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,\  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 


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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. 


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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. 

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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. 


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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 


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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 


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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  0  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  0  ^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 


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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 


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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. 


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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  Ocular  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. 


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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. 


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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 


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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- 


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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  Program  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. 


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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 


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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 


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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 


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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 


PHS  6040  (Rev.  5/92) 


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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 


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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. 


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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: 


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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 


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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  and  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 


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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 


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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  Cell  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. 


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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 


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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. 


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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. 


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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. 


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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 


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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. 


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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. 


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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 


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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 


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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 


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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  Genetics  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 


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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 


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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 


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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  Choroid  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  Clinical  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