day 15 of embryonic development, 
was followed by expulsion of the lens through the 
cornea and detachment of the imdifferentiated retina. 
The newborn mice presented a "scabby eye" pheno- 
type. Ectopic expression of the growth factor int-2 
during the embryonic development of the lens 
affected the differentiation of the entire eye, high- 
lighted by the appearance of intraocular secretory 
glandular epithelium, similar to dermoid-like patholo- 
gy, in the adult eye. 

aFGF. — In collaboration with Drs. Overbeek and 
Robinson , as well as Dr. Yves Courtois (Institute for 
Gerontological Research, INSERM), we injected into 
mouse embryos a recombinant DNA containing the 
aA-crystallin promoter (-366/+46) fused to the 
bovine aFGF cDNA. Three lines of transgenic mice 



174 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



were obtained; no particular phenotype was observed. 
We currently are analyzing these mice for expression 
of the transgene. 

Significance to Biomedical Research and the 
Program of the Institute 

The aberrant expression of IFN-y or int-2 will allow 
us to elucidate the mechanisms underlying eye 
development. At the same time, it opens new 
avenues in the development of animal models for 
studies of eye pathologies and of gene regulation in 
the eye. 

Proposed Course 

The following studies will continue during Fiscal 
Year 1994: 

1. We will characterize further the effect of BFN- 
Y on the regulation of gene expression in the eyes of 
the transgenic mice. 

2. We will continue to study the effect of int-2 
on gene expression in the eyes of the transgenic mice 
and try to understand its role in eye growth and 
differentiatioa 



NEI Research Program 

Cataract — Molecular Genetics 

Publications 

Chepelinsky AB, Overbeek PA, Chan C-C, Jamieson 
S, Dickson C, Parker DM, Robinson M: Int-2 
ectopic expression induces differentiation of 
secretory epithelia in the eyes of transgenic mice. 
Invest Ophthalmol Vis Sci 34(4)(suppl):1222, 
1993. 

Egwuagu CF, 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 pathology and 
MHC class n gene expression. Invest Ophthal- 
mol Vis Sci, in press. 

Egwuagu CF, Sztein J, Chan C-C, Reid W, Mahdi R, 
Nussenblatt RB, Chepelinsky AB: Gamma 
interferon expression in the eyes of transgenic 
mice disrupts differentiation of the lens and 
retina. Invest Ophthalmol Vis Sci 34(4)(suppl): 
1455, 1993. 



175 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00253-05 LMDB 



PERIOD COVERED 



October 1. 1992 to September 30, 1993 



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

Regulation of Expression of Lens Fiber Membrane Genes 



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

PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI 

Regulation of Gene 
Expression 

Others: Chiaki Ohtaka-Maruyama 
Xiaoyan Wang 
LaShawn R. Drew 
Devonne M. Parker 



Ph.D. 


Visiting Fellow 


LMDB, NfEl 


M.D. 


IRTA Fellow 


LMDB. NEI 


B.S. 


Chemist 


LMDB, NEI 


B.S. 


Biologist 


LMDB, NEI 



COOPERATING UNITS (il any) 

Harvard University (David Paul, Ph.D.); Columbia University (Jorge Fischbarg, M.D.) 



LAB/BRANCH 



Laboratory of Molecular and Developmental Biology 



SECTION 



Section on Regulation of Gene Expression 



INSTITUTE AN^ LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



3.2 



PROFESSIONAL: 



2.0 



OTHER: 



1.2 



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 studies the regulation of expression of genes encoding lens fiber membrane chaimel proteins. We 
are focusing on the regulation of expression of the gene encoding MIP, the major intrinsic protein of the lens 
fiber membrane, which is specifically expressed in the ocular lens fibers and belongs to an ancient superfamily 
of transmembrane channel proteins. 

We characterized 2,840 bp of 5' flanking sequence of the human MIP gene to study the cis regulatory 
elements responsible for the tissue specificity and developmental regulation of the MIP gene. We found that 
a DNA fragment containing 253 bp of 5' flanking sequence and 42 bp of exon 1 of the human MIP gene fused 
to the reporter chloramphenicol acetyltransferase (CAT) gene is able to express the CAT gene in cultured lens 
cells. We are studying the interaction of transcription factors with the cis regulatory elements of the MIP gene 
and its effect on the in vitro transcription of the MIP gene in Drosophila nuclear extracts. Purified human 
Spl and AP2 interact with cis regulatory elements of the MIP promoter and activate the in vitro transcription 
of the MIP promoter, suggesting their involvement in the regulation of MIP gene transcription. These studies 
will further our understanding of the role of general u-anscription factors on the tissue-specific expression of 
the MIP gene. 



176 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



Project Description 

Objectives 

The objective of this project is to elucidate the 
mechanisms involved in the regulation of expression 
of fiber membrane genes involved in cell-cell com- 
munication in the lens. The identification of the cis 
regulatory elements of these genes and their inter- 
action with trans-acting factors is essential for 
understanding the regulation of gene expression in 
the lens. 

Methods 

Recombinant DNA techniques used in this study 
include screening genomic libraries, subcloning, 
plasmid construction, oligonucleotide synthesis. 
Southern and Northern hybridizations, DNA sequenc- 
ing, primer extension, polymerase chain reaction 
(PCR), reversed transcription PCR, gel mobility shift 
assays, DNA footprinting, methylation interference, 
subcellular fractionation to obtain nuclear extracts, in 
vitro transcription, tissue culture techniques (includ- 
ing transfection of primary lens explants and cell 
lines), and analysis of transgenic mice. 

Major Findings 

Cis regulatory elements of the human major intrinsic 
protein (MIP) gene promoter. — We characterized 
2,840 bp of 5'-flanking sequence of the human NOP 
gene to identify the cis regulatory elements responsi- 
ble for the tissue specificity and developmental 
regulation of the MIP gene. We found that a DNA 
fragment containing 253 bp of 5'-flanking sequence 
and 42 bp of exon 1 of the human MIP gene fused 
to the reporter gene chloramphenicol acetyltrans- 
ferase (CAT) directs CAT gene expression to leas 
cells in transient assays. Therefore, the -253/-i42 
sequence of the human MIP gene contains informa- 
tion for lens cell expression, suggesting that cis 
regulatory elements responsible for the lens-specific 
expression of the MIP gene are localized within this 
domain. 

Several motifs known to bind transcription factors 
in other genes are present in the 5'-flanking sequence 
of the MIP gene. To elucidate whether those motifs 
are involved in the regulation of MIP gene expres- 
sion, we are studying their interaction with several 
transcripton factors. Analysis by DNA footprinting 
and gel mobility shift assays show that purified 
human Spl and AK interact with several domains of 
the MIP promoter. They also activate the in vitro 



transcription of the MIP promoter in Drosophila 
nuclear extracts. We found that the initiation site of 
transcription of the human MIP gene was the same 
in vivo and in vitro. These results suggest that SPl 
and AP2 are involved in the regulation of transcrip- 
tion of the MIP gene. 

We have generated several lines of transgenic 
mice containing 253 bp of the human MIP gene 5'- 
flanking sequence with 42 bp of exon 1 fused to the 
CAT gene as a transgene. In one transgenic line, the 
CAT gene expressed specifically in the ocular lens. 
Expression of the CAT gene was observed in the 
ovaries of the females of four additional transgenic 
lines, although no expression was observed in any 
tissue of the male progeny. We currentiy are map- 
ping other regulatory elements localized between 
-2,8(X) bp and the initiation site of transcription of 
the MIP gene that may be required for proper tissue 
specificity. We also are cloning the murine MIP 
gene in order to study the mouse MIP promoter in its 
homologous in vivo environment 

3' untranslated sequence of the MIP gene. — We 
are sequencing the 3'-flanking region of the human 
MIP gene to characterize the polyadenylation sites 
and the processing of the two MIP transcripts ob- 
served in vivo. 

Cloning of the connexin 46 gene. — ^We are 
cloning the coimexin 46 gene, which encodes one of 
the lens fiber g^ junction proteins, to be able to 
study how its expression is regulated in the lens. 

CHIP28 expression in cornea endothelial cells. — 
In collaboration with Dr. Jorge Fischbarg (Columbia 
University), we are characterizing the member of the 
MIP family responsible for the CHIP28-like water 
channels observed in primary cultures of bovine 
cornea endothelial cells. Sequencing data indicate 
that it is CHIP28. 

Significance to Biomedical Research and the 
Program of the Institute 

Since the differentiation of lens epithelial cells into 
fiber cells results in a dramatic increase of new 
plasma membrane synthesis by elongating cells, 
proper membrane biosynthesis and physiology are of 
utmost importance in maintaining the transparent 
state of the lens. Membrane protein synthesis is 
regulated in a temporal and spatial manner in the 
lens. Alterations in lens membranes, particularly 
involving MIP, have been observed during cataracto- 
genesis and aging. Therefore, studies on MIP gene 



177 



Laboratory of Molecular and Developmental Biologj' 



NEI Annual Report— FY 1993 



expression should further our understanding, not only 
of the mechanisms involved in the regulation of gene 
expression in the normal lens, but also of its disrup- 
tion during disease. 

Proposed Course 

The following studies will continue during Fiscal 
Year 1994: 

1 . Charaaerization of the cis regulatory elements 
of the human MIP promoter. 

2. Isolation of the murine MIP gene promoter 
and its comparison with its human homolog. 

3. Study of the interaction of the MIP gene cis 
regulatory elements with transcription factors. 

4. Characterization of the 3'-flanking region of 
the human MIP gene. 

NEI Research Program 

Cataract — Molecular Genetics 



Publications 

Chepelinsky AB: The MIP transmembrane chaimel 
family, in Peracchia C (ed): Handbook of Mem- 
brane Channels: Molecular and Cellular Physiol- 
ogy. Academic Press, in press. 

Ohtaka-Maruyama C, Drew LR, Pisano MM, Chepe- 
linsky AB: Regulatory elements of the MIP gene 
promoter. Invest Ophthalmol Vis Sci 34(4) 
(suppl):1342, 1993. 

Ohtaka-Maruyama C, Drew LR, Pisano MM, Chepe- 
linsky AB: Transcriptional regulation of the 
human MIP gene. J Cellular Biochem 17A 
(suppl):167, 1993. 

Tomarev SI, Zinovieva RD, Weis VM, Chepelinsky 
AB, Piatigorsky J, McFall-Ngai MJ: Abundant 
mRNAs in the squid light organ encode proteins 
with a high similarity to mammalian peroxidases. 
Gene 132:219-226, 1993. 



178 



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



PROJECT NUMBER 



ZOl EY 00285-01 LMDB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

NEI Central Transgenic Animal Production Facility 



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 
R. Steven Lee 
Mariana Gonzalez-Baez 



B.S. 


Chemist 


LMDB, NEI 


B.S. 


Biologist 


LMDB, NEI 




Biological Science 


LMDB, NEI 




Lab Aide, Stay-in- 






School Program 


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: 



2.7 



PROFESSIONAL: 



0.5 



OTHER: 



2.2 



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.) 

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 providing transgenic 
animal support to 18 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 52 DNA constructs 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 analysis on the mice bom from tliese 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. Over the 
year we have generated 129 transgenic mice from 26 DNA constructs; set up 231 matings of transgenic mice; 
weaned, tagged, and tail-biopsied 3,033 mice; and isolated DNA and performed DNA analysis on 2,313 biopsy 
samples. We are working toward creating an embryo cryopreservation and banking program to provide long- 
term storage of important transgenic lines to eliminate the need to maintain live mice. In addition to service 
functions, we collaborate with NEI researchers on transgenic animal projects. This year we collaborated with 
Dr. Igal Gery (Laboratory of Immunology, NEI) in creating DNA constructs, and subsequently transgenic 
mice, to examine the immunologic consequences of expressing foreign antigens in the encapsulated ocular 
lens. 



179 



PHS 6040 (Rev. 5/92) 



Laboratory of Molecular and Developmental Biology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

This project has been established to (1) produce 
transgenic animals for use in NEI's eye research, 
(2) supply ancillary services related to maintenance 
of transgenic animals, (3) provide advice and exper- 
tise in matters of transgenic animal projects to all 
NEI intramural researchers using this technology in 
their research, and (4) act as a central facility for all 
transgenic animal work conducted in the NEI Intra- 
mural Research Program in order to coordinate and 
conserve resources and utihze severely limited 
animal housing space with maximum efQciency. We 
provide a comprehensive program for short- and 
long-term storage of transgenic animal lines, both as 
live animals and as frozen embryos. 

Methods 

Standard methods are used for microinjecting DNA 
into the pronucleus of one-celled mouse embryos and 
surgically reimplanting the injected embryos into 
foster mothers for development. Conventional 
molecular biology techniques are used to isolate and 
analyze DNA from biopsy samples of transgenic 
mice. Data on all transgenic mice are maintained in 
a computerized relational data base accessed by 
programs written within our group. 

Major Findings 

Production of new transgenic mouse lines. — We have 
generated 129 new transgenic founder mice from 26 
constructs submitted by researchers in 4 NEI intra- 
mural laboratories. These constructs are quite 
diverse in nature, reflecting the diversity of research 
being performed in the NEI. Some of the general 
categories of constructs are (1) promoter/reporter 
constructs in which the promoter of an eye gene is 
fused to a reporter gene to assess transcriptional 
activity in the transgenic mouse, (2) eye-specific or 
ubiquitous promoters driving expression of genes 
believed to be involved in eye pathologies to as.sess 
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 lines are derived by mating of the 
original transgenic founder mice and derivation of 
successive generations of progeny, which are then 



used in biomedical research. To generate lines of 
transgenic mice from our transgenic founder mice, 
we have set up 231 mouse matings and weaned, 
tagged, and biopsied 3,033 mice resulting from 
matings and microinjection procedures. 

DNA analyses. — Approximately 10-25% of mice 
bom from microinjected embryos are transgenic; 
similarly, approximately 50% of mice resulting from 
a transgenic mouse mating are transgenic. A r^id, 
efficient, and rehable method of identifying transgene 
positive and negative mice is in place in our group. 
We have processed 2,313 biopsy samples to obtain 
DNA and have performed analyses on these samples 
to determine whether the mice were transgene 
positive. 

Embryo cryopreservation and banking. — ^We have 
been experimenting with freezing mouse embryos for 
banking of important lines of ttansgenic mice. Slow 
freezing of embryos in vials and in sfraws has been 
attempted. The sfraw method has the advantages of 
being simpler and more time efficient and will be 
pursued further. Reconstitution of mice has been 
accomplished by transferring thawed embryos into 
the oviduct or into the uterus of foster mothers. We 
have had the best results with oviductal transfers and 
will pursue this methodology. Our reproducibility 
in freezing and thawing embryos and in reconstitut- 
ing mouse lines from thawed embryos is not yet 
sufficient to begin large-scale banking of embryos 
with confidence that we can regenerate the banked 
lines. 

Significance to Biomedical Research and the 
Program of the Institute 

Transgenic mice are currently the only readily 
attainable system for studying gene expression in the 
context of an entire, intact animal. While tissue 
culture can yield a great deal of information in many 
studies, true understanding of how a particular gene 
affects an organ (such as the eye) or an entire organ- 
ism can be obtained only by studying that gene in 
tlie intact organism. We play a pivotal role in many 
NEI inframural research projects by providing the 
technology and expertise to insert into the mouse 
genes related to normal eye development and patho- 
logical eye conditions. 

Proposed Course 

In Fiscal Year 1994 we will continue our research as 
follows: 



180 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



1. We will continue producing new transgenic 
mice for NEI researchers as required for their re- 
search projects. 

2. We will continue breeding and maintaining the 
existing transgenic mouse lines needed for ongoing 
NEI research. 

3. We will continue our efforts to reproducibly 
freeze and thaw mouse embryos and reconstitute 
mouse lines from the thawed embryos. Once we are 
certain that we can regenerate transgenic mouse lines 



from frozen embryos, we will begin cryopreservation 
and banking of some existing transgenic mouse lines, 
which are important to keep but which are not 
currently in use. This will free some of our limited 
animal housing space and ensure that important lines 
of mice will not be lost due to aging and loss of 
fertility. 

NEI Research Program 

Cataract — Molecular Genetics 



181 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 



PROJECT NUMBER 



ZOl EY 00286-01 LMDB 



October 1. 1992 to September 30. 1993 



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

g-Crystallin Gene Disruption in the Mouse 



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

PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI 



COOPERATING UNITS (il 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, MD 20892 



TOTAL STAFF YEARS: 



0.5 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 
n (a1) Minors 
□ (a2) Interviews 



PROFESSIONAL: 



0.5 



OTHER: 



0.0 



n (b) Human tissues [x] (c) Neither 



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

The a-crystallins, which comprise a large fraction of the soluble protein in the vertebrate lens, where they are 
beUeved to function solely as structural proteins, are the first crystallins to be expressed in the developing 
mouse lens and are a relatively small family of crystaUins encoded by only two genes, the oA- and oB- 
crystalhn genes. The a-crystallins exhibit molecular chaperone activity and, at least in the case of oB- 
crystallin, have been shown to be expressed in a variety of nonlenUcular tissues, where their function is 
unknown. Toward understanding the role of the a-crystallins in lens and nonlens tissues, we are attempting 
functional deletion of a-crystallins by disrupting the a-crystallin genes in mice. We are employing the 
technique of homologous recombination in pluripotent mouse embryonic stem cells, followed by the generation 
of chimeric mice containing the altered stem cells. We have isolated and mapped 15-kb clones containing 
the OA- and oB-crystallin gene loci from a mouse 129SV library (the same strain as most of the embryonic 
stem cell Lnes currently in use). Construction of the aA-crystallin "knockout vector" is near completion In 
collaborauon with Dr. Nicholas Ambulos (University of Maryland Medical School), we have nearly completed 
double-stranded sequencing of the mouse aA-crystallin gene (5 kb) and single-stranded sequencing of an 
addiuonal 4 kb of the 5 -flanking sequence. We currently are generating constructs to look for enhancer 
regions m the aA-crystallin introns and in the 5'- and 3'-flanking regions. 



182 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Molecular and Developmental Biology 



Project Description 

Additional Personnel 

Ellen Liberman 



Ph.D. Division of Basic 
Vision Research, 
NEI 



Objectives 

This project was designed to disrupt the a-crystallin 
genes (otA and oB) in the mouse to study their effect 
on normal lens and eye development. Disruption of 
the genes essentially will delete these proteins from 
the mouse, enabling us to analyze the effects these 
proteins have on expression of other lens proteins, 
developmental regulation and morphology of the lens 
and other eye structures, and the role of these pro- 
teins in nonlenticular tissues. 

Methods 

Standard molecular biology techniques are used to 
clone the a-crystallin genes and construct "gene 
knockout" vectors. Disruption of the genes will be 
accomplished by the newly developed technology of 
homologous recombination in pluripotent mouse 
embryonic stem cells, followed by insertion of the 
genetically altered cells into blastocyst mouse embry- 
os to generate chimeric mice with the gene disrup- 
tion. Chimeric "knockout" mice will be bred to 
generate mice with heterozygous and homozygous 
knockouts. 

Major Findings 

Cloning of mouse a-crystallin genes. — To maximize 
the success of homologous recombination in the 
planned experiments, we have isolated clones for 
clA- and oB-crystallin from a mouse 129 SV genom- 
ic library. This is the mouse strain from which most 
currently used embryonic stem cells are derived. 
Use of isogeneic DNA has been shown to improve 
greatly the yield of correctly targeted gene disrup- 
tions. Two overlapping aA clones and five overlap- 
ping (xB clones have been isolated and mapped. A 
15-kb oA clone with 9 kb of 5'- and 2 kb of 3'- 
flanking sequence and a 16-kb ctB clone with 7 kb 
of 5'- and 6 kb of 3'-flanking sequence will be used 
in construction of the knockout veaor. 

Construction of knockout vectors. — Construction 
of the aA-crystallin gene knockout vector is nearly 
complete. The vector contains 9 kb of aA 5'-flank- 
ing sequence, a selectable neomycin resistance gene 



cassette disrupting the aA gene, 1.3 kb of oA 
sequence, and a negative selectable marker (HSV tk). 

Sequencing of mouse aA-crystallin gene. — ^We are 
now sequencing the mouse aA-crystallin gene locus 
in collaboration with Dr. Nicholas Ambulos. A 
double-stranded sequence of the gene is nearly 
complete, and a single-stranded sequence of 4 kb of 
5'-flanking sequence has been done. Having the 
sequence of the locus will enable easy interpretation 
of DNA analysis of stem cells and mouse biopsies 
carrying the gene knockout. It also will facilitate 
identification of binding sites for regulatory protein 
in portions of the gene not yet studied. 

Transcriptional regulation of mouse aA-crystallin 
gene. — We are constructing vectors containing 
portions of the aA-crystallin gene locus to search for 
transcriptional enhancer elements. A base vector 
containing the oA-crystalUn promoter (-366 to +46) 
fused to the bacterial CAT reporter gene is under 
construction. Large pieces of the aA locus will be 
inserted into the base vector and tested for enhancer 
activity in transient transfection assays in cultured 
cells. 

Significance to Biomedical Research and the 
Program of the Institute 

Deletion of the a-crystallin proteins, individually or 
together, will provide a fundamental understanding of 
how these proteins function during normal lens 
development and how they may influence the struc- 
ture and function of the lens and the entire eye. In 
addition, it would give us insight into the function of 
these proteins in nonlenticular tissues, which in turn 
could help us imderstand some of their more subtle 
roles in the eye. 

Proposed Course 

In Fiscal Year 1994 we will continue our investiga- 
tion as follows: 

1 . We will continue construction of aA and aB 
knockout vectors so that mice deficient in either of 
these genes can be created. We also will perform 
the gene knockouts by introducing the targeting 
vector into mouse embryonic stem cells, selecting 
appropriately altered cells, and then creating chimeric 
mice from these cells. Deletion of a single allele of 
either aA or aB can be studied to assess the gene 
dosage effect (50% reduction of protein). Breeding 
to homozygosity (deletion of both alleles) will allow 
us to study the consequences of complete absence of 



183 



Laboraton of Molecular and Developmental Biolog> 



NEI Annual Report — FY 1993 



the individua] protein. Eventually we will mate aA 
and oB icnockout mice to produce mice totally 
devoid of a-crystallin. 

2. We will complete sequencing of the aA- 
crystallin gene locus. Although much is known 
about regulation of the mouse ocA-crystallin gene, the 
complete sequence of the gene has not yet been 
detennined. Knowing the complete sequence of the 
gene and flanking regions will be beneficial in the 
location of possible regulatory sites not in the imme- 
diate 5'-flanking region of the gene. This knowledge 



will be invaluable in analysis of gene knockouts in 
stem cells and mice. 

3. We will continue construction of vectors for 
use in transient transfection assays to locate addition- 
al regulatory elements in and around the oA-crystal- 
lin gene. This, along with the sequence of the locus, 
will help us to identify potential sites influencing the 
levels of gene expression. 

NEI Research Program 

Cataract — Lens Development and Aging 



I 

1 



184 



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 exam- 
ining aldose reductase inhibitors (ARIs) and anticata- 
ract agents. In pursuing the development of more 
effective and less toxic ARIs, the efforts are pro- 
gressing toward the development of an inhibitor 
unrelated to previous ARIs. A patent application has 
been made, and efforts are concentrated on further 
characterizing this inhibitor using biochemical, 
pharmacological, and computer molecular design 
techniques. Studies designed to elucidate the specific 
mechanism(s) by which aldose reductase initiates 
diabetic complications also are being conducted. 



In studies utihzing galactose-fed dogs, LOT 
investigators have established that retinal changes 
associated with diabetic retinopathy progress to the 
proliferative stage. The dog represents the first 
animal model that demonstrates clinical and histolog- 
ical changes found in all stages of retinopathy. 
Studies are now focused on the development of 
proliferative retinopathy in long-term galactose-fed 
dogs. In addition, investigators are analyzing the 
specific role of aldose reductase in neuropathy, 
thyroid changes, and immune system responses. 
Models for the evaluations of lens changes by 
magnetic resonance imaging also are being 
developed. 



187 



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



PROJECT NUMBER 



ZOl EY 00003-20 LOT 



PERIOD COVERED 



October 1, 1992 to September 30. 1993 



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

Pharmacology of Ocular Complications 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, 
PI: Peter F. Kador 

Others: WiUjam Greentree 

JuD Inoue 

YoDg Lee 

Martii] Lizak 

Amta Bartoszko- Malik 

Kazuhiko Mori 

Heike Neuenschwander 

Libaniel Rodriguez 

Maneo Scbaffbauser 



Ph.D. 


Chief 


D.V.M. 


IRTA FeUow 


M.S., Phann. 


Special Volunteer 


Ph.D. 


Staff Fellow 


Ph.D. 


Staff Fellow 


Ph.D. 


Visiting Fellow 


M.D., Ph.D. 


Visiting Fellow 


M.D. 


Special Volunteer 


Ph.D. 


Staff Fellow 


Ph.D. 


Visiting Fellow 



and institute affiliation) 
LOT, NEI 
LOT, NEI 
LOT. NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT, NEI 
LOT. NEI 
LOT. NEI 
LOT. NEI 



COOPERATING UNITS (it any) 



LAB/BRANCH 



Laboratory of Ocular Therapeutics 



SECTION 



INSTITUTE AND LOCATION 

NEI, NIH. Bethesda, MP 20892 



TOTAL STAFF YEARS: 



6.75 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



PROFESSIONAL: 



6.75 



OTHER: 



0.0 



[x] (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. Specific studies include 
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. 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. 

Also being studied are events leading to the formation of several types of cataracts, as well as methods for 
controlling the onset of these cataracts through pharmacological intervention. 



188 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Ocular Therapeutics 



Project Description 

Additional Personnel 

Robert Balaban Ph.D. 



Duane Miller 



Ph.D. 



National Heart, Lung 
and Blood Institute 
Ohio State University 
College of Pharmacy, 
Columbus, OH 



Objectives 

This project is designed to (1) gain insight into the 
mechanisms by which polyol-induced ocular diabetic 
complications and cataracts are formed and (2) devel- 
op methods for their regulation. 

Methods 

Diabetes can be induced experimentally in animals 
through the injection of streptozotocin. Diabetes- 
related complications linked to the sorbitol pathway 
also can 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 photogra- 
phy. Biochemical smdies used for the purification of 
enzymes include column chromatography, polyacryl- 
amide gel electrophoresis (PAGE), isoelectric focus- 
ing, chromatofocusing, and high-pressure liquid 
chromatography (HPLC). Polyol levels were deter- 
mined by gas-liquid chromatography (GLC). Immu- 
nological analyses include the use of enzyme-linked 
immunosorbent assay (ELISA), radioimmunoassay 
(RIA), Western blots, and immunohistochemical 
techniques employing the coupled antibody DAB- 
PAP technique. Computational methods for enzyme 
analysis, inhibitor structure-activity studies, and 
pupil-function changes require the use of the NIH 
PROPHET computer system and Charm and Quanta 
computer systems from Molecular Design. 

Major Findings 

Biochemical studies. — Studies on defining the inhibi- 
tor site of aldose reductase and aldehyde reductase 
are continuing with the evaluation of a number of 
Michael addition affinity-labeled aldose reductase 
inhibitors (ARIs). Analogs of the ARIs AL1576 and 
alrestatin possessing sterically diverse substituents, 
Michael addition adducts, and haloalkyl groups have 
been synthesized and evaluated in vitro for their 



ability to inhibit and irreversibly bind rat lens aldose 
reductase and rat kidney aldehyde reductase. 

Inhibitory potency in general was reduced in a 
series of alrestatin analogs in which the carboxyl acid 
was condensed with sulfonamide groups; however, 
inhibition increased with 5-substitution with either 
nitro or Michael addition adducts. In the spirofluor- 
enehydantoin series, substitution in the 2 position 
with similar open- or constrained-ring Michael 
addition adducts did not result in increased inhibi- 
tion. The selective introduction of Michael addition 
adducts can result in increased inhibitory activity and 
irreversible binding of aldose reductase but not 
aldehyde reductase. Specific nucleophilic residues 
on the enzyme also are being identified by subjecting 
the alkylated enzyme to trypsin digestion and subse- 
quent chemical ionization mass spectroscopy. 

The pharmacophore requirements of the inhibitor 
site and the location of reactive nucleophilic and 
electrophilic sites are being refined through the use 
of molecular modeling. The theoretical tools utilized 
for this study include the AMI quantum chemical 
method and the fitting methods of Quanta 3.3 con- 
ducted on an SGI Crimson computer. These results 
were then correlated to observed inhibitions of rat 
lens aldose reductase. Geometry optimization and 
energetics calculations have been conducted for a 
series of carboxyl acid-containing analogs of 9- 
spirofluorenehydantoin. 

These calculations were conducted on the charged 
(-1) rather than neutral species because they are 
assumed to be charged at physiological pH. Super- 
position of these geometry-optimized compounds on 
AL1576 were conducted by utilizing both torsional 
flexible and rigid body fits, and the spatial regions at 
which reversible nucleophihc siibstimtion takes place 
were then compared. The spirohydantoin AL1576 
was chosen as a template in this study because (1) it 
contains a rigid hydantoin ring having two probable 
pharmacophores in which the relative position in 
space is fixed and (2) it has been shown to have high 
affinity for aldose reductase. These studies indicate 
that a negative charge center resides in the vicinity of 
the 2-oxygen atom of the hydantoin ring while the 4- 
carbonyl represents a region where a nucleophilic 
substitution likely takes place. Understanding the 
pharmacophore requirements will lead to the rational 
development of new ARIs. New, potent ARIs have 
been uncovered, and patent application has been 
made. 



189 



Laborator) of Ocular Therapeutics 



NEI Annual Report— FY 1993 



Recent studies have demonstrated that the dog is 
an excellent animal model for investigating ocular 
diabetic complications. Cataract development in the 
dog is similar to cataract development in humans 
with diabetes. The cataracts are characterized by 
formation of anterior and posterior superficial cortical 
opacities with the posterior polar region being more 
advanced. Furthermore, the dog develops advanced 
retinal changes similar to those clinically observed in 
human diabetics. Polyol formation initiated by the 
NADPH-dependent enzyme aldose reductase has 
been demonstrated to initiate these lens and retinal 
changes. 

Magnetization transfer contrast (MTC) is a 
method in magnetic resonance imaging (MRI) that 
generates high-contrast images based on characteris- 
tic tissue differences resulting from the interaction of 
water and macromolecules. We are applying the 
MTC to document cataract formation and other 
structural changes of the anterior segment associated 
with diabetes eye complications in galactose-fed 
dogs. Dogs, sedated with acepromazine (i.m. injec- 
tion), intubated, and then ventilated with 1.0-1.5% 
halothane are administered succinylcholine chloride 
to prevent eye movements and placed in a General 
Electric 2-T Omega MRI system. 

M^ and M„ images are acquired using gradient- 
recalled echo sequences, with and without the satura- 
tion pulses, respectively, consisting of rf-irradiation 
10 kHz off-resonance from the free-water proton 
signal. The Ti^^, image data are obtained using one- 
short T,-imaging, employing an inversion pulse 
followed by a series of small tip-angle pulses that 
sample the relaxation curve. These images are then 
compared with photographs obtained with photo-slit- 
lamp and retroillumination photography. The results 
indicate that MTC not only generates excellent 
images of the lens but also aids in the visualization 
of fine structures in the anterior segment, including 
the cornea, iris, ciliary bodies, choroid membrane, 
and Schlemm's canal. 

"F-NMR spectroscopy also is 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-fluorosorbitol. This work 
is an extension of the in vivo evaluation of aldose 
reductase activity in rabbit lenses. Initial spatial 
coordinates for lenses are calculated from 'H-images 
determined on a 2.0 Tesla GE Omega-CSI spectrom- 
eter. The SLOOP (spectral localization with optimal 



pointspread function) technique is then used with a 
proton decoupler to measure the accumulation of 
sorbitol in the rabbit lens. A double spin-echo 
sequence is utilized with selective excitation and 
refocusing pulses and with optimized phase-encoding 
gradient pulses using 1-sec repetition times and 25- 
msec echo times. SLOOP experiments indicate that 
3-deoxy-3-fluorosorbitol can be observed in spectra 
of the anterior portion of the lens when adequate 
amounts of 3-deoxy-3-fluoroglucose are adminis- 
tered. 

Retinal studies. — Vascular changes associated 
with diabetic retinopathy can be produced experi- 
mentally in beagles fed a 30% galactose diet In 
studies designed to clarify the initiating lesions and 
progression of diabetic retinopathy, we have docu- 
mented 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-linked formation of pericyte 
ghosts and subsequent development of acellular 
capillaries, microaneurysms, and intraretinal hemor- 
rhages. This early retinopathy progresses to include 
the appearance of occluded vessels, areas of nonper- 
fusion, and intraretinal microvascular abnormalities 
(IRMA). Finally proliferative retinopathy develops, 
including the formation of fibrovascular membranes 
seen histologically on both the retinal surface and the 
posterior hyaloid membrane. 

Pericyte ghost formation and the subsequent 
appearance of microaneurysms, intraretinal hemor- 
rhages, and acellular capillaries associated with 
background retinopathy have been arrested in a dose- 
dependent maimer in 36- to 38-month prevention 
studies utilizing 0.5, 5, 10, and 16 mg/kg/day of the 
ARI M79175. The dog represents the first animal 
model to demonstrate all the clinical and histological 
retinal vessel changes observed in human diabetics. 

Significance to Biomedical Research and the 
Program of the Institute 

Loss of vision from cataract and diabetic retinopathy 
are significant; therefore, methods for the pharmaco- 
logical control of these ocular complications are 
required. We have developed an animal model that 
demonstrates advanced retinal vessel changes that are 
virtually identical both clinically and histologically to 
those observed in advanced diabetic retinopathy. 
Our present studies in dogs demonstrate for the first 



190 



NEI Annual Report— FY 1993 



Laboratory of Ocular Therapeutics 



time that loss of retinal pericytes, associated with 
aldose reductase, initiates retinal changes associated 
with both background and advanced diabetic retinop- 
athy and that adniinistration of ARIs in prevention 
studies can ameliorate the loss of pericytes and 
subsequent microaneurysms and retinal hemorrhages 
in a dose-dependent manner. The successful devel- 
opment of noninvasive methods for monitoring 
aldose reductase activity by nuclear magnetic reso- 
nance (NMR) procedures may have a direct impact 
on ongoing and plaimed clinical trials in which this 
procedure could serve as a quantitative indicator of 
drug efficacy. Cataract is one of the major causes of 
bhndness in the developing world. In addition, loss 
of vision due to cataract is one of the major health 
problems of both people with diabetes and the aging 
population in the United States. 

Proposed Course 

These studies will be continued. Currently discov- 
ered ARIs will be evaluated and developed pharma- 
cologically. The inhibitor site will be probed further 
through the use of affinity labels so that more potent 
and specific inhibitors may be developed. Studies 
will be continued on the mechanisms through which 
aldose reductase induces diabetic complications in 
various tissues. 

NEI Research Program 

Retina] Disease — Diabetic Retinopathy, Sickle Cell 
Retinopathy, and Other Vascular Abnormalities 

Publications 

Bartoszko-Malik A, Schaffhauser M, Ghany-Abdel 
Y, Miller DD, Kador PF: Evaluation of novel 
aldose reductase inhibitor site probes. Invest 
Ophthalmol Vis Set 34(suppl):280, 1993. 

Fukase S, Mori K, Sato S, Kador PF: Comparison 
of NADPH-dependent reductases in dog lens and 
leukocytes. Invest Ophthalmol Vis Sci 34(suppl): 
757, 1993. 

Kador PF: Intermediary metabolism of the lens, in 
Raviola E, Dowling J (eds): Principles and 
Practice of Ophthalmology. New York, Wiley 
Press, in press. 

Kador PF, Takahashi Y, Schaffhauser M: Vorbeug- 
ung diabetischer Komplikationen im Auge mit 
Aldosereduktase-Hemmem. Diabetes und Stojf- 
wechsel, in press. 



Kador PF, Takahashi Y, Sato S, Wyman M: Retinal 
vessel changes in galactose-fed dogs treated with 
the aldose reductase inhibitors M79175 and 
FK366. Invest Ophthalmol Vis Sci 34(suppl):64, 
1993. 

Kador PF, Takahashi Y, Wyman M, Ferris F III: 
Arch Ophthalmol 111:585, 1993. 

Lee YS, Peralstein R, Kador PF: Molecular model- 
ing of aldose reductase inhibitors. J Med Chem, 
in press. 

LI Q, Lopez JS, Caspi RR, Roberge FG, Nussenblatt 
RB, Kador PF, Chan C-C: Suppression of S- 
antigen induced experimental autoimmune 
uveoretinitis in Lewis rats by oral administration 
with COS- 13080, a thromboxane synthetase 
inhibitor. Exp Eye Res 57:601-608, 1993. 

Mori K, Ceckler TL, Kador PF, Balaban RS: Mag- 
netic resonance imaging of the galactosemic dog 
eye using magnetization transfer contrast. Invest 
Ophthalmol Vis Sci 34(suppl):1061, 1993. 

Ogawa K, Yamawaki I, Matsusita Y, Nomura N, 
Kador PF, Kinoshita JH: Synthesis of substituted 
2,4-dioxo-thienopyriniidine-l -acetic acids and 
their evaluation as aldose reductase inhibitors. 
Eur J Med Chem, in press. 

Okamoto S, Terubayashi H, Tsutsumi M, Ikebe H, 
Nishimuna C, Kador P, Akagi Y: Localization of 
aldose reductase mRNA in the rat lens. Nippon 
Ganka Gakkai Zasshi 96:1373-1378, 1992. 

Reddy VN, Lin L-R, Giblin FJ, Lou M, Kador PF, 
Kinoshita JH: The efficacy of aldose reductase 
inhibitors on polyol accimiulation in human lens 
and retinal pigment epithelium in tissue culture. 
J Ocular Pharmacol 8:43-52, 1992. 

Smar MW, Ares J, Nakayama T, Itabe H, Kador PF, 
Miller DD: Selective irreversible inhibitors of 
aldose reductase. J Med Chem 35:1117-1120, 
1992. 

Takahashi Y, Augustin W, Wyman M, Kador PF: 
Quantitation of retinal vessel changes associated 
with diabetic retinopathy in galactose-fed dogs. 
J Ocular Pharmacol, 9:257-269, 1993. 

Waldbillig 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: Implications for under- 



191 



Laboratory of Ocular Therapeutics 



NEI Annual Report — ^FY 1993 



standing diabetic retinopathy. J Cliri Invest, in 
press. 

Woel V, Kuszak JR. Takahashi Y, Wyman M, Kador 
PF: An ultrastructural analysis of posterior 
migrating lens epithelial cells in cataracts of 
galactose-fed dogs. Invest Ophthalmol Vis Sci 
34(suppl):916, 1993. 



192 



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



PROJECT NUMBER 

ZOl EY 00275-02 LOT 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (BO characters or less. Title must tit or one line betweer) tt\e borders.) 

Role of NADPH-Dependent Reductases 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 Ph.D. Chief LOT, NEI 

Shigeru Fukase M.D. Visiting Associate LOT, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 



Laboratory of Ocular Therapeutics 



SECTION 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 

1.35 



PROFESSIONAL: 

1.35 



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 increased influx of glucose into the sorbitol pathway in diabetes results in the accumulation of sugar 
alcohol sorbitol, which is linked to the pathogenesis of various diabetic complications such as retinopathy, 
neuropathy, and nephropathy. The role of aldose reductase and related enzymes in polyol formation and the 
subsequent onset of these complicadons are being investigated. 



193 



PHS 6040 (Rev. 5/92) 



Laboratory' of Ocular Therapeutics 



NEI Annual Report — FY 1993 



Project Description 

Objectives 

In diabetes, excess glucose results in an increased 
influx of glucose into the polyol pathway. The 
accumulated sugar alcohol, sorbitol, has been linked 
to the onset of various diabetic complications such as 
cataract formation, retinopathy, neuropathy, and 
nephropathy. The development of potent aldose 
reductase inhibitors (ARIs) represents a new pharma- 
ceutical approach to the treatment of diabetic compli- 
cations. Understanding NADPH-dependent enzymes 
in target tissues is a required first step in the design 
of ARIs. This project is designed to investigate 
aldose reductase and its related enzymes in various 
tissues where diabetic changes occur. 

Methods 

Biochemical techniques include gel filtration, affinity 
chromatography, electrophoresis, immunoblotting, 
and isoelectric focusing on high-pressure liquid 
chromatography. Gas chromatography is used to 
identify and quantitate sugars. In vitro culture 
techniques include the culture of retinal capillary 
pericytes and endothelial cells, leukocytes, and 
fibroblasts. The results, including enzyme kinetic 
evaluations, are calculated using the NIH PROPHET 
computer system. 

Major Findings 

Human kidney. — Like that of the rat and dog, human 
kidney cortex contains predominantly aldehyde 
reductase rather than aldose reductase, whereas the 
medulla contains aldose reductase. The kinetic 
properties of aldose and aldehyde reductases purified 
from human kidney are essentially identical to those 
of the rat and dog kidney enzymes. Moreover, as 
demonstrated with rat kidney enzymes, aldehyde 
reductase — in addition to aldose reductase — ^generates 
polyols from both glucose and galactose in an in 
vitro incubation system. As in rat kidney, aldehyde 
reductase contributes to polyol formation in human 
kidney cortex, where diabetic changes occur. The 
use of animal models is based on the premise that 
similar pathological changes occur in both experi- 
mental animals and humans. Evidence that both 
human kidney aldose and aldehyde reductases are 
similar to the rat and dog enzymes in kinetic proper- 



ties and inhibition by aldose reductase inhibitors 
gives enzymatic rationale for this approach. 

Rat lens. — Rat lens displays dehydrogenase 
activity with naphthalene dihydrodiol as substrate. 
This dehydrogenase activity corresponds to aldose 
reductase throughout all purification procedures, 
which include gel filtration, affinity chromatography, 
and chromatofocusing. The dehydrogenase activity 
is observed with the highly purified aldose reductase 
and also with recombinant enzymes from the rat lens 
aldose reductase clone. The evidence indicates that, 
in rat lens, dehydrogenase activity in the presence of 
naphthalene dihydrodiol comes from aldose reduc- 
tase. 

Significance to Biomedical Research and the 
Program of the Institute 

Despite the establishment of insulin therapy, the risk 
of loss of vision due to diabetic complications is still 
significantly high. Based on evidence that excess 
amounts of polyols are linked to the onset of diabetic 
complications, worldwide efforts have been made to 
develop ARIs. The fiill understanding of NADPH- 
dependent reductases and polyol formation wall 
provide essential information on their clinical poten- 
cy and contribute to further development of more 
potent inhibitors. Evidence that aldose reductase 
responds to naphthalene dihydrodiol with dehydroge- 
nase activity may expose new facets of the role(s) of 
aldose reductase in certain types of toxic cataract 

Proposed Course 

We will continue our evaluation of the location and 
enzymatic properties of aldose and aldehyde reduc- 
tases in various tissues in which diabetic complica- 
tions occur. To investigate retinal pericytes and 
endothelial cells as keys in diabetic retinopathy, we 
will utilize cell culture techniques. Leukocytes and 
various leukemia cells also will be investigated. 

NEI Research Program 

Diabetic Complications 
Cataract Research 

Publications 

Fukase S, Mori K, Sato S, Kador PF: Comparison 
of NADPH-dependent reductases in dog lens and 



194 



NEI Annual Report — ^FY 1993 Laboratory of Ocular Therapeutics 

leukocytes. Invest Ophthalmol Vis Sci 34(4) Sato S, Kador PF: Human kidney aldose and alde- 

(suppl):757, 1993. hyde reductases. J Diab Compl 7:179-187, 1993. 

Sato S: Aldose reductase is the major protein associ- Sato S, Lin L-R, Reddy V, Kador PF: Aldose 

ated with naphthalene dihydrodiol dehydrogenase reductase in human retinal pigment epithelium, 

activity in rat lens. Invest Ophthalmol Vis Sci Exp Eye Res 57:235-241, 1993. 
34:3172-3178, 1993. 



195 



Laboratory of Retinal Cell and Molecular Biology 



Report of the Chief, Laboratory of Retinal Cell and Molecular Biology 

Gerald J. Chader, Ph.D. 



The research focus of members of the Laboratory 
of Retinal Cell and Molecular Biology 
(LRCMB) is on elucidating new genes and biochemi- 
cal mechanisms and learning the underlying causes 
of ocular diseases. In this way, we hope to intervene 
in the disease process before substantial damage to 
vision has been done or to ^ply rational methods of 
gene therapy before the terminal stages of the disease 
have been reached. Most of the approaches taken 
are molecular biological, molecular genetic, and/or 
candidate gene approaches. 

The work of the Laboratory members is within 
the following National Institutes of Health strategic 
initiatives and National Eye Institute priorities: 
(1) molecular medicine, (2) gene research and gene 
therapy, and/or (3) research of high clinical 
relevance. 

Within the LRCMB, the following three areas are 
emphasized: 

1. Molecular biology and molecular genetics, 

2. Gene therapy, and 

3. Immunopathology. 



Molecular Biology and Molecular 
Genetics 

Specific advances made in the area of molecular 
biology and molecular genetics are discussed 
below. 

Retina-specific genes. — Several genes that are pre- 
dominantly or exclusively expressed in ocular tissues 
have been identified by subtractive cloning. Retina- 
specific genes and genes located on the short arm of 
the X-chromosome are pinpointed. These genes are 
being localized chromosomally to determine whether 
they are linked to eye diseases. Concurrently, 
genomic cloning and sequencing are being done to 
generate appropriate markers and polymorphisms. 
Cell lines are being immortahzed in tissue culture 
such that subsequent laboratory experiments can be 
conducted. 



Genes specific to retinal pigment epithelium 
(RPE). — ^Little is known about the specific comple- 
ment of genes in the RPE or how these could be 
involved in diseases of the visual system. Thus, 
cloning of genes unique to RPE and its functioning 
is of importance. A new 65-kD protein of potential 
immunologic importance has been isolated from the 
human RPE. The gene has been cloned, allowing for 
smdy of tissue-specific expression. This gene is the 
first RPE-specific gene to be reported and character- 
ized. 

Photoreceptor-specific genes. — ^An effort has been 
made to identify and characterize genes for proteins 
and enzymes that are critical in functioning of the 
photoreceptor outer segment. For example, two 
proteins of the phototransduction cascade, S-antigen 
(S-Ag) and phosducin, have been well characterized 
as to their expression control. Both proteins are 
specific to the rod neuron and interact with visual 
cycle components (e.g., opsin). cDNAs and 
genomics for S-Ag and phosducin have been cloned 
and thoroughly analyzed, allowing for current advan- 
ces in our understanding of expression, function, and 
pathology of the gene products. 

Interphotoreceptor retinoid-binding protein 
(IRBP). — ^IRBP also is a critical link in the chain of 
enzymes and proteins that make up the visual cycle. 
Because of the huge size of the gene, however, it is 
very difficult to sequence in potential disease cases. 
We are thus cloning the homolog of the human IRBP 
gene from Drosophila megalogaster (i.e., fhiitfly). 
Interestingly, it maps to an area of the Drosophila 
genome that is rich in mutants of ocular disease. 
Knowing the characteristics (e.g., erg) of the dif- 
ferent diseases in the fly, we hope to pinpoint a 
specific human population with similar characteristics 
and examine the gene for defects in specific human 
famihes. 

Pigment epithelium-derived factor (PEDF). — We 
have identified a novel neurotrophic protein, PEDF, 
synthesized by fetal human RPE cells that may be 
critical in the development of retinal photoreceptors. 
At very low concentration, PEDF causes the exten- 



199 



Laboratory of Retinal Cell and Molecular Biologj 



NEI Annual Report— FY 1993 



sion of elaborate neuronal processes from cultured 
retinoblastoma cells. Since these cells are thought to 
be derived from photoreceptor cone cells, we hope 
that PEDF can be as effective on cone neuron 
development in vivo. An important possibility is 
that, in the Royal College of Surgeons rat, a defect 
in the PEDF gene could cause retinal degeneration. 
Also, we are continuing evaluation of the clinical use 
of PEDF in retinal transplantation in collaboration 
with Dr. M. del Cerro. The molecular biology of 
this potentially very important neurofrophic agent is 
now being studied for application to retinal dysfunc- 
tions. 

Fatty acid and tubulin defects in retinal degenera- 
tion. — In collaboration with Dr. Muriel Kaiser- 
Kupfer (Ophthalmic Genetics and Clinical Services 
Branch), we are investigating fatty acid uptake and 
metabolism in Bietti's crystalline retinopathy and a 
tubulin acetylation defect in a form of atypical 
retinitis pigmentosa (RP) for which we hope to 
elucidate the specific defects. Significant progress 
has been made in pinpointing the metabolic problems 
expressed in both these hereditary conditions. 



Gene Therapy of Retinal Diseases 

Points of focus and advances made witliin the area 
of gene ther^y are discussed below. 

Transgenic studies. — ^The IRBP and S-Ag genes 
are the best studied retinal genes other than rhodop- 
sin. Ci5-acting elements controlling the IRBP and 
S-Ag promoters, and thus their protein expression, 
have been identified in transfected human cells and 
in ttansgenic mice. Transgenic studies, in particular, 
have helped to uncover factors controlling gene 
activation in the embryonic period, specifically in the 
photoreceptor cell. 

Gene analysis systems in transgenic mice and in 
fransient fransfections in cultured himian retinoblasto- 
ma cells have been established for IRBP. Much of 
the 5'-flanking region of IRBP has been thoroughly 
examined to date. Similarly, a good deal of progress 
has been made with the S-Ag promoter. Enhancer 
elements necessary for expression are being defined 
through target mutagenesis studies. Tissue- and 
stage-specific elements, including TATA and CAAT 
boxes, are being defined as to retinal expression. 



This work is important in that specific molecules can 
be "gene-targeted" to the retina with precision. 

Gene therapy. — Ribozymes are specifically 
constructed RNA species that can control expression 
of proteins within cells. By linking these simplified 
gene forms to appropriate promoters and utilizing a 
suitable transfer vector, we can construct new thera- 
peutic modalities. Gene therapy can then be planned 
to freat autosomal dominant disorders that are cur- 
rently uimianageable. 

Ribozyme constructs for IRBP have been de- 
signed and are being studied in a ttansfected human 
retinoblastoma cell system. Concurrently, fransgenic 
mice have been reared to determine if an RP-like 
condition is produced through down-regulating IRBP 
synthesis. Once perfected, ribozymes should be 
useful, not only with IRBP-related retinopathies but 
in conditions such as diabetic retinopathy and reti- 
nopathy of prematurity, disorders which probably 
involve overexpression of normal proteins such as 
growth factors. 



Immunopathology 

Most of this work is in collaboration with inves- 
tigators in the Laboratory of Immunology. 
The focus is on the induction of experimental auto- 
immune uveitis. 

Immunopathology. — Collaborative work with 
Dr. Igal Gery continues on the study of uveitis. The 
immunopathological site(s) of IRBP are being 
dissected in the Lewis rat and in the human with the 
final goal of controlling or preventing the disease 
process in man. 

Immunogenetics. — Collaborative work with Dr. 
Rachel Caspi has established the IRBP-mouse model 
for experimental autoimmune uveoretinitis as very 
useful for studying the genetics of the disease and its 
relapsing characteristics. 

Antigen presentation. — Collaborative work with 
Drs. Gery, Marc de Smet, and Robert Nussenblatt 
has demonsfrated the presence of a 70-kD cell 
surface protein of B cells that specifically binds the 
major immunopathological determinant of IRBP. It 
is thought that this protein may function as a molec- 
ular chaperone in antigen presentation. It is a likely 
candidate for gene therapy in uveoretinitis. 



200 



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



PROJECT NUMBER 



ZOl EY 00070-16 LRCMB 



PERIOD COVERED 



October 1, 1992 to September 30. 1993 



TITLE OF PROJECT (BO characters or less. Title must fit on 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 frf 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.); U. Maryland Med. Sch. (M. Rodrigues, M.D., Ph.D.); Medical College of Georgia (S. Smith, 
Ph.D.); Emory Eye Center (J. Nickerson, Ph.D.) 



LAB/BRANCH 

Laboratory of Retinal Cell and Molecular Biology 

SECTION 

Section on Biochemistry 

INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



5.0 



PROFESSIONAL: 



3.0 



OTHER: 



2.0 



CHECK APPROPRIATE BOX{ES) 

□ (a) Human subjects 
□ (a1) Minors 
n (a2) Interviews 



[x] (b) Human tissues □ (c) Neither 



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

Studies on the physicochemical characteristics of a fatty acid-binding site on the interphotoreceptor retinoid- 
binding protein (IRBP) with fluorescent fatty acid analogs demonstrated that fatty acids were bound in a 
hydrophobic environment, that there was a single, specific fatty acid-binding site for each molecule of IRBP, 
and that there was nonradiative energy traasfer from tryptophan residues to bound ligand. Probing the 
microenvironment of bound fluorophore with a quencher indicated a liighly structured binding site. 

Studies of the formation and release of ll-m retinal by the retinal pigment epithelium at a physiological 
concentration of IRBP demonstrated that a sequential (i.e., unbranched) pathway mediates the processing of 
all-rran^ retinol to ll-cis retinal and its transfer to IRBP. 

In the mi'"'mi"' mutant mouse model of retinal degeneration, retinyl palmitate was elevated fourfold and IRBP 
was elevated twofold in the eyes of affected mice, as compared with that in controls at 6-8 weeks of postnatal 
development. At the same time, IRBP mRNA was not elevated. The elevation in retinyl palmitate may be 
a significant factor in the retinal degeneration in this mutant, and IRBP turnover may be affected by an 
aberration in retinoid metabolism. 

A 72-kDa heat shock protein (hsp) which bound specifically to peptide 1169-1191, a potent uveitogenic 
determinant of IRBP, has been identified in Lewis rat B cells and Epstein Barr virus-transformed B cells from 
normal human donors and uveitis patients. This hsp has a potential role in antigen processing and presentation 
by antigen-presenting cells. 



201 



PHS 6040 (Rev. 5/92) 



Laboratory' of Retinal Cell and Molecular Bioloe>' 



NEI Annual Report— FY 1993 



Project Description 

Additional Personnel 
Igal Gery Ph.D. 



Rachel Caspi Ph.D. 

Tatiana Putilina Ph.D. 

Mark de Smet M.D. 



Head, Section on 

Experimental 

Immunology, LI, 

NEI 

Visiting Associate, 

LI, NEI 

Visiting Associate, 

LRCMB, NEI 

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 spectroscopy, 
high-performance liquid chromatography, SDS- 
polyacrylamide gel electrophoresis. Western blotting. 
Northern blotting, slot-blotting, and the enzyme- 
linked immunosorbent assay were used to study 
retinoid-binding proteins. 

Major Findings 

The physicochemical characteristics of a fatty acid- 
binding site on IRBP were examined using a set of 
fluorescent fatty acid analogs with an anthracene 
moiety attached at different positions along the 
hydrocarbon chain. The results demonstrated that 
fatty acids were bound in a hydrophobic environ- 
ment, as indicated by a blue shift in fluorescence 
maxima and by an increase in quantum yield of the 
bound ligand. A single, specific fatty acid-binding 
site existed for each molecule of IRBP with an 
apparent K^3.6xlO'^M. There was nonradiative 
energy transfer from tryptophan residues to bound 
ligand, as well as fluorescence energy transfer to all- 
trans retinol when this ligand was bound to IRBP. 

The interactions of IRBP and bound fatty acids 
were sensitive to denaturation by increasing concen- 
trations of urea as judged by changes in nonradiative 
energy transfer efficiency and the quantum yield of 
the bound probe. Quantum yields of bound fatty 



acid analogs varied with position of the fluorophore 
along the hydrocarbon chain and had the lowest 
values for the fluorophore located at the midpoint. 
Probing the microenvirorunent of bound fluorophore 
with a quencher indicated a highly structured binding 
site. 

In studies of the formation and release of 11 -cis 
retinal by the retinal pigment epithelium (RPE) in the 
toad eyecup preparation, the time course of the 
specific activity of radio-labeled l\-cis retinal at a 
fixed, near-physiological IRBP concentration demon- 
strated that a sequential (i.e., unbranched) pathway 
mediates the processing of zl\-trans retinol to ll-cis 
retinal and its transfer to IRBP. 

In the mutant mouse model of retinal degenera- 
tion, levels of retinyl palmitate in the eyes were 
elevated fourfold greater than controls by 8 weeks, 
and the levels remained elevated through 42 weeks. 
Levels of ll-cis retinal, dll-trans retinol, and all- 
trans retinal were similar to those of controls, as 
were plasma retinol levels. Levels of IRBP at 2-4 
weeks were similar in affected and control animals, 
but by 6 weeks IRBP levels were twofold greater in 
the affected mice than in controls and remained high 
for several weeks until degeneration of photoreceptor 
cells took place. At the same time, IRBP mRNA 
was not increased in the affected mice, indicating 
that IRBP turnover probably decreased in the disease. 
The elevation of retinyl palmitate may be a signifi- 
cant factor in the retinal degeneration seen in the 
mouse mutant, and IRBP turnover may be affected 
by an aberration in retinoid metabolism. 

Peptide 1169-1191 is a potent uveitopathogenic 
determinant of IRBP. Recently a new class of 
proteins known as chaperones, which are part of the 
heat shock protein (hsp) family, have been implicated 
in antigen presentation and appear to prevent further 
degradation of antigen by lysosomes. A 72-kD 
protein that bound specifically to peptide 1169-1191 
was found in Lewis rat B cells and EBV-transformed 
B cells from normal human donors and uveitis 
patients. This protein reacted with antibodies 
specific for both constituitively expressed and induc- 
ible 72/73-kD HSP 70 proteins and could have a 
potential role in antigen processing and presentation 
by antigen-presenting cells. 

Large-scale purification of IRBP was continued 
for studies on the production of experimental auto- 
immune uveitis (EAU) in rats and mice and possible 
modes of suppression of the disease. 



202 



NEI Annual Report— FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



Significance to Biomedical Research and the 
Program of the Institute 

Because of its importance in normal photoreceptor 
cell physiology (i.e., in facilitating the transport of 
retinoids during the visual cycle as well as the 
transport of fatty acids that are essential to normal 
function), abnormalities in IRBP function resulting 
from changes in concentration, distribution, or 
affinity for retinoids or fatty acids could be important 
either directly or indirectly in visual cell pathogen- 
esis. 

Proposed Course 

Studies on the fatty acid- and retinoid-binding sites 
on IRBP will be continued to elucidate the relation- 
ship between the two ligands and the possible effect 
of fatty acid binding on the binding of retinoids and 
the function of IRBP in the interphotoreceptor 
matrix. We also will continue to smdy the physio- 
logical role of ERBP in the visual cycle, particularly 
the effect of removing IRBP during retinal detach- 
ment on regeneration of visual pigment. Continued 
studies on the mi^'mi"" mouse model of retinal 
degeneration will include studies of retinoid metabo- 
lism in the RPE to determine the mechanism causing 
large elevations in retinyl palmitate in mutant mice. 
Further work also will be carried out to characterize 
the 72-kD hsp, which binds the uveitogenic peptide 
1169-1191 of IRBP, and its possible role in human 
disease. We will continue to conduct large-scale 
purification of IRBP protein for studies of EAU. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and Other 
Inherited Disorders 



Publications 

Hara Y, Caspi RR, Wiggert B, Chan CC, Streilin 
JW: Use of ACAID to suppress interphotore- 
ceptor retinoid binding protein-induced experi- 
mental autoinunune uveitis. Curr Eye Res 1 1:97- 
100, 1992. 

Kutty RK, Kutty G, Duncan T, Nickerson J, Chader 
GJ, Wiggert B: Radioanalytic estimation of 
amplification products generated by RT-PCR 
using (alpha-"P) deoxynucleotide triphosphate. 
Biotechniques, 15:808, 811-812, 1993. 

Pepperberg DR, Okajima TL, Wiggert B, Ripps H, 
Crouch RK, Chader GJ: Interphotoreceptor 
retinoid-binding protein (IRBP), in Molecular 
Neurobiology. Clifton, NJ, Humana Press Inc, 
1993, in press. 

Putilina T, Sittenfeld D, Chader GJ, Wiggert B: 
Smdy of a fatty acid binding site of interphotore- 
ceptor retinoid-binding protein using fluorescent 
fatty acids. Biochemistry 32:3797-3803, 1993. 

Rajagopalan S, Rodrigues MM, Wiggert B, Advani 
SH, Nair CN, Nickerson JM: Retinoblastoma: 
Interphotoreceptor retinoid-binding protein mRNA 
analysis by polymerase chain reaction. Ophthal- 
mic Paediatr Genet, in press. 

Sasamoto Y, Kawano YI, Bouligny R, Wiggert B, 
Chader GJ, Gery I: Immunomodulation of exper- 
imental autoimmune uveoretinitis by intravenous 
injection of uveitogenic peptides. Invest Ophthal- 
mol Vis Sci 33:2641-2649, 1992. 



203 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00196-10 LRCMB 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



TITLE OF PROJECT (SO crmraclars or lass Tilla must In on one line between the borders j 

Molecular Genetics of the Eve and Ocular Diseases 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, latmralory. and institute alliliation) 

Ph.D. Senior Staff Fellow LRCMB, NEI 



PI 



Others: 



Diane E. Borst 
Steven Bernstein 



Ph.D.. M.D. Senior Staff Fellow 



LRCMB, NEI 



COOPERATING UNITS (if any) 

Emor> Universit>. Atlanta, GA (J.M. Nickerson, Ph.D., J-S. Si, M.D.); University of Michigan, Ann Arbor 
(E. Farr, M.D.); University of Texas. Dallas (R. Hammer. Ph.D.) 



LAB/BRANCH 



Laboratory of Retinal Cell a nd Molecular Biology 



SECTION 

Section on Gene Regulation 



INSTITUTE AND LOCATION 

NEI. NIH. Bethesda. MP 20892 



I TOTAL STAFF YEARS: 



PROFESSIONAL: 



2.0 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 
n (a1) Minors 
□ (a2) Interviews 



2.0 



OTHER: 



0.0 



[x] (b) Human tissues □ (c) Neither 



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

Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycolipoprotein that is expressed in the 
reuna and pineal gland. IRBP mRNA is synthesized by the photoreceptor cells of the retina. We are 
charactenzing the cw-elements regulating IRBP expression, using a transient transfection assay and transgenic 
I^o^t; '^"^ ^^ ^° conserved areas of sequence in the 5'-flanking regions of the bovine, human, and mouse 
IRBP genes, one from -1 to -350 and another at -1200 to -1410. Tlie 5'-flanking region is necessary for 
expression of IRBP in transient transfection assays in Y79 retinoblastoma cell cultures. In transgenic mice 
the same region also shows promoter activity in the retina and pineal, demonstrating that tissue specificity is 
engendered within the tested 5'-flanking regions of the gene 



204 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



Project Description 

Additional Personnel 

Eric Wawrousek Ph.D. 



Research Biologist, 
OSD, NEI 



Objectives 

This research is designed to (1) define the cw-acting 
elements and trans-acting factors that regulate inter- 
photoreceptor retinoid-binding protein (IRBP) gene 
expression in a tissue and/or developmentally specific 
manner, (2) down-regulate eye-specific genes by 
exogenously derived genetic elements introduced 
either as antisense/catalytic RNA or antisense DNA 
oligonucleotides, and (3) use antisense technology in 
determining the pathophysiological role of aldose 
reductase in diabetic pathology. In addition to the 
obvious gene therapeutic possibilities, these ap- 
proaches also can be used when total deletion of a 
target gene would be deleterious to the siu^'ival of 
the organism. 

Methods 

These studies use conventional techniques for clon- 
ing and analysis of nucleic acids. Transgenic mice 
have been made using standard techniques. Trans- 
genic rats containing an antisense construct for 
aldose reductase are being produced in collaboration 
with Dr. Robert Hammer. Chloramphenicol acetyl 
transferase (CAT) activity is measiu^ed by an en- 
zyme-linked immunosorbent assay (ELISA) or the 
biphase assay. 

Catalytic RNA/antisense constructs (ribozymes) 
have been used for permanent transfection of cell 
lines that actively transcribe the messenger for IRBP. 
In addition, the transgenic animals produced express 
high levels of ribozymes targeted against endogenous 
IRBP mRNA. IRBP mRNA levels are measured 
quantitatively by techniques based on polymerase 
chain reaction (PCR), and by Northern analysis. 

Major Findings 

Gene expression. — Constructions containing pre- 
sumptive elements of the IRBP promoter joined to an 
indicator gene (CAT) were made with both bovine 
and mouse IRBP promoters for study of the expres- 
sion of the IRBP gene. Two sites in the 5'-flanking 
(promoter) region show significant homologies across 
species, and we have made constructions containing 



(1) both conserved blocks, (2) only one of the two 
blocks, or (3) neither blocks. These constructions 
were tested in several systems, including retinoblas- 
toma cells (Y79 and WERI), frog oocytes, mixed 
pinealocyte primary cultures, transformed pinealo- 
cytes, and normal mouse fibroblasts. There is 
promoter activity in Y79 cells transfected with the 
IRBP promoter-CAT constructs containing both 
coaserved blocks of sequence. 

This is the first report of transfection of any 
retinoblastoma cell line yielding successful transient 
expression. In each block there is gel-shift experi- 
mental evidence for the binding of rron^-acting 
factors confirmed in the proximal upstream area by 
DNAse footprinting experiments (collaboration with 
Drs. John Nickerson and Jing-Sheng Si). Southwest- 
em blot analysis reveals a 120,(KK)-MW protein that 
binds to the -300 region of the promoter. This 
binding activity is not unique to the retina, being 
present also in the heart, kidney, and lung; however, 
it is not ubiquitous. 

DNA methylation. — DNA methylation is known 
to play a role in the regulation of gene expression. 
Experiments were done to determine the methylation 
state of the IRBP promoter in various tissues. DNA 
isolated from different tissues was digested with 
either Msp I or Hpa II and size-fractionated on 
agarose gels. Msp I and Hpa II are isoschizomers, 
but Msp I will digest the sequence when the 3' 
cytosine is methylated, whereas Hpa II will not. 
Southern blots of mouse tail, liver, and retina DNA 
were probed with a labeled 1.6-kb piece of the 
mouse IRBP promoter. The autoradiographs show 
that the IRBP promoter is hypomethylated in the 
retina but not in the liver or the tail. This indicates 
that DNA methylation may somehow be involved in 
the tissue-specific regulation of IRBP gene expres- 
sion. 

Downregulation of gene expression. — The effect 
of site specificity and varying complement length on 
ribozyme activity in vitro has been studied using in 
vitro partial duplex transcription, cloned ribozyme 
templates, and substrate fragments. Ribozyme activ- 
ity can be "tuned" in vitro by varying complement 
length. This tuning is unique and target site specific. 

We have developed ribozymes, targeted against 
different sites in the IRBP mRNA, which have high 
in vitro activity. These ribosymes have been used to 
generate transgenic mice that express these ribo- 
zymes in ocular and other tissues. Preliminary data 



205 



Laboraton ot Retinal Cell and Molecular Biology 



NEI Annual Report— FY 1993 



show that there are apparently significant differences 
in the embryonic survival and tissue-specific expres- 
sion of transgene and IRBP mRNA in these different 
constructs. 

We are also using mouse retinoblastoma cell lines 
derived from mice transfected with SV-40 large T 
antigen. We have characterized these lines in terms 
of photoreceptor-specific gene expression; they also 
express high levels of IRBP mRNA. 

Sequence analysis of the mouse IRBP genome. — 
Sequencing the genomic clones encoding the mouse 
IRBP gene has shown that the mouse IRBP gene is 
similar in the coding regions to both human and 
bovine genes. They differ, however, in that the 
mouse fourth exon contains a 3'-untranslated region 
that is intermediate in length (1.0 kb) between bovine 
(2.4 kb) and human (0.7 kb) orthologs. We are 
examining the sequence to determine alternative 
splice sites that may explain the imique appearance 
of two IRBP mRNA-size classes, as well as the 
difference between the forms of uveitis in rat and 
mouse species. 

Significance to Biomedical Research and the 
Program of the Institute 

Elucidation of the gene sequences of IRBP is funda- 
mental to understanding normal retina development 
and function. Fmdings from the transgenic mice 
carrying the IRBP ribozyme construct will yield 
much useful information on the role of IRBP in 
development, as well as the function of the retina 
during relative IRBP deficiency. 

Proposed Course 

We have finished the major structural studies on the 
IRBP gene. With this foundation of information and 
battery of cloned genes, we have begun to study the 
regulation of IRBP gene expression. Related ques- 
tions about the consequences of abnormal or absent 
IRBP function can be investigated in transgenic mice 
and in vitro systems. 

Gene expression.— A deletion series of IRBP- 
promoter plasmids has been made, and preliminary 
experiments indicate that both the distal and proxi- 
mal conserved sequences are important for expres- 
sion of the IRBP gene in Y79 cells. However, fewer 



than 205 bases of the 5'-flanking region are needed 
for basal promoter activity in the Y79 cells. Some 
of these constructions have been injected into fertil- 
ized mouse eggs, and offspring are being examined 
for the expression of the constructions. We will 
compare the expression of these constructions in 
transgenic animals and Y79 cells under different 
culture conditions. Preliminary studies show that the 
transgene is active in development as early as embry- 
onic Day 9, but high levels of expression coincide 
with the beginning of outer segment elongation. 
Steady state adult levels are not reached until about 
postnatal Day 30. 

In future studies, we will examine in other trans- 
genic mouse lines gene expression in both the retina 
and several other tissues during development. We 
will characterize the cw-acting DNA sequences that 
bind proteins in the promoter region by making 
alterations to these sequences. We plan to isolate the 
proteins that bind to these elements by screening 
retina cDNA expression libraries by the established 
Southwestern blotting procedure. Preliminary 
screenings have yielded two potential clones. 

Downregulation of gene expression. — Mouse lines 
containing the IRBP ribozyme constructs are being 
analyzed concurrently with ocular histology and 
electrophysiological studies to assess the role of 
IRBP deficiency in ocular pathology. Transgenic 
rats expressing antisense for aldose reductase are 
currently being analyzed. A downregulation of 
aldose reductase in these animals should yield 
delayed galactose-induced cataractogenesis and 
resistance to diabetes-induced histopathology, con- 
firming the importance of AR in these pathologic 
states. 

NEI Research Program 

Retinal Diseases — Photoreceptors and Retinal Pig- 
ment Epithelium 

Publications 

Humayun M, Bernstein SL, Gould HB, Chavis RM: 
Orbital childhood acute lymphoblastic leukemia 
as the initial presentation. J Pediatr Ophthalmol 
Strabismus 29:252-255, 1992. 



206 



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



PROJECT NUMBER 



ZOl EY 00124-13 LRCMB 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



TITLE OF PROJECT (BO characters or less. Title must fit art arte Ime between the borders.) 

Metabolism 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: Robert Waldbillig 

Bruce Pfeffer 

Joyce Tombran-Tink 

Stephen Gaudet 

S. Patricia Becerra 
Timothy Schoen 



Ph.D. 


Expert 


LRCMB, NEI 


Ph.D. 


Senior Staff Fellow 


LRCMB, NEI 


Ph.D. 


Staff Fellow 


LRCMB, NEI 


Ph.D. 


Staff Fellow 


LRCMB, NEI 


Ph.D. 


Visiting Scientist 


LRCMB, NEI 


B.S. 


Biologist 


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, MP 20892 



TOTAL STAFF YEARS: 



6.5 



PROFESSIONAL: 



OTHER: 



5.5 



1.0 



CHECK APPROPRIATE BOX(ES) 

r~| (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.) 

Studies are focused on an understanding of the molecular biology and molecular genetics of the retina and 
hereditary retinal degenerations. The retina and pigment epithelium are neuroepithelial tissues that work in 
close cooperation. Specific growth and differentiating factors found in the eye guide development and 
interactions of individual ocular tissues to form a functional visual system. For example, ocular tissues 
synthesize a number of growth factors. There now appear to be several systems that could self-regulate 
growth and metabolic activity in the retinal pigment epithelium and that could be involved in eye diseases. 
In this regard, we have cloned and characterized a unique differentiating protein secreted from fetal human 
pigment epithelial cells, called pigment-epithelium-derived factor, that is neurotrophic to cultured human 
retinoblastoma cells and may affect neural retinal development in vivo. This protein maps to chromosome 17p, 
where there is a cluster of cancer-related genes. It is a prime candidate in the hereditary retinal dystrophy 
observed in the Royal College of Surgeons rat 



207 



PHS 6040 (Rev. 5/92) 



Laborator> of Retinal Cell and Molecular Biolo}!:^ 



NEI Annual Report — FY 1993 



Project Description 

Objectives 

Our objective is to obtain a better understanding of 
the molecular biology and molecular genetics of 
ocuJar tissues in health and disease. Study of growth 
and differentiation factors, be they protein (e.g., 
pigment epithelium-derived factor fPEDn) or poly- 
p)eptide (e.g., insulin-like growth factor [IGF]-!), is 
critical in obtaining a view of the events that control 
the early development of the eye and in maintaining 
normal function in the adult. 

Methods 

Molecular biological, genetic, and immunocytochem- 
ical techniques are used. Tissue culture is used to 
grow cells. In particular, the human retinoblastoma 
cell line, Y79, is used as a test system for differenti- 
ating agents. 

Major Findings 

Hereditary diseases often occur in the presence of 
genes important in cell division and differentiation. 
PEDF seems to be such a gene product. It is se- 
creted by cultured fetal human pigment epitiielial 
cells and appears to be present in the normal adult 
interphotoreceptor matrix. The protein migrates at 
approximately 54 kD on SDS-polyacrylamide gels. 
PEDF causes marked differentiation of human Y79 
retinoblastoma cells in cultiire. This differentiation 
is charaaerized by an extensive elongation of neu- 
rite-like processes and a gatiiering of cells into 
"rosette-like" aggregates. Immunocytochemisti7 
shows that the expression of specific neuronal 
markers also is enhanced. Thus, PEDF is a unique 
protein, synthesized and secreted by retinal pigment 
epithelial cells, tiiat could direct early development, 
even early in embryogenesis. It may be that PEDF 
also is present after the important developmental 
period and may help to maintain retinal cell viability 
in the adult retina. 

We have cloned tiie cDNA for tiie PEDF gene, 
and have determined tiiat the protein is a member of 
tiie SERPIN (serine protease inhibitor) superfamily of 
genes. Some members of tiiis family are known to 
promote cellular differentiation, making it more 
probable that PEDF has a major, similar role in tiie 
retina. Using fluorescent in situ hybridization, 
polymerase chain reaction, and Soutiiern blottin", we 



have localized the PEDF gene to the short arm of 
human chromosome 17. Through analysis of somatic 
cell hybrids containing only specific regions of 17p 
and 17q, we have further pinpointed PEDF to 17pl3- 
.1. It is important that PEDF colocalizes to the 
chromosomal area that contains the Li-Fraumeni 
cancer gene and a number of yet unknown cancer 
genes. Thus, PEDF may be part of an important 
cluster of genes involved in cellular proliferation and 
cancer as well as a prime candidate gene in the 
retinal dystrophy in the Royal College of Surgeons 
rat. The recombinant protein, which has now been 
expressed in Escherichia coli cells, has been shown 
to be an active neurotrophic agent. The availability 
of relatively large amounts of recombinant PEDF 
should allow for more direct studies on its r61e(s) in 
ocular development and disease. 

In parallel work, we have evidence implicating 
IGF-1 in visual development. In the eye, IGF-1 
seems to participate in the attainment of overall size 
of the eye and in the function of individual ocular 
tissues and cell types. Specific IGF-binding proteins 
(IGFBPs) are known to control the bioavailability of 
the IGFs and thus are important regulators of IGF 
activity in health and disease. The vitreous and 
several ocular tissues contain very high levels of 
IGFBPs that are not derived from extraocular 
sources. The ciliary body is the probable source of 
synthesis of at least one of the vitreal binding pro- 
teins (BPs), specifically IGFBP2. We believe that 
the ciliary body probably secretes the BP into the 
vitreous, where it could be a major factor in regulat- 
ing developmental programs in the eye. Interesting- 
ly, the cornea exhibits exceptionally high amounts of 
IGFBP activity. Its role in corneal metabolism is yet 
unknown but, because of their growth-regulating 
potential, BPs could be involved in important pro- 
cesses such as wound healing and corneal complica- 
tions of diabetes. 

Significance to Biomedical Research and the 
Program of the Institute 

Determining the genes tiiat conti-ol normal ocular 
growth, differentiation, and function and studying 
tiiem on molecular biological and molecular genetic 
levels will aid us in understanding eye diseases, 
especially tiiose of a hereditary, early developmental 
nature. With such knowledge, we can apply rational 
metiiods of gene tiierapy to ocular diseases. 



208 



IVEI Annual Report— FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



Proposed Course 

The molecular biology and molecular genetics of 
ocular development will be further examined. We 
will investigate the factors that affect normal and 
abnormal growth. Examination and analysis of the 
full PEDF gene will help to elucidate its presumptive 
role(s) in retinal development The recombinant 
PEDF protein will be used to elucidate the role of 
the novel new protein in retinal disease processes. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and Other 
Inherited Disorders 

Publications 

Becerra SP, Palmer I, Kumar A, Steele F, Shiloach 
J, Notario V, Chader GJ: Overexpression of fetal 
human pigment epithelium-derived factor in 
Escherichia coli: A functionally active neuro- 
trophic factor. J Biol Chem, 268:23148-23156, 
1993. 

Boje KM, Skolnick P, Raber J, Fletcher RT, Chader 
GJ: Strychrine-insensitive glycine receptors in 
embryonic chick retin: characteristics and 
modulation of NMDA neurotoxicity. Neurochem 
Int 20:473-486, 1992. 



Gaudet SJ, Chader GJ: Partial purification and 
characterization of arylamine-N-acetyltransferase 
in bovine retina. Curr Eye Res 11:1185-1192, 
1992. 

Gaudet SJ, Hayden BJ, Chader GJ, Namboodiri MA: 
Differentia] regulation of arylamine and arylalkyl- 
amine N-acetyltransferases in human retinoblas- 
toma (Y-79) cells. Neurochem Int 22:271-275, 
1993. 

Schoen TJ, Beebe DC, Clemmons DR, Chader GJ, 
Waldbillig RJ: Local synthesis and develop- 
mental regulation of avian vitreal insulin-like 
growth factor-binding proteins: A model for 
independent regulation in extravascular and 
vascular compartments. Endocrinology 131:2846- 
2854, 1992. 

Steele FR, Chader GJ, Johnson LV, Tombran-Tink J: 
Pigment epithelium-derived factor (PEDF): 
Neurotrophic activity and identification as a 
unique member of the serine protease inhibitor 
(SERPIN) gene family. Proc Natl Acad Sci USA 
90:1526-1530, 1993. 

Tombran-Tink J, Li A, Johnson MA, Johnson LV, 
Chader GJ: Neurotrophic activity of interphotore- 
ceptor matrix on human Y79 retinoblastoma cells. 
J Comp Neurol 317:175-186, 1992. 



209 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



PROJECT NUMBER 



ZOl EY 00148-20 LRCMB 



TITLE OF PROJECT (80 characters or lass Title must In on one line between the boraers.) 

Visual Control Mechanisms 



PRINCIPAL INVESTIGATOR (Lisl oiner protessional personnel below me Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI 



Others: Paul Wong 

Tatiana Putilina 
Ignacio Rodriguez 
Jun Li 
Susan Gentleman 

R. Theodore Fletcher 



Ph.D. 
Ph.D. 
Ph.D. 
M.D. 
Ph.D. 
M.S. 



Visiting Fellow 
Visiting Associate 
Staff Fellow 
Visiting Associate 
Biologist 
Chemist 



LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 
LRCMB, NEI 



COOPERATING UNITS (il any) 

School of Vetennao' Medjcme, University of Pennsylvania (G. Aguuie. D.V.M., Ph.D.); Department of Anaiomy. Erasmus Univeisity, 
Roaerdani, The Netherkmds (S. Sanyal. Ph.D.); Department of Zoology. University of Lund, Lund, Sweden (T- van Veen, Ph.D.); Instituto 
Nazionale per la Ricera sul Cancro, Genova, Italy (A. Albmi. Ph.D., D. Noonan, Ph.D.) 



LAB/BRANCH ' 

Laboratory of Retinal Cell and Molecular Biolopy 

SECTION '^^- 



Section on Gene Regulation 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MP 20892 



I TOTAL STAFF YEARS: 



6.5 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (al) Minors 
□ (a2) Interviews 



PROFESSIONAL: 



5.5 



OTHER: 



1.0 



[x] (b) Human tissues □ (c) Neither 



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

hJTw'th.T.If"'"^^- °^.'^' '''^°' '^'P'"'^ °" knowledge of normal complements of tissue-specific genes and 
how they change m disease, we are studying the expression of specific gene products rdated to several 

redS^f^o '?'• ''.r'^f "^'^'^^'^ '^'' '^"'^^ diseases of the retina'such^ etTbl^ma^r 
retmitis pigmentosa will result We have developed new techniques to clone and sequence retinH^dfic 

S : S' ^^^7, ^' ^" '"^ 'r' "^^ '^'''^' "^ ™P^^^ '^^^''^^ matrix p"et"^5 
S^fi'c^lv f V S H r"'"''^!' ''"^ retinoblastoma cell growth and promote differentiation. 



210 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



Project Description 

Objectives 

Normal expression of genes in the retinal photorecep- 
tor neuron is crucial to visual function in the adult. 
Thus, the factors that code for normal gene control 
and expression in himian retina and in animal models 
of retinal degeneration are of primary interest. We 
also have mounted a major effort to develop new 
molecular biological techniques such that unique 
retinal and retinal pigment epithelial genes can be 
identified, cloned, and sequenced for ultimate use in 
screening human populations with inherited diseases 
of the visual system. 

Methods 

Standard molecular biological, biochemical, and 
neurochemical techniques are employed. Histochem- 
ical techniques are used when necessary. 

Major Findings 

1. Laminin is a ubiquitous extracellular matrix 
protein that has profound effects on a variety of cell 
types. For example, both gene and protein expres- 
sion in culnired human Y79 retinoblastoma cells are 
switched from a photoreceptor to a conventional 
neuronal pathway by addition of this basement 
membrane glycoprotein in culture. Unlike other cell 
systems where laminin influences differentiation, 
Y79 cells cannot attach to or chemotactically respond 
to laminin. Cyclic AMP (cAMP) is also an intracel- 
lular messenger that can influence differentiation in 
several cell types. Using cultured human retinoblas- 
toma cells as a model system, we have found both 
laminin and cAMP to have major positive influences 
on photoreceptor differentiation. 

2. We are interested in developing new molecular 
biological techniques that will allow for more effi- 
cient identification of highly expressed genes of the 
retina-pigment epithelium complex. Each tissue of 
the body expresses a unique complement of genes 
that are transcribed 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 process can take place. 
Similarly, it is often a genetic defect in these tissue- 
specific genes that results in a hereditary degenera- 
tion such as retinitis pigmentosa We have devel- 
oped and are using new methods for rapid polymer- 



ase chain reaction-based construction of specifically 
enriched libraries from very small retinal samples. 
This is especially important because tissue samples 
are limited for studying early development and rare 
pathology samples. An important methodological 
advance involves subtractive cloning on an immobi- 
lizing base. We are now applying these techniques 
to the study of apoptosis (i.e., programmed cell 
death) in the retina and to the elucidation of fatty 
acid-binding proteins in normal and degenerating 
retinas. In apoptosis, in particular, it now appears 
that programmed cell death may be a common 
mechanism by which many hereditary defects initiate 
photoreceptor cell death. 

Significance of Biomedical Research and the 
Program of the Institute 

To control a hereditary disease process in a tissue 
and to reverse it through gene therapy, one must 
identify the normal complement of unique genes 
expressed in that tissue. This is especially true in an 
early degenerative process (e.g., retinitis pigmentosa) 
and in other hereditary diseases, such as retinoblas- 
toma, in which abnormal changes are subtie and can 
be masked by normal developmental switches in 
gene expression. Thus, studying apoptosis and 
similar processes in the retina will lead to better 
methods for gene ther^y in the neural retina 

Proposed Course 

Molecular biological and developmental control 
mechanisms in the retina and pigment epithelium 
will continue. In particular, we will investigate gene 
expression in normal retinas and in retinas affected 
with specific genetic diseases. Apoptosis will 
continue to be a focus, since futiu-e gene therapy in 
retinal degenerations may depend on understanding 
how to prevent death of the photoreceptor neuron. 

NEI Research Program 

Retinal Diseases — Retinitis Pigmentosa and Other 
Inherited Disorders 

Publications 

Albini A, Melchiori A, Garofalo A, Noonan DM, 
Basolo F, Taraboletti G, Chader GJ, Gavazzi R: 
Matrigel promotes retinoblastoma cell growth in 
vitro and in vivo. Int J Cancer 52:234-240, 
1992. 



211 



Laboraton of Retinal Cell and Molecular Biology 



NEI Annual Report— FY 1993 



Hooks JJ, Robbins S, Wiggen B, Chader G, Detrick 
B: Can viruses trigger retinal degenerative 
processes? in Hollyfield JG, Anderson RE, LaVail 
MM (eds): Progress in Clinical Biological 
Research, Degenerative Retinal Disorders: Clini- 
cal and Laboratory' Investigations, in press. 

Kutty G, Duncan T, Nickerson J, Si JS, van Veen T, 
Chader GJ, Wiggen B: Light deprivation pro- 
foundly affects gene expression of interphotore- 
ceptor retinoid-binding protein in the developing 
mouse eye. Exp Eye Res, in press. 

Pepperberg DR, Okajima TI, Wiggert B, Ripps H, 
Crouch RK, Chader GJ: Interphotoreceptor reti- 



noid-binding protein (IRBP) — oiolecular-biology 
and physiological role in the visual cycle of 
rhodopsin. Mol Neurobiol 7:61-85, 1993. 

Putilina T, Smith S, Gentleman S, Chader GJ: Rapid 
PCR-based construction of specifically enriched 
libraries firom small retina samples. Exp Eye Res 
54:825-826, 1992. 

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 chromo- 
some 8. Am J Human Genet, in press. 



212 



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



PROJECT NUMBER 



ZOl EY 00260-04 LRCMB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Molecular Biology of Outer Retina-Specific Proteins 



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

PI: T. Michael Redmond Ph.D. Research Biologist LRCMB, hfEI 



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, MP 20892 



TOTAL STAFF YEARS: 



2.0 



PROFESSIONAL: 



2.0 



OTHER: 



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 are characterizing RPE65, a developmentally regulated, conserved 65- 
kD RPE-specific microsomal membrane-associated protein. We have cloned the cDNA for RPE65 and found 
that it encodes a novel protein. This protein does not have any predicted transmembrane segments, yet it has 
a strong affinity for phospholipids, which may be related to its function. The cDNA sequence is being used 
to overexpress RPE65 protein for functional studies. The potential role of the protein in inducing uveitis also 
will be studied using recombinant protein. 

The lack of translation of RPE65 mRNA in cultured RPE cells is being investigated as a possible mechanism 
of |X)Sttranscriptional regulation that may have a bearing on the RPE-retina relationship as well as on RPE 
transplantation studies. We have isolated a full-lengtJi genomic clone for RPE65. It is at least 22 kb in 
length. We have used the cDNA and genomic sequences to localize the human gene for RPE65 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-invoIved disease. 



213 



PHS 6040 (Rev. 5/92) 



Laboratory of Retinal Cell and Molecular Biology 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

The retinal pigment epithelium (RPE) and the photo- 
receptor cell layer of the neural retina form a func- 
tionally and developmentally interdependent com- 
plex. Dysfunction of the RPE, accordingly, is 
deleterious to the photoreceptors and, hence, to 
vision itself. Despite these important considerations, 
little is known about the RPE at the molecular level. 
In this laboratory, we are cloning proteins specific- 
ally or preferentially expressed in the RPE with the 
aim of understanding mechanisms important to the 
RPE. Our major emphasis is on a 65-kD protein that 
we have named RPE65. We also are studying other 
RPE-expressed proteins. 

Methods 

Molecular cloning and biochemical and protein 
chemistry techniques are employed in this study. In 
addition, we are performing automated fluorescent 
DNA sequencing and gene mapping. 

Major Findings 

1. RPE65 is a developmentally regulated, mem- 
brane-associated, nonglycosylated 65-kD protein 
restricted to and conserved in vertebrate RPE. It is 
the major protein of the RPE microsomal fraction. 
This protein displays a calcium-independent affinity 
for phospholipids. 

2. We have cloned a composite 3,1 15-bp cDNA 
for this protein and have shown it to encode a novel 
protein of 533 aa that matches exactly the authentic 
protein sequences from peptide fragments of RPE65. 
Recombinant protein expressed in Escherichia coli 
has the same molecular weight as native RPE65 and 
is recognized by the RPE9 monoclonal antibody. 

3. mRNA for the protein, which is restricted to 
RPE, is abundant in primary cultures of RPE. 
However, these cultures do not express the protein, 
suggesting that the message is posttranscriptionally 
regulated in vitro. 

4. We have isolated a human genomic clone for 
RPE65. It is at least 22 kb in length. We are now 
mapping and sequencing it 

5. We have localized the gene for the RPE65 to 
human chromosome lp31 and to the far distal end of 



mouse chromosome 3. Neither of these loci matches 
tliat of a known ocular disease or phenotype. 

Significance to Biomedical Research and the 
Program of the Institute 

TTie RPE is poorly characterized at the molecular 
level, despite its pivotal role in the maintenance of 
photoreceptor function and, hence, in vision itself. 
We have identified RPE65 as a conserved, RPE- 
specific molecule that is developmentally expressed. 
cDNA sequencing demonstrates that it is a novel 
protein. The function of this protein, while not yet 
clear, may be related to its affinity for phospholipids. 
Elucidation of the basis for its posttranscriptional 
regulation in vitro may have significant bearing on 
the culture of RPE cells. This has some clinical 
significance because RPE cell transplantation is 
receiving much attention as a possible mode of 
intervention 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 its mouse chromosomal locations match 
those of any mapped disease loci. As more disease 
loci are matched, this may change. Again, in view 
of its RPE-specific expression, elucidation of its gene 
structure may uncover RPE-specific regulatory 
elements. Finally, in view of the involvement of the 
RPE in uveitis, it is possible that RPE65 is uveito- 
genic. Now that we have cloned the cDNA, it will 
be possible to overexpress the protein to test this 
hypothesis. 

Proposed Course 

1 . The basis for the posttranscriptional regulation 
of RPE65 will be investigated. 

2. The structure of the human RPE65 gene will 
be studied. The gene will be sequenced, and its 
regulatory regions will be analyzed. The mouse gene 
RPE65 will be compared with that of the human. 

3. RPE65 will be tested as a possible RPE auto- 
antigen. RPE65 protein will be overexpressed for 
this purpose. 

4. Elucidation of the structure and function of 
RPE65 will continue. This will involve use of a 
variety of approaches. 

5. Other RPE proteins will be cloned. 



214 



NEI Annual Report — ^FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



NEI Research Program 

Retina] Diseases — Photoreceptors and Retinal Pig- 
ment Epitlielium 

Publications 

Hamel CP, Tsilou E, Harris E, Pfeffer BA, Hooks JJ, 
Detrick B, Redmond TM: A developmentally 
regulated microsomal protein specific for the 
pigment epithelium of the vertebrate retina. 
J Neurosci Res 34:414-425, 1993. 



Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick 
B, Redmond TM: Molecular cloning and expres- 
sion of RPE65, a novel retinal pigment epitheli- 
um-specific microsomal protein that is post- 
transcriptionally regulated in vitro. J Biol Chem 
268:15751-15757, 1993. 

Redmond TM, Tsilou E, Pfeffer BA, Detrick B, 
Hooks JJ, Hamel CP: Cloning and expression of 
a novel retinal pigment epithelium-specific 65 
kDa microsomal protein. Invest Ophthalmol Vis 
Sci 34(suppl):982, 1993. 



215 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



PROJECT NUMBER 



ZOl EY 00132-12 LRCMB 



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

Molecular Biology of Phototransduction 



PRINCIPAL INVESTIGATOR (List otner prolessionai personnel below tne Principal Investigator.) (Name, title, laboratory, and institute atlilialion) 

PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI 

Molecular Biology 



Others: Takanobu Kikuchi 



Ph.D. 



Visiting Associate 



LRCMB, NEI 



COOPERATING UNITS (il 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 Cel l and Molecular Biology 



SECTION 



Section on Molecular Biology 



INSTITUTE AND LOCATION 

NEI, NTH. Bethesda, MD 20892 



TOTAL STAFF YEARS: 

1.5 

CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 
n (a1) Minors 
□ (a2) Interviews 



PROFESSIONAL: 



1.5 



OTHER: 



0.0 



n (b) Human tissues jx] (c) Neither 



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

We have characterized the S-antigen genes from human and mouse and the 33-K protein genes from mouse 
and human. ITie S-antigen genes were approximately 50 kbp in length, contained 16 exons and 15 introns 
and comprised 97% mtron and 3% exon. The 5'-flanking regions of the genes, approximately 1.5 kbp bn^ 
had no known regulatory elements for transcription, such as TATA, GC, or CCAAT boxes. 

Regulatory sequences and nuclear factors governing ti.ssue-restricted expression of the mouse airestin gene 

direct low evels of retina-specific gene expression, sequences extending upstream to position -209 suorxm 
Sn the f ^Tr^^'" " "^^ "'"^' " "^" " '^^^^^^'^'^ expression in'the lens, pS^eTZl^TS 
h^ ?mer TGACCT thTr^'f''" '''l^' -'''' ' "^^°" "^'^^ ^'^^^ ^' ^-'^ repeats of Te 
a^d Bd3 thm^oh n r P™"""'"' ^'°'^' ^'' "PP^^^^'y ^^tina-specific nuclear factors-Bpl, Bp2, 

auid Bp3-through overiapping sequences centered between positions -25 and -15 Bpl and Bd3 also 

P^cffrjnef m' "''^'' T"" '°""' " "^^ ^"™°^" ^^^^°°^ °f --^^ «*- vertebrrt pltoretptor- 
specific genes. Moreover, the consensus binding site for Bpl, designated PCEl is identical to RCSl an 
element known to play a criUcal role in eliciting photoreceptor-s^ific gene express^nln dS.^ 
2'J^ogaster. The results suggest that PCEl and RCSl are function^ly, as well as s^^crally S2 
that, despite marked differences in the fly and venebrate visual system, th transcriplnTiSL^Tnv^I^d 
in photoreceptor-specific gene expression has been strongly conserved evolutioT^^y ™"'^"''^ '"'"'^^^ 



216 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



Project Description 

Objectives 

The objectives of this project are (1) to understand 
the basic mechanism of phototransduction in the 
retina and (2) 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 being used include protein purification 
and RNA and DNA isolation, characterization, and 
sequence determination. Various recombinant DNA 
techniques also being used include a Baculovirus 
expression vector system, synthesis of point mutation 
clones, characterization of promoters, and transgenic 
animals. We also have synthesized and used purified 
oligopeptides and oligonucleotides. 

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 small- 
est exon encodes for three amino acids. 

2. The intron-exon map sequence of the moase 
S-Ag gene has been well conserved. Approximately 
97% of the S-Ag gene is intron and 3% is exon. 

3. The human and mouse S-Ag cDNAs have 
been subcloned into two expression vectors and have 
been expressed. The products of S-Ag cDNA were 
purified by column chromatography and prepared for 
crystallization. 

4. The 5'-flanking sequence of the human and 
mouse S-Ag genes were determined. Promoter 
activity was demonstrated in the in vivo and in vitro 
transcriptional assays. 

5. Although the S-Ag promoter sequences are 
highly conserved between human and mouse, pro- 
moter activity was found at different 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 chloroamphenicol acetyl- 
transferase (CAT) gene, and that gene was intro- 
duced 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-specific enhancer and promoter activity. 

7. The opsin promoter was fused with a diph- 
theria toxin gene, and that fusion gene was intro- 
duced into transgenic mice, which subsequentiy lost 
only the photoreceptor rod cell layer. 

8. Several cDNAs of Shuzin, a retinal photore- 
ceptor protein, were isolated from human and cow 
retinal cDNA libraries (k-gtll), and the entire DNA 
sequences were determined. The deduced protein 
has sequence similarity with TFIID. Its gene also 
was isolated from a genomic library and its DNA 
sequence was determined. It is composed of two 
introns and three exons. 

9. Two genes of 33-kD ROS-specific proteins 
have been isolated from the retinal libraries of human 
and mouse, and the entire DNA sequence of these 
genes have been determined. They have four exons 
and three introns. 

10. The proximal promoter sequence positions 
-38 to -1-304 are sufficient to direct low levels of 
retina-specific gene expressioiL 

11. The proximal promoter binds three retinal 
specific nuclear factors (Bpl, Bp2, and Bp3) through 
overlapping sequences centered between positions 
-25 and -15. 

12. The distal promoter sequence positions -205 
to -185, a region which contains two direct repeats 
of the hexamer, TGACCT. 

13. We found a consensus retinal photoreceptor- 
specific site (PCEl). 

14. The transcriptional machinery involved in 
photoreceptor-specific gene expression has been 
strongly evolutionarily conserved. 

Significance to Biomedical Research and the 
Program of the Institute 

Eyes have remarkable properties in functioning 
efficientiy over a wide range of illuminations. Rod 
cells, having photosensitive rhodopsin, are more 
sensitive to dim light; they adapt in the dark to 
increase their sensitivity. However, rod cells cease 
tiieir sensitive phototransduction in bright light. In 
contrast, cone cells do not operate in dim light but 
are operative in bright light Rhodopsin, transducin, 
phosphodiesterase, rhodopsin kinase, and S-Ag have 
been known to be associated with the phototransduc- 
tion cascade. Rhodopsin kinase and S-Ag are 



217 



Laboratory of Retinal Cell and Molecular Biologj' 



NEI Annual Report — FY 1993 



considered to be the imponant proteins for light- 
dependent modulation of phototransduction. To 
understand this light-dependent modulatory mecha- 
nism in rod outer segments, we have characterized 
S-Ag, Shuzin, and 33K protein as well as their 
genes. Interestingly, other signal transduction 
systems have cascades similar to that of phototrans- 
duction (one of the best characterized receptor-medi- 
ated signaJ transduction processes). In the photo- 
transduction cascade, the shutoff mechanism appears 
to be modulated by the phosphorylation and dephos- 
phorylation of rhodopsin. Studying this modulation 
mechanism is important for understanding photo- 
transduction as well as for understanding signal 
transduction in general. In addition, we think that 
the night blindness of vision may in part be associ- 
ated with light adaptation. 

Proposed Course 

The following studies are in progress or have been 
proposed for Fiscal Year 1993: 

1. Identification of the S-Ag promoter using 
transgenic animals. 

2. Identification of m-acting factors of the S-Ag 
and 33K protein promoter. 

3. The knockout of genes of S-Ag and phos- 
ducin. Investigation of a functional role for S-Ag 
and 33K protein, the homologous recombination 
between a mutant gene and a normal gene will be 
induced in ES cell culture. The recombinant ES 
cells will be introduced into a transgenic animal 
system in order to produce a mutant mouse. 



NEI Research Program 

Retinal Diseases — Photoreceptors and Retinal Pig- 
ment Epithelium 

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. 

Danciger M, Kozak CA, Abe T, Shinohara T, Farber 
DB: The gene for retinal rod 33-kDa protein is 
on mouse chromosome 2, near lamb2. Cytogenet 
Cell Genet, 56:202-205, 1991. 

Kikuchi T, Raju K, Breitman ML, Shinohara T: The 
proximal promoter of the mouse arrestin gene 
directs gene expression in photoreceptor cells and 
contains an evolutionarily conserved retinal 
factor-binding site. Mol Cell Biol 13:4400-4408, 
1993. 

Shinohara T, Kikuchi T, Tsuda M, Yamaki K: A 
family of retinal S-antigen (arrestin) and their 
genes: Comparative analyses of human, mouse, 
rat, bovine, and Drosophila. Camp Biochem 
Physiol, 103:505-509, 1992. 

Usukura J, Khoo W, Abe T, Shinohara T, Breitman 
ML: Abnormal development of cone cells in 
transgenic mice ablated of cone rod photoreceptor 
cells. Ann N Y Acad Sci, in press. 

Usukura J, Khoo W, Abe T, Shinohara T, Breitman 
M: Cone cells fail to develop normally in trans- 
genic mice ablated of rod photoreceptor cells. 
Tissue Cell, in press. 



218 



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



PROJECT NUMBER 



ZOl EY 00250-06 LRCMB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or less. Title must fit on or\e Ime 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 
Siradanahalli Guru 
Shirley Yu 



Ph.D. 
Ph.D. 
B.S. 



Visiting Associate 
Visiting Fellow 
Biologist 



LRCMB, NfEI 
LRCMB, NEI 
LRCMB, NEI 



COOPERATING UNITS (if any) 

Department of Ophthalmology, Miami University, Miami, FL (D. Hamasaki, Ph.D.); Department of Anatomy, 
Nagoya University School of Medicine, Tsuramai, Showa-ku, Nagoya, Japan (Jiro 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: 



3.5 



PROFESSIONAL: 



2.5 



OTHER: 



1.0 



CHECK APPROPRIATE BOX(ES) 

[xj (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.) 

We had previously determined amino acid sequences of liuman, mouse, rat, and bovine retinal S-antigen (S-Ag) and rat 
pineal gland S-Ag. Immunogenic sites and four uveitopathogenic sites of S-Ag also were determined; two immunogenic 
sequences were highly conserved among the species. Many proteins in the National BiomediC£il Research Foundation 
data base have a sequence similar to that of a uveitopathogenic site. We chemically synthesized many peptides, some 
of which induced experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis rats. 
In addition, we found native yeast histone H3 enable of inducing EAU. 

To understand the role in autoimmunity of infectious microorganisms which have cross-reactive antigens, we injected 
Lewis rats with peptide M, together with one of six different killed bacteria, with or without incomplete Freund's adjuvant 
(IF A). The rats injected with IFA developed EAU. To assess the impact of infection by live microorganisms, we 
injected low doses of live Escherichia coli expressing S-Ag and baker's yeast with a cross-reactive antigen into the rats 
several times. The rats injected with either Uve E. coli or live yeast developed EAU. We conclude that infection by 
microorganisms which 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 conducted 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 5 weeks from the date of the first injection. 
The rats injected with a synthetic peptide derived from Salmonella typhimurium protein, which has five amino acid 
residues identical to rat P-crystallin (P-B2), also induced similar damage. Infection by microbes having antigens 
homologous to the lens antigens can induce high levels of auto;intibodies that provoke lens epithelial cell damage. Thus, 
autoimmune insult in lens epitheUal cells may be an etiology of an initial stage of cataractogenesis. Our future research 
will focus more on autoimmunity in lens cataractogenesis. 



219 



PHS 6040 (Rev. 5/92) 



Laboraton of Retinal Cell and Molecular Bioloj^ 



NEI Annual Report — FY 1993 



Project Description 

Objectives 

The objectives of this project are to understand the 
basic etiology of autoimmune inflammation including 
uveitis and to find possible treatments for human 
uveitis. 

Methods 

The conventional methods for analysis of proteins 
and nucleic acids used include the following: protein 
purification; RNA and DNA isolation, characteriza- 
tion, and sequencing; molecular cloning; screening of 
clones; in situ hybridization; immunocytochemistry; 
and chromosome mapping. We also have synthe- 
sized and used oligopeptides and oligonucleotides. 
Bovine, murine, primate, and human materials are 
used. Animal experiments are carried out with 
Lewis rats and monkeys. T-cell response and adop- 
tive transfer are 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, includ- 
ing potato proteinase inhibitor Da, Escherichia coli 
hypothetical protein, hepatitis B virus probable DNA 
polymerase, Moloney murine sarcoma virus gag- 
polyprotein, Moloney murine leukemia virus gag-pol 
polyprotein, 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 Lewds rats; its pathology was similar to 
that of EAU induced by pepUde M or native S-anti- 
gen (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 administration of histone H3 peptide 
suppressed EAU in the Lewis rats. 

5. The suppression of EAU by histone H3 also 
was found in the EAU induced by the S-Ag. Thus, 
the tolerance also cross-reacted with the ''peptide, 
which has molecular mimicry. 

6. The T-lymphocytes obtained from rats immu- 
nized with peptide M or yeast histone H3 transferred 



disease (i.e., EAU) in the naive rats (adoptive 
transfer) when stimulated either with peptide M or 
histone H3. In addition, oral tolerance was adop>- 
tively transferred from rats fed peptide M or histone 
H3 to the naive rats. 

7. Infection by microorganisms which have 
cross-reactive antigens can break immune tolerance 
to a self-antigen and induce inflammatory auto- 
immune 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 worid. For many 
decades, physicians have suspected some types of 
uveitis to be induced by bacterial and viral infec- 
tions; however, there is no clear link between infec- 
tion 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, ctoss- 
reacts with a normal host protein — may play a role 
in autoimmunity. Here we have proposed the idea of 
molecular mimicry and showed evidence that molec- 
ular mimicry may play a role in the pathogenesis of 
EAU. In addition, we have provided evidence that 
infection is a possible cause of autoimmune inflam- 
mation. These fmdings provide an important clue for 
understanding the etiology of autoimmune inflamma- 
tory diseases in humaa 

Proposed Course 

The following studies are in progress or proposed for 
Fiscal Year 1993: 

1 . We will conduct further evaluation of foreign 
proteins similar to S-Ag that induce EAU. 

2. We will characterize peptide M with respect 
to the minimum number of amino acids required for 
induction of EAU. 

3. We will study the induction of EAU in trans- 
genic mice that express foreign proteins in photo- 
receptor cells. 

4. We will further characterize molecular mimic- 
ry and its role in EAU and human uveitis. 

NEI Research Program 

Retina] Diseases— Inflammatory Diseases 



220 



NEI Annual Report— FY 1993 



Laboratory of Retinal Cell and Molecular Biology 



Publications 

Chan CC, Li Q, KiJkuchi T, Shinohara T, Nussenblatt 
RB: Enhancement of S-antigen and its mRNA in 
the irides of uveitic patients. J Autoimmun 5:719- 
732, 1992. 

Eto K, Suzuki S, Singh VK, Shinohara T: Immuni- 
zation with recombinant Escherichia coli express- 
ing retinal S-antigen induced experimental auto- 
immune uveitis (EAU) in Lewis rats. Cell Immu- 
nol 147:203-214, 1993. 

Hamasaki DI, Sato H, Santhanakrishnan S, Shinohara 
T: Correlation between the physiological and 
morphological changes in the experimental auto- 



immune uveitis induced by peptide G of S-anti- 
gen. Exp Eye Res, in press. 

Nityanad S, Singh VK, Shinohara T, Paul AK, Singh 
VK, Agarwal PK, Agarwal SS: Cellular immune 
response of patients with uveitis to peptide M, a 
retinal S-antigen fragment. J Clin Immunol, in 
press. 

Sunil S, Eto K, Singh VK, Shinohara T: Oligopep- 
tides of three to five residues derived fi^om uve- 
itopathogenic sites of retinal S-antigen induce 
experimental autoimmune uveitis (EAU) in Lewis 
rats. Cell Immunol 148:198-207, 1993. 



221 



Laboratory of Sensorimotor Research 



Report of the Chief, Laboratory of Sensorimotor Research 

Robert H. Wurtz, Ph.D. 



One of the most admired human abilities is that 
of skilled motor control — be it hitting a baseball 
in Baltimore or returning a teimis serve on Long 
Island. These abilities are highly sophisticated 
sensory motor tasks; they depend heavily on vision. 
The Laboratory of Sensorimotor Research concen- 
trates on such sensory motor tasks, particularly in 
relation to the visual control of eye movements. Our 
goal is to understand the systems within the brain 
that process visual information and produce these eye 
movements and to understand what happens when 
disease or trauma leads these to fail. While our main 
interests are the systems in humans, we are fortunate 
to have a superb animal model, the Rhesus monkey, 
which allows us to explore not only the exact behav- 
ioral mechanisms related to visual motor behavior 
but also the underlying brain mechanisms controlling 
such behavior. Our investigations are best illustrated 
by a selection from the work of each of the five 
sections within the Laboratory. 



It previously has been shown that humans who 
wear spectacle lenses are able to generate saccades 
that differ in amplitude between the two eyes exactly 
as required by the different magnifications of the 
lenses, and usually it has been assumed that this 
ability results from some neural adaptive mechanism 
that adjusts for this over time. I>r. Miles' group has 
found that such an ad^tation period is not necessary. 
These investigators found that humans immediately 
adjusted the amplitude of the eye movement in ways 
appropriate for the size of the stimulus. They 
hypothesize that it is not adaptation that is control- 
ling binocular alignment of the eyes in this case but 
rather the use of the horizontal disparity in the image 
detected by the visual system. They were able to 
show exactly the same phenomena in the monkey, 
opening the way for extensive quantitative analysis 
of the parameters confrolling these saccadic eye 
movements and the possibility of determining the 
brain mechanisms imderlying this control. 



Section on Oculomotor Control 



Section on Yisuomotor Integration 



One of the most frequent uses of vision for the 
control of eye movement is in the generation of 
rapid or saccadic eye movements — those eye move- 
ments that move the eyes from one part of the field 
to another. Such shifts allow us to look from one 
area of the field of interest to another. Both eyes 
move together to maintain binocular alignment, 
which is critical for good depth vision. Dr. Fred 
Miles and his collaborators are able to measure these 
eye movements with great accuracy in both humans 
and monkeys to determine how we solve a problem 
in generating these saccades (i.e., what happens when 
humans or monkeys are first confronted with images 
that differ in size for the two eyes). Such a differ- 
ence in image size results when spectacle lenses 
cause the two eyes to see images that differ in size 
by several percent for each diopter of difference in 
correction between the eyes. 



Another case illusfrating the strong visual control 
of movement is from the work that my collab- 
orators and I have done on the control of our move- 
ments through the environment and the stabilization 
of our posture. It has been shown in humans that 
motion through the visual field produces a specific 
pattern of large field visual motion, referred to as 
"optic flow." The nature of this large field visual 
motion is thought to provide information about our 
direction of movement through the envirormient. It 
also provides information to control our posture; 
humans sway back and forth substantially more with 
their eyes closed than with their eyes open. 

In the past year we have tested whether monkeys 
use such visual information to control posture by 
training the monkey to stand oh a small platform that 
measures how much the monkey sways and in what 
direction. By projecting onto a screen a pattern of 



225 



Laboratorv of Sensorimotor Research 



NEI Annual Report— FY 1993 



motion simulating the motion that would occur as the 
monkey leaned forward or back or side to side, we 
have been able to measure the monkey's postural 
changes. We have shown that the monkey responds 
to this visual stimulation and that the response is, in 
most respects, similar to that reported for humans. 
This now provides us with the ability to investigate 
further the regions of the brain that we know process 
this type of visual motion and to see whether alter- 
ations of these regions alter the monkey's use of the 
visual stimulation for the control of posture. 



Section on Neural Modeling 

The way neurons convey visual information has 
been studied by Dr. Lance Optican and his 
collaborators over the past several years. While in 
most studies of neurons within the brain the neuronal 
activity has been taken as the total number of action 
potentials or spike discharges emitted in response to 
a visual stimulus. Dr. Optican has shown that more 
information is conveyed if one looks at the temporal 
patterning of the action potentials as well as their 
total number. An understanding of this extra visual 
information may lead to a better understanding of 
visual perception. Dr. Optican and his collaborators 
previously have shown that neurons in a number of 
visual areas (i.e., VI, V2, V3, V4, and the inferior 
temporal cortex) both encode and transmit informa- 
tion about patterns that vary in form, color, bright- 
ness, and duration by a temporal code that represents 
these stimulus-dependent messages. In their present 
experiments. Dr. Optican's group trained monkeys to 
choose a particular stimulus according to whether it 
matched a previously given cue stimulus. They 
found that the temporal pattern of cell discharge 
varied with not only the stimulus falling on the 
receptive field of the cell but also with the nature of 
the cue stimulus. Furthermore, they found that each 
stimulus could be represented as the product of two 
wave forms that were specific for the features paired 
in each stimulus— for example, color and pattern. 
Thus, unique codes for every possible combination of 
visual features are not required. These experiments 
address the major issue in understanding information 
processing within the brain— the code by which this 
information is transmitted. This work clearly shows 
that neurons convey information about visual features 
by using a temporal code. This work may provide a 



key to understanding how the brain processes infor- 
mation to form a visual perception. 



Section on Visual Behavior 

Determining which visual stimulus is of impor- 
tance for visual processing is one of the critical 
functions of the visual system. We look at only one 
part of the visual field at a time; the selection of the 
region of the visual field to look at next is the 
function referred to as "selective visual attention." 
Dr. David Lee Robinson has continued to explore the 
neurons in the brain that give evidence of participat- 
ing in this attention process. He and his colleagues 
have conducted experiments on the superior collicu- 
lus to understand its role in visual attention. They 
have discovered that neurons there discharge at the 
appearance of certain visual stimuli and that these 
signals help indicate a change in the direction of 
attention. Other cells located in parts of the collicu- 
lus that are connected to the fovea also respond to 
visual stimuli, and here their activity starts the 
engagement of attention by images on the fovea 
These are the first data to demonstrate a visual 
function of the superior colliculus that is not related 
to eye movements. 



Section on Neuro-Ophthalmologic 
Mechanisms 

One of our most effective methods of studying 
the systems within the brain is the alteration of 
that system by electiical stimulation or chemical 
injection (as in Dr. Robinson's experiments). Such 
modification allows us to test hypotiieses about the 
contribution of a given set of cells within the system 
to the brain's function in controlling behavior. 
Dr. Michael Goldberg and his collaborators recentiy 
have been able to use such a technique, not only in 
the monkey model of the control of eye movements 
but also in humans. Dr. Goldberg previously had 
shown the characteristics of cells in a part of die 
frontal cortex of tiie monkey referred to as tiie firontal 
eye fields. Recent work using PET scanning identi- 
fied the approximate region in the human where such 
frontal eye fields are located. By using the technique 
of focal magnetic stimulation, which changes the 



226 



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



PROJECT NUMBER 



ZOl EY 00256-05 LSR 



PERIOD COVERED 

October 1. 1992 to September 30, 1993 



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) 



PI: Lance M. Optican 

Others: John W. McCIurkin 
Arthur V. Hays 
Brad J. Zoltick 
Jennifer A. Zarbock 
Merk Na Chee-Orts 
Marc H. Cohen 



Ph.D. 

Ph.D. 

B.A. 

M.A. 

B.A. 

Ph.D. 

M.S£. 



Chief, Neural Modeling 
Section 
Staff Fellow 
Electronics Engineer 
Computer Programmer 
Electronics Engineer 
Visiting Associate 
Visiting Associate 



LSR, NEI 

LSR, NEI 
LSR, NEI 
LSR, NEI 
LSR, NEI 
LSR, NEI 
LSR, NEI 



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: 



5.6 



PROFESSIONAL: 



4.0 



OTHER: 



1.6 



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 neuroas 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 carried by a temporal code. Now we have extended those 
studies to show that neurons in the visual cortex (areas VI, V2, V3, and V4) carry information about the form, 
color, luminance, and size 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 
on the neuron's response in a separable way. 



228 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Sensorimotor Research 



electrical activity of the brain in a small region 
below the skull, they have been able to show that 
stimulation of the frontal eye field of humans alters 
saccadic eye movements. Furthermore, they have 
been able to show that the type of alteration depends 
on the time at which the stimulation is given before 
the onset of the saccade. Thus, identification of a 
region in the brain of the monlcey has led to the 
localization, and now the modification, of a similar 
area in human subjects. 



A part of the activity of the Laboratory this year 
involved the move of those laboratories devoted 
primarily to research on monkeys into the new Silvio 
O. Conte Building (Building 49). This move was 
completed by June 22. The new building provides 
superb facilities for nonhuman primates, fine labora- 
tories for investigation, and a few small rooms for 
offices. 



227 



NEI Annual Report— FY 1993 



Laboratory of Sensorimotor Research 



Project Description 

Objectives 

Perception and recognition of complex visual pic- 
tures depend on the normal function of interconnect- 
ed brain regions extending from the retina through 
the inferior temporal cortex. The properties of these 
regions are derived from the function of the single 
neurons within them. Thus, to understand how 
visual perception occurs, we must learn how infor- 
mation is encoded by the neurons in each stage of 
processing. If we could understand this neuronal 
code, it might be possible to distinguish between 
information related to the physical properties of a 
stimulus (e.g., form, luminance, color, and size) and 
information related to its behavioral significance 
(e.g., leading to a reward). 

Individual neurons in all the visual areas smdied 
thus far (retinal ganglion cell fibers; lateral geniculate 
nucleus neurons; pulvinar neurons; cortical neurons 
in visual areas VI, V2, V3, and V4; and inferior 
temporal cortical neurons) encode and transmit 
information about stationary, two-dimensional 
pictures that vary in form, color, brightness, and 
duration. The neurons use a multidimensional 
temporal code to represent and transmit their stimu- 
lus-dependent messages. We now have shown that 
visual neurons convey complex messages about (1) a 
stimulus' physical parameters and (2) its behavioral 
significance. 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, atten- 
tion, and memory. 

Major Findings 

We have developed a new approach to studying 
single neurons in which they are treated as communi- 
cation channels that transmit information about visual 
pictures in their responses. This has allowed us to 
apply methods from signal processing, statistics, 
systems analysis, and information theory to under- 
stand single neurons. 

According to a commonly held view of neuronal 
function, the strength of a neuron's response repre- 
sents 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 informational analysis, we have 
shown that information about different stimulus 
parameters is not confounded but is carried across 
tlie different parts of the multidimensional neuronal 
code. 

In recent experiments, we recorded responses of 
neurons in four visual cortical areas — VI, V2, V3, 
and V4 — of a monkey trained to choose one of three 
parafoveal stimuli on the basis of 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, 
stimuli consisted of either colored bars that were 
isoluminant with the background or black or white 
bars that varied in size. Information about stimulus 
features developed continuously, but not uniformly, 
throughout the time-coiu"se of the neuronal responses. 
Most of the information was encoded in the initial 
50-60 msec of the response. Some neurons also 
encoded a large amount of information in a second 
50-msec interval, beginning 20-30 msec after the 
first 

These results show that neiirons in VI -V4 carry 
information about the color, pattern, contrast, and 
size of stimuli. Rnally, the development of informa- 
tion over time in different areas suggests that temp- 
orally modulated 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, contrast 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 waveforms predicted the 
neuronal responses to stimuli with feature combina- 
tions not used to train the neural net (e.g., novel- 
colored patterns). 

Feature waveforms were often similar for all 
neurons within a cortical area. To compare these 
waveforms across cortical areas, we pooled all the 
responses from neurons within each area. Waveforms 
encoding pattern were strikingly similar across all 
areas, irrespective of the behavioral task. Waveforms 
encoding color in the color/pattern task differed 
between cortical areas, but there was a striking 
similarity for waveforms encoding color in the 



229 



Laborator\' of Sensorimotor Research 



NEI Annual Report— FY 1993 



isoluminant-color/orientation paradigm. These resulLs 
suggest that neurons convey information about 
compound visual features by multiplexing feature- 
specific messages together. The invariance of the 
code waveforms suggests that information about a 
stimulus feature is represented similarly in all visual 
areas. 

Significance to Biomedical Research and the 
Program of the Institute 

This project studies how visual information is en- 
coded and transmitted by neurons. Knowledge of 
these fundamental processes is important for under- 
standing deficits of visual processing, such as those 
occurring in amblyopia, and for developing visual 
prosthetic devices to compensate for field defects or 
blindness. 

Proposed Course 

Discovering that the responses of visual system 
neurons are multidimensional led to the discovery 
that information about multiple stimulus features may 
not be confounded by single neurons, a result with 
important, even revolutionary, consequences. We 
now know that a substantial part of the temporal 
modulation 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 properties 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 information 
processing is required. This theory will treat the 
visual system more as a concurrent processing 
system than as a hierarchical cascade of independent 
areas. Both these issues are being pursued. 

In addition, our findings have suggested previous- 
ly unconsidered principles as the basis for interac- 
tions among neurons. To investigate these princi- 
ples, we need to collect and analyze data from 
several simultaneously recorded neurons. Thus, we 
have been developing the apparatus needed to make 
multiple, simultaneous single-neuronal recordings. 
The apparatus should be completed sometime during 
the next year. The simultaneously recorded respons- 
es will be related to each other through use of recent 



extensions to methods of signal identification, which 
should allow us to develop models that will describe, 
relatively rapidly, the roles of single neurons as 
components of larger networks. These studies should 
yield a better understanding of the information 
transmission mechanisms, such as pattern perception 
and recognition, used for cognitive functions. 

Our findings suggest a completely new concep- 
tional framework in which to investigate neuronal 
function. One presumed reason for the huge number 
of single neurons has been the necessity to uncon- 
found stimulus features. However, we propose that 
the simultaneous messages about different features 
can be used as tags, so that the messages which 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, we are exploring this 
hypothesis both experimentally and theoretically. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Processing — 
Visual Processing and Functional Organization 
(Structure and Function of Central Visual Pathways) 

Publications 

Chee-Orts MN, Optican LM: Cluster method for 
analysis of transnutted information in multivariate 
neuronal data. Biol Cybem 69:29-35, 1993. 

Eskandar EN, Optican LM, Richmond BJ: Role of 
inferior temporal neurons in visual memory: II. 
Comparing tempwral waveforms arising fi-om 
vision and memory. J Neurophysiol 68: 
1296-1306, 1992. 

Eskandar EN, Richmond BJ, Optican LM: Role of 
inferior temporal neurons in visual memory: I. 
Temporal encoding of information about visual 
images, recalled images, and behavioral context. 
J Neurophysiol 68:1277-1295, 1992. 

Kapoula Z, Robinson DA, Optican LM: Visually 
induced cross-axis postsaccadic eye drift. J 
Neurophysiol 69: 1 03 1 - 1 043 , 1 993. 

McClurkin JW, Zarbock JA, Optican LM: Temporal 
codes in monkey sQ-iate cortex for colors, patterns 
and memories, in Peters AA, Rockland KS (eds): 
Primary Visual Cortex of Primates, Cerebral 
Cortex. New York, Plenum, 1993, vol 10, in 
press. 



230 



NEI Annual Report— FY 1993 



Laboratory of Siensorimotor Research 



Zee DS, FitzGibbon EJ, Optican LM: Saccade- 
vergence interactions in humans. J Neurophysiol 
68:1624-1641, 1992. 



231 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00049-15 LSR 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



TITLE OF PROJECT (80 charaaars or less Tula must lit on one line between the borders.) 

Cerebral Cortical Mechanisms for Eye Movements and Visual Attention 



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

PI: Michael E. Goldberg M.D. Chief, Neuro-Ophthalmologic LSR, NEI 

Mechanisms Section 



Others: Edmond J. FitzGibbon 
Carol L. Colby 
Suzanne Y. Musil 



M.D. 
Ph.D. 
Ph.D. 



Medical Officer 
Senior Staff Fellow 
Staff Fellow 



LSR, NEI 
LSR, NEI 
LSR, NEI 



COOPERATING UNITS (il any) 

Medical Neurology Branch, National Institute of Neurological Disorders and Stroke 



LAB/BRANCH 



Laboratory of Sensorimotor Research 



SECTION 

Neuro-Ophthalmologic Mechanisms Section 



INSTITUTE AND LOCATION 

NEI. NIH. Bethesda, MD 20892 



TOTAL STAFF YEARS: 



5.3 



PROFESSIONAL: 



4.0 



OTHER: 



1.3 



n (b) Human tissues [x] (c) Neither 



CHECK APPROPRIATE BOX(ES) 

□ (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 

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

Two Unes of inquiry were followed to determine how the cerebral cortex and its efferent regions control eye 
movements and visuospatial attention. 

In one, focal transcranial magnetic stimulation of the human fi-ontal eye field was used to determine the effect 
that frontal eye field activity can have on the generation of saccadic eye movements. Depending on the 
relationship of the exogenous stimulation to the ongoing processes of saccade initiation, such stimulation can 
facilitate or interfere with saccade generation. 

In the other, single neuron recording was used to probe the mechanisms whereby the parietal cortex of the 
monkey achieves spatial accuracy. Neuronal behavior in a double-step task is entirely predicted by presaccadic 
and predictive-shift activity in a single-step task. Tlie activity of parietal neurons is most consistent with their 
specifying a shift of visual attention of particular amplitude and direction. 



232 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Sensorimotor Research 



Project Description 

Objectives 

This section has concentrated on two aspects of the 
physiology and phenomenology of higher visual and 
oculomotor processing in the monkey and man, 
especially as these functions relate to the parietal and 
frontal regions of the cerebral cortex, their afferent 
regions, and their efferent targets. Previous work in 
this laboratory has shown that neurons in the parietal 
cortex have neiu"ons that discharge in response to 
visual stimuli and before saccadic eye movements. 
There is a predictive aspect of these neurons' visual 
responses: Neurons respond to stimuli in which 
spatial location will be brought into their visual 
receptive fields by impending saccadic eye move- 
ments. 

The double-step task of Hallett and Lightstone has 
been considered the paradigmatic example of accur- 
ate spatial behavior. Humans and monkeys perform 
this task accurately, making accurate eye movements 
to successive, briefly flashed targets, despite a 
dissonance between the retinal location of the target 
and the amplitude and direction of the saccade 
necessary to fixate it Work in the laboratory during 
this year has concentrated on comparing the activity 
of neurons in the double-step task to see whether that 
activity could be explained by their activity in more 
simple tasks. 

We have demonstrated a neuronal mechanism for 
the initiation and targeting of saccadic eye move- 
ments in the monkey frontal eye field, where neurons 
discharge predictively before purposeful saccades and 
intracortical electrical miaostimulation elicit sac- 
cades. In this period, we used the technique of 
transcranial magnetic stimulation in human subjects 
to stimulate the frontal eye field selectively at 
various times in the generation of visually guided 
saccades in order to see whether electrical stimula- 
tion of this region affected saccades in a manner 
consonant with the single-neuron data from the 
monkey. 

Methods 

Monkeys were implanted with magnetic search coils 
for the measurement of eye position, along with 
devices for temporary restraint and electrophysiologi- 
cal recording and stimulation. The monkeys were 
trained to perform a number of visuomotor tasks. 



including fixation, saccades, and smooth pursuit. 
Microelectrodes placed in the lateral intraparietal area 
single neurons enabled smdy while the monkey 
performed various visuomotor tasks. 

Normal volunteers were instructed to fixate a 
central target and make a saccade as quickly as 
possible in response to the appearance of a peripheral 
light Eye movements were measured by an infrared 
eye tracker. In randomly interleaved trials, focal 
transcranial magnetic stimulation was delivered 
through a figure-eight-shaped coil over the presumed 
site of the frontal eye field, which was located with 
reference to the hand motor representation. 

Major Findings 

Transcranial magnetic stimulation j^jplied long 
before the onset of the expected saccade produced 
shorter saccadic reaction times and increased saccade 
acceleration. Conversely, transcranial magnetic 
stimulation ^plied shortiy before the onset of the 
expected saccade yielded longer saccadic reaction 
times and decreased saccade acceleration. Similar 
effects were observed when subjects were instructed 
to perform antisaccades. Independent of the effect 
on saccadic reaction times, transcranial magnetic 
stimulation produced transient divergence of the eyes 
immediately preceding saccade onset. When trans- 
cranial magnetic stimulation occurred during an 
ongoing saccade, it transientiy arrested or slowed the 
eye movement. In summary, transcranial magnetic 
stimulation can facilitate or retard saccadic reaction 
time and can affect the metrics of saccadic eye 
movements. When it occurs at a time at which it 
might interfere with ongoing frontal eye field pro- 
cessing, it slows saccades. When it appears at a time 
at which it could reasonably be expected to substimte 
for normal processing, it speeds up saccadic reaction 
times. 

Neurons in the lateral intraparietal have visual and 
sometimes presaccadic responses. The visual re- 
sponses are sometimes predictive, occurring before a 
saccade that will bring the spatial location of a visual 
target into the neuron's receptive field. Neurons that 
discharge in the double step tasks have presaccadic 
responses, predictive responses, or both. The activity 
in the double-step task approximates the sum of the 
presaccadic and predictive response. These data 
illustrate that the response in the double-step task 
emerges as a consequence of the neuron's response 
in relation to single eye movements and that it is 



233 



Laborator>' of Sensorimotor Research 



NEI Annual Report— FY 1993 



unnecessary to postulate a special mechanism to 
account for activity in the double-step task. 

Significance to Biomedical Research and the 
Program of the Institute 

Understanding the way in which the cerebral cortex 
and its afferent regions guide eye movement; and 
modulate visual attention and learning is useful as a 
model for the neural control of other, more compli- 
cated behaviors. It is also a key to understanding 
and developing treatments for disorders of the neural 
control of vision, eye movements, and attention. 

Proposed Course 

TTie frontal eye fields will be examined to see 
whether they have predictive responses. The activity 
of neurons in both the frontal eye fields and the 
parietal cortex will be examined under the more 
natural condition of visual search. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Processing — 
Visual Processing and Functional Organization 
(Structure and Function of Central Visual Pathways) 

Publications 

Colby CL, Duhamel JR, Goldberg ME: The analysis 
of visual space by the lateral intraparietal area of 
the monkey: The role of extraretinal signals. 
Prog Brain Res 95:307-316, 1993. 



Colby CL, Duhamel JR, Goldberg ME: Ventral 
intraparietal area of the macaque: Anatomic 
location and visual response properties. J Neuro- 
physiol 69:902-914, 1993. 

Duhamel JR, Goldberg ME, FitzGibbon EJ, Sirigu 
A, Grafman J: Saccadic dysmetria in a patient 
with a right frontoparietal lesion: The importance 
of corollary discharge for accurate spatial behav- 
ior. Brain 115:1387-1402, 1992. 

Goldberg ME, Musil SY, FitzGibbon EJ, Smith MK, 
Olson CR: The role of the cerebellum in the 
control of saccadic eye movements, in Mano N 
(ed): Cerebellum and Basal Ganglia in the 
Control of Movement. Amsterdam, Elsevier, 
1993, in press. 

Olson CR, Musil SY, Goldberg, ME: Superior 
cingulate cortex and visuospatial cognition: 
Properties of single neurons in the behaving 
monkey, in Vogt BA, Gabriel M (eds): The 
Neurobiology of Cingulate Cortex and Limbic 
Thalamus. Boston, Birkhauser, 1993, in press. 

Segraves MA, Park K: The relationship of monkey 
frontal eye field activity to saccade dynamics. J 
Neurophysiol 69:lSS0-nS9, 1993. 

Stanton GB, Bruce CJ, Goldberg ME: Topogr^hy 
of projections to the fi-ontal lobe fi-om macaque 
frontal eye fields. J Comp Neurol 330:286-301, 
1993. 



234 



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



PROJECT NUMBER 



ZOl EY 00153-11 LSR 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or lass. Title must fit on arte 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: Urs Schwarz 

Claudio Busettini 



M.D. 
Ph.D. 



Visiting Associate 
Visiting Fellow 



LSR, NEI 
LSR, NFEI 



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: 



2.5 



PROFESSIONAL 



1.3 



OTHER: 



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.) 

It has been known for some time that corrected anisometropes who wear spectacle lenses of different power 
in front of the two eyes are able to generate saccades that differ in amplitude in the two eyes exactly as 
required by the differing magnifications of the spectacle lenses. A common assumption is that this ability 
results from the operation of a neiu^al adaptive mechanism, which, over time, gradually adjusts the relative 
amplitudes of the saccades produced by the two eyes. We now report that when the scenes viewed by the two 
eyes suddenly differ in size, the two eyes produce saccades that inmiediately differ in amplitude without any 
prior period of adaptation. Two slide projectors and an arrangement of orthogonal polarizing filters were used 
to present overlapping stationary random dot patterns simultaneously yet separately to the two eyes. The right 
eye always saw the same pattern while the left eye saw a pattern that was initially identical (pretest) and later 
replaced by one that was 8% smaller (test). The positions of both eyes were recorded with the electromagnetic 
search coil, and horizontal saccades were elicited by target spots projected onto the pattern through polarizing 
filters so as to be visible only to the right eye. Subjects were three humans and three rhesus monkeys. 
Immediately upon viewing the smaller pattern, the left eye produced horizontal saccades that were significantly 
smaller than those produced by the right eye. This indicates that the saccadic system has some ability to cope 
immediately with aniseikonia, the compensation being almost complete in some subjects. We suggest that the 
important cue in these experiments is horizontal disparity and that the saccadic system uses this to scale the 
relative amplitudes of the saccades produced by the two eyes. 



235 



PHS 6040 (Rev. 5/92) 



Laboraton of Sensorimotor Research 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

In recent years there has been considerable interest in 
the binocular alignment of the two eyes because it is 
critical for good stereoscopic depth vision. 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 
varying distances in a three-dimensional world. We 
have used the high-resolution electromagnetic search 
coil to examine the saccadic eye movements of both 
monkeys and humans when they are first confronted 
with images that differ in size for the two eyes. 

Correction of anisometropia with spectacle lenses 
causes the two eyes to see images that differ in size 
by 2-3% for each diopter of difference. It has been 
previously shown by others that human subjects who 
wear such spectacles are able to generate saccades 
that differ in amplitude between the two eyes exactly 
as required by the differing magnifications of the 
spectacle lenses. It has been commonly assumed that 
this ability results from the operation of a neural 
adaptive mechanism that over time gradually adjusts 
the relative amplitudes of the saccades produced by 
the two eyes. However, previous investigators had 
not recorded eye movements immediately after the 
subjects put on the spectacles; hence, they did not 
know whether this behavior was immediate or 
required a period of adaptation. Therefore, we 
looked at the saccadic eye movements produced by 
both human and monkey subjects when first con- 
fronted with the challenge of aniseikonia (i.e., un- 
equally sized images seen by the two eyes). 

Methods 

The subjects (i.e., three humans and three rhesus 
monkeys) faced a tangent screen onto which were 
projected two superimposed images, each of which 
was visible to only one of the two eyes. This we 
achieved using a special screen and polarizing filters 
so that one of the images was polarized in a plane 
orthogonal to the other. When viewing the screen 
through goggles with cross-polarizing filters, each 
eye could see only one of the images, which were 
computer-generated random dot patterns. We record- 
ed the positions of both eyes using the electromag- 
netic search coil method while the subjects made 



saccadic eye movements between target spots pro- 
jected onto the screen through polarizing filters so as 
to be visible only to the right eye. The targets were 
located 10 degrees to the right and left of straight 
ahead. The right eye always saw the same random 
dot pattern, whereas the image seen by the left eye 
could be either the same or 8% smaller, resulting in 
a gradient of binocular disparities across the scene 
(i.e., slight differences in the locations of the images 
on the two retinas). Fifty leftward and fifty right- 
ward saccades were recorded when the patterns seen 
by the two eyes were idenfical (pretest) and another 
50 each when the two patterns differed in size (test). 

Major Findings 

When the patterns were identical in size, the two 
eyes made saccades that were essentially identical in 
amplitude, though not velocity. Immediately on 
viewing the smaller pattern, the left eye produced 
horizontal saccades significantly smaller than those 
produced by the right eye. Similar observations were 
made using scenes that differed only in their horizon- 
tal dimensions, when the pattern of disparity was like 
that experienced by an observer who views a vertical 
surface slanting away from him or her. A striking 
feature of such stimuli was that, at best, the human 
observers had only a very weak perception of such a 
slanting surface. However, the horizontal saccades 
of all subjects immediately showed considerable 
compensation for the aniseikonia; in some subjects it 
was almost complete. For example, when the left 
eye saw a pattern that was 8% smaller horizontally, 
the saccadic amplitude ratios (left eye/right eye) of 
the three humans during the test period were on 
average smaller than those during the pretest by 
7.3%, 4.0%, and 7.3% for rightward saccades and by 
5.5%, 3.8%, and 7.4% for leftward saccades. Similar 
saccadic data were obtained from the three rhesus 
monkeys. 

Significance to Biomedical Research and the 
Program of the Institute 

These data indicate that, when suddenly confi-onted 
with aniseikonic images, the saccadic systems of 
both monkeys and humans are able to make saccades 
that differ appropriately in size. Furthermore, in the 
experiments, the only cue provided was (horizontal) 
disparity, indicating that the saccadic system can 
directly utilize such cues to adjust the relative 
amplitudes of the saccades produced by the two eyes. 



236 



NEI Annual Report— FY 1993 



Laboratory of Sensorimotor Research 



It also is interesting that the motor system responded 
to the disparity cues, even though human subjects 
failed to perceive them. 

Proposed Course 

Future experiments will ftuther examine the saccadic 
eye movements associated with aniseikonia in both 
human subjects and monkeys. The research will 
involve parametric studies of these asymmetric 
saccades to establish the limits of the system and 
their impact on saccadic dynamics. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Processing — 
Image Formation and Stabilization (Ocular Motility) 



Publications 

Kimmig HG, Miles FA, Schwarz U: Effects of 
stationary textured backgrounds on the initiation 
of pursuit eye movements in monkeys. J Neuro- 
physiol 68:2147-2164, 1992. 

Miles FA: The sensing of rotational and transla- 
tional optic flow by the primate optokinetic 
system. Rev Oculomot Res 5:393-403, 1993. 

Miles FA, Busettini C, Schwarz U: Ocular responses 
to linear motion, in Shimazu H, Shinoda Y (eds): 
Vestibular and Brain Stem Control of Eye, Head 
and Body Movements. Tokyo, Japanese Scientific 
Societies Press/Karger 1992, pp 379-395. 

Miles FA, Wallman J: Prologue. Rev Oculomot Res 
5:v-viii, 1993. 



237 



KHUJtO I rNUMDtn 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



ZOl EY 00045-15 LSR 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



TITLE OF PROJECT (80 characters or loss. Title must In on one line berween the borders.) 

Visuomotor Properties of Neurons in the Thalamus 



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

PI: David Lee Robinson Ph.D. Section Chief LSR, NEI 



Others: Alexander A. Kustov 



Ph.D. 



Fogarty Fellow 



NEI 



COOPERATING UNITS (il 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: 



2.4 



PROFESSIONAL: 



1.0 



OTHER: 



1.4 



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.) 

Visual stimuli excite the retina and often produce a shift of attention. To understand some of the brain 
mechanisms involved in shifts of attention, we recorded the activity of neurons in the superior colliculus while 
monkeys performed various tasks. We discovered that neurons in the representations of the peripheral visual 
fields respond uniformly to targets, regardless of the direction of the animal's attention. This occurs whether 
attention is shifted by visual cues or by more cognitive processes. In contrast, neurons within parietal cortex 
are modulated by the direction of the animal's attention. Neurons within the foveal representation of the 
colliculus respond differentially to fixation targets, depending on the behavioral task. While the animal is 
simply fixating, these neurons are only weakly responsive; during more attention-demanding tasks, there are 
more brisk responses of these same cells. We injected the colliculus with muscimol, a GABA-agonist, and 
discovered that it produced a slowing of responses to all targets within the injected visual field. The neurons 
recorded at the injection site had increased spontaneous activity and were also more responsive to visual 
stimuli. These data are some of the first to demonstrate an attentional contribution of the colliculus, 
independent of eye movements. The results suggest that the superior colliculus provides a visual trigger signal 
for shifts of attention. 



238 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Laboratory of Sensorimotor Research 



Project Description 

Objectives 

Visual stimuli continually excite the eye, and some 
of these elicit a shift of attention. The present 
studies were conducted to understand some of the 
mechanisms the brain uses to mediate attentional 
shifts. We sought to discover the physiological 
mechanisms used within the superior coUiculus to 
produce visual activity that initiates shifts of atten- 
tion. In addition, we sought to learn the functional 
contributions this collicular activity might make to 
the whole organism. Because we previously had 
studied the contributions of pareital cortex to atten- 
tion, this smdy was intended to compare these two 
areas. 

Methods 

To study the activity of the superior colliculus during 
attentional behavior, we trained monkeys to enter a 
primate chair, sit quietly,. fixate spots of light, and 
make eye movements to them. After preliminary 
training, the monkeys were implanted with several 
recording devices during sterile surgery. Scleral 
search coils were implanted in the monkeys for 
recording eye movements and eye position. The 
monkeys learned to contact a bar at the beginning of 
each trial. They then fixated on a spot of light 
projected onto a screen and released the bar when- 
ever target lights were flashed onto the screen. The 
monkeys also learned to make eye movements to 
spots of light flashed onto the screen, as well as 
make fine discriminations of selected fixation targets. 
At the sites of interesting cellular activity, small 
marking lesions were made for later localization on 
histological sections. 

Major Findings 

As we previously had demonstrated, monkeys re- 
spond faster to visual targets that are preceded by 
visual cues at the same location than to visual targets 
that follow cues at other points. It is hypothesized 
that the cue shifts attention to that point and thereby 
improves visual performance. We studied neurons 
within the superficial layers of the superior colliculus 
while monkeys performed this and related tasks. 

Collicular neurons compose a uniform population 
of cells with consistent latencies and response 
magnitudes. Data from our previous studies of 



parietal cortex show that cells in that region make up 
at least three subgroups; most of these neurons could 
be driven by our collicular afferents. All collicular 
neurons responded to both the onset and offset of 
visual cues that controlled the monkeys' attention. 
In addition, the responses to these cues were uniform 
across the temporal intervals used. When these cues 
excited collicular neurons, they produced a very 
stong period of refractoriness that was much more 
intense than the one measured for parietal cells. 

When collicular neurons were tested with the 
attentional cue outside the visual receptive field but 
positioned to produce attentional effects, collicular 
cells responded equivalently to all targets, regardless 
of where attention was directed. Cells in parietal 
cortex tested under these same conditions were 
differentially modulated, depending on the direction 
of the animals' attention. When collicular neurons 
were tested via tasks that cognitively controlled the 
direction of the animals' attention, there also was 
consistent response independent of the attentional 
direction. 

We also studied neurons within the foveal repre- 
sentation of the colliculus. When these cells were 
analyzed at the time that attention was shifted to the 
cue, there were no changes in the activity levels of 
the cells. These data suggest that the foveal parts of 
the colliculus do not participate in the shifting of 
attention. However, the cells had different activity 
patterns, depending on the behavioral task. When 
the animals simply had to fixate a spot of light to 
obtain a reward, there was only weak responding to 
the onset of the fixation point. These cells were 
excitable by other lights, so there was no overall 
suppression of their excitability. When the animal 
fixated the identical tight during the performance of 
the attentional cuing task, there was a much stronger 
response from collicular neurons. The cells respond- 
ed as if the act of attending had facilitated their 
visual responsiveness. Comparably strong responses 
were obtained when the animal actively attended to 
the same fixation point during a special foveal 
attention task. These data suggest that the foveal 
region of the colliculus is under attentional control, 
enhancing visual excitability, whereas the peripheral 
collicular representation is not attentionally modu- 
lated. 

To evaluate the contribution of these collicular 
signals to the monkeys' behavior, we altered collicu- 
lar activity by microinjections of muscimol, a 



239 



Laboratory' of Sensorimotor Research 



NEI Annual Report — FY 1993 



GABA-agonist. After an injection of muscimol, the 
moniceys were slow to respond to all visual targets 
that appeared within the affected region of visual 
space. However, collicular neurons at the site of the 
injection were more responsive after the injections, 
contrary to observations obtained from microionto- 
phoresis. Now there was an increase in spontaneous 
activity of the collicular neurons. In addition, these 
cells discharged more intensely to the cues and 
targets. No changes in cellular activity were ob- 
served with injections of saline. 

These data suggest that the effects of limited 
injections of muscimol are not always inhibitory and 
do not decrease transmission through a structure. 
They also suggest that the discharge of collicular 
neurons provides a visual trigger signal that can lead 
to a shift of attention. When this signal is modified, 
in this case by the production of increasing and 
distracting discharges, there is a generalized break- 
down in performance. 

Significance to Biomedical Research and the 
Program of the Institute 

Various disorders of the brain produce abnormal 
attention and eye movements. Some of these, such 
as progressive supranuclear palsy, involve dysfunc- 
tion of the superior colliculus. The data obtained 
this year help to define the contribution of the 
colliculus to these symptoms. Understanding these 
processes might facilitate early detection of such 
disease processes. For rehabilitation of people with 
damage to parts of the brain, it is important to know 
the normal capacities of certain neural centers and 
the ways in which one brain area can take over the 
functions of damaged areas. By gaining a clearer 
understanding of the contribution of the colliculus to 
visual behavior, we can gain insights into its capacity 
to acquire other functions. 

Proposed Course 

One of the major interests in this Section is visual 
attention. We recently have discovered that saccadic 



eye movements can be initiated at very shon laten- 
cies. Under certain conditions, including manipula- 
tion of the state and direction of attention, saccadic 
eye movements can begin rapidly. These movements 
are termed "express saccades." Whereas our previ- 
ous studies have demonstrated that certain regions of 
tlie parietal cortex and pulvinar are related to visuo- 
spatial attention, our future studies will attempt to 
determine the conoibutions of the pulvinar and 
parietal cortex to express saccades. Rhesus monkeys 
will be trained on a variety of eye movement tasks 
that will reliably evoke express saccades. Subse- 
quently, we will electrically excite or chemically 
inactivate these portions of the brain to determine 
how their attentional mechanisms contribute to the 
initiation of saccadic eye movements. 

NEI Research Program 

Strabismus, Amblyopia, and Visual Processing — 
Visual Processing and Functional Organization 
(Structure and Function of Central Visual Pathways) 

Publications 

Bowman EM, Brown VJ, Kertzman C, Schwarz U, 
Robinson DL: Covert orienting of attention in 
macaques. I. Effects of behavioral context J 
Neurophysiol 70:431-443, 1993. 

Brown VJ, Schwarz U, Bowman EM, Fuhr P, 
Robinson DL, Hallett M: Dopamine dependent 
reaction time deficits in patients with Parkinson's 
disease are task specific. Neuropsychologia 
31:459-469, 1993. 

Robinson DL: Functional contributions of the 
primate pulvinar. Prog Brain Res 95:371-380, 
1993. 

Robinson DL, Cowie RJ: Attentional engagement 
and the pulvinar. Behav Brain Sci, in press. 



240 



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



PROJECT NUMBER 



ZOl EY 00109-13 LSR 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Visuomotor Processing in the Primate Brain 



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

PI: Robert H. Wurtz Pli.D. Chief LSR, NEI 

Others: Charles J. Duffy 
Hiroshi Aizawa 
Gregg H. Recanzone 



M.D., 


Ph.D. 


Staff Fellow 


LSR, NEI 


Ph.D. 




Visiting Fellow 


LSR, NEI 


Ph.D. 




Guest Researcher 


LSR, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Sensorimotor Research 



SECTION 

Visuomotor Integration Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



3.5 



PROFESSIONAL: 



2.0 



OTHER: 



1.5 



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.) 

Saccadic or rapid eye movements shift the direction of gaze from one part of the visual field to another, but 
most of normal vision occurs during the period of visual fixation between these saccades. This year we tested 
the hypothesis that fixation cells in the rostral superior colliculus (SC) suppress the activity of saccade-related 
cells in the posterior colliculus and thus also suppress saccades. Electrical stimulation of fixation cells 
interrupted saccades and the burst of activity in the posterior colliculus that precedes saccades. Stimulation 
of the rostral pole also lengthened the interval between a series of saccades that result from stimulation of the 
posterior SC. Both results are consistent with the hypothesis that the fixation cells in the rostral SC regulate 
the generation of saccades. 

As we move through the environment, we generate a full-field visual motion — a pattern of optic flow. We 
have previously studied cells in the cerebral cortex of monkey that appear to be selective for such flow. This 
year we began experiments to test the extent to which monkeys respond to this visual flow stimuli. When 
the monkey stood on a platform that allowed postural changes, we could detect in the monkey a sway related 
to the optic flow. Vibration of the platform increased reliance on flow. These experiments demonstrate the 
sensitivity of the monkey to flow stimuli and open tlie way to testing the effect of removal of the flow 
sensitive cortical neurons on the use of optic flow stimuli. 



241 



PHS 6040 (Rev. 5/92) 



Laboratory of Sensorimotor Research 



NEI Annual Report— FY 1993 



Project Description 

Objectives 

Saccadic eye movement shifts the eyes rapidly from 
one item of interest in the visuaJ field to another. 
The neuronal organization underlying this system has 
been studied intensively in the monJcey during the 
past 20 years, particularly in this Laboratory. Our 
work this year centered on a brainstem structure that 
is related to the generation of these saccadic eye 
movements — the superior coUiculus. As we move 
through the environment, we use the resulting motion 
of the visual field as a whole, referred to as "optic 
flow," to provide stabilization of posture and possible 
guidance of movement. We had previously studied 
the visual processing related to such control in an 
area of the cerebral cortex, the medial superior 
temporal region of the superior temporal sulcus. Our 
experiments this year concentrated on the demonstra- 
tion of the use of these stimuli by the monkey. 

Methods 

The Rhesus monkey, Macaca mulatta, is an incom- 
parable model of the visual system of humans. Its 
saccadic eye movements are nearly identical to those 
of humans. The response of cells to optic flow field 
stimulation suggests that the monkey also uses such 
full-field stimulation in its behavior, although this is 
less well understood. In the study of saccadic eye 
movements, the monkey is awake and trained to 
perform a visual motor task that involves making 
saccadic eye movements from one target to another. 
Because the cooperation of the monkey is required to 
obtain a high level of visual performance, the mon- 
key is rewarded for making these eye movements. 

The entire experiment is managed by an online 
computer that turns on the stimuli, rewards the 
monkey, collects the neuronal and behavioral data, 
and stores that data on magnetic disks for later 
computer analysis. In the experiment on the effects 
of full-field visual stimulation, the monkey is free to 
move within a cage; his posture is measured by 
strain gauges attached to a flat platform on which the 
monkey is trained to stand. Full-field visual stimula- 
tion is projected on one side of the cage, which the 
monkey faces. 



Major Findings 

We had determined previously that damage to the 
superior colliculus produces immediate deficits in the 
generation of saccadic eye movements. We infer, 
therefore, that cells within the colliculus are related 
to the generation of saccades. This year we com- 
pleted a detailed study of the types of cells within 
the superior colliculus. We had detailed the two 
types of cells related to the generation of saccades — 
(1) burst cells and (2) preparatory or buildup cells— 
in last year's annual report This year we completed 
experiments and analysis on the remaining major cell 
type within the superior colliculus — fixation cells. 
These cells do not discharge with a saccadic eye 
movement. Rather, they increase their discharge rate 
when the monkey actively fixates on an object and 
decrease their discharge during saccadic eye move- 
ments. The period of this decrease is roughly 
equivalent to the duradon of the saccadic eye move- 
ment, suggesting that the pause of these cells is 
necessary for the generation of the saccadic eye 
movement. 

Two studies demonstrated that this interaction 
between fixation cells and saccade-related cells does 
occur. In the first, we electrically stimulated the 
fixation cells while recording both the eye movement 
and discharge of the saccade-related cells within the 
colliculus. We found that the stimulation of the 
fixation zone interrupted the saccadic eye movement 
and reduced the discharge of the saccade-related 
cells. The reduction in activity, a presumed indica- 
tion of inhibition, was greater for the burst cells than 
for the buildup cells. This action on the saccade 
cells is consistent witii our hypothesis that the 
fixation cells within the colliculus act to inhibit tiie 
generation of saccadic eye movements while the 
monkey is actively fixating. 

The second set of experiments on the action of 
tile fixation cells was to test tiie effect of tiieir 
stimulation on tiie generation of a "staircase" of 
saccades. Ttiis staircase occurs when the saccade- 
related cells of tiie superior colliculus are stimulated 
over a period of several hundreds of milliseconds. 
The result of tiiis stimulation is a series of individual 
saccades witii a pause between each saccade. 



242 



NEI Annual Report— FY 1993 



Laboratory of Sensorimotor Research 



Why there would be such a series of identical 
saccades rather than one saccade alone has been a 
puzzle for more than 20 years. One explanation 
might be that, when the saccade-related cells are 
stimulated, they are active in the absence of a pause 
in the fixation cells such that the eye is not held in 
its new position after the saccade by the activity of 
the fixation cells. To test this notion, we electrically 
stimulated the fixation zone to see whether it altered 
the time between saccades; it did. As we stimulated 
the fixation cells, we increased the period between 
successive saccades in the staircase, indicating that it 
was the activity in the fixation zone that determined 
the spacing between the saccades. This finding is 
also consistent with the hypothesis that the fixation 
cells, when active, inhibit the generation of saccadic 
eye movements. 

Our second set of investigafions on the effect of 
large-field visual stimulation has concentrated not so 
much on the activity of cells within this area but on 
the consequence of the visual stimulation to the 
monkey's behavior. One of the goals of our research 
is not only to determine the relationship of cell 
activity to visual stimulation and behavior but to 
determine whether removal of these cells leads to a 
change in visual motor behavior. Thus, in the 
current set of experiments, we have begun to test the 
effect of the large-field stimulation on the monkey's 
behavior in order to eventually test whether removal 
of these cells affects that behavior. 

Our strategy has been to use the well-known 
effect of full-field visual stimulation on human 
posture. Whether such stimulation is used by mon- 
keys has never been determined. We measured the 
monkey's posture while full-field stimulation was 
given by using strain gauges attached to a platform 
on which the monkey was trained to stand. We 
oscillated the full-field motion over a period of 
several seconds and observed a swaying motion of 
the monkey synchronous with the moving visual 
field. We were struck, however, that the monkey 
was able to compensate, using other mechanisms, so 
that repeated presentations of the visual stimulation 
no longer produced the sway. 

One mechanism that could contribute to this 
compensation is proprioception, the sense that 
indicates the position of muscles and joints. To 
reduce this sensation in the monkey, we attached a 
low-frequency vibrator to the platform, thereby 
reducing the amount of information proprioception 



and increasing the monkey's sensitivity to visual 
stimulation. This increased sensitivity persisted over 
time. These experiments seem to show that (1) mon- 
keys are sensitive to ftill-field stimulation, as are 
humans, and (2) the use of the visual stimuli is 
coupled with the use of proprioception, and probably 
vestibular sensation, to control posture. 

Significance to Biomedical Research and the 
Program of the Institute 

The saccadic eye movement system usually has been 
taken in isolation as a system that moves the eye 
from one point of the visual field to another. Our 
studies on the fixation cells within the superior 
colliculus and their effect on the generation of 
saccadic eye movements demonstrate that a system 
for visual fixation is as important as the generation 
of saccades. Thus, the saccade moves the eye from 
one point to another, and the fixation system holds 
the eye in position. All of our vision results from 
the period of fixation, so that understanding this 
fixation system is essential to understanding normal 
vision. Just as the study of this visual fixation is not 
generally studied in primates, it is not frequentiy 
smdied in the clinic. We hope that clarification of 
the interaction of the saccadic and fixation systems 
in the monkey will stimulate analysis of fixation in 
the human as well. 

The effect of full-field or optic flow stimulation 
smdied in the monkey on postural responses attempts 
to establish the monkey as a model of the effect of 
vision on postural control. These experiments move 
the study of the monkey from one of just eye move- 
ment control to one of control of movement within 
tiie experiment, and it is increasingly recognized that 
one very important aspect of the use of visual 
stimulation is the control of movement in the envi- 
ronment, including posture. Our experiment estab- 
lished the monkey as a model for the study of these 
systems. Again in humans, damage to the parietal 
cortex is known to produce certain types of disorien- 
tation, and our studies in the monkey open the 
possibility of dissecting the types of disorientation, 
particularly those related to full-field visual stimula- 
tion. 

Proposed Course 

In the analysis of the fixation and saccadic systems, 
a major next step will be the development of a 
precise model of the saccadic-fixation control system. 



243 



Laboratory of Sensorimotor Research 



NEI Annual Report— FY 1993 



This research, to be conducted in this Laboratory in 
collaboration with Dr. Lance Optican, of LSR, will 
serve a heuristic function — summarizing the explo- 
sive growth of knowledge of the superior colliculus 
in the past several years. It also will produce a 
computer-based simulation that will allow tests not 
only on the visual saccadic-fixation system of the 
monkey but on that of the human. In experiments on 
full-field visual stimulation, we will attempt to 
determine the effect of damage to the cortical areas 
most likely to be related to the monkey's use of 
optic flow. These experiments will indicate whether 
we can identify a specific system that relates optic 
flow to the behavioral use of this optic flow. 

NEI Research Program 

Strabismus, Ambylopia, and Visual Processing — 
Visual Processing and Functional Organization 
(Structure and Function of Central Visual Pathways) 



Publications 

Duffy CJ, Wurtz RH: An illusory transformation of 
optic flow fields. Vis Res 33:1481-1490, 1993. 

Munoz DP, Wurtz RH: Role of the rostral superior 
colliculus in active visual fixation and execution 
of express saccades. J Neurophysiol 67:1000- 
1002, 1992. 

Munoz DP, Wurtz RH: Fixation cells in the monkey 
superior colliculus. I. Characteristics of fixation 
cells. J Neurophysiol, 70:559-575, 1993. 

Munoz DP, Wurtz RH: Fixation cells in the monkey 
superior colliculus. II. Reversible activation and 
deactivation. J Neurophysiol, 70:576-589, 1993. 

Wurtz RH, Munoz DP: Role of monkey superior 
colliculus in control of saccades and fixation. 
Cog Neurosci, in press. 



244 



Ophthalmic Genetics and Clinical Services Branch 



Report of the Acting Chief, Ophthalmic Genetics and CUnical Services 
Branch 

Muriel I. Kaiser-Kupfer, M.D. 



The Ophthalmic Genetics and Clinical Services 
Branch within the National Eye Institute (NEI) 
Intramural Research Program has been operational 
since February 1989. The Branch is organized into 
four sections: Ophthalmic Genetics, Acting Chief 
Muriel I. Kaiser-Kupfer, M.D.; Cataract and Corneal 
Diseases, Chief Manuel B. Datiles, M.D.; Ophthal- 
mic Pathology, Acting Chief W. Gerald Robison, Jr., 
M.D., Ph.D.; and Clinical Services, Acting Chief 
Rafael C. Caruso, M.D. 

The purpose of the Branch is to conduct clinical 
and laboratory research on gene expression 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 visual system. Such disorders 
include corneal and retinal diseases, cataract, and 
visual pathway abnormalities. 

The Branch is responsible for the essential 
psychophysical and electrophysiological diagnostic 
tests of visual function required by clinical intra- 
mural research programs of all the Institutes. In 
addition, it processes ocular clinical biopsy and 
autopsy materials. The Branch differs from other NEI 
laboratories engaged in molecular investigations 
because its emphasis is the translation of appropriate 
research findings directly to the clinical setting. This 
Branch is also a point of focus for the trans-National 
Institutes of Health (NIH) emphasis on research in 
genetics, more effectively aligning its organizational 
structure within the Institute's Intramural Research 
Program. Since beginning its operation, the Branch 
has shown considerable growth and productivity. 



Section on Cataract and Corneal 
Diseases 

The Section on Cataract and Corneal Diseases 
continued to pursue research on the anterior 
segment, especially the short- 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 morphology is being studied by 
specular microscopy to compare the endothelial 
status in patients wearing different types of lenses. 
The development of automated computer analysis is 
under way to facilitate data analysis, which currently 
is performed by hand and is therefore time consum- 
ing and laborious. 

This Section has been particularly productive in 
studies using different systems to develop objective 
and subjective methods of monitoring and document- 
ing opacities in the human lens. Reproducibility 
studies on objective systems include the use of the 
Scheimpflug cameras (Zeiss and Oxford) and the 
retroillumination cameras (Neitz and Oxford). Sub- 
jective systems or methods — such as the LOCS n 
grading system and the effects of cataracts on visual 
perception, contrast sensitivity, and glare — may be 
useful in identifying additional parameters. These 
systems are being used to study the natural history of 
various cataracts, such as presenile, senile or age- 
related, steroid-induced, radiation, diabetic, retinitis 
pigmentosa, gyrate atrophy (GA), and neurofibroma- 
tosis 2 (NF2). Genetic linkage studies are under way 
to pursue the gene(s) of congenital cataracts. Moni- 
toring 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. 

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). Through the 
collaborative efforts of cataract surgeons and basic 
researchers, efforts are under way to develop and 
modify techniques to study materials that become 
available at surgery and can be well documented 
clinically. We are carefully documenting the cataract 



247 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



preoperative! y, using clinical and photographic LOGS 
II grading, as well as Zeiss Scheimpflug and Oxford 
retroilluniination videophotography and image 
analysis. Cataracts are extracted extracapsularly, 
followed immediately by implantation of intraocular 
lenses. Specimens obtained are examined histologi- 
cally via light and electron microscopy and biochem- 
ically via two-dimensional gel electrophoresis 
(PHAST and LSB systems). Cataractous specimens 
are compared with normal tissues obtained from eye 
bank eyes. Abnormal proteins are identified by 
immunoblotting 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 crystallins. These aging changes need to be 
differentiated from changes occurring in cataract 
formation. 

Investigators in this Section have been in the 
forefiront of recognizing the role of the neural crest 
in normal and abnormal development of the anterior 
segment. Studies continue on anterior chamber 
abnormalities and iridocorneal endothelial syndrome 
patients. 



Section on Ophthalmic Genetics 

Studies by the Ophthalmic Genetics Section have 
emphasized retinal degeneration and ophthalmic 
involvement in systemic genetic diseases. This 
Section has been a leader in studying GA of the 
choroid and retina The accumulation of natural 
history data and the work on definition of the genetic 
abnormalities have been unique. Evidence for bio- 
chemical, clinical, and molecular heterogeneity 
continues to be confirmed. There appear to be many 
different single-point mutations in the ornithine 
aminotransferase gene in GA patients. Dietary 
intervention studies utilizing an arginine deficient 
diet have been promising, especially in young 
patients, in whom a delay in the onset of pathologic 
changes has been demonstrated. 

Foveal cone sensitivity (assessed by measurement 
of increment thresholds) and orientation (esfimated 
by measurement of the Stiles-Crawford effect) were 
found to be abnormal in a group of patients with 
GA. These results suggest that foveal cones are 



altered in their orientation and sensitivity before the 
encroachment on the foveal area by the atrophic 
lesions of GA. 

Albinism has been associated in animals with an 
anatomic anomaly of the visual pathways character- 
ized by excessive crossing of the retinogeniculate 
fibers, with two different modes of geniculocortical 
projection. In humans, indirect evidence of the same 
anomaly is demonstrated by asymmetry in visually 
evoked potentials (VEPs) elicited by pattern-reversal 
stimulation. Recent studies using appearing-disap- 
pearing patterns claimed VEP asymmetry to be 
diagnostic and proposed a uniform type of asym- 
metry. We used the same recording conditions to 
determine the diagnostic value of VEP in albinism 
and to attempt to correlate the VEP results with 
clinical features. 

This study shows that there are two different 
patterns of VEP asymmetry in albinism, which may 
be explained by differences in reorganization of the 
geniculocortical pathway. VEP asymmetry occurs 
frequently but may not be constant in this condition. 
However, its value is decreased in some cases in 
which the low amplitude of the responses makes 
interpretation difficult. Furthermore, there is no 
correlation of the type of asymmetry with any other 
feature of albinism. 

Collaboration with the Interinstitute Genetics 
Program has continued, with active participation by 
the Genetics Clinic. During the past year, we have 
seen approximately 200 individuals representing 
approximately 60 different disease categories. Be- 
cause of the high frequency of ocular involvement in 
these cases, almost all of these patients were evalu- 
ated by the Ophthalmic Genetics staff. 

NF2, otherwise known as bilateral acoustic 
neuroma, is inherited as an autosomal dominant 
disorder. Multiple members of several large pedi- 
grees, as well as a large number of unrelated fami- 
lies, have been studied in collaboration with Dr. 
Dilys Parry (National Cancer Institute). An important 
original observation was the striking frequency of 
posterior capsular cataract in patients with NF2 (BO- 
SS %). In addition, 30% of the patients have shown 
associated cortical cataracts. These findings are 
helpful in establishing a diagnosis of NF2 in at-risk 
patients. The etiology of die cataract is unclear; 
however, it is interesting that the gene locus for 
bilateral acoustic neuromas is on chromosome 22, as 
is the gene for pB -crystalline. Combined pigment 



248 



NEI Annual Report— FY 1993 



Ophthalmic Genetics and Clinical Services Branch 



epithelial and retinal hamartomas appear to be 
another ocular marker for some patients with severe 
NF2. 

GA is a condition amenable to gene therapy; 
preliminary laboratory studies are under way toward 
that goal. Usher's syndrome, congenital deafness, 
and retinitis pigmentosa patients are being studied; 
molecular techniques are used to map the gene and 
to identify the responsible mutation. 

Finally, the results from the continuing double- 
masked, controlled clinical trial of topical cysteamine 
patients with nephropathic cystinosis are exciting. 
Since confirming the usefulness of 0.5% cysteamine 
eye drops in the young patients, we expanded our 
study to include older patients, with similarly striking 
results. Particularly 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 in their quality of 
Ufe. 



The results of VEP studies showed that two 
waveforms, which frequently show the same surface- 
positive polarity but are generated by stimulation of 
each hemifield, combine to generate peaks of the 
full-field VEP. 

Our results indicate that the sum of the asymmet- 
rical contributions of both eyes (either hemisphere of 
each) is responsible for the symmetrical VEP elicited 
by binocular stimulation with a full-field stimulus. 
An asynmietrical, full-field VEP may occur in 
normal subjects and does not imply an abnormality 
in the visual pathways. 

Studies of dark adaption (DA) in patients with 
retinal dystrophies indicated that a complete evalua- 
tion of DA should include, in addition to measure- 
ment of DA, the time constant of adaptation, which 
provides information about the rate at which this 
final threshold is reached. The time constant serves 
as a clinically relevant parameter in both the diagno- 
sis of retinopathies and the followup of individual 
patients over time. 



Section on Clinical Services 

The Clinical Services Section has been active in 
characterizing psychophysical and electrophysio- 
logical findings in patients with diseases that affect 
the eye and the visual system. Continued documenta- 
tion by noninvasive techniques has shown that more 
and more refined and accurate classification of 
diseases is possible. Psychophysical and electrophysi- 
ological information is particularly helpful in under- 
standing the pathogenesis of disease, as well as being 
available for use as a marker in various treatment 
modalities. 



Section on Ophthalmic Pathology 

The Section on Ophthalmic Pathology has pro- 
vided technical support services to investigators 
involved in clinical and basic research as well as to 
those performing routine pathology. Careftil monitor- 
ing of the volume of material handled shows a 
steady increase in processing by the Laboratory, with 
excellent results. Considerable savings to the Institute 
have resulted from the elimination of costly contract 
services. 



249 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1, 1992 to September 30. 1993 



PROJECT NUMBER 



ZOl EY 00187-10 OGCSB 



TITLE OF PROJECT (80 cheracters or less Tillg must lit on one line between the borders ) 

The Effects of Corneal Contact Lenses on the Cornea 



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



PI: 


Manuel B. Datiles 


M.D. 


Medical Officer 


OGCSB, NEI 


Others: 


Gregory P. Kracher 


O.D. 


Expert 


OGCSB, NEI 




Lessie McCain 


R.N. 


Nurse Specialist 


OGCSB, NEI 




Louella Lopez 


M.D. 


Visiting Associate 


OGCSB, NEI 




Doretha Leftwood 


B.A. 


Computer Specialist 


OGCSB, NEI 




Anup Mahurkar 


B.S. 


Visiting Associate 


OGCSB, NEI 



COOPERATING UNITS (if any) 

Image Processing and Analysis Laboratory, Division of Computer Research and Technology NIH (Mark 
Vivino, B.S.) 



LAB/BRANCH 



I 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEI. NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.575 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



PROFESSIONAL; 



0.450 



OTHER: 



0.125 



n (b) Human tissues □ (c) Neither 



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

This investigation of the short-term as well as the long-term effects of contact lens wear on the cornea includes 
specular microscopy smdies of changes in corneal curvature, corneal epitheUal morphology, and corneal 
endothehal cell morphology. Analysis of the data obtained will help us understand the dynamics involved in 
Uie mteraction between a contact lens and the cornea, the risk to corneal tissues, and how a systemic or local 
disorder may increase these risks. In addition, we are studying the differences in corneal endothelial status 
or wearers of soft compared with hard contact lenses. 

Animal models showing corneal endothelial abnormalities similar to those in long-term contact lens wearers 
also are bemg explored in diabetic and galactosemic animal models. Treatment with aldose reductase 
inhibitors helps prevent these corneal abnormalities. 



250 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 

Clinical Protocol Number 

84 EI-133 

Objectives 

The objective of this project is to investigate the 
effects of contact lens wear on corneal tissues, 
including the study of factors that increase or de- 
crease the potential risk of injury to corneal tissues 
by contact lens wear. 

Methods 

We used each patient's complete history, ophthalmo- 
logic examination, photography, keratotometry, 
pachymetry, and specular microscopy of the corneal 
endothelium. 

Major Findings 

We have found that diabetes may increase the risk of 
complications from contact lenses in the first 6 
months of wear. In addition, we have found changes 
in the corneal endothelium after long-term wear of 
contact lenses. These changes include polymegathism 
and pleomorphism. Furthermore, 2 years after some 
of our patients discontinued wearing contact lenses, 
we found a trend toward recovery but no statistically 
significant change. 

In addition, we found that diabetic and galactos- 
emic animals have these endothelial abnormalities 



and that treatment with aldose reductase inhibitors 
prevented these abnormalities. 

Significance to Biomedical Research and the 
Program of the Institute 

Contact lenses are commonly used for correction of 
errors of refraction as well as for ther^y. However, 
our knowledge of the interaction of contact lenses 
with the cornea and tears is limited. In addition, risks 
associated with wearing contact lenses are poorly 
understood. Understanding the interaction between 
contact lenses and corneal tissues will allow us to 
determine why some patients cannot wear contact 
lenses and provide methods to avoid some of the 
complications associated with contact lens wear. 

Proposed Course 

The following studies are in progress or proposed for 
next year: (1) continued examination of patients who 
have worn hard contact lenses and have now shifted 
to gas-permeable or soft contact lenses, (2) recruit- 
ment of patients who plan to discontinue contact lens 
wear or to shift from one type of contact lens to 
another; and (3) development of automated computer 
analysis of all types to facilitate the analysis of data. 

NEI Research Program 

Corneal Diseases — Corneal Edema, Endothelial 
Dysfunction, Dystrophies and Inherited Disease 



251 



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



PROJECT NUMBER 



ZOl EY 00188-10 OGCSB 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



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

Documentation and Monitoring of Opacities in the Human Lens 



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

PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI 



Others: 



Benjamin Magno 


M.D. 


Maria Susan Lasa 


M.D. 


Louella Lopez 


M.D. 


Anup Mahurkar 


B.S. 


Doretha Leftwood 


B.A. 


Joan Lee 





Visiting Associate 
Visiting Associate 
Visiting Associate 
Visiting Associate 
Computer Specialist 
Clinic Coordinator 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (il any) 

Image Processing and Analysis Laboratory, Division of Computer Research and Technology (DCRT), NIH (Benes Trus, Ph.D.; Mark 
Vivino, B.S.); Biomedical, Engineering and Instrumentation Branch. DCRT, NIH (Michael Unser, Ph.D.); Epidemiology Branch, NEI 
NIH (Michael Unser, Ph.D.); Clinical and Diagnostic Trials Section, National Cancer Institute, NIH (Sylvan Green, M.D.); Nuclear 
Medicine, CImical Center. NIH (Joseph Frank, M.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: 



3.725 



PROFESSIONAL: 



2.30 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 

□ (a1) Minors 

□ (a2) Interviews 



OTHER: 



1.425 



n (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 
studies on the objective systems— the Scheimpflug (Zeiss and Oxford) and retroillumination (Neitz and 
Oxford) cameras. Our study of subjective systems or methods, such as the LOGS H 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. 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, retiniUs pigmentosa, gyrate atrophy, and neurofibromatosis U cataracts. This study will 
prepare the way for eventual clinical trials of anticataract drugs. 

Genetic linkage studies under way are pursuing tlie gene(s) of congenital cataract 



252 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 



Additional Personnel 



Malina A. Drews- 


M.D. 


Visiting Fellow, 


Bankiewicz 




OGCSB, NEI 


Yvonne Duglas-Tabor 


B.S. 


Biologist, 
OGCSB, NEI 


Marvin Podgor 


M.D. 


Epidemiologist, NEI 


Robert Sperduto 


M.D. 


Chief, Epidemiology 
Section, NEI 


Laura Wozencraft 


B.S. 


Genetic Counselor, 
OGCSB, NEI 


Mark H. Scott 


M.D. 


Senior Staff Fellow, 
OGCSB, NEI 


J. Fielding 


M.D., 


Staff Scientist 


Hejtmancik 


Ph.D. 


LMOD, NEI 



Clinical Protocol Number 

84-EI-132 

Objectives 

The objective of this project is to formulate means of 
documenting cataract formation and progression. This 
is an important step prior to 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, including 
contrast sensitivity, glare testing, and potential acuity 
testing, are performed for each person in the study. 
Techniques used to measure and evaluate cataracts 
include Scheimpflug photography, retroillumination 
photogr^hy, specular microscopy, and laser light- 
scanning spectroscopy. 

Major Findings 

We have found that clinical grading of cataracts 
using the LOCS II system is a means of quantitative- 
ly detecting the progression of age-related cataracts 
within 1 year. In addition, we found that in various 
types of cataracts, glare and contrast sensitivity 
testing show abnormal results only in the severe or 
more advanced grades. The only exception was in 
posterior subcapsular cataracts, which showed an ab- 
normality in contrast and glare sensitivity in the early 



stages, based on LOCS II grading. In a study of pure 
nuclear cataracts, we found a significant correlation 
between lens nuclear density (assessed by either 
LOCS II 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 perform quickly densi- 
tometry of Scheimpflug nuclear cataract images and 
compare the results with previous images to detect 
significant changes, which are expressed in optical 
density units. For posterior subcapsular and cortical 
cataract, we also have developed a semiautomated 
method of quantitating the cataracts in square milli- 
meters using retroillumination photography. 

Significance to Biomedical Research and the 
Program of the Institute 

The monitoring and documenting of human cataract 
progression is a crucial step toward the ultimate 
testing of several medications believed capable of 
preventing or reversing human cataracts. This step is 
also important in categorizing types of cataracts in 
various parts of the world and correlating them with 
physical and genetic factors iii specific geographic 
regions. 

Subjective methods of determining visual function 
are also important to determine the degree of handi- 
cap that cataract patients have in coping with daily 
activities. Since in our studies none of the subjective 
methods could demonstrate subjective experiences in 
association with early cataracts, research is needed to 
develop more sensitive techniques. 

Proposed Course 

We will continue the study and development of 
subjective and objective methods of documenting and 
monitoring human cataracts. We will pursue the 
improvement and automation of the present system 
of lens photography (e.g., Scheimpflug, retroillu- 
mination, and laser-light spectroscopy), as well as 
exploration of possible applications of new techno- 
logical advances. Appropriate population groups for 
study will be identified. 

A^£/ Research Program 

Cataracts — Epidemiology of Cataract 



253 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Publications 

Datiles M: Clinical evaluation of cataracts, in 
Tasman W, Jaeger E (eds): Duane's Clinical 
Ophthalmology. Philadelphia, Lippincon Co., 
1992, Vol. I 73B. pp 1-16. 

Datiles M, Magno B, Leftwood D, Friedlin V, 
Vivino M: Longitudinal study of age related 
nuclear cataracts using the NEI Scheimpflug 
Imaging System. Investig Ophthalmol Vis Sci 
34:4a, 943, 1993. 

Fox PC, Datiles M. Atkinson JC, Scott J, Fletcher D, 
Valdez IH, et al: Prednisone and piroxicam for 
treatment of primary Sjogren's syndrome. Clin 
Exp Rheumatol 11:149-156, 1993. 

Genhart M, Kelly K, Coursey R, Datiles M, Rosen- 
thal N: Effects of bright light on mood in normal 
elderly women. Psychiatry Res 41 -.SI -91, 1993. 

Kashima K, Trus BL, Unser M, Datiles MB, Ed- 
wards PA, Sibug M: Aging studies on normal 
volunteer lenses using the Scheimpflug slit lamp 
camera. Invest Ophthalmol Vis Sci 34:263-269, 
1993. 



Kashima K, Unser M, Datiles MB, Trus BL, Ed- 
wards PA: Minimum views required to charac- 
terize cataracts when using the Scheimpflug 
camera. Graef Arch Ophthalmol, 231: 687-691, 
1993. 

Lasa S, Datiles MB: Longitudinal study of cortical 
cataracts using retroillumination photographs. 
Investig Ophthalmol Vis Sci 34:4a, 943, 1993. 

Lasa S, Podgor M, Datiles M, Magno B: Glare sensi- 
tivity in early cataracts. Br J Ophthalmol 77:489- 
491, 1993. 

Lopez ML, Datiles M: Longitudinal study of age 
related posterior subcapsular opacities using ret- 
roillumination photographs. Investig Opthalmol 
Vis Sci 34:4a, 943, 1993. 

Magno B, Datiles M, Friedlin V: Reproducibility of 
the NEI Scheimpflug Cataract Imaging System. 
Investig Ophthalmol Vis Sci 34:4a, 943, 1993. 

Magno BL, Datiles MB, Lasa SM: Senile cataract 
progression studies using the Lens Opacity Clas- 
sification System. Invest Ophthalmol Vis Sci 
34:2138-2141, 1993. 



254 



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



PROJECT NUMBER 



ZOl EY 00212-08 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30. 1993 



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

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: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI 



Others: 



Maria Susan Lasa 


M.D. 


Benjamin Magno 


M.D. 


Yvonne Tabor 


B.S. 


Louella Lopez 


M.D. 


I*ushpa Sran 


M.D. 



Visiting Associate 
Visiting Associate 
Biological Technician 
Visiting Associate 
Medical Officer 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 

Laboratory of Mechanisms of Ocular Diseases, NEI CDonita 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 



OTHER: 



1.30 



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.) 

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 lenses. 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 LOCS II 
grading and Scheimpflug (Zeiss and Oxford) retroillumination (Oxford) 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 
elecfrophoresis (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. 



255 



PHS 6040 (Rev. 5/92) 



Ophtbalmjc Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Additional Personnel 

Samuel Zigler Ph.D. 



Head, Section 
on Cataracts, 
LMOD, NEI 



Clinical Protocol Number 

84-EI-194 

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 maximally used for biochemical 
and genetic research. 

Methods 

We examined patients who have different forms of 
cataracts and documented their cataracts, as described 
in project ZOl EY 00188-10, Documentation and 
Monitoring of Opacities in the Human Lens. After 
extracapsular cataract extraction, fragmented lens 
materials — which include the anterior lens capsule, 
lens nucleus, and aspirated lens cortical material — 
undergo protein and biochemical analyses. Some of 
the specimens are frozen for futtire genetic studies. 
We also have undertaken 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 
clinician-surgeon and the basic researcher. Although 
the two professionals have different immediate 
priorities (one being patient care; the other, adequacy 
of tissue samples for laboratory studies), the ultimate 
goal of alleviating human suffering is the same. 

The two-dimensional gel electrophoresis technique 
may be extremely useful in determining lens protein 
changes using very small amounts of tissue (300 
meg). Aging results in acidic shifting of proteins, an 
increased number of polypeptide species in the 
molecular weight range of the crystallins, and cova- 
lent cross-linking of crystallins — changes that need to 
be differentiated from changes occurring in cataract 



formation. We also have found that lens material 
aspirated through the irrigator-aspirator or phako- 
emulsifier lose some crystallins; optimum samples 
are those we obtain separately, thus avoiding 
oxidation. 

Significance to Biomedical Research and the 
Program of the Institute 

A severe setback is being dealt many cataract proj- 
ects because of the lack of human cataract material 
available for basic research studies. While the current 
technique, which involves fragmenting the cataract 
during extraction, is extremely successful and effec- 
tive, there is no foreseeable change back to utiliza- 
tion of the intracapsular method (i.e., removal of the 
lens in toto). Hence, it is imperative that we modify 
our basic research methodology to adapt to the use 
of fragmented lens materials in order to continue 
basic lens research projects that deal with human 
materials. 

Proposed Course 

We will continue to pursue the development of the 
use of firagmented lens material in basic research 
experiments. Using two-dimensional gel electro- 
phoresis, we will study ways in which surgeons can 
modify their surgical techniques without compromis- 
ing 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 handling lens materials to maximize 
the quality of specimens for basic research. 

NEI Research Program 

Cataract — ^Treatment of Cataract and Correction of 
Aphakia 

Publications 

Datiles M, Kinoshita J: Pathogenesis of cataracts, in 
Tasman W, Jaeger E (eds): Duane's Clinical 
Ophthalmology. Philadelphia, Lippincott Co., 
1991, 72B, pp 1-14. 

Garland D, Tabor Y, Zigler JS, Datiles MB: Protein 
analysis of lens cortical aspirates obtained at 
surgery. Investig Ophthalmol Vis Sci 34:4a, 1335, 
1993. 



256 



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



PROJECT NUMBER 



ZOl EY 00281-01 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (BO characters or less. Title must lit or) one \ir\e 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 alliliation) 

PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEl 



Others: J. Fielding Hejtmancik 
Mark H. Scott 



M.D., Ph.D. 
M.D. 



Senior Staff Fellow 



LMOD, NET 
OGCSB, NEl 



COOPERATING UNITS (it any) 

Marshall Parks, M.D. (Private Practice), Washington, DC 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Cataract and Corneal Diseases 



INSTITUTE AND LOCATION 

NEl, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



1.325 



PROFESSIONAL: 



1.325 



OTHER: 



0.0 



CHECK APPROPRIATE BOX(ES) 

[x] (a) Human subjects 
jx] (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, littie 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 opacities of the lens. Animal studies have 
suggested that alterations in lens crystalline can cause hereditary cataracts, making them reasonable candidate 
genes for causing hereditary cataracts in humans. In addition, it is ^parent that hereditary lesions that mimic 
or contribute additively to envirormiental 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. 



257 



PHS 6040 (Rev. 5/92) 



Ophtbalmjc Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

84 EI -194 A 

Objectives 

The objective of this study is to do linkage analysis 
using molecular genetic techniques in families with 
congenital hereditary cataracts. 

Methods 

Linkage analysis (i.e., gene mapping) will be carried 
out by following the inheritance of genetic markers 
in families with hereditary cataracts. In informative 
families, blood specimens will be obtained and 
analyzed for gene marker linkage analysis. 

Major Findings 

Under way at present is the enrollment of families 
with congenital cataracts. A large family with 
known autosomal dominant congenital cataract has 
been analyzed. This analysis is the first to provide 
evidence of intraocular phenotypic heterogeneity in 
a family with autosomal dominant congenital cata- 
ract. 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 
responsible for the development of congenital 
cataracts. 

Proposed Course 

More famiUes who have congenital cataract will be 
recruited, and linkage analysis will be performed on 
these families. 

NEI Research Program 

Cataract — Treatment of Cataract and Correction of 
Aphakia 

Publications 

Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky J: 
Molecular biology and inherited disorders of the 
eye lens, in Scriver CR, Beaudet AL, Sly WS, 
Valle D (eds): Metabolics Basics of Inherited 
Disease. McGraw-Hill Inc., submitted. 

Scott MH, Hejtmancik JF, Wozencraft LA, Reuter 
LM, Parks MM, Kaiser-Kupfer MI: Autosomal 
dominant congenital cataract: Interocular pheno- 
typic heterogeneity. Ophthalmology, submitted. 



258 



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



PROJECT NUMBER 



ZOl EY 00084-15 OGCSB 



PERIOD COVERED 

October 1. 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or /ess. Title must fit on one tine 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 McCain 

Manuel B. Datiles 

Maria Susan 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: 



1.0 



PROFESSIONAL: 



0.7 



OTHER: 



0.3 



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 coimective tissues 
anterior to the lens epithelium. Therefore, the group of developmental anomalies of the anterior chamber with 
glaucoma or ocular hypertension is being reviewed. 



259 



PHS 6040 (Rev. 5/92) 



Ophtbalmjc Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

77-EI-119 

Objectives 

The object of this study is to determine whether 
congenita] or developmental anomalies of the anteri- 
or chamber are related to faulty migration or terminal 
differentiation of neural crest tissue. 

Methods 

Patients of all ages with congenital or developmental 
anomalies of the anterior chamber are being exam- 
ined to determine involvement of cornea, trabecular 
meshwork, iris stroma, lens, and ciliary body. When 
intractable glaucoma cannot be controlled with 
medication, surgery is performed and the specimens 
are examined histologically. When the lenses become 
cataractous, cataract extractions are performed and 
the lens epithelium is grown in tissue culture. When 
the cornea is opaque and corneal transplantation is 
indicated, that procedure is performed and 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 mem- 
brane, and trabecular meshwork endothelium. 



We recently performed trabeculectomies on 
patients with the irido-comeal-endothelial syndrome. 
Histopathologically, we found the presence of a 
membrane covering 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 diagnosis and 
treatment The presence of this membrane may 
explain the 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 cham- 
ber, including congenital cataracts, will be examined 
for abnormalities in tissue derived 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— Other Glaucoma (Developmental, Con- 
genital, and Infantile Glaucoma) 

Publications 

Kupfer C, Chan C-C, Bumier M Jr, Kaiser-Kupfer 
MI: Histopathology of the ICE syndrome. Trans 
Am Ophthalmol Sac 90:149-160, 1992. 



260 



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



PROJECT NUMBER 



ZOl EY 00123-13 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Clinical Psychophysics of the Visual System 



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

PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI 



Others: Muriel I. Kaiser-Kupfer M.D. 

Malina Drews-Bankiewicz M.D. 

Doris J. Collie A.A. 

Patricia A. Mercer M.P.A. 

Leanne M. Reuter B.S. 



Chief 

Visiting Associate 
Ophthalmic Technician 
Ophthalmic Technician 
Ophthalmic Technician 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Eye Services Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.49 



PROFESSIONAL: 



0.41 



OTHER: 



1.08 



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.) 

The visual function of patients with ocular diseases or lesions in the visual pathways and of normal subjects 
is measured using psychophysical techniques. The data are correlated with those obtained with 
electrophysiological tests of visual fiinction. The results contribute to the diagnosis of ocular and neural 
disorders that affect vision and are needed to characterize their nature and evolution. They are also valuable 
in the assessment of how different forms of treatment affect the outcome of these diseases. 



261 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

93-EI-0131 

Objectives 

The aims of this project are (1) to apply and develop 
psychophysical techniques for the study of vision in 
the clinical setting, (2) to characterize the human 
visual system's normal function, and (3) to analyze 
the patterns of its alteration in ocular diseases and 
lesions of the visual pathways. 

Methods 

Several psychophysical techniques are employed as 
discussed below: 

Perimetry. — Visual fields are explored with 
kinetic quantitative perimetry and static quantitative 
perimetry. 

Color vision. — Central color vision is estimated 
using HRR pseudoisochromatic plates, Ishihara 
pseudoisochromatic plates, Famsworth's Tritan plate, 
Famsworth-Munsell D-15 panel, Lanthony's desatur- 
ated D-15 panel, Famsworth-Munsell 100 hue test, 
and the Nagel anomaloscope. 

Adaptometry. — Dark-adapted rod and cone thresh- 
olds are measured with a modified Goldmann-Week- 
ers adaptometer. 

Spatial vision. — ^The spatial contrast sensitivity 
function is determined using sinusoidal luminance 
gratings. A two-alternative temporal forced-choice 
technique is used for a criterion-free judgment of 
threshold visibility. 

Luminance and chromatic increment thresholds. — 
These are measured with a two-channel Maxwellian 
view instrument. This instrument also is used to 
assess retinal receptor orientation by measuring the 
Stiles-Crawford effect (SCE of the first kind). 

Major Findings 

The diagnostic value of the time constant of the dark 
adaptation (DA) function as an estimator for the 
evaluation of visual function was studied in patients 
with retinal degenerations. Seven groups (70 sub- 
jects) were included in this study: pa'ients with 
retinitis pigmeniosa TIP) (12), gyrate atrophy (GA) 
(15), cone dystrophy (11), juvenile macular dystro- 



phy (9), fundus flavimaculatus (FF) (33), other 
retinal degenerations (6), and normal subjects (14). 
The results obtained on one eye chosen at random in 
each subject were analyzed. Measurements were 
made with a modified Goldmann-Weekers adaptome- 
ter. The subjects were initially light adapted with a 
2,550 cd/m^ background for 5 minutes. DA func- 
tions were obtained by measuring the change in 
absolute threshold with time (using von Bekesy 
tracking) for 30 minutes. The following model 
(Pugh, 1975) was used to fit each of the two limbs 
of the DA function: 

time 

Threshold = a + b • e " 

In this model, "a" is the final DA threshold (in dB), 
"b" is the magnitude of DA (in dB), and "c" is the 
time constant of DA (in minutes). Analysis of 
variance was used to examine differences between 
the means of each parameter among the study 
groups. Statistically significant differences in final 
threshold ("a") and time constant ("c") were seen in 
both the cone- and rod-mediated limbs of the DA 
function. Abnormally high time constants in the 
cone-mediated limb were observed in RP and GA 
patients. FF patients who had a normal final thresh- 
old had abnormal elevation in the time constant of 
rod-mediated limbs. The usual DA estimator is the 
final absolute threshold. These findings suggest that 
a complete evaluation of DA should include, in 
addition to a measurement of the final threshold, the 
time constant of adaptation, which provides informa- 
tion about the rate at which this final threshold is 
reached. The time constant serves as a clinically 
relevant parameter in both the diagnosis of retinopa- 
thies and the followup of individual patients over 
time. 

Significance to Biomedical Research and the 
Program of the Institute 

Psychophysical techniques are noninvasive methods 
useful in the diagnosis and management of ocular 
diseases and visual pathway lesions. In addition to 
the application of validated tests, the development of 
new techniques contributes to the elucidation of the 
pathophysiological mechanisms of visual disorders. 

Proposed Course 

Clinical psychophysical studies of visual fiincfion in 
diseases of the eye and visual pathways will be 



262 



NEI Annual Report— FY 1993 Ophthalmic Gen etics and Clinical Services Branch 

continued. We will introduce modifications that are Publications 

expected to enhance the diagnostic value of the r^ d i- • »** ^ t>^ r^ • t^ ^ 

techniques described. Drews-Bankiewicz MA, Caruso RC, Kaiser-Kupfer 

MI: The clinical relevance of the time course of 
NEI Research Program '^^*' adaptation in reUnal degenerative diseases. 

* Invest Ophthalmol Vis Sci 34(4)(suppl):1077, 

Retinal and Choroidal Diseases — Noninvasive 1993. 

Techniques in the Study of Retinal Disorders 

Strabismus Amblyopia, and Visual Processing — 
Visual Processing and Amblyopia 



263 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00144-12 OGCSB 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



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

Clinical Electrophysiology of the Visual System 



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

PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI 



Others: Muriel I. Kaiser-Kupfer M.D. 

Malina Drews-Bankiewicz M.D. 

Patricia A. Mercer M.P.A. 

Doris J. Collie A.A. 

Leanne M. Reuter B.S. 



Chief 

Visiting Associate 
Ophthalmic Technician 
Ophthalmic Technician 
Ophthalmic Technician 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Eye Services Section 



INSTITUTE AND LOCATION 

, NEI, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



1.41 



PROFESSIONAL: 



0.41 



CHECK APPROPRIATE BOX{ES) 

[x] (a) Human subjects 
[x] (a1) Minors 
□ (a2) Interviews 



OTHER: 



1.0 



□ (b) Human tissues □ (c) Neither 



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

The visual function of patients with ocular diseases or lesions in the visual pathways and of normal subjects 
is measured objectively using electrophysiological techniques. The data are correlated with those obtained 
with psychophysical tests of visual function. The results contribute to the diagnosis of ocular and neural 
disorders that affect vision and are needed to characterize their nature and evolution. They are also valuable 
in the assessment of the effects of different forms of treatment on the outcome of these diseases. 



264 



PHS 6040 (Rev. 5/92) 



NEI Annual Report— FY 1993 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 



Clinical Protocol Numbers 

91-EI-26 

82-EI-55 



Objectives 

The aims of this project are (1) to apply and develop 
electrophysiological techniques for the study of 
visual function in the clinical setting, (2) to charac- 
terize the normal electrical activity of the human 
visual system, and (3) to analyze the patterns of its 
alteration in ocular diseases and lesions of the visual 
pathways. 

Methods 

The electrophysiological techniques employed 
involve recording potentials generated by the retinal 
pigment epithelium (electrooculogram), the neural 
retina (electroretinogram), and the central visual 
pathways (visually evoked potentials [VEPs]). These 
potentials are elicited by unstructured stimuli (Ganz- 
feld full-field or focal stimulation) and spatially 
structured stimuli (sinusoidal gratings or checker- 
board patterns). 

Major Findings 

The transient pattern reversal VEP elicited by stimu- 
lation of the central visual field is characterized by a 
typical negative-positive-negative (NPN) waveform. 
Previous reports using hemifield stimulation suggest 
that the peaks similar to those elicited by full-field 
stimulation arise primarily from the ipsilateral 
electrode sites. In this study, we investigated the 
contributions of both ipsilateral and contralateral 
electrode sites to the origin of the full-field VEP 
peaks. Fifty normal human subjects were studied. 

VEPs elicited by the contrast reversal of a 4 
cycles/degree sinusoidal grating were recorded over 
five electrode sites 5 cm above the inion (midline) 
and 5 cm (occipital) and 10 cm (temporal) to the left 
and right of the midline. VEPs were elicited by 
binocular and monocular stimulation of the full (26° 
X 20°) field and of the left and right (13° x 20°) 
hemifields. 

VEPs elicited by stimulation of a hemifield have 
been described as consisting of NPN waveforms over 
the ipsilateral electrodes and variable low-amplitude 



responses of opposite polarity (i.e., surface-negative) 
over the contralateral electrodes. However, in our 
study, hemifield stimuli elicited asymmetrical sur- 
face-positive waveforms on both sides of the midline 
in most cases (92%). The amplitude of these wave- 
forms was larger over the electrodes ipsilateral to the 
stimulated hemifield ("paradoxical" lateralization). 
Their main positive peak occurred earlier over the 
electrodes contralateral to the stimulated hemifield. 
The peaks of the fiill field VEP at each electrode site 
were generated by the algebraic sum of the peaks of 
the hemifield VEPs. This sum of two hemifield 
responses closely matched the full-field VEP, with 
negative peaks being frequently generated by a single 
hemisphere. A summation of two waveforms, which 
very frequently show the same surface-positive 
polarity and are generated by stimulation of each 
hemifield, generates the peaks of the full-field VEP. 

The symmetry of pattern-reversal VEPs recorded 
to the left and right of the midline has been proposed 
as a valuable estimator of the functional integrity of 
the visual pathway. We explored the variability of 
VEP symmetry and analyzed the contribution of both 
eyes and both hemispheres to this symmetrical 
response. Fifty normal human subjects underwent an 
ophthalmologic examination, including a 30° visual 
field test (static perimetry). Transient VEPs elicited 
by the contrast reversal of a 4 cycles/degree sinu- 
soidal grating were recorded over five electrode sites 
5 cm above the inion. VEPs were elicited by binoc- 
ular and monocular stimulation of the fijll (26° x 
20°) field and of the left and right (13° x 20°) 
hemifields. 

Full field stimulus. — After binocular stimulation, 
mean VEP amplitudes were symmetrica] about the 
midline. However, asymmetries in the amplitude of 
VEPs were seen in individual subjects. Therefore, 
95% tolerance intervals about the mean amplitude 
differences (left lead minus right lead) are large 
(-11.42 to 9.55 nV for the occipital sites and -4.80 
to 5.18 |iV for the temporal sites). Monocular 
stimulation of either OD or OS generated larger 
mean amplitudes over the ipsilateral electrodes. This 
voltage difference was not large but significant (p < 
0.001). 

Hemifield stimulus. — VEPs elicited by stimulation 
of a hemifield were asymmetrical. They consistently 
showed larger amplitudes over the ipsilateral elec- 
trodes (paradoxical lateralization). The sum of the 
two asymmetrical hemifield responses was synmietri- 



265 



Ophthalmjc Genetics and Ciinical Services Branch 



^fEI Annual Report — FY 1993 



ca] and closely matched the full-field VEP. Our 
results indicate that the sum of the asymmetrical 
contribution of either hemisphere and either eye are 
responsible for the symmetrical VEP elicited by 
binocular stimulation with a full-field sfimulus. An 
asymmetrical full-field VEP may be seen in normal 
subjects and does not imply an abnormality in the 
visual pathways. 

Significance to Biomedical Research and the 
Program of the Institute 

Electrophysiological techniques are noninvasive 
methods useful in the diagnosis and management of 
ocular diseases and visual pathway lesions. In addi- 
tion to validating tests, the new techniques developed 
contribute to the elucidation of the pathophysiologi- 
cal mechanisms of visual disorders. 

Proposed Course 

We will continue clinical electrophysiological studies 
of visual function in diseases of the eye and visual 
pathways, introducing modifications expected to 
enhance the diagnostic value of the techniques 
described. 



NEI Research Program 

Rednal and Choroidal Diseases — Noninvasive 
Techniques in the Study of Retinal Disorders 

Strabismus, Amblyopia, and Visual Processing — 
Visual Processing and Amblyopia 

Publications 

Caruso RC, Reuter LM, Muller SF, Drews- 
Bankiewicz MA, Kaiser-Kupfer Ml: Origin of 
the peaks of the human transient pattern reversal 
visual evoked potential. Invest Ophthalmol Vis 
5a34(4)(suppl):1276, 1993. 

Reuter LM, Caruso RC, Muller SF, Drews- 
Bankiewicz MA, Bouzas EA, Kaiser-Kupfer MI: 
The pattern reversal visual evoked potendals: 
Symmetrical or asymmetrical. Invest Ophthalmol 
Vis Sci 34(4)(suppl):1276, 1993. 



266 



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



PROJECT NUMBER 



ZOl EY 00257-05 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Visual Function Diagnosis Service 



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

PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI 



Others: Miuiel I. Kaiser-Kupfer M.D. 

Tracy T. Nolan M.A. 

Dessie Koutsandreas C.O.A. 

Anne Randall C.O.M.T. 

Linda Goodman C.O.T. 



Chief 

Health Technician 
Ophthalmic Technician 
Ophthalmic Technician 
Ophthalmic Technician 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 



Eye Services Section 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



6.81 



PROFESSIONAL: 



0.15 



OTHER: 



6.66 



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 general service project provides diagnostic support for all research protocols conducted by the Clinical 
Sections of the NEI and other Institutes that require an assessment of visual function. Psychophysical and 
electrophysiological techniques are used to detect and quantify visual loss due to disorders of the ocular media, 
uvea, retina, optic nerve, and central visual pathways. 



267 



PHS 6040 (Rev. 5/92) 



OpfathalmJc Genetics and Clinical Services Branch 



NEI Annual Report — FY 1993 



Project Description 

Additional Personnel 

R. Patrick McDaniel Ophthalmic Technician, 

OGCSB, NEI 
Antoinette LaReau Ophthalmic Technician, 

OGCSB, NEI 
Sueli Muller Special Volunteer, 

OGCSB, NEI 

Objectives 

The aim of this project is to provide accurate mea- 
surements of visual function for the differential 
diagnosis of visual loss. The first step in this process 
is detection of a visual deficit (i.e., determining 
whether visual loss has occurred). The second step is 
the quantification of a detected deficit The third is 
an analysis of the characteristics of the visual deficit 
to determine the site of the lesion responsible for this 
symptom (topographic diagnosis). The final step is 
correlation with other clinical findings to ascribe the 
visual deficit to a given pathological process. 

Methods 

The psychophysical techniques employed include 
commercially available and laboratory-developed 
techniques for the measurement of visual acuity, 
visual fields, color vision, dark adaptation, spatial 
contrast sensitivity, and glare disability. 

The electrophysiological techniques used include 
recording potentials generated by the retinal pigment 
epithelium (electrooculogram), the neural retina 
(electroretinogram), and the central visual patiiways 
(visually evoked potentials). 

Major Findings 

During the period October 1, 1992, through Septem- 
ber 30, 1993, we performed tiie following tests: 
Kinetic perimetry 312 

Static perimetry 413 



Screening perimetry 102 

Manifest refraction 1,313 

Color vision tests 193 

Adaptometry 45 

Contrast sensitivity tests 18 

Glare disability tests 5 

Ganzfeld electroretinography I81 

Focal electroretinography 50 

Electrooculography 102 

Visually evoked potentials 117 

This represents an increase of 57% over the tests 
performed diuing the same period in Fiscal Year 
1992. 

Significance to Biomedical Research and the 
Program of the Institute 

This project provides all tests of visual function for 
patients who visit the NEI Eye Clinic. In the major- 
ity of ophthalmologic diseases, visual loss is the 
most meaningful finding. In most clinical research 
protocols involving diseases of the eye and visual 
pathways, visual deficit is used as an indicator of die 
progress of a disease or the effect of a treatment. 
Therefore, sensitive and accuriate measurements of 
visual function are essential for tiiese clinical re- 
search projects. 

Proposed Course 

The provision of clinical electrophysiological and 
psychophysical tests of visual function for patients 
with diseases of the eye and visual pathways will be 
continued. We will introduce modifications that are 
expected to enhance the diagnostic value of the 
techniques described. 

NEI Research Program 

Retinal and Choroidal Diseases — Noninvasive 
Techniques in the Study of Retinal Disorders 

Strabismus, Amblyopia and Visual Processing 

Visual Processing and Amblyopia 



268 



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



PROJECT NUMBER 



ZOl EY 00011-19 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Pigment Dispersion With and Without Glaucoma 



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: Lessie McCain 
Mark H. Scott 



R.N. 

M.D. 



Nurse Specialist 
Senior Staff Fellow 



OGCSB, NEI 
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: 



0.25 



PROFESSIONAL: 



0.15 



OTHER: 



0.1 



CHECK APPROPRIATE BOX(ES) 

[x| (a) Human subjects 
\x\ (a1) Minors 
[x] (a2) Interviews 



□ (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 without 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. 



269 



PHS 6040 (Rev, 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 
Additional Personnel 



Marvin Podgor 



M.S. 



Statistician, 
Biometry and 
Epidemiology 
Program, NfEI 



Clinical Protocol Number 

76-EI-189 

Objectives 

This project was designed (1) to compare patients 
with and without glaucoma who have pigment 
dispersion by documenting and following the clinical 
features and courses of disease and by evaluating 
performance on a variety of diagnostic tests; (2) to 
determine the presence of abnormal aqueous humor 
dynamics in glaucoma and nonglaucoma patients 
with pigmentary dispersion; and (3) to compare the 
association of pigment dispersion, with and without 
glaucoma, with possible genetic markers. 

Methods 

A complete evaluation includes the following: 
complete family history with detailed pedigree; best- 
corrected visual acuity with manifest refraction; slit- 
lamp biomicroscopy; visual field examination 
(Goldmann I-,e and l4e); q)planation Goldmann 
tension; photography of iris color, iris transillumina- 
tion, and Krukenberg spindle; A-scan, anterior 
chamber depth, and anterior chamber volume mea- 
surements; goniophotography; static perimetry; 
baseline tonography and water-drinking tonography 
1 hour later, when indicated; fasting blood sugar, 
when indicated; dilated ophthalmoscopic examination 
(using 2.5% phenylephrine and 1% cyclogel); and 
stereophotographs of the optic nervehead. 

Major Findings 

One hundred sixty-four patients were classified into 
three groups: (1) pigment dispersion syndrome (PDS) 
without abnormal ocular pressure, (2) PDS witli 
ocular hypertension, and (3) PDS with glaucoma 
(PDS-(-GL). Analysis of baseline characteristics with 
respect to anatomical and physiological parameters 
has yielded the following conclusions: 

1 . It appears that the majority of patients recruited 
have PDS with a benign course: They do not develop 
ocular hypertension or glaucoma. 



2. Consequently, family members of PDS patients 
should be alerted and appropriately screened. PDS 
may be familial and can show a dominant inheritance 
pattern. 

3. Analyses of graded iris transillumination, the 
amount of pigment deposited on the trabecular 
meshwork, and the anterior chamber depth have 
demonstrated no significant differences among the 
three categories of PDS. Thus, pigment deposited in 
the angle may be only a secondary factor adversely 
affecting an already compromised outflow facility 
that is primarily a result of open-angle glaucoma. 

4. It also appears that those patients who develop 
ocular hypertension and demonstrate early field 
changes can be managed medically by control of 
intraocular pressure and reversal of early field loss. 
Patients who develop glaucoma do not appear to be 
more difficult to treat than patients with open-angle 
glaucoma. 

5. The phenomenon of unilateral or asymmetric 
PDS, in which there is little difference between the 
measurements of the two eyes, is being investigated 
in followup studies. 

6. In our series, refinal detachment does not 
appear to occur with any greater frequency than with 
high myopia. A history of asymptomatic and nonpro- 
gressive peripheral retinal holes was noted in two 
patients. 

7. The data from this study have been computer- 
ized, and an indepth analysis is under way. 

Significance to Biomedical Research and the 
Program of the Institute 

These results may facilitate determination of the risk 
of the development of glaucoma for patients with 
pigment dispersion. Specifically, it may be possible 
to identify which features have predictive value in 
forecasting which PDS patients will develop visual 
field defects. In addition, the data will aid investiga- 
tion of the relationship of "pigmentary" glaucoma to 
tile known characteristics of open-angle glaucoma. 

Proposed Course 

This project will be continued for 3 more years to 
obtain additional data on the patients enrolled in the 
study and to recruit more patients to add to the 
knowledge about PDS. 

NEI Research Program 

Glaucoma — Other Glaucomas (Developmental, 
Congenital, and Infantile Glaucomas) 



270 



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



PROJECT NUMBER 



ZOl EY 00060-15 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Visual Function and Ocular Pigmentation in Albinism 



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: Lessie McCain R.N. 

Rafael Caruso M.D. 

Evrydiki Bouzas M.D. 

Malina Drews-Bankiewicz M.D. 



Nurse Specialist 
Visiting Scientist 
Visiting Scientist 
Visiting Associate 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
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, MD 20892 



TOTAL STAFF YEARS: 



0.31 



PROFESSIONAL: 



0.26 



OTHER: 



0.05 



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.) 

Patients with hypomelanotic disorders, such as ocular albinism, oculocutaneous albinism, Chediak-Higashi 
disease, Hermansky-Pudlak syndrome, and iris traasillumination defects, are being recruited to determine visual 
fiinction with these conditions and to evaluate the changes in visual function over time. Family members are 
evaluated in an attempt to determine factors which may identify the heterozygous state. 



271 



PHS 6040 (Rev. 5/92) 



Opfathalmjc Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

76-EI-207 

Objectives 

The objectives of this study are (1) to relate the level 
of visual function to the amount of ocular pigmenta- 
tion, especially iris and retinal pigmentation; (2) to 
correlate the amount of nystagmus with visual acuity 
and iris pigmentation; (3) to determine whether 
ocular pigmentation, visual acuity, and nystagmus 
change with age; (4) to identify the heterozygous 
state of family members; and (5) to determine 
whether abnormalities of crossing of the optic nerve 
fibers can be correlated with the lack of pigmentation 
and whether previous reports of crossing abnormali- 
ties can be confirmed. 

Methods 

For each patient, a complete family history with 
detailed pedigree is compiled and the following 
procedures are performed: best-corrected visual 
acuity near and at a distance with refraction; slit- 
lamp examination; psychophysical testing, including 
D-15 and Munsell 100 hue as well as rod and cone 
thresholds; dilated ophthalmoscopic examination; 
photography to document hair color, eye color, iris 
transillumination, and the status of the disc and 
macula; visually evoked response testing; and, in 
selected patients, contrast sensitivity measurements. 
Information on family members is collected by 
examination of best-corrected visual acuity, slit-lamp 
examination of iris, photography of iris transillumina- 
tion, and fundus examination when vision is not 
corrected to 20/20. 

Major Findings 

Major findings were as follows: 

1. Examination of 82 patients and family mem- 
bers indicated that transillumination of the iris may 
occur in the absence of recognized albinism. The 
pattern, which appears to be punctate, may be present 
in a diffuse manner or limited to the 6 o'clock 
sector. The finding is not associated with nystagmus. 



2. These patients presented with marked iris 
transillumination, reduced pigmentation of the 
fundus, and no nystagmus, but they had decreased 
visual acuity, which has improved in conjunction 
with an increase in the pigmentation of the fundae. 

3. Visually evoked responses were normal in 
some patients, but in a subset of albinos, there was 
evidence of nonceniform pattern of asynmietry due 
to the miswiring of the visual pathways. The low 
amplitude of the visually evoked potentials recorded 
in a consecutive series of patients shows the difQ- 
culties of studying the phenomenon in a clinical 
setting. 

Significance to Biomedical Research and the 
Program of the Institute 

These data may allow identification of the carrier 
state of albinism, which would be important in 
genetic counseling. Determinafion of whether the 
development of the fovea is abnormal in albinism, 
whether this abnormal foveal development is the 
cause of the decreased visual acuity in albinism, or, 
alternatively, whether decreased visual acuity is 
secondary to hypopigmentation and the resultant light 
scatter and glare may be possible. Collection of these 
data also will facilitate ascertainment of whether 
visual acuity improves with age and whether this 
correlates with changes in pigmentation. 

In addition, studies are being conducted to verily 
the reported findings of abnormalities of the aossing 
fibers, as measiu-ed by visually evoked responses, 
contrast sensitivity, degree of nystagmus, and amount 
of pigmentation. 

Proposed Course 

This project will be continued for 5 more years to 
obtain additional data. 

NEI Research Program 

Retinal and Choroidal Diseases — Developmental and 
Hereditary Disorders 

Publications 

Bouzas EA, Caruso RC, Drews-Bankiewicz MA, 
Kaiser-Kupfer MI: Evoked potential analysis of 
visual pathways in human albinism. Ophthalmol- 
ogy, submitted. 



272 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1. 1992 to September 30. 1993 



PROJECT NUMBER 



ZOl EY 00083-16 OGCSB 



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

Gyrate Atrophy of the C horoid and Retina and Other Retinal Degenerations 



PRINCIPAL INVESTIGATOR (List other professional personr,el below the Prmapal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI 



Others: Evrydiki Bouzas 
Lessie McCain 
Rafael Caruso 
Pushpa K. Sran 
Doris Collie 



M.D. 
R.N. 
M.D. 
M.D. 

A.A. 



Visiting Scientist 
Nurse Specialist 
Visiting Scientist 
Medical Officer 
Ophthalmic Technician 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) ' " " ' — 

Howard Hughes Medical Institute Laboratory and Department of Pediatrics, The Johns Hopkins University 
School of Medicine, Baltimore, MD (David L. Valle, M.D.) 



LAB/BRANCH ~~ ' 

Ophthalmic Genetics and Clinica l Services Branch 

SECTION 

Section on Ophthalmic Genetics 

INSTITUTE AND LOCATION "^ 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



1.15 



CHECK APPROPRIATE BOX(ES) 

HJ (a) Human subjects 
[x] (a1) Minors 
Q (a2) Interviews 



PROFESSIONAL: 



0.75 



OTHER: 



0.40 



n (t>) Human tissues □ (c) Neither 



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

Patients with gyrate atrophy of the choroid and retina are examined systematically to confirm the diagnosis. 
Skin fibroblasts from affected patients and family members are grown in tissue culture and assayed for 
ornithine aminotransferase activity. The results are evaluated for correlation with the presence of 
homozygosity or heterozygosity for the disease trait. Each patient is given a trial of pyridoxine to see whether 
serum concentration of ornithine can be reduced; if so, the patient is classified as a "responder," and treatment 
with pyridoxine is continued. Nonresponder and responder patients are then placed on a low-arginine, low- 
protein diet with supplemental amino acids and observed for arrest or improvement of die disease. If patients 
are not considered eligible for the diet, or if they ^pear unable to comply with the dietary regimen, we follow 
them to record the natural progression of the condition. Patients with other forms of retinal degeneration, such 
as retinitis pigmentosa, fundus flavimaculatus, juvenile retinoschisis, and Usher's syndrome, also are examined. 
The courses of their diseases are compared with those of gyrate ati-ophy patients. 



273 



PHS 6040 (Rev. 5/92) 



OpbtbalmJc Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 



Additional Personnel 

Laura Wozencraft 

J. Fielding 
Hejtmancik 

Susan Gentleman 



M.S. Genetic Counselor, 
OGCSB, NEI 

Ph.D. Medical Officer, 



LMOD, NEI 
Ph.D. Biologist, 

LRCMB, NEI 



Clinical Protocol Number 

78 EI-01 

Objectives 

This project is being conducted (1) to determine the 
biochemical processes responsible for the elevated 
plasma ornithine and the chorioretinal lesions that 
occur in gyrate atrophy (GA) of the choroid and 
retina; (2) to determine which patients respond to 
pyridoxine treatment with a decrease in plasma 
ornithine concentration; (3) to determine whether 
treating "responders" with pyridoxine and nonrespon- 
ders with an arginine-deficient diet will arrest the 
progress of chorioretinal atrophy; (4) to study the 
natural history of this condition when intervention is 
not undertaken and to determine the degree of 
heterogeneity; (5) to define the molecular mutations 
and compare the molecular defect with the clinical 
features of the disease; and (6) to characterize and 
follow the progression of lens opacities, obtaining 
lens specimens at the time of cataract extraction for 
protein analysis. 

Methods 

Patients suspected of having GA of the choroid and 
retina are examined according to a standard set of 
procedures to confirm the diagnosis. Plasma ornithine 
concentration is measured periodically. Punch biop- 
sies of the skin are grown in tissue culture, their 
ornithine aminotransferase activity is measured, and 
patient molecular defect is characterized. Complete 
evaluation of ocular function in these patients in- 
cludes best-corrected visual acuity, Goldmann visual 
fields, color vision, cone thresholds, dark adaptation, 
electroretinogram (ERG), foveal electroretinogram 
(FERG), electrooculogram (EOG), contrast sensitiv- 
ity, and Stiles-Crawford effect. 



Major Findings 

Gyrate atrophy (GA), a rare autosomal recessive 
disorder, is associated with hyperornithinemia, 
overflow ornithinuria, and a deficiency of activity of 
the mitochondrial enzyme ornithine-6- 
aminotransferase (OAT). Although rare, the condition 
has been described worldwide in all races. Thirty-six 
patients have been recruited and evaluated in this 
study. The patients' ethnic origins vary and include 
African-American, Asian Indian, English, Fiimish, 
German, Israeli, Lebanese, Portuguese, Scottish, 
Turkish, and Welsh. 

In this study, among 44 patients, 22 females and 
22 males range in age from 2.5 to 65 years, with 10 
children less than age 12 at the time of recruitment. 
Observations of these patients have enabled docu- 
mentation of both clinical evidence and laboratory 
heterogeneity. 

Analysis of the mutation that causes GA of the 
choroid and retina has been undertaken by 
Drs. David Valle and Grant Mitchell and colleagues 
of The Johns Hopkins University. They have ana- 
lyzed probands fi-om 72 GA pedigrees. No gross 
structural alterations of the OAT gene have been de- 
tected; 85% of the probands express nearly normal 
amounts of normal-sized OAT roRNA. The remain- 
der express little or no OAT mRNA (n = 5) or an 
mRNA with an altered size (n = 2). Western blot 
studies showed the OAT antigen to be absent in 67% 
of the mRNA+ mutants and all of the mRNA- 
mutants. A total of 14 mutations have been deline- 
ated at the molecular level: 10 missense mutations 
(Mil, R180T, L402P, C93F, Y55H, R154L, A270P, 
R271KL, G375V, and P417L/L437F); a single 
nucleotide deletion at cDNA position +159 (H53fs); 
an interesting in-fi-ame three-nucleotide deletion of 
A la- 184 (A185F0), and a nonsense mutation at a 
CpG dinucleotide (R396ter). 

The functional consequences of several mutations 
have been examined by substituting the mutations 
into otherwise wild-type OAT cDNA in the expres- 
sion vector P9 1023b and transfecting the recombi- 
nant constructs into CHO-Kl cells that lack endoge- 
nous OAT mRNA or protein. Three (R180T, L402P, 
A184D0) have been shown to encode a CRM+, 
enzymatically inactive protein, while Mil — as ex- 
pected for an initiation codon alteration — has a 
CRM- phenotype. Studies are under way to correlate 
mutational heterogeneity with clinical and biochemi- 
cal heterogeneity. 



274 



NEI Annual Report — ^FY 1993 



Ophthalmic Genetics and Clinical Services Branch 



The earliest clinical and electrophysiologic fea- 
tures were documented in the two youngest patients 
(ages 2.5 and 3 years). The minimal evidence of 
clinical retinal changes when significant reduction of 
rod and cone function is seen by electroretinographic 
studies is noteworthy. 

Clinical and biochemical evidence of genetic 
heterogeneity is present in these patients. Fewer than 
10% of patients have been reported to have a 
30-50% decrease in plasma ornithine following 
treatment with vitamin Bg. Only one of our patients 
showed an in vivo response to this treatment. Com- 
parisons of sibships reveal that there is a greater 
degree of interfamilial variability than intrafamilial 
variability. 

Whereas arginine is the precursor of ornithine in 
the metabolic pathway of ornithine metabolism, we 
have undertaken a dietary intervention study limiting 
arginine. Of 25 patients placed on a low-protein (i.e., 
low arginine) diet, all sustained significant reduction 
of ornithine during hospitalization; however, the diet 
was discontinued in 4 Finnish patients following 
their discharge because of poor compliance and in 7 
other patients because of a variety of factors. Of 15 
patients remaining on the diet, 4 have excellent 
control; 4, fair control; and 4, erratic control. One 
young child was followed for too short a period of 
time to assess control. Ophthalmologic evaluations 
are performed on all patients every 6 to 12 months, 
travel permitting. 

In the two patients with the best biochemical 
control for the longest time (11 and 12 years old, 
respectively), there was evidence of improved visual 
function. One patient, after being on the diet for 14 
months, showed improved dark adaptation and aver- 
age ERG and color vision. This improvement was 
sustained for 30 months, then the ERG amplitude 
showed a small but definite reduction. The second 
patient who had lowered plasma ornithine levels and 
who had been on the diet for 11 years showed 
progressive improvement in visual field and color 
vision and has since remained stable. A third patient, 
despite fair control, was stable for 36 months but has 
deteriorated for the past 18 months. It should be 



noted that she was the oldest patient and had the 
most advanced disease at the outset. Other patients 
followed for various periods of time currently appear 
stable. Of particular interest are the children who 
were ages 2.5 to 9 years old at the outset of diet. The 
results indicate that as a result of dietary intervention 
the course of the disease in the younger of each 
sibship has been improved, compared with that of the 
older sibling. 

All but one patient over age 1 1 have had progres- 
sive cataracts in the posterior capsule. They present 
a uniform histologic picture and can be identified by 
their characteristic pattern in image analysis. 

Significance to Biomedical Research and the 
Program of the Institute 

GA of the choroid and retina is the first isolated of 
the genetically determined severe retinal degenera- 
tions for which a specific biochemical marker and 
concomitant enzyme defect have been demonstrated. 
Designed to test the efficacy of treatment for this 
bUnding eye disease, this smdy will serve as a model 
for the investigation of other genetically determined 
retinal degenerations. Smdy of the two young 
patients is the best opportunity for the evaluation of 
diet control. This disease is a likely candidate for 
future smdies to begin gene therapy. 

Proposed Course 

This project will be continued for 3 more years to 
assess further the knowledge concerning the reduc- 
tion of ornithine to halt chorioretinal degeneration. 

NEI Research Program 

Retinal and Choroidal Disease — Development and 
Hereditary Disorders 

Publications 

Brody LC, Mitchell GA, Obie C, Michaud J, Steel 
G, Fontaine G, Robert M-F, Sipila I, Kaiser- 
Kupfer M, Valle D: Ornithine o-aminotransferase 
mutations in gyrate atrophy. J Biol Chem 
267:3302-3307, 1992. 



275 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl EY 00163-11 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

NIH Interinstitute Genetics Program: The Genetics Clinic 



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

PI: Muriel 1. Kaiser-Kupfer M.D. Chief OGCSB, NEI 



Others: 



Evrydiki Bouzas 


M.D. 


Mark Scott 


M.D. 


Lessie McCain 


R.N. 


Anren Li 


M.D. 


Laura Wozencraft 


M.S. 



Visiting Scientist 
Senior Staff Fellow 
Nurse Specialist 
Visiting Associate 
Genetic Counselor 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 

Interinstitute Medical Genetics Program, NIH 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 



Section on Ophthalmic Genetics 



INSTITUTE AND LOCATION 

NEI, NIH. Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.8 



PROFESSIONAL: 



0.3 



OTHER: 



0.5 



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.) 

The Interinstitute Genetics Program and the Genetics Clinic supported by the Clinical Center offer a 
multidisciplinary approach to patients with genetic disease (ZOl CP 05139-06 CEB). Involved in the program 
are researchers from all Institutes. Patients evaluated in the clinic represent a broad spectrum of genetic 
diseases. During the past year, approximately 200 persons seen represented about 60 distinct disease 
categories. Due to the high frequency of ocular involvement in many of the cases, almost all the patients were 
evaluated by Clinical Branch staff or were discussed in consultation. The clinic serves as a source of 
interesting case material concerning patients with inherited or developmental abnormalities of the visual 
system. 



276 



PHS 6040 (Rev. 5/92) 



NEI Annual Report — FY 1993 



Ophthalmic Genetics and Clinical Services Branch 



Project Description 

Clinical Protocol Number 

Interinstitute Medical Genetics Program 

Objectives 

The objectives of this Program are (1) to evaluate 
patients with ocular abnormalities associated with 
genetic disease in the context of a multidisciplinary 
approach to the patient; (2) to provide genetic 
counseling to patients at risk for inherited ocular 
disease; (3) to recommend and advise appropriate 
evaluation for the ocular problem; and (4) to provide 
training in the diagnosis, counseling, and treatment 
of individuals with or at risk for genetic disease, as 
well as in the research approach to genetic disease. 

Methods 

Referred patients are examined, and the appropriate 
diagnostic ophthalmologic workup is recommended. 

Major Findings 

1. Iris nodules were seen commonly in the classic 
cases of neurofibromatosis (NFl) and less frequently 
seen in patients with less-well-defined disease. They 
were seen rarely in patients with bilateral acoustic 
neuroma (BAN or NF2). Interestingly, in a series of 
14 consecutive patients with Cushing's disease, two 
patients (14%) had typical, unilateral lisch nodules. 
To our knowledge, the association of NFl on lisch 
nodules with Cushing's disease has not been de- 
scribed. The association of Cushing's disease and 
lisch nodules may represent a mild form of multiple 
endocrine neoplasia of the mixed type. It is possible 
that a common underiying mechanism leads to the 
overgrowth of melanocytes in the iris and cortico- 
trophs in the pituitary. Patients with NF2 showed 
increased frequency of posterior capsular cataracts, 
which serve as an excellent marker, being present in 
80% of individuals with NF2. A new finding is the 
association of peripheral cortical cataracts in 37.8% 
of NF2 patients. In a group of severely affected ^fF2 
patients, it appears that combined pigment epithelial 
and retinal hamartomas are also an ocular marker for 
NF2. In fact, there may be a predilection for the 
macula in some cases. 

2. Serious ocular complications were observed in 
13 long-term postrenal transplantation nephropathic 
cystinosis patients. These complications included 



decreased visual acuity and visual function, as 
measured by psychophysical and electrodiagnostic 
tests, band keratopathy, and posterior synechia. 
Corneal transplantation may be necessary in cases 
with debilitating symptoms from recurrent erosion 
after all other treatment modalities have failed. In 
two such patients, the corneal grafts have remained 
clear for as long as 6 years. 

3. Ophthalmic studies performed in a population 
of patients with endogenous Cushing's syndrome 
revealed that posterior subcapsular cataracts were an 
infrequent phenomenon compared with exogenous 
Cushing's syndrome. Although an uncommon find- 
ing, central serous chorioretinopathy was seen in 3 of 
60 patients (5%), suggesting that glucocorticoids may 
play a role in the development of the disease. 

Significance to Biomedical Research and the 
Program of the Institute 

Genetic and developmental anomalies of the eye are 
a major cause of blindness and visual disability; they 
are responsible for about 35% of the cases of blind- 
ness in developed nations. Involvement with the 
Interinstitute Genetics Program affords a systematic 
approach to studying these and other conditions 
associated with genetic diseases. 

Proposed Course 

The project is in a growth phase and will be expand- 
ing in future years. 

NEI Research Program 

Retina] and Choroidal Disease — Development and 
Hereditary Disorders 

Publications 

Bouzas EA, Kransewich D, Koufroumanidis M, 
Papadimitriou A, Marini JC, Kaiser-Kupfer MI: 
Ophthalmological examination in the diagnosis of 
Proteus syndrome. Ophthalmology 100:334-338, 
1993. 

Bouzas EA, Freidlin V, Parry DM, Eldridge R, 
Kaiser-Kupfer MI: Lens opacities in neurofibro- 
matosis 2: Further significant correlation. Br J 
Ophthalmol 77:354-357, 1993. 

Bouzas EA, Mastorakos G, Chrousos GP, Kaiser- 
Kupfer MI: Lisch nodules in Gushing disease. 
Arch Ophthalmol 111:439, 1993. 



277 



Opbtbaimjc Genetics and Clinical Services Branch 



^fEI Annual Report— FY 1993 



Bouzas EA, Mastorakos GM, Chrousos GP, Kaiser- 
Kupfer MI: Posterior subcapsular cataract is 
infrequent in endogenous Gushing syndrome. 
Invest Ophthalmol Vis Sci 34(4)(suppl):1064, 
1993. 

Bouzas EA, Mastorakos G, Friedmann T, Scott MI, 
Chrousos GP, Kaiser-Kupfer MI: Posterior 
subcapsular cataracts in endogenous Cusing 
Syndrome: An uncommon manifestation. Invest 
Ophthalmol Vis Sci 34:3497-3500, 1993. 

Bouzas EA, Parry DM, Eldridge R, Kaiser-Kupfer 
MI: Visual impairment in patients with neuro- 
fibromatosis 2. Neurology 43:22-623, 1993. 



Bouzas EA, Scott MH, Mastorakos GP, Chrousos 
GP, Kaiser-Kupfer MI: Central serous chorioreti- 
nopathy in endogenous hypercortisolism. Arch 
Ophthalmol, 111: 1229-1233, 1993. 

Kaiser-Kupfer MI, Bouzas EA: Ocular manifestations 
of metabolic disorders. Curr Opin Ophthalmol 
3:221-227, 1992. 

Mastorakos G, Bouzas EA, Burnier MN, Chrousos 
GP, Chrousos GA: Presence of immunoreactive 
corticotropin releasing hormone in the optic nerve 
but not the inflammatory infiltrate of allergic 
optic neuritis/encephalomyelitis. Invest Ophthal- 
mol Vis Sci 34(4)(suppl):1000, 1993. 



278 



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



PROJECT NUMBER 



ZOl EY 00211-08 OGCSB 



PERIOD COVERED 



October 1, 1992 to September 30, 1993 



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

A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine 



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

PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI 



Others: Lessie McCain 
Manuel Datiles 
Evrydiki Bouzas 
Mark Scott 
Anren Li 



R.N. 

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



Nurse Specialist 
Medical Officer 
Visiting Scientist 
Senior Staff Fellow 
Visiting Associate 



OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 

Human Genetics Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 
(William Gahl, M.D., Ph.D.) 



LAB/BRANCH 



Ophthalmic Genetics and Clinical Services Branch 



SECTION 

Section on Ophthalmic Genetics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.70 



PROFESSIONAL: 



0.45 



OTHER: 



0.25 



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.) 

Nephropathic cystinosis is an autosomal, recessively inherited storage disease in which noiy)rotein cystine 
accumulates within cellular lysosomes due to a defect in lysosomal cystine transport. Ocular manifestations 
include photophobia; crystal deposits in the cornea, conjunctiva, and iris; and depigmentation of the retina. 

Ten years ago, cysteamine, a free thiol that depletes cystine from cells, was introduced in the therapy of 
cystinotic patients. Although patients had improved growth and stabilized renal function, there was no 
noticeable effect on the accumulation of corneal crystals. Recent studies showed that corneal cells in tissue 
culture are readily depleted of cystine by the introduction of cysteamine, making feasible the use of topical 
ophthalmic cysteamine to circumvent the humoral route. After appropriate animal smdies to test for 
complications revealed none, we began a double-masked chnical trial to test the efficacy of topical cysteamine 
(0.1% and 0.5%) in humans. 

To date, in 14 of 29 young patients the code was successfully broken; of the 15 remaining, 2 died, 1 
discontinued medication, and 12 are still in the trial with poor compliance and have not been seen for 
followup. Because of the success in the younger patients, this study was expanded to include older patients, 
3 to 31 years of age. The findings have been most exciting: Twenty-three patients have shown a significant 
decrease in crystals in treated eyes as well as improvements in comfort, i.e., relief of pain and photophobia. 
This study has resulted in significantly improved quality of life for the successfully treated patients. Because 
of the success of this clinical trial, and evidence from the cysteamine-benzalkonium trial (Protocol Number 
93 EI-0230), the Food and Drug Administration has requested that all patients in this protocol be switched 
to cysteamine plus benzalkonium and receive medication in both eyes. Each patient then will be judged by 
a comparison with his or her own natural history. 



279 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 



Additional Personnel 
Ernest M. Kuehl 



Chief, Photography Section, 
OGCSB, NEI 



Clinical Protocol Number 

86-EI-62 

Objectives 

The purpose of this project is to test the efficacy of 
topical cysteamine in patients with nephropathic 
cystinosis. 

Methods 

Slit-lamp examination and photography of the cornea 
are performed by a masked observer to determine 
whether there is a difference in the quantity of 
crystals seen in the cornea. 

Major Findings 

Topical cysteamine eyedrops (0.5%) are well toler- 
ated. The crystal accumulation is reversible in very 



young patients, who do not have crystals packing the 
cornea, as well as in older patients in which the 
crystals pack the cornea. 

Significance to Biomedical Research and the 
Program of the Institute 

The continued accumulation of crystals in the cornea 
appears to lead to increasing discomfort in cystinosis 
patients, who develop severe photophobia with 
recurrent corneal erosions. Topical cysteamine 
treatment, which has been found to halt the process, 
has led to an improvement in the quality of life of 
these patients. 

Proposed Course 

This study will be replaced by a study in which the 
crystal accumulation will be compared with the 
natural history of the condition. 

NEI Research Program 

Corneal Diseases — Ocular Surface Problems GDrug 
Delivery and Toxicity) 



280 



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



PROJECT NUMBER 



ZOl EY 00282-01 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between t/ie borders.) 

Usher's Syndrome — Clinical and Molecular Studies 



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, NEl 



Others: J. Fielding Hejtmancik 
Mark H. Scott 
Rafael C. Caruso 
Laura A. Wozencraft 
Anita Pikus 



M.D., Ph.D. 

M.D. 

M.D. 

M.S. 

M.A. 



Medical Officer 
Senior Staff Fellow 
Visiting Scientist 
Genetic Counselor 
Chief, Audiology Unit 



LMOD, NEI 
OGCSB, NEI 
OGCSB, NEI 
OGCSB, NEI 
HS/NOB, NIH 



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: 



0.6 



PROFESSIONAL: 



OTHER: 



0.6 



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.) 

The purpose of this project is to document the clinical features of Usher's syndrome, to refine the localization, 
and eventually to isolate the genes causing this disease. 



281 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

93 EI-0161 

Objectives 

The objectives of this study are (1) to relate the level 
of visual fimction to the amount of ocular pigmenta- 
tion, especially iris and retinal pigmentation; (2) to 
correlate the amoimt of nystagmus with visual acuity 
and iris pigmentation; (3) to determine whether 
ocular pigmentation, visual acuity, and nystagmus 
change with age; (4) to identify the heterozygous 
state of family members; and (5) to determine 
whether abnormalities of crossing of the optic nerve 
fibers can be correlated with the lack of pigmentation 
and whether previous reports in abnormalities of 
crossing can be confirmed. 

Methods 

Included in the evaluation will be audiometric, 
vestibular, ophthalmologic, and electrophysiologic 
and electrodiagnostic testing. These clinical findings 
will help classify the features of the different types 
of Usher's syndrome, as well as correlate the 
phenotypic features with the genetic mutation. To 
identify the genetic mutation, we will study informa- 
tive families, collecting blood specimens from all 



available family members for studies that will utilize 
molecular technology developed for linkage analysis. 
In cases in which there are no other affected family 
members, blood specimens will be obtained to study 
specific gene mutations when the specific gene or 
genes for Usher's syndrome are identifiable. 

Major Findings 

The recruitment for this project has begun: 40 
patients have been recruited. Patients are being 
evaluated, and their blood specimens are being 
collected and maintained in the laboratory. Linkage 
analysis on these families has not yet begun. 

Significance to Biomedical Research and the 
Program of the Institute 

By molecular studies of patients with Usher's syn- 
drome, mutations may be correlated with clinical 
findings and genes responsible for Usher's syndrome 
may be defined, leading to the possibility of genetic 
therapy at some point. 

Proposed Course 

Patient recruitment into the study will be continued. 

NEI Research Program 

Retinal and Choroidal Disease — Development and 
Hereditary Disorders 



282 



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



PROJECT NUMBER 



ZOl EY 00283-01 OGCSB 



PERIOD COVERED 

July 13, 1992 to September 30, 1993 



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

A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine 



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, NEl 



COOPERATING UNITS (if any) 

Human Genetics Branch, National Institute of Child Health and Human Development, NIH (William A. Gahl, 
M.D., Ph.D.) 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 



Section on Ophthalmic Genetics 



INSTITUTE AND LOCATION 

NEI, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 



0.2 



PROFESSIONAL: 



0.1 



OTHER: 



0.1 



CHECK APPROPRIATE BOX(ES) 

Fxl (a) Human subjects 
[x] (a1) Minors 
□ (a2) Interviews 



□ (b) Human tissues □ (c) Neither 



SUMMARY OF W/ORK (Use standard unreduced type. Do not exceed the space provided.) 

Cysteamine ophthalmic drops prepared for commercial availability must pose no risk for contamination and 
subsequent infection. This study is designed to demonstrate conclusively that benzalkonium chloride plus 
cysteamine is a safe preparation that is effective when administered every waking hour to patients who have 
nephropathic cystinosis in corneal crystals. 



283 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

93 EI-0230 

Objectives 

The purposes of this study are to determine whether 
the addition of benzalkonium ctiloride to cysteamine 
eyedrops is a safe preparation and whether this 
preparation is effective in removing crystals from 
patients with cystinosis. 

Methods 

Thirty patients were to be entered into this study. 
These were nephropathic cystinosis patients for 
whom the code had been successfully broken in 
conjunction with protocol 86 EI-62. Each patient 
was randomized, with one eye always serving as a 
comparison to the fellow eye. One eye was treated 
with cysteamine alone; the second, with cysteamine 
0.5% plus benzalkonium 0.101%. The primary 
outcome parameter was the safety of the additive 
benzalkonium. There were periodic checks of retinas 
for irritation attributable to benzalkonium. Efficacy 
for cysteamine was evaluated over a 6-month period. 



Major Findings 

In the 20 patients who have been em-olled in this 
protocol so far, there is no evidence of toxicity from 
the addition of benzalkonium to the cysteamine eye 
drops. Furthermore, if used as required, the cyste- 
amine plus benzalkonium appears to be as effective 
as the cysteamine without benzalkonium. 

Significance to Biomedical Research and the 
Program of the Institute 

Ensuring that benzalkonium added to cysteamine 
eyedrops is not toxic but still effective will move this 
drug one step closer to new drug approval by the 
FDA. 

Proposed Course 

Since the toxicity has been proven to be nil and the 
drug is still effective, this protocol will be termi- 
nated. 

NEI Research Program 

Corneal Diseases— Ocular Surface Problems (Drug 
Delivery and Toxicity) 



284 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PHOJECT NUMBER 



ZOl EY 00284-01 OGCSB 



PERIOD COVERED 

October 1, 1992 to September 30, 1993 



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

Characteristics of Macular Scotomas in Patients With Primary Monofixation Syndrome 



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

PI: Mark H.Scott M.D. Senior Staff Fellow OGCSB, NEI 



Others: Rafael Caruso M.D. 

Muriel I. Kaiser-Kupfer M.D. 



Visiting Scientist 
Chief 



OGCSB, NEI 
OGCSB, NEI 



COOPERATING UNITS (if any) 

Marshall M. Parks, M.D. (Private Practice), Washington, DC 



LAB/BRANCH 

Ophthalmic Genetics and Clinical Services Branch 



SECTION 



Section on Ophthalmic Genetics 



INSTITUTE AND LOCATION 

NEI, NTH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 



0.175 



PROFESSIONAL: 



0.175 



OTHER: 



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.) 

The monofixation syndrome (MPS) is a defective form of binocular vision characterized by preservation of 
extramacular function with absence of macular fusion. Fusion is defined as the ability to perceive 
simultaneously similar images projected onto corresponding areas of each retina. The fusing of images is a 
binocular phenomenon that occurs in the higher-order parastriate and peristriate areas of the prestriate 
visuomotor cortex (Brodmann areas 18 and 19, respectively). Patients in this protocol have been examined 
by Goldmann perimetry and the Lancaster red-green test to map the facultative macular scotoma in the 
nonfixating eyes in patients with primary MFS, surgically corrected congenital esotropia, and anisometropic 
amblyopia. The characteristics of the scotomas in each population of patients will be compared. The results 
of this study will contribute to the understanding of primary MFS by testing the hypothesis that primary MFS 
is a mild expression of a gene or series of genes that causes congenital esotropia and that these genes exert 
their variable expression on the binocular neurons of the central macular fusion area. 



285 



PHS 6040 (Rev. 5/92) 



Ophthalmic Genetics and Clinical Services Branch 



NEI Annual Report— FY 1993 



Project Description 

Clinical Protocol Number 

93 EI -0067 

Objectives 

The objectives of this study are to plot the character- 
istics of macular scotomas in patients with primary 
monofixation syndrome (MPS) and to gain data to 
test the hypothesis that such MPS scotomas may 
result from expression of a particular gene or series 
of genes that cause congenital esotropia. 

Methods 

Patients entering the study undergo a complete 
ophthalmic examination by Goldmann perimetry. By 
use of the Lancaster red-green test, the facultative 
macular scotoma is mapped out in the nonfixating 
eyes of patients with primary MPS, surgically cor- 
rected congenital esotropia and anisometropic 
amblyopia. The kinetic mode of the perimeter is 
used to plot the size and shape of the scotoma; the 
static mode of the perimeter is used to determine the 
depth of suppression within the scotomatous region. 
Lancaster red-green tests both standard and auto- 



mated versions also are used to plot the size of the 
scotomas. The characteristics of the scotomas in 
each population of patients can then be compared 
with each other. 

Major Findings 

Although the study is in its preliminary phases, it has 
been possible to plot the scotomas as planned. 
Analysis of the data awaits further recruitment. 

Significance to Biomedical Research and the 
Program of the Institute 

A better understanding of mechanisms of develop- 
ment of scotomata will help in elucidating the 
etiologies of certain potential blinding conditions 
such as amblyopia 

Proposed Course 

Work will continue with the completion of the 
analysis of data from patients who have been re- 
cruited into the study. 

NEI Research Program 

Developmental and Strabismus 



286 



Index 



Index 



Acetazolamide 99 

Acidic fibroblast growth factor 174 

Action potentials 226 

Acyclovir 83 

Adaptometry 262 

Adhesion molecules 59 

Aggregation 139 

AIDS (acquired immune deficiency syndrome) 

43, 45, 57 
Albinism 248, 272 
Aldehyde 

dehydrogenase 168 
reductase 189 
Aldose reductase 43, 131, 133, 189, 194 

inhibitor(s) 44, 131, 149, 187, 189, 194, 251 
Animal model 126 

autoimmune disease 44 
diabetic 251 
dog 44 

beagle 190 
diabetic 194 
galactose-fed 187, 190 
galactosemic 251 
monkey 220, 239, 242 
macaque 48 

retinas 50 
Rhesus 43, 225 
mouse 

Balb/c 82 

C3H-hen, EAU 104 
CD-I 82 
chromosome 214 
chromosomal locations 214 
EAU model 58, 108 
mutant 202 

ocular toxoplasmosis 59 
transgenic 67, 168, 174, 205 
rat 

diabetic 194 

retinopathy 43, 131 
EAU 108 
galactose-fed 150 
galactosemic model 149 
lens 194 

Lewis 116, 126,220 
B cells 202 



EAU 58, 61, 104, 119 

Royal College of Surgeons (RCS) 52 

transgenic 44, 67, 155, 180 

uveitis 99 
Anterior chamber 

anomalies 260 
Anticataract agents 44, 143, 146, 187 

drugs 131 
Antigen(s) 

cross-reactive 220 

S- 115, 116 
Antisense ribozymes 205 
Antiviral therapy 82 
Apoptosis 211 

Aqueous humor dynamics 270 
Arginine-deficient diet 274 
Autoimmune diseases 

predisposition 67 
Autoimmune inflammation 

etiology 220 
B 

Benzalkonium chloride 284 
Bilateral acoustic neuroma 248, 277 
Binocular alignment 225 
Biochemical 52 
Bionutrition 52 
Brain 

behavior control 226 

mechanisms 43, 225 

c 

Cadherins 171 

Cataract(s) 43,45, 131, 135, 139, 189, 247, 
256 (see Congenital cataracts) 

clinical grading 253 

development 146 

formation 143, 253 

Marner 171 

mature proteins 52, 136 

sugar 149 
Cataractogenesis 247 
Cell 

adhesion 59 

molecules 104, 171 

cycle regulatory protein, cyclin B 158 

flare meter 45 
Cell-cell communication in the lens 177 
Cellular differentiation 44, 155 



289 



Index 



NEI Annual Report— FY 1993 



Cerebra] cortex 233 
Chaperones 202 
Chromosomal locations 

human 214 

mouse (see Animal model, mouse) 
Clinical 

immunology 57 

services 249 
Collagen dysgenesis 92 
Color vision 262 
Combination therapy 61 
Complications from contact lenses 251 
Cone neuron development 45 
Cones 

blue-sensitive 50 

green-sensitive 50 

red-sensitive 50 
Congenita] cataracts 133 

esotropia 286 

hereditary cataracts 258 
Contrast sensitivity 45 
Cornea 168 

cyrstals in 280 

disease 247 

endothelium 168 

epithelium 168 

healing defects 149 
Coronaviruses 82 
Corticosteroids 126 
Cortisol 99 

Crossing of the optic nerve fibers 272, 282 
Cryopreservation 181 
Crystallin 45 (see Human A-crystallin) 

a-crystallin 131, 146, 162, 183 

oB-crystallin 59 

p-crystalUn(s) 43, 131, 138, 163 

PB2 crystallin 143 

6-crystallin gene 163 

^-crystallin 146 

Q-crystalUn 164 

aggregation 135 

enzyme- 168 

J- 164 

S- 164 
Cushing's syndrome 277 
Cyclins 44 
Cyclosporine 123, 126 

A 61 
Cysteamine 249 

eyedrops 284 

topical 280 



Cytokines 59, 76. 104 
Cytomegalovirus (CMV) 82 

retinitis 43,45, 57, 113 
Cytoskeletal proteins 165 
Cytotoxic agents 126 
D 

Depth vision 225 
Dexamethasone 61 
Diabetes 67, 133, 189 
Diabetic complications 131, 187, 194 

retinopathy 44, 149, 190 
Diagnosis 99 

noninvasive methods 262 
Dideoxyinosine(ddl) 

2', 3'- 102 
Diet 

low-arginine 275 
Dietary intervention 45, 248 
Distal promoter (see Promoter, distal) 
E 

EBV-transformed B cells 202 
Electrical 

activity of the human visual system 265 

stimulation 242 
Electrodiagnostic information 249 
Electrophysiological techniques 265, 268 
Embryonic stem cells 183 
Embryos 180 

Endothelial abnormalities 251 
Endotoxin-induced uveitis (EIU) 61, 92, 99 
Enzyme digestion procedure elastase 149 
Experimental autoimmune uveitis (EAU) 45, 
58, 64, 69, 92, 104, 108, 126, 202, 220 
Extracapsular cataract extraction 256 
Eye development 175 

physiology 67 
Eye movement(s) 239 

control 44 

rapid 43 

saccadic 43 
F 
Fatty acid-binding 

proteins 211 

site on IRBP 202 
FK 506 99, 120 

Fluorescence energy transfer 202 
Fluorescent fatty acid analogs 202 
Fluorouracil 99, 119 
Frontal eye field 227 



290 



NEI Annual Report— FY 1993 



Index 



GABA-agonist 240 
Galactose 44 
Galactosemia 189 
Ganciclovir 43, 113 

slow-release implant 45, 57 
Gene(s) 

Y-crystallin 171 

expression 44, 59, 205, 247 

knockout 183 

lens 177 

regulation 45, 155, 162 
mechanism of 158 

retina-pigment epithelium complex 21 1 

retina-specific genes 45, 199 

therapy 43, 45, 46, 199, 208 (see Gyrate 
atrophy) 
Genetic(s) 

counseling 272, 277 

disease 277 

disorders 43 

engineering 44 

ophthalmic 247 
Genomic manipulation 155 
Genotyping 139 
Glare testing 45 
Glaucoma 260, 270 
Glutathione 143, 146 
Graft rejection 

immunology of 119 

suppression of 1 19 
Growth factors 89 
Gyrate atrophy 45, 46, 247, 274 

gene therapy 58 
H 
Heat shock protein 202 

HSP70 116,202 
Hereditary diseases 211 
Herpes simplex virus type 1 (HSV-1) 82 
Histidine 43, 133 
HIV (human inmiunodeficiency virus) infection 

102 
Homologous recombination 183 
Horizontal disparity in the images 43 
Human otA-crystallin 170 

chromosome 214 

frontal eye field 233 

kidney cortex 194 

retinal pigment epithelial (RPE) cell 89 

S-antigen 69, 123 



Immune responses 69 
Immunology 

experimental 58 
Immunomodulation 126 
Immunopathology 

experimental 59 
Immunoregulation 58 
Immunosuppressive agents 92 

treatment 61 
Immunotherapy 108 
Implantable slow-release device 113 

(see ganciclovir slow-release implant) 
Inflammatory mediators 89 
lasulin-like growth factor [IGF]-1 208 
Intercellular adhesion molecule 1 (ICAM-1) 59 
Interferon 76 

gamma (IFN-y) 59, 67 
Interinstitute Genetics Program 248 
Interleukin 1 (IL-1) 59 

2(E.-2) 76 
receptor 45 

6(IL-6) 59,76 
Interphotoreceptor 

matrix 208 

retinoid-binding protein (IRBP) 69, 116, 
199, 205 
Intraocular 

inflammation 1 19 

lymphoma 99, 119 

promoter 205 

turnover 202 
Irido-comeal-endothelial (ICE) syndrome 260 
K 

J 

J-crystallin (see Crystallin, J-) 

L 

Laser examination 113 

Lens 44, 52, 143 

biology 131 

cell differentiation 158, 170 

crystallins 44, 131, 146, 162 

development 162 

materials 256 

opacities 247, 274 

organ culture 131 

structure and function 170 
Lewis rat (see Animal model, rat) 
Leukoregulin 83 



291 



Index 



^fEI Annual Report— FY 1993 



Linkage analysis 258, 282 

Linomide 58, 70 

Lipid peroxidation 52 

Lipopolysaccharide G-PS) . -,116 

LOCS-2 grading system 45 

Long QT syndrome 139 

Luminance and chromatic increment thresholds 

262 
Lymphocyte function-associated antigen 1 

(LFA-1) 59 
Lymphokines 92 
M 

Macaque retinas (see Monkey, Macaque) 
Macrophage migration inhibitory factor 171 
Macular scotomas 286 
Magnetic resonance imaging 187 
Magnetization transfer contrast 190 
Marner cataract (see Cataract(s)) 
Metals 131 

Methylprednisolone 120 
Microinjecting DNA 180 
Miniosmotic pump 61 
Molecular 

biology 57, 168, 170, 208 

function 155 

genetic techniques 258 

genetics 155, 208, 209 

interactions 247 

markers of differentiation 171 

structure 155 
Molteno glaucoma implant 57, 1 19 
Monoclonal 

antibodies 104 

antibody therapy 45 
Monofixation syndrome 286 
Mouse chromosome (see Animal model, mouse) 
Movement 

visual control 43 
Myotonic dystrophy 139 
N 
NADPH 147 

-dependent enzymes 194 
Nephropathic cystinosis 249, 277, 280, 284 
Neural crest 260 
Neurofibromatosis 247, 277 
Neuronal discharge 226 
Nitric oxide 61 

Nonradiative energy transfer 202 
Nystagmus 272 



O 

Objective systems 45 

Ocular autoimmune diseases 64 

complications of diabetes 43 

development 209 

diseases 199 
inherited 277 

hypertension 270 

immunomodulation 58 

inflammation 104 

inflammatory diseases 45 

pigmentation 282 
Opacities in the human lens 45 
Ophthalmic drugs 187 
Oral tolerization 57 
Ornithine aminotransferase 

gene 248 
Ornithine-6-aminotransferase (OAT) 274 

gene 57 
Oxidation 131 

thiol-dependent metal-catalyzed 135 
Oxidative stress 143, 146 
P 

Parietal cortex 233, 239 
Pathology 

ophthalmic 249 
Peptide 1169-1191 202 
Perimetry 262 
Peripheral vision fields 239 
PET scaiming 226 
Phospholipids 214 
Photophobia 280 
Photoreceptor 

cell 214 

-specific genes 199 

site 217 
Pigment dispersion syndrome (PDS) 270 

epithelium 211 

derived factor (PEDF) 45, 199, 208 
Pigmentation 

iris 272 

retinal 272 

ocular 272 
Plasma ornithine 274 
Polyol 133 

formation 190 

pathway 194 
Post-transcriptional regulation 214 
Primate visual system 50 



292 



NEI Annua] Report — ^FY 1993 



Index 



Proliferative retinopattiy 187 
Promoter 

oA-crystallin 174 

distal 217 

proximal 217 
Protein 72-kD 202, 203 
Proto-oncogene(s) 44, 158 

int-2 174 
Psychophysical 

diagnostic information 249 

techniques 262, 268 
Pyridoxine treatment 274 

Q 
R 

Rapamycin 45, 61 
Regulatory 

cell in the retina 76 
element 44 
Retina 52,211 

-specific genes (see Genes) 
Retinal 

11 -CIS 202 

antigen-specific T-cell lines 108 
degeneration 52,202,211 
degenerative disorders 79 
disease 247 
fatty acid defea 200 
genetic 248 
tubulin defect 200 
vasculature 149 
Retinal pigment epithelium(al) (RPE) 45 52 
59, 102, 202, 214 
cell(s) 76,79, 119 

transplantation 79 
-specific 

expression 214 
gene(s) 45, 199 
transplants 45 
Retinitis 119 

CMV (see Cytomegalovirus (CMV), retinitis) 

pigmentosa 247 
Retinoblastoma 59 
Retinoic acid 171 
Retinoid metabolism 202 
Retinyl palmitate 202 
Rostral superior colliculus 242 
RPE (see Retinal pigment epithelium[al]) 



S-Ag 115, 116 

promoter sequences 217 
-induced uveitis (see uveitis, S-Ag-induced) 
Saccadic eye movements 225, 233, 242 
Sampling intraocular tissue 1 19 
Sarcoidosis 99 
Selective visual attention 226 
Shift of attention 239 
Single neuron recording 233 
Site-directed mutagenesis 43 
Sorbitol dehydrogenase 43, 131, 133 
Spatial vision 262 
Spectral property transformations 48 
Stabilization of posture 44, 225 
Stimulus-dependent messages 226 
Sugar cataracts (see Cataract(s)) 
Superior colliculus 239 
T 
T lymphocyte(s) 

role of 64, 92 
T-cell receptor (TCR) 
genes 58 
therapies 64 
Targeting veaor 183 
Temporal code 44 
patterning 226 
Temporally modulated code 229 
Tolerance 220 
Toxic compounds 89 
Toxoplasmosis 45 
ocular 59 
model 45 
Trabeculectomy 99 
Tumor necrosis factor (TNF-a) 59, 76 
U 
Usher's syndrome 139, 282 

type I 43, 131, 139 
Uveitis 67, 92, 99, 123, 126, 214, 220 
IRBP-induced 58 
S-Ag-induced 58 
Uveitogenic antigens 126 
Uveitopathogenic determinant 202 
Uveoretinitis 61 
V 

Virology 59 
Virus infections 82 



293 



Index 



NEI Annual Report— FY 1993 



Visual 

cortex (areas VI, V2. V3, and V4) 229 

deficit 268 

loss 

differential diagnosis 268 

motion 226 

motor behavior 225 

pathway 48 

abnormalities 247 

perception 44, 226 

sense 43 

stimuli 242 
Visually evoked potentials (VEP) 248, 265 
Visuospatial attention 233 
Vitritis 119 

w 

X 

X-linked agammaglobulinemia 139 
Y 



294 




Hoft '?^^"'er Drive 



3 1