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PROGRAM ACTIVITIES

MTIOML INSTITUTE OF NEUROLOGICAL DISEASES AND STROKE

Fiscal Year I97I

PART II

ANNUAL REPORT

July 1, 1970 through June 30, 1971

Associate Director, Collaborative and Field Research

National Institute of Neurological Diseases and Stroke

National Institutes of Health

Through October 31, 1970, leadership of this program area was provided
on an acting basis by Dr. Eldon L. Eagles, Deputy Director, NINDS, On
November 1, Dr. Warren V, Ruber was appointed Associate Director. Dr. Ruber
was formerly Project Director of the American Neurological Association's
Joint Conmiittee for Stroke Facilities, and had served as Chief of Neurology
in the Veteran's Administration's Department of Medicine and Surgery.
Dr. Ruber's appointment marked a re-emphasis of the role of collaborative,
directed, and field- type research in the overall effort to accomplish the
scientific objectives of this Institute. The intent of this report is not
to encapsulate items of information found in the separate branch summaries
but to provide an overview of the significant accomplishments and problems
of the Institute's Collaborative and Field Research programs during Fiscal
Year 1971, and to look briefly, though broadly, toward the future of these
programs .

PROGRESS AND ACCOMPLISHMENTS, FY 1971

Increased funds provided by the President's budget and congressional
supplements permitted orderly program expansion in epilepsy, funding for a
pilot study of cerebral death, and sufficleut support to prevent dislocation
in other programs. Without these increases significant program opportunities
would have been lost.

The Special Projects Branch continued to pursue studies of neurologic
disorders, concentrating on controlled clinical studies for the evaluation
of two experimental anticonvulsants and one marketed agent. These efforts
have demonstrated the value of blood level determinations as an adjunct to
anticonvulsant therapy and established methodologies for further anticonvul-
sant studies. The successful transition of Epilepsy Abstracts from a fully
supported Institute publication to a partially subsidized periodical available
by subscription has demonstrated that periodical's scientific merit and
value. The completion of the evaluation of the 15-year field follow-up of
head injured veterans of the Korean campaign, and the evaluation of data from
Phase I of the Head Injury Model Construction Program, mark the conclusion
of two important aspects of the Institute's directed Head Injury Program.
Funds must now be obtained and leadership recruited for continued efforts
in this important program area.

The process of planning and developing a collaborative study of the
criteria of cerebral death was accomplished by staff of the Office of the
Associate Director and the Head Injury Section of the Special Projects
Branch. With expert guidance from an ad hoc advisory committee, an
acceptable protocol was developed and a coordinator and collaborators were
selected. Contracts for the study will be awarded before July 1, 1971.

Results of controlled and limited studies using the assembled data base
have continued to come in to the Perinatal Research Branch. Subject area
task forces, in collaboration with the Epidemiological and Statistical
Advisory Committee, are redirecting staff effort and attention to larger,
more significant statistical analyses to identify major trends, developmental
patterns, and risk factors. Interesting and significant studies of the
influence of infectious diseases in pregnancy and childhood upon the
development of the child's central nervous system (CNS) continue.

A multi-institutional study to determine the feasibility of developing
an effective sensory prosthesis for the blind has been launched. Currently,
the study is focused on the problems of direct stimulation of visual centers
of the brain, including the safety thereof, and the reproducibility of
stimulation results. The future success of this project is enchanced in
large part by the careful, expert, and conscientious project direction being
provided by the Chief, Laboratory of Neural Control, Intramural Research,
and his assistant.

Epidemiologic studies of a selected group of acute and chronic nervous
system disorders continue. Short term studies, particularly of multiple
sclerosis, sub-acute sclerosing panencephalitis, and CNS tumors have pointed
the way toward a better understanding of the etiology of these diseases.

Collaboration with scientists working in a broad range of the basic
biological sciences and in clinical medicine has improved considerably our
understanding of the mechanisms and etiology of slow, latent and temperate
virus infections. Successful transmission of two sub-acute and chronic
degenerative diseases of the nervous system from man to primates has led to
the implication of suspect viral agents — a significant step in the march
toward understanding the etiology and thereby, hopefully, being able to
successfully treat or prevent such types of central nervous system disease.

PROBLEMS - FY 1971

Despite the successes and progress of the past year there are problems
affecting program development which remain to be solved.

Tight controls on full-time and permanent employment restricts the
recruitment of staff needed to implement and develop important new programs.
This has necessitated the most careful allocation of positions as vacancies
occur. Only through this mechanism can needed and important shifts in
priority be implemented.

Head and spinal cord injury has been recognized as a serious problem
for several years. Despite generous Extramural support and a vigorous
Intramural program, important scientific questions remain unanswered; these
must be answered if the goal of improved treatment, coupled with improved
prevention, is to be attained. Directed collaborative studies aimed at
elucidating the pathophysiologic responses of brain and spinal cord tissues
to trauma are urgently needed. Until additional funds are available and
adequate leadership can be found — and the two needs are interrelated — studies
in this important problem area cannot be developed.

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The significant FY 1972 budget decrease that has been imposed on the
Perinatal Research Branch (PRB) makes it exceedingly difficult to accomplish
two important tasks. The first is an orderly completion of the Collaborative
Perinatal Project. As a result of the cut, important activities in follow-up,
sample maintenance, and quality control at the collaborating institutions
and at the coordinating center in the PRB will be curtailed. The second task
which must be foregone is the planning for research projects on neurological
diseases of childhood; preliminary results of the Collaborative Perinatal
Project indicate these would be productive. Although an orderly decrease
in budget support and effort by the PRB was planned as the study phased out,
the immediate and drastic decrease at this time is quite damaging.

The Collaborative and Field Research Programs of NINDS are, by their
nature, heavily dependent upon continuing and active biostatistical
participation. The need for biostatisticians in these varied programs
goes deeper than simple part-time consultation. To be effective, the
statistical staff must be part of the research teams and relate closely to
them. Scientific program leaders, charged with the responsibility of
promptly producing evidence of research results to fully collaborate with
scientists outside of NINDS, must have the explicit and constant control of
the processing and analysis of the data upon which they rely. Without such
control they have experienced an inability to discharge these responsi-
bilities promptly and effectively. Therefore, it is imperative that such
a realistic relationship with the Office of Biometry, NINDS, be established.

LOOKING AHEAD

Fiscal Year 1972 promises to be a difficult year in terms of avail-
ability of resources to move forward with existing and planned programs.
Budget cuts will effect the Epilepsy, Head Injury, and Cerebral Death
Programs as well as the Collaborative Perinatal Project. Considerable
resourcefulness will be required to maintain programs which have been so
laboriously built and organized. In no way will it be possible to mount
new programs in stroke research, in disorders of communication, or to
pursue significant head and spinal cord injury research.

As data collection for the Collaborative Perinatal Project moves toward
completion, and is supplanted by less expensive, but promising, investi-
gations of neurologic disorders of childhood, funding required by the Peri-
natal Research Branch will diminish. It is anticipated that this will permit
the resumption of active, directed, collaborative research in the field of
head and spinal cord injury, and the commencement of similar efforts in
the fields of communicative disorders and stroke, with only relatively
minor increases in the overall funding level over the next five years .

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ANNUAL REPORT

July 1, 1970 through June 30, 1971

Special Chronic Disease Studies for Collaborative and Field Research

National Institute of Neurological Diseases and Stroke

National Institutes of Health

Study of Child Growth and Development and Disease Patterns in Primitive
Cultures, and Slow, Latent and Temperate Virus Infections

This section has continued to focus its attention on long term
studies of the human biology of the many vanishing primitive societies. Our
laboratory research and that of our numerous collaborating investigators is
directed to problems which have been phrased under this theme. The neuro-
logical development and learning patterns in children in diverse cultural
experiments in the human condition has been the major focus of attention.
The laboratory studies on human biology, genetics and associated molecular
biology, immunology, virology, and biochemistry have all been directed at
solving problems which have been carefully chosen from small isolated bands
still living in the primitive situation in which these problems may be more
appropriately studied than in larger civilized societies.

Our efforts to document the development and neurological patterning
in disappearing primitive cultures has resulted in the largest archive of
such documentation in the world. The collection and preservation of exist-
ing cinema data from Australian aborigines, New Guinean, Oceanic and African
aborigine groups, and American Indians, as they live as hunter-gatherers or
primitive hoe-and-digging-stick agriculturists provides the only such docu-
mentation of the life and behavior of man, as he most probably lived and
evolved for over 997c, of his evolutionary history, or about one million years.

Kuru, a new disease which we discovered and described in the New
Guinea Highlands fourteen years ago, has been the first subacute and chronic
degenerative disease of the nervous system of man with a transmission model
in animals and an established virus etiology. From analogy with kuru, we
selected Creutzfeldt-Jakob disease and other forms of spongiform encephalo-
pathy as likely slow infections. The "hunch" proved right: Creutzfeldt-
Jakob disease has now been transmitted to chimpanzees, independently from
ten patients, and serial chimpanzee to chimpanzee transmission has also been
successful through the third passage. The agent, as that of kuru, is fil-
terable. During this year significant breakthroughs were achieved in the
study of these two CNS diseases with the successful transmission of both
kuru and Creutzfeldt-Jakob disease from human patients to 3 species of New
World monkeys — the spider monkey (Ateles geof froyi) , the squirrel monkey
(Saimiri sciureus) and the capuchin monkey (Cebus sp.) . Creutzfeldt-Jakob
disease was transmitted from affected chimpanzee to yet another New World
monkey, the woolly monkey (Lagothrix lagothricha) , as well as to squirrel
monkeys. Serial passage and viral pathogenesis and characterization studies
have been expanded utilizing these more economically purchased and con-
veniently handled primates. These animals are also being used in isolation
studies being conducted on other human slovj-infections suspected of being
caused by viruses. It is significant to note that during the past year we
have received greater than 400 specimens from patients with CNS diseases.

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Of this number, approximately 50% (200) are brain biopsy and autopsy speci-
mens and between 50-60 of these are from patients with Creutzfeldt-Jakob
disease. We now believe that the two human diseases, kuru and Creutzfeldt-
Jakob disease, and the two similar animal diseases, scrapie and mink enceph-
alopathy, which we are also studying, form a group of diseases of similar
pathogenesis, which we have called the spongiform viral encephalopathies.

Our work has continued to incriminate the measles virus as the cause
of SSPE, but we have not yet found evidence of a second virus in this disease,
With the return of Dr. David Asher, from 17 months work in various insti-
tutes in the Soviet Union, work has been begun on the problem of chronic
progressive forms of encephalitis caused by tick-borne encephalitis virus.
Several strains of the virus known to induce chronic infections have been
inoculated into rhesus monkeys. The continuation of these studies, in
collaboration with our colleagues in the Soviet Union, are providing working
models for the in depth studies to demonstrate chronic virus infection as
the cause of epilepsy partialis continua or focal epilepsy in children in
which viral- like inclusion bodies are found in brain cells. Further, we are
pursuing with our Soviet colleagues our studies on epidemic hemorrhagic
fever, of the nephro-nephritis type, which occurred in epidemic proportions
in civilians and troops during the Korean War and which still occurs at a
somewhat lower level of incidence at the present time.

Encouraged by our success with one of the presenile dementias and
several cases in the borderland between Alzheimer's and Creutzfeldt-Jakob
diseases, we are concentrating on the other presenile dementias and senility
with particular emphasis on Alzheimer's and Pick's diseases, parkinsonism-
dementia and senile dementia. Our work on the presenile and senile demen-
tias, the isolation of latent viruses from the central nervous system, and
comparisons of the many aspects of the pathological processes in kuru and
Creutzfeldt-Jakob disease (including the presence of amyloid (senile)
plaques and extensive glial hypertrophy with fibrillary tangles in the astro-
cytes) has led us to be increasingly concerned with possible virus involve-
ment in certain aspects of senile dementias and normal aging.

In our attempts to establish infection as the etiology of other dis-
eases of the CNS of man we have successfully isolated in tissue culture a
strain of adenovirus from the brain of a patient with lymphosarcoma. We
have shown the virus to be closely related to candidate adenovirus type 32.
Furthermore, we have recently demonstrated Cowdry type A intranuclear inclu-
sion bodies, vjhich by EM contain papovavirus-like virions, in in vitro ex-
plant cultures of human brain from a patient with progressive multifocal
leucoencephalopathy .

We are trying to establish the range of cases with spongiform enceph-
alopathy which may be transmitted; other diseases we are studying by in vitro
cultivation of brain and other tissues, and with animal (including monkeys
and apes) inoculations, are multiple sclerosis, amyotrophic lateral scler-
osis (both sporadic and familial types) , parkinsonism-dementia complex, in
both the U.S. and Guam, essential parkinsonism, myoclonic epilepsy,
Schilder's disease, metachromatic leucodystrophy, and progressive supra-
nuclear palsy. In collaboration with Drs ., Michael Alpers and Malcom Simons

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of Australia investigations to seek the agent of kuru in cultivated leuco-
cytes of patients and of experimental chimpanzees are under way, and the
evaluation of the humoral (thymic) and delayed hypersensitivity (splenic)
immune status in kuru in man and in the experimental disease have been made.
Most significant has been our continued isolation of further strains
of hidden, masked, or latent viruses from surgically sterile brain and
other tissues of both our chimpanzees with transmitted neurological diseases
and of human patients. The list of these virus isolates from chimpanzees is
now over one hundred virus strains. These fall thus far into ten identified
new virus species. Six of these have been thoroughly investigated virolo-
gically and have been the subject of both graduate theses and papers in the
specialty journals.

All six viruses have been deposited in the American Type Culture
Collection. Two of the six viruses. Pan 1 and Pan 2, have now been class-
ified as simian foamy viruses type 6 and type 7, respectively. We have
demonstrated that these RNA viruses have an RNA-DNA dependent polymerase
suggesting a close relationship to the RNA oncogenic group of viruses.

In 1969 we isolated two strains of virus from the lungs and brain,
respectively, from sheep with progressive pneumonia in Montana. We have now
demonstrated that both viruses are serologically indistinguishable from
maedi, a lymphoreticular progressive pneumonia, and visna, a primary demye-
linating disease of sheep in Iceland. Moreover, visna, maedi and the virus
of Montana sheep progressive pneumonia are all RNA viruses associated with
an RNA-DNA dependent polymerase. Further characterization of these viruses
and their relationship to the RNA oncogenic group of viruses having these
properties are under way.

In many cases, two or more viruses have been isolated from the same
brain. We are forced to speculate that activation of these latent agents
may be a process responsible for some subacute or chronic neurological
diseases. We are attempting to study the activation of measles virus to
produce SSPE, of chickenpox virus to produce herpes zoster in later life,
of herpes simplex virus and the simian herpes viruses to produce central
nervous disease, and of a papova-like virus to produce progressive multifo-
cal leucoencephalopathy. Cytomegalo virus and EB (Epstein-Barr) virus
activation to produce the post-transplantation of "pump" syndrome in most
subjects receiving organ transplants has been studied by Dr. Lang at Duke
University. We are now interested in using this work as well as a model for
the chronic and slow infections with which we work. Our current hypothesis
is that similar processes may underlie the pathogenesis of kuru, Creutzfeldt-
Jakob disease, and other spongiform encephalopathies and, perhaps, even be
involved in some presenile and senile dementias.

We therefore continue attempts at isolating virus strains from long-
maintained sterile tissue explants and trypsinized cell suspensions, using
the techniques of cell fusion and co-cultivation. These are being applied
to the degenerative central nervous system disorders listed above, as well
as to other chronic diseases. The electron microscopy, in collaboration
with Dr. Peter Lampert, and the immunological techniques, including fluores-
cent antibody, are being used to localize virus-like particles, gamma glob-

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ulin, components of complement, and antigen-antibody complexes in tissue
and in sera of patients with these diseases; and extensive serological in-
vestigations of serum and spinal fluid from patients with these diseases for
antibodies against a wide range of microbial antigens has been continued.

Nutritional studies, and studies on reproduction and fertility, on
the selective advantage and establishment of genetic polymorphisms, of un-
usual and odd alternative ways of employing the central nervous system in
its higher cerebral function of language learning and use, computation
(number sense and calculation with a numbers system), psychosexual cultur-
ally-modified behavior, and cognitive style, are providing data on alterna-
tive forms of possible neurological functioning for man, of which we would
remain unaware and for moral, ethical and political reasons be unable to
produce or investigate in the clinic or laboratory, once the natural cul-
tural experiments in primitive human population isolates had all finally
been amalgamated into the modern civilized cultural veneer which is now
imposed upon almost all members of the community of man.

Growth and development studies in primitive cultures have yielded new
evidence for incredibly delayed puberty and slow growth rates in certain
peoples, particularly the short-of-stature populations in the Highlands of
New Guinea. Here we find people with the mean age of menarche over eighteen
years, and with male puberty of eighteen at twenty years. These are thus
the slowest growing populations on earth, with the most delayed puberty; two
thirds of their life span is spent in reaching maturity. Preliminary evi-
dence suggests high levels of pituitary growth hormone in such populations,
in both pituitary glands obtained at autopsy and in the serum. Growth rate
seems to be proportional and age of puberty inversely proportional to mean
adult stature.

Congenital mental defect and a wide range of neurological problems
associated with the most severe foci of endemic cretinism in the world, in
the Highlands of West New Guinea (West Irian, Indonesia) , are under further
investigation. Our investigation of an accumulation of cases of familial
periodic paralysis in certain of the Pacific Islands continues.

The discovery of new genetic factors, including haptoglobins and
hemoglobins, with elucidation of their biochemical structure and their later
use as markers in human population genetic studies, has been a by-product
of these investigations. Similarly, the discovery of immunologically virgin
populations without exposure to respiratory and enteroviruses which are
ubiquitous in the civilized world, has permitted: 1) fundamental investiga-
tion on the immune response in man; 2) investigations possible no where
else on the persistence of immune response after natural measles infection
and live attenuated measles virus immunization in the absence of circulating
virus, yielding data important to the diagnosis of understanding of delayed
slow measles encephalitis (subacute sclerosing panencephalitis, SSPE) , and
3) establishing the serological identity of the agent of the 1918 influenza
pandemic to aid in the genetic-historical elucidation of the serial mutation
of influenza virus. By virtue of limited travel during their entire life-
time and intensive exposure to their natural ecology, members of primitive
groups serve as unequaled sentinel populations for revealing the focal micro-

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bial agents that infect man in their environment. Thus, for infections
ranging from Chagas disease and toxoplasmosis to arbovirus infections, prim-
itive groups offer unusually fruitful subjects for investigation. In
toxoplasmosis, filariasis, yaws and malaria, as well as in the arbovirus
encephalitides , we have studies in progress.

Our studies on the antigenic composition of the virus causing the
1918-22 influenza pandemic and on the immunologic response to various close-
ly related influenza viruses, including the new 1968 Hongkong Asian influenza
(the latest A2 variant which might better have been designated A3) have now
been published. However, the opportunity of supervising and participating
in investigations of the influenza epidemic which swept through the Terri-
tory of Papua and New Guinea, and through West New Guinea in late 1959 has
brought additional material for further study on virus evolution of influen-
za into our laboratory. Incidental to this work our field investigations
also provided the opportunity to study new virgin soil measles epidemics
and to find new isolated populations free of many nearly ubiquitous res-
piratory viruses.

We planned, organized and selected participants for Theme III on
Pathogenesis of Slow Virus Diseases of the Central Nervous System at the
Vlth International Congress of Neuropathology in Paris in September, 1970.
This theme provided reviews and new concepts on virus-host relationships
and significant new data on the pathogenesis of cellular injury associated
with persistent viral infections, immunological aspects of slow infections
and characterization and properties of subacute spongiform virus enceph-
alopathies. New and significant findings of the neuropathology of these
diseases were presented and reviewed. The papers presented were published
in the Proceedings of the Vlth International Congress of Neuropathology,
Masson and Cie, Paris.

Our various laboratory investigations have thus evolved around the
theme of elucidating medical problems in the cultures of primitive man, or
man living in small, isolated traditional communities, by cautiously
selecting such problems of immunological, virological, biochemical, genetic
and nutritional interest which are pertinent to the long-term studies of
growth, behavior and human biology in these groups.

Serial No. NDS (CF)-65 OAD 1282

1. Collaborative & Field Research

2. Office of Associate Director

3. Bethesda, Maryland

PHS - NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Study of Child Growth and Development and Behavior, and
Disease Patterns in Primitive Cultures.

Sub-Project I: Study of the developmental patterning of the human nervous
system (cybernetics of human development).

a. Analysis of culturally determined methods of approach to
symbolic representation from drawings and art forms of child-
ren and adults in primitive societies.

b. Analysis of the development in writing of adolescents
in preliterate cultures.

c. Analysis of child care and behavior patterns in primi-
tive cultures from photographic recording (development of
techniques and methods).

d. Investigation of nonrecurrent phenomena (objectives and
selectivity used in documentation of aperiodic phenomena to
preserve maximum information) .

e. A research archive for ethnopediatric film investigation
of styles in the patterning of the nervous system.

Sub-Project II: Human evolutionary studies in isolated primitive groups.

a . Kuru .

b. Motor neuron disease and other degenerative diseases in
East and West New Guinea, and in other inbred Pacific Islands
populations.

c. Blood group genetic studies of Australasian (Melanesia
and Micronesia), and South American indigenous groups.

d. Red cell enzyme, serum factor and leucocyte type pleo-
morphisms among these groups.

e. Analysis of dermatoglyphic variance.

f. Epidemiologic and ecologic investigations of kuru: with
expanding, manipulatable computer records system.

Sub-Project III: Studies of isolated Micronesian populations.

a. Child development and behavior on Ulithi, Ifalik and
Lamotrek atolls, and on Pais Island.

b. Response to live measles virus vaccine in immunological
virgin populations without circulating measles virus (special
attention to response in susceptible adults and pregnant
women and their offspring). Follow-up studies to determine
persistence of antibody response and to observe these popula-
tions for possible long-term neurological sequellae following
use of attenuated live vaccine virus.

Serial No. NDS (CF)-65 OAD 1282

c. Influenza A2 virgin soil epidemics (epidemiological,
clinical and immunological response and discovery of popula-
tions without previous experience with Type A or Type B
influenza. Follow-up studies of these populations as sentinel
populations for detection of epidemics due to new strains of
influenza virus.

d. Studies of infectious disease patterns in remote indivi-
dual populations.

e. Genetic characterization of the population of the Western
Caroline Islands.

f. The antigenic identification of the 1918-29 influenza
virus from residual antibody in aged persons in isolated popu-
lations free of influenza since the pandemic.

Sub-Project IV: Studies of isolated New Guinea populatioiB.

a. Slow growth, delayed puberty and early aging in Melane-
sians of short stature.

b. CNS defects in areas of intense endemic goitrous creti-
nism in Highland populations.

c. Child development and behavior patterns in the Asmat,
Tjitak, Auyu, Kayagar, Western Dani, Kaure, Toure groups of
West New Guinea; the Kukukuku (Anga) , Eastern Highlands
peoples, the Biami, Etoro and Waragu people of the Great
Papuan Plateau of Papua and New Guinea; and, the West Nakanai,
Mangsing and Mamusi of New Britain.

d. Infectious disease — including encephalitis--studies in
diverse, ecologically isolated New Guinea populations.

e. Study of the pattern of the A2 Hongkong influenza epide-
mic in East and West New Guinea in 1969-70.

f . Genetic and demographic characterization of New Guinea
populations.

g. Hereditary and genetic disease patterns in New Guinea,
h. The Anga (Kukukuku): an intensive longitudinal study of

growth and development, behavior, disease patterns, human
genetics, communication, in an archaic mountain population of
New Guinea.

i. Research cinema films of behavior patterns of children
in New Guinea.

j. Study of the hazards of lowland resettlement of Highland
groups in New Guinea.

Sub-Project V: Studies of isolated New Hebrides and Solomon Islands popu-
lations,

a. Child development and behavior in Tongariki, the Banks
and Torres Islands and Espiritu Santo.

b. Tongariki: an intensive study of human evolution in the
Shepherd Islands.

c. Human genetics and disease patterns survey in the Banks
and Torres Islands.

d. Seroepidemiology of infectious disease in New Hebrides.

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Serial No. NDS (CF)-65 OAD 1282

Sub-Project VI: Studies of Australian Aborigines.

a. Arbovirus seroepidemiological studies of Aboriginal
groups in Cape York.

b. Survey patterns of infectious disease in Aboriginal
groups in the Haast Bluff, Cape York, the Kimberley and
Bentnik-Mornington Islands groups.

Sub-Project VII: Studies of Central and South American Indians.

a. Human genetics and disease patterns of Guayaki and Chaco
Indian tribes, and the Mennonite colonists in the Chaco of
Paraguay .

b. Child growth and development of Guayaki and Ai'yore
Indians of Paraguay.

c. Child growth and development of Aroyo (Moro) Indians of
Bolivia and Paraguay.

d. Population genetics, disease patterns and child growth
and development of the Waunan Indians of Columbia and
Ecuador.

Sub-Project VIII: Developmental, genetic and disease patterns in primitive
populations of Asia, Africa and in Polynesia.

Sub-Project IX: Experimental developmental neuropediatrics in infantile

programming: an empirical approach to the language of infor-
mation input into the nervous system.

Sub-Project X: Ciphers and notation for the coding of sensory data for
neurological information processing:

a. Notational systems for human movement.

b. Ciphers and notation for human form (physiognomy,
physique, palm printing, ear form, hair).

c. Theoretical studies in notational problems in mathema-
tics, linguistics, music, dance.

d. Alphabets: a theoretical investigation into their rela-
tion to linguistics, and the application to non-linguistic
information.

e. Form recognition: neuroanatomic and genetic determina-
tion of preferential recognition, and interrelationships of
the problems in computer programming and in the arts.

f. The ciphering and coding of visual data as solved in the
visual arts (drawing, painting and sculpture).

Sub-Project XI: Racial distribution and neuroanatomic variations in the
structure of the human brain.

Principal Investigator: D. Carleton Gajdusek, M.D.

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Serial No. NDS (CF)-65 OAD 1282

Other Investigators: Clarence J. Gibbs, Jr., Ph.D. ^ Paul W. Broira, M.D. ,
Raymond Rods, M.D. , David M. Asher, M.D. , Michael Alpers,
M.D., Vincent Zigas, M.D. , Francoise Cathala, M.D. , Richard
Marsh and Robert Cornelius, D.V.M, , John Hooks, Ph.D.,
E. Richard Sorenson, Nancy Rogers, Mint Basnight, Helena
Gilbert, Judith Meyer and Richard Benfante.

Project Description:

The Section for the Study of Child Growth and Development, and Behavior,
and Disease Patterns in Primitive Cultures has continued all projects listed
in the previous Annual Reports, with expansion of collaborating investigators
as reflected in the authorship and studies of the publications listed. The
titles of sub-projects and their subdivisions are sufficiently explicit to
constitute the project description.

Sub-Project I. Study of the developmental patterning of the human nervous

system (cybernetics of human development).

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: Michael Alpers, M.D. , Paul Bro^<m, M.D. , Vincent Zigas,
M.D. , E. Richard Sorenson, Judith Meyer, Peter Fetchko and
Donald Rubinstein.

Cooperating Investigators: Dr. Margaret Mead, American Museum of Natural
History, New York; Dr. Ted Schwartz, Univ. California, Los
Angeles; Alan Lomax, Columbia Univ., New York; Mr. and Mrs.
Mark Jablonko, Columbia Univ.; Dr. Paul Ekman and W.V.
Friesen, Langley Porter Neuropsychiatric Inst. , San Franciscq
Dr. Peter Kundstadter, Univ. Washington, Seattle; Kal Muller,
Univ. Arizona, Tucson; Thomas Kiefer, Dr. Edwin Cook, Univ.
California, Davis; Dr. Gordon Gibson, Smithsonian Inst.; Dr.
Robert MacLennan, Inter. Agency for Cancer Res., France; Dr.
Maurice Godelier, Sorbonne; William H. Bloxam, Wayne Dye,
John MacGregor, Father David Gallus, Father F. Trenkenshuh,
0. Kooyers, Dr. C.K. Dresser, West New Guinea; Timothy Asch,
Brandeis Univ.; N. Chagnon, Univ. Michigan; James Bruce,
Pasadena, Calif.; Elizabeth and Perry Kennedy, Univ. Buffalo.

Sub-Project II. Human evolutionary studies in isolated primitive groups.

Principal Investigator: D. Carleton Gajdusek, -M.D.

Other Investigators: Paul Brown, M.D. , C.J. Gibbs, Jr., Ph.D., M. Alpers,
M.D., F. Cathala, M.D. , D. Asher, M.D. , N. Rogers, M. Bas-
night, J. Hooks, Ph.D.

10,

Serial No. NDS (CF)-65 OAD 1282

Cooperating Investigators: Dr. Stephen Fazekas, CSIRO, Sydney; Eric French,
and Cyril Curtain, M.D. , CSIRO, Melbourne; Dr. Malcolm Simons,
Royal Children's Hospital, Melbourne; Dr. R.T. Simmons, John
J. Graydon, Alan Duxbury and Frank Warbuton, Commonwealth
Serum Laboratories, Melbourne; and Dr. Robert Kirk, John Cur-
tain School of Medicine, Canberra, Australia; Dr. Vincent
Zigas, Dr. Richard Hornabrook and Dr. Adolph Suweri, Public
Health Department, Territory of New Guinea.

Sub-Project III: Studies of isolated Micronesian populations.

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: Paul Brown, M.D. , C.J. Gibbs, Jr. and J. Anthony

Morris, Ph.D., David Asher, M.D., Francoise Cathala, M.D. ,
MJchael Alpers, M.D., Vincent Zigas, M.D. , John Hooks, Ph.D.,
Nancy Rogers, Mint Basnight, Richard Benfante.

Cooperating Investigators: Dr. Leon Rosen, Gordon W. Wallace, NIAID , Honolu-
lu, Hawaii; Dr. Jacob Brody, NINDS; Chris Plato, NICHD; Dr.
Kwang Ming Chen, National Taiwan Univ. , Tapei; Newton Morton,
Population Genet. Laboratory, Honolulu; Dr. Stephen Fazekas,
Sydney; Dr. Antonio Golbuu, Jose Torres, Eddie Iderug, NINDS
Research Center, Guam.

Sub-Project IV: Studies of isolated New Guinea populations.

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: Drs. Paul Brown, Rajmiond Roos, Michael Alpers, Vincent
Zigas, C.J. Gibbs, Jr., David Asher, Francoise Cathala and
John Hooks; Judith Meyer, Richard Benfante, Mint Basnight
and Helene Gilbert.

Cooperating Investigators: Dr. C.K. Dresser, West New Guinea; William Bloxam,
John MacGregor, Richard Hornabrook, John Mathews, Territory
of New Guinea; Edwin Cook, Univ. California, Davis; Ted
Schwartz, Los Angeles; Dr. R. MacLennon, France; Dr. John
Hotchin, Department of Health, New York; Paul Ekman, San
Francisco; R.T. Simmons, J.J. Craydon, C.C. Curtain, Aust-
ralia; David Kitchin, M.D. , Columbia Univ.; Alexander Beam,
M.D., Rockefeller Inst., New York; Maurice Godelier, Ph.D.,
Paris; E. Beck, P. Daniel, Inst. Psychiatry, London; Chris
Plato, NICHD; Roger Rodrigue, M.D. , Temple Univ.; and J. van
Delden, New Guinea.

lis

Serial No. NDS (CF)-65 OAD 1282

Sub-Project V: Studies in Isolated New Hebrides and Solomon Islands popu-
lations.

Principal Investigator: D. Carleton Gaidusek, M.D.

Other Investigators: Paul Brown, M.D., David Asher, M.D., C.J. Gibbs, Jr.,
Ph.D., Nancy Rogers, Mint Basnight and Helene Gilbert.

Cooperating Investigators: Dr. H. Lehraann, Univ. Cambridge; Dr. Robert Kirk,
Australian National Univ., Canberra; Dr. Jean Guiart, Sor-
bonne, France; Dr. James MacGregor, Honiara; Dr. Roger Green-
ough. Dr. William Rees, New Hebrides; Chris Plato, NICHD.

Sub-Project VI: Studies of Australian Aborigines.

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: Paul Brown, M.D. , Michael Alpers, M.D., C.J. Gibbs, Jr.,
Ph.D., Nancy Rogers, Mint Basnight and Helene Gilbert.

Cooperating Investigators: Drs. R.T, Simmons, J.J. Graydon, A. Duxbury and
F. Warbuton, Commonwealth Serum Laboratories, Melbourne;
C. Curtain, Sydney; E. Beck, London; and W.C. Leyshon, NIDR.

Sub-Project VII: Studies of Central and South Araerican Indians.

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: C.J. Gibbs, Jr., Michael Alpers, M.D., Mint Basnigh t,
Nancy Rogers and Helene Gilbert.

Cooperating Investigators: W.C. Leyshon, NIDS; J.L. Sever, NINDS; K. Walls,
CDC, Atlanta; R.L. Anderson, WRAIR; J. A. Morris, DBS, Arthur
Steinberg, Western Reserve Univ., Cleveland; C.C. Curtain,
CSIRO; Elizabeth and Perry Kennedy, Buffalo.

Sub-Project VIII: Developmental, genetic, and disease patterns in primitive
populations of Asia, Africa and Polynesia.

Principal Investigator: D. Carleton Gajdusek, M D.

Other Investigators: Frank D. Schofield, M.D.,. Nairobi; Peter Kundstadter,
Seattle; Thomas Kiefer, Univ. California; L. Vogel, M.D. ,
Nairobi; Kok Ann Lira, M.D. , Singapore; Chris Plato, NICHD.

^2l

Serial No. NDS (CF)-65 OAD 1282

Sub-Project IX: Experimental developmental neuropediatrics in infantile

programming: an empirical approach to the language of inform-
ation input into the nervous system.

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: Michael Alpers, M.D., E. Richard Sorenson, Judith Meyer;
Don Rubinstein.

Cooperating Investigators: Paul MacLean, M.D. , NINDS; Paul Ekman, San
Francisco

Sub-Project X: Ciphers and notation for the coding of sensory data for
neurological information processing.

Principal Investigator: D. Carleton Gajdusek, M.D.

Other Investigators: E. Richard Sorenson, Michael Alpers, M.D., Judith Meyer

Cooperating Investigators: Dr. R.L. Kirk, Canberra; Patricia Hunt, Univ.

California, Berkeley; Paul MacLean, M.D. , NINDS; Chris Plato,
NICHD; Alan Lomax, New York.

Sub-Project XI: Racial distribution and neuroanatomic variations in the
structure of the human brain.

Principal Investigator: D, Carleton Gajdusek, M.D.

Cooperating Investigators: Elisabeth Beck, Peter Daniel, M.D. , Institute of
Psychiatry, London; Paul Yakovlev, M.D. , Richard Sidman, M.D.,
Dr. Kemper, Dr. H. Hamlin, Harvard Univ., Boston; Dr. Peter
Lampert, Univ. California, La Jolla; Dr. K. Earle, AFIP;
Dr. P. MacLean, NINDS; Drs, R. Hassler and H. Stephan, Max-
Plank Inst., Frankfurt; Dr. G. Inke, Stony Brook, New York;
Dr. A, Hopf, Neustadt, Germany.

13c

Publications:

Gajdusek, D.C., and Bro^m, P. (Eds.): Isolated and Migratory Population
Groups. Health Problems and Epidemiologic Studies. In Amer. J. Trop. Med.
Hyg. 19: 127-175, 1970.

Plato, C.C.. and Gajdusek, D.C.: Dermatoglyphics of the natives of Tonga-
riki in the New Hebrides. J. Archaeol . Phys . Anthrop. Oceania, in press.

Gajdusek, D.C., Gibbs, C.J., Jr., and Lim, K.A. : Prespects for the control
of chronic degenerative disease with vaccines. Proc. Inter. Conf. Applica-
tion of Vaccines Against Viral, Rickettsial and Bacterial Diseases of Man.
Pan American Health Organization, World Health Organization, in press.

Fedson, D.S., Gibbs, C. J. , Jr., and Brown, P.: Problem of antibody response
in man following initial exposure to A2 influenza neuraminidase. Lancet,
in press.

Zigas, v., and Benfante, R.J.: Recent knowledge of human toxoplasmosis.
Trop ■ Geogr. Med. , in press.

Zigas, v., and Gajdusek, D.C.: The role of serological survey: with special
reference to New Guinea. Papua and New Guinea Med. J., in press.

Gajdusek, D.C., and Alpers, M. : Genetic studies in relation to kuru.
I. Cultural and historical background. Amer. J. Hum. Genet., in press.

Simmons, R.T., Graydon, J.J., Gajdusek, D.C., Alpers, M.P., and Hornabrook,
R.W.: Genetic studies in relation to kuru. II. Blood group genetic patterns
in kuru patients and populations of the Eastern Highlands of New Guinea.
Amer. J. Hum. Genet., in press.

Kitchin, F.D., Beam, A.G., Alpers, M. , and Gajdusek, D.C.: Genetic studies
in relation to kuru. III. The distribution of the inherited serum group
specific protein (Gc) phenotypes in New Guineans: an association of kuru and
the GcAb phenotype. Amer. J. Hum. Genet., in press.

Plato, C.C, and Gajdusek, D.C.: Genetic studies in relation to kuru.
IV. Dermatoglyphics of the Fore and Anga populations of the Eastern High-
lands of New Guinea. Amer. J. Hum. Genet., in press.

Hamlin, H. , Gajdusek, D.C., Kemper, T.L., and Yakovlev, P.I.: How differ-
ent might be the brain of Stone Age man? Amer. J. Phys. Anthrop., in press.

14s

Serial No, NDS (CF) -62 OAD 969

1. Collaborative-Field Research

2. Office of Associate Director

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Slow, Latent, and Temperate Virus Infections of the Central
Nervous System of Man and Animals

Sub-Project I: Attempts to isolate transmissible agents from sub-
acute and chronic diseases of the nervous system

Sub-Project II: Characterization and pathogenesis of kuru virus

Sub-Project III: Characterization and pathogenesis of Creutzfeldt-
Jakob disease virus

Sub-Project IV: Studies on the characterization and nature of scrapie,
mink encephalopathy and visna virus and the relationship
of visna virus to the virus of progressive pneumonia of
sheep in Montana

Sub-Project V: Studies on Australian antigen and antibody in chimpan-
zees, monkeys and primate handlers

Sub-Project VI: Fluorescent antibody studies on the intracellular lo-
calization and identification of viral antigens in vivo
and _in vitro in tissues from patients with subacute
diseases of the CNS

Sub-Project VII: Tissue and cell culture Jji vitro studies of viral
induced slow infections of man and animals

Sub-Project VIII: Characterization and distribution of two new foamy

viruses isolated from chimpanzees tissues grown in. vitro
and their relationship to the RNA oncogenic group of
viruses

Sub-Project IX: Characterization of newly identified adenoviruses
isolated from chimpanzee tissues grown j^ vitro

Sub-Project X: Attempts to demonstrate a viral etiology for chronic
encephalitis with focal epilepsy

Sub-Project XI: Isolation, epidemiology and pathogenesis of mourning
dove pox

15s

Serial No. NDS (CF) -62 OAD 969

Sub-Project XII: Infectious and contagious disease management in a
primate colony

Sub-Project XIII: Slow Virus Symposia, Vlth International Congress of
Neuropathology, Paris, France

Sub-Project XIV: Studies on the ecology, epidemiology and pathogenesis
of arbovirus infections of man and animals

Principal Investigators: D. Carleton Gajdusek, M.D., and
Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: Paul Brown, M.D. , John Hooks, Ph.D., Nancy Rogers, M.S.,
Mint Basnight, M.S., Robert Cornelius, D.V.M. ,
Raymond Roos , M.D., Francoise Cathala, M.D. , Vincent
Zigas, M.D., Ronald DiGiacomo, D.V.M. , Larry Fry, Ph.D.

Technical Assistants: Michael Sulima, Alfred Bacote, Helena Gilbert,

Richard Thomas, Paul Martin, Lloyd Horst, Paul Horst,
James Webster, Judith Meyer, Sarah Andersen, Monica Lewis,
Galen Miller, Amos Fox, James Noll and Albert Bontrager

Students: By special arrangement this department participates in the co-
operative college programs of the University of South
Florida, Tampa and Antioch College, Ohio. During the
past year the following students have spent 3-6 months in
the laboratory: Michael Proctor, Henry Theiss,
Jerome Kurent and Mark Littlewood

Administrative: Marion Poms, Juliette Harvey and Sharon Williams

Project Description:

The studies reported in this section are being conducted in the NINDS
Laboratory of Slow, Latent and Temperate Virus Infections on the NIH campus
and at the Patuxent Wildlife Research Center, Laurel, Maryland. They are in
part being conducted in collaboration with the Bureau of Wildlife and Sports
Fisheries, U.S. Department of Interior. Additional associated collaborators
are the Departments of Neurology and Neurovirology, Johns Hopkins University
School of Medicine and the Departments of Epidemiology and Pathobiology,
Johns Hopkins School of Public Health and Hygiene. In house collaborators
are Jacob Brody, M.D. , Epidemiology Branch, John Sever, M.D. , Perinatal
Research Branch, NINDS; K. Takemoto, S. Baron, H. Levy and W. Hadlow, NIAID;
L. Barker, M.D. , DBS. Contractural phases of this work are being conducted
at Gulf South Research Institute, New Iberia, Louisiana; National Center for
Primate Biology, University of California, Davis,. California; and Public
Health Research Institute of New York, Otisville, New York, This project is
part of the Study of Child Growth and Development and Disease Patterns in
Primitive Cultures and is under the director of that study. (see Project
Report Serial No. NDS (CF)-1282 (I- IX.)

16 s

Serial No. NDS (CF)-62 OAD 969

SUB-PROJECT I: Attempts to isolate transmissible agents from subacute
and chronic diseases of the nervous system

Principal Investigators: D. Carleton Gajdusek, M.D. , and
Clarence J. Gibbs , Jr., Ph.D.

Other Investigators: Raymond Roos , M.D., Paul Brown, M.D. , David Asher, M.D. ,
John Hooks, Ph.D., Mint Basnight, M.S., Larry Fry, Ph.D.,
Nancy Rogers, M.S., Vincent Zigas, M.D. ,
Robert Cornelius, D.V.M. , Francoise Cathala, M.D. ,
Ronald DiGiacomo, D.V.M. , and Richard Sorenson, Ph.D.

Cooperating Investigators: J.D. Mathews and R.W. Hornabrook, Kuru Research
Office, Okapa, New Guinea; J.C. Steele, M.D. , University
of Toronto, Canada; David Poskanzer, M.D., Massachusetts
General Hospital, Boston; Richard T. Johnson, M.D. , Johns
Hopkins University Hospital, Baltimore; J.T. Sever, M.D. ,
NINDS; A.M. Gardashyan, M.D. , Mayo Clinic, Rochester,
Minnesota; J. A. Morris, Ph.D., and Hope E. Hopps, DBS;
B.H. Dessel, M.D. , Veterans Hospital, Wood, Wisconsin;
Elisabeth Beck and Professor P.M. Daniel, Department of
Neuropathology, The Maudsley Institute, London;
P. A. Palsson, D.V.M., and M. Gudnadottir, M.D. , Institute
for Experimental Pathology, Keldur, Iceland; W. Hadlow,
D.V.M., Rocky Mountain Laboratory, NIAID; J. Hourrigan,
D.V.M., and H.A. McDaniel, D.V.M., Animal Research Section,
USDA; E. Dustman, Ph.D., and CM. Herman, Sc.D., Patuxent
Wildlife Research Center, Department of Interior; King
Engel, M.D. , Medical Neurology, NINDS; J. A. Brody, M.D. ,
Epidemiology, NINDS; C. Colson, M.D. , Columbia Presby-
terian Hospital, New York; R. Katzman, M.D., Yeshiva
University, New York; M. Schaeffer, M.D. , City of New York
Department of Health, and Dr. Ward, Otisville, New York;
G. McKhann, M.D. , R.T. Johnson, M.D. , Leslie Weiner, M.D.,.
Robert Herndon, M.D., Neurology-Neurovirology, Johns
Hopkins University School of Medicine, Baltimore; Dr.
Marino, Grady Memorial Hospital, Emory University, Atlanta;
I.H. Pattison, D.Haig, D.V.M., and D. Hunter, Ph.D., ARS ,
CoiTipton, England; J.T. Stamp, D.V.M., Moredun Institute,
Edinburgh; E.J. Field, Medical Research Council, Newcastle-
upon-Tyne; F. Dixon, M.D. , and M. Oldstone, M.D. , Scripps
Clinic and Research Foundation, La Jolla, California;
N. Nathanson, M.D. , and F. Bang, M.D., School of Public
Health, Johns Hopkins University Hospital, Baltimore;
W. Zeman, M.D., Indiana University Medical Center, Indiana-
polis; G. ZuRhein, M.D., University of Wisconsin, Madison;
H. Koprowski, M.D., and M. Katz, M.D. , Wistar Institute,
Philadelphia; K. Earle, M.D. , and R. Heffner, Armed Forces
Institute of Pathology; P. Lampert, M.D. , University of

17s

Serial No, NDS (CF)-62 OAD 969

California, La Jolla; P. Van Nuls, M.D. , Grand Rapids;

T. Rasmussen, M.Do , University of Montreal, Canada;

W. Greer, D.V.M. , Gulf South Research Institute, Louisiana;

C. Espana, Ph.D., and R.E. Stowell, M.D. , National Center

for Primate Biology, University of California, Davis:

A. Lowenthal, M.D. , Foundation Born-Bunge for Research,

Antwerp; F. Cathala, M.D., and C. Chany, M.D. , Hopital de

la Salpetriere, Paris; H. Thormar, Ph.D., and R. Carp, Ph.D.,

Institute for Basic Research in Mental Retardation, Staten

Island

Technical Assistants: Michael Sulima, Alfred Bacote, Helena Gilbert,
Paul Martin, Lloyd Horst, Paul Horst, Judith Meyer,
Galen Miller, Amos Fox, James Noll, and Albert Bontrager

Student Assistants and Part-time Temporary Employees: Robert Rhorer,
Wesley Russell, Henry Theiss, Michael Proctor,
Mark Littlewood, and Jerome Kurent

Project Description:

Objectives: The objectives of these long-term studies were established
in 1957 with the discovery of kuru in the Eastern Highlands of New Guinea.
They were fully promulgated in 1962 with the activation of the Laboratory of
Slow, Latent and Temperate Viruses of the Nervous System. A.s established the
objectives remain unchanged as follows: (1) to establish infection as the
etiology of chronic and subacute progressive degenerative diseases primarily
of the nervous system of man and animals; (2) to characterize and determine
the nature of viruses isolated during these studies by eliciting physical,
chemical, biological and morphological properties; (3) to study the etio-
logical role of viruses in presenile and senile dementias as well as in nor-
mal aging processes; (4) to study the ecology of disease, disease processes,
and the nature of their causative agents in primitive and frequently virgin
populations; (5) to determine the presence and interactions of "helper" or
"hinderer" viruses in the pathogenesis of diseases under study; (6) to de-
termine the nature of viral masking, latency, temperateness , incompleteness,
persistence, interference, eclipse and immunopathological processes as they
contribute to subacute degenerative processes of the CNS. The major diseases
under study are kuru, Creutzfeldt-Jakob disease, multiple sclerosis, Alper's
disease, Alzheimer's disease, Schilder's disease, subacute sclerosing panen-
cephalitis, amyotrophic lateral sclerosis, parkinsonism-dementia, Parkinson's
disease, systemic lupus erythematosus, dermatomyositis , epilepsy partialis con-
tinua, ataxia-telangiectasia, progressive supranuclear palsy, polymyositis,
Behcet's disease, post-infectious encephalitis, chronic tick-borne hemorrhagic
fever, nephro-nephritis , hemorrhagic fever, Guillain-Barre syndrome, scrapie,
mink encephalopathy, visna, progressive pneumoni.a of sheep in Montana, and
diabetes mellitus.

Methods Employed: Basic approaches established in 1962 and which have
thusfar proved successful are being pursued. Essentially they consist of
standard and classical techniques, supplemented by developmental techniques
for the isolation of viruses with certain notable exceptions: (1) the
demonstration that the genetic mechanism of the host influences the host

183

Serial No. NDS (CF) -62 OAD 969

resistance or susceptibility to disease requires inoculation of specimens in-
to an extensive array of animals, including higher apes and many species of
old and new-world monkeys, birds and cell and tissue culture lines in vitro;
(2) exceedingly long asymptomatic incubation periods require experiments to
be held, in isolated facilities to prevent cross infections, for periods of
from 5 to 10 years before negative conclusions can be determined; (3) estab-
lishment and utilization of cell culture lines iji vitro prepared by explanta-
tion and trypsinization of biopsy and autopsy tissues from humans and animals
with CNS diseases - these lines are maintained under rigid requirements of
temperature and C02 for unmasking latent and endosymbiotic viruses, with
considerable emphasis being placed on viral genome rescue employing co-acti-
vation, fusion, Sendai virus medicated fusion; (4) utilization of fluores-
cent, phase and electron microscopy to elicit intracellular viral genome; and
(5) histochemical and clinical chemistry procedures to detect significant
changes in humans and animals affected with natural and experimental diseases.
An elaborate system for optimally obtaining specimens has been established
with scientific collaborators in the United States and throughout the World.
In addition to studying diseases in search of viruses this laboratory is
investigating the nature of over 100 viruses either known or suspected of
inducing fatal human diseases particularly those affecting the brain of man
and animals. In these studies attempts are made to develop animal or tissue
culture models for determining physical, chemical and biological properties
and pathogenesis of the virus.

Major Findings: During this year our studies on neurological diseases of
man and animals provided data for the classification of a new group of infec-
tious agents which we have called the Subacute Spongiform Virus Encephalo-
pathies: kuru, Creutzfeldt-Jakob disease, scrapie and mink encephalopathy.
Over 200 chimpanzees, 1600 smaller monkeys of 27 species or sub-species, more
than 200,000 small laboratory animals including over 25 selectively inbred
lines of mice, a variety of pure bred and randomly bred domestic animals and
avian hosts as well as more than 50 established tissue and cell culture lines
of human, animal and avian origins have, since the beginning of these studies,
been inoculated with suspensions of tissues obtained at biopsy or early
autopsy from humans and animals affected with neurological and systemic dis-
eases. During the last 6 months alone, more than 450 human tissue specimens
for virus isolation have been received into the laboratory; of these approxi-
mately 260 are brain specimens and 507o of those submitted were brain biopsy
specimens. Significant findings include the continued isolation, characteri-
zation and passage of kuru through 4 serial passages in chimpanzees, the
isolation and serial passage of the virus of Creutzfeldt-Jakob disease from
10 patients to chimpanzees, successful primary and serial transmission of
kuru and Creutzfeldt-Jakob disease to 4 species of new-world monkeys: spider,
squirrel, capuchin and woolly monkeys; isolation of adenovirus type 32 from
the brain of a patient with subacute encephalitis; isolation of virus from
brain and lung of sheep with fatal progressive pneumonia in Montana and
demonstration that both these viral isolates are antigenically closely re-
lated or identical to visna and maedi viruses of Iceland; thus for the first
tim.e establishing these viruses in the United States, and the demonstration
that two viruses (Pan 1 and Pan 2) isolated from the brains of many chimpan-
zees in our colony are RNA viruses associated with an RNA dependent DNA

19s

Serial No. NDS (CF)-62 OAD 969

polymerase, not serologically related to known simian foamy or monkey
mammary tumor viruses and which morphologically resemble viruses belonging
to the RM oncogenic group.

Kuru; Kuru virus continues to serve as the prototype virus causing fatal
subacute degenerative disease of the CNS of man and is a dramatic model for
the study of other spongiform encephalopathies particularly those referred to
as the presenile dementias. A major breakthrough occurred during this report-
ing year with the successful primary transmission of the disease to spider
monkeys, squirrel monkeys and a capuchin monkey inoculated with suspensions
of brain from fatal human cases. Although the incubation periods were some-
what longer on primary passage in these animals as compared to serial passage
in the chimpanzee it is anticipated that serial passage of the virus in the
more readily available, easier housed and more economically purchased new-
world monkeys will be associated with a reduction in the incubation period.
Further, we now have conclusive evidence that neuropathological lesions are
present in pre-clinical experimentally inoculated animals even as early as 5
months following inoculation. For the first time we have evidence that by
millipore filtration the virus of kuru passes through a membrane with an
average pore diameter of lOOnm. Details on the characterization of kuru
virus are presented in Sub-Project II.

Creutzfeldt-Jakob disease: This disease has now been transmitted from 10
humans to 12 chimpanzees and, like kuru, from humans to two species of new-
world monkeys: spider monkeys and squirrel monkeys, and from Creutzfeldt-
Jakob disease affected chimpanzees to squirrel monkeys and a brown woolly
monkey. Successful transmissions have been obtained with brain tissues that
have varied greatly in the degree and intensity of status spongiosis, a find-
ing which lends support to the unitarian hypothesis of the disease. Further-
more, the transmissible cases have been worldwide in their distribution
coming from the United States, England, Canada, and Belgium. Although pre-
viously thought to be a rare disease, since our first report on the trans-
missibility we have received human specimens from over 60 cases. A critical
review of the case histories revealed that 13 of 39 patients had liver
abnormalities and at least two cases, as yet not transmitted, had onset of
their neurological diseases during immunosuppressive therapy following organ
transplantations. During the past year employing the complement- fixation and
he'magglutination-inhibition techniques sera from human patients and experi-
mentally infected chimpanzees were tested for antibodies against 10 major
groups of viruses and 58 arboviruses representing Group A, Group B, Bun-
yamwera group and a number of ungrouped arboviruses to determine whether or
not any one or more of the viruses tested were involved in the disease. No
etiological relationship was elicited. Details on the characterization of
the Creutzfeldt-Jakob disease virus are presented in Sub-Project III.

Other CNS diseases: Although we have been successful in (1) trans-
mitting kuru and Creutzfeldt-Jakob disease to several species of primates,
(2) isolating adenovirus type 32 from the brain of a patient with subacute
encephalitis and (3) isolating measles virus from the brain of a patient with
SSPE we are also accumulating negative data on animals inoculated with the
following numbers of cases of specified disease: Alper's disease (4), amyo-
trophic lateral sclerosis of the sporadic and familial types (15), Alzheimer's
disease (15) , SSPE (65) , chronic focal epilepsy or epilepsia partialis

20 s

Serial No. NDS (CF)-62 OAD 969

continua (16), progressive supranuclear palsy (10), Schilder's disease (7),
multiple sclerosis (14), Alzheimer's disease of the sporadic and familial
types (15), Parkinson's disease (6), parkinsonism-dementia (4), progressive
multifocal leucoencephalopathy (5) , Reyes syndrome (4) , endomyocardial
fibrosis (22), hemorrhagic fever nephro-nephritis syndrome (9), Werdnig-
Hoffmann disease (1), rheumatoid arthritis (7), dermatomyositis (3), poly-
myositis (3), leukemia (3), and 134 cases of subacute neurological diseases in
which final diagnosis have not yet been established. These are in addition
to the 56 cases of kuru and 68 cases of Creutzfeldt-Jakob disease currently
under study.

Significance: The results thusfar obtained in these long-term studies
have firmly established a new group of infectious filterable viruses - which
we have classified as subacute spongiform virus encephalopathies. The success
of these transmissions warrant continued and increased efforts to demonstrate
infection as the etiology of other spongiform encephalopathies of the gray
matter of the brain in human diseases. Isolation of the kuru and Creutzfeldt-
Jakob disease viruses now provides for the ultimate development of iinmuno-
prophylactic measures for humans at risk, chemotherapeutic measures for
treatment of affected humans and definition of the pathogenesis of these dis-
eases in the human host. These studies further are providing the optimal
techniques for the successful elucidation of the infectious nature of mul-
tiple sclerosis, ALS , ALS-PD, Parkinson's disease, Alzheimer's disease, pro-
gressive multifocal leucoencephalopathy, the presenile and senile dementias as
well as the role of infectious viruses in aging processes.

Proposed Course: 1) Identification and characterization of the agent
causing disease in chimpanzees; 2) continued long-term observation of
inoculated animals. Continued serial studies on the fractionation of serum
specimens from these animals for the determination of shifts in the electro-
phoretic patterns, as well as their antibody status which may be indicative
of sub-clinical infections; 3) continued effort to develop suitable antigen
antibody system for the study of established strains of 'slow' viruses;
application of these new techniques to the study of human diseases; 4) in-
tensification of the development and application of fluorescent antibody
techniques with the model virus and other chronic viruses, such as LCM and
rabies, which may remain latent for many years before clinically apparent
disease becomes manifest; 5) greater emphasis on growth, cultivation and
establishment of cell culture lines of "target organ", nervous tissue, from
humans and animals with degenerative diseases of the nervous system, as well
as from cases of "auto-immune" diseases in an effort to isolate an etio-
logical agent in a controlled in vitro environment, detection of abnormal
antigenic fractions giving indirect evidence of disease and possible associa-
tion with known viruses and establishment of new cell lines for the study of
viral growth, maturation, and measurement of interferon or interferon- like
substances; 6) increased efforts to adapt strains of 'slow' viruses to
growth, serial propagation and characterization in tissue and cell culture
systems employing viral genome rescue techniques; 7) continued efforts
toward the development of procedures for the successful isolation of etio-
logical agents responsible for degenerative diseases of the CNS, such pro-
cedures to include cell culture blocking techniques, detection of endosym-
biotic relationship of masked, latent, or temperate viruses in the intact host

21 s

Serial No. NDS (CF)-62 OAD 969

and host cells grown jjj vitro and chemotherapeutic and immunosuppressive
regimens to lower animal resistance to infection; 8) continued efforts to
broaden experimental host range for kuru, Creutzfeldt- Jakob, SSPE, visna,
and, at the same time, seek out those experimental hosts whose genetic mech-
anisms render them susceptible to other human diseases under study. 9) fol-
low-up studies on Pan 1 and Pan 2 viruses (simian foamy virus 6 and 7,
respectively) from chimpanzees to determine their distribution in man, pri-
mates, and other mammalian species; to further elicit oncogenic and tumori-
genic properties they may have; and, to further characterize what role they
may have in the pathogenesis of experimental disease.

22

Serial No. NDS (CF)-62 OAD 969

SUB -PROJECT II: Characterization and pathogenesis of kuru virus

Principal Investigators: D. Carleton Gajdusek, M.D. , and
Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: John Hooks, Ph.D., Nancy Rogers, M.S., Paul Brown, M.D. ,
Mint Basnight, M„S., Robert Cornelius, D.V.M. ,
Raymond Roos, M.D. , Ronald DiGiacomo, D.V.M. , and
Larry Fry, Ph.D.

Cooperating Investigators: William Greer, D.V.M., New Iberia, Louisiana;
Carlos Espana, Ph.D., University of California, Davis;
Peter Lampert, M„D. , University of California, La Jolla;
Morris Schaeffer, M.D., and Gerald Ward, D.V.M., Public
Health Research Institute, New York; Peter Daniels, M.D. ,
and Elisabeth Beck, The Maudsley Institute, London;
K. Earle, M.D. and R. Heffner, M.D., Armed Forces Insti-
tute of Pathology, Washington, D.C. ; Byron Kakulas, M.D. ,
Perth, Australia; Michael Alpers, M.D. , Perth, Australia;
Charles Chany, M.D., and Francoise Cathala, M.D., Paris,
France; Vincent Zigas, M.D., New Guinea; Sam Baron, M.D.,
and Hilton Levy, Ph.D., NIAID

Technical Assistants: Michael Sulima, Helena Gilbert, Monica Lewis,

Alfred Bacote, Judith Meyer, Paul Martin, Lloyd Horst,
Richard Thomas, Paul Horst, Amos Fox, Galen Miller,
James Webster, James Noll, and Albert Bontrager

Project Description:

Ob iectives: (1) To elucidate the morphological, biological, chemical,
immunological and physical properties of kuru virus in experimentally in-
fected animals; (2) to broaden experimental host range of the virus;
(3) to determine the pathogenesis of the disease in the experimental host;
and, (4) to develop techniques for the elucidation of the nature of kuru
virus.

Methods Employed: (1) Inoculation of chimpanzee adapted kuru virus into
chimpanzees, lesser primates, small laboratory animals, embryonated hens'
eggs and a variety of commercially available tissue and cell cultures;
(2) experiments to determine the size of kuru virus by filtration through
millipore filters; (3) effects of temperature, lyophilization procedures
and organic solvents on kuru virus; (4) attempts to demonstrate neutralizing
antibody to kuru virus in human and animal sera; (5) in vitro growth of
explanted and trypsinized tissues from kuru affected chimpanzees to demon-
strate viral antigens and virions through FA, EM and techniques to unmask
latent agents.

Major Findings: Kuru virus has now undergone 4 serial passages in chim-
panzees. Of the 96 animals inoculated in these studies kuru has developed
in 26 of 46 inoculated on primary passage with human tissues, and on serial
passages in chimpanzees the disease has occurred in 11 of 15 on second pass-
age, 9 of 13 on third passage and 8 of 19 on fourth passage. The virus can

23£

Serial No. NDS (CF)-&2 OAD 969

be directly isolated in squirrel, spider and capuchin monkeys inoculated with
human brain. In addition to serial passage in the chimpanzee the virus is
serially transmissible from chimpanzees to spider monkey, spider monkey to
spider and squirrel monkeys and back to chimpanzees. Asymptomatic incubation
periods on primary passage to chimpanzees have ranged from 14-39 months
whereas in capuchin, squirrel and spider monkeys it has been 45 months, 25
months, and 23 months, respectively. In chimpanzees serial passage is asso-
ciated with a reduction in incubation period to 10-18 months. A similar
reduction was noted on passage in spider monkeys when on second passage it
was 16 months. In suspensions of human brain the virus has an infectivity
titer of ^ 105 and in chimpanzee brain the virus titer is ^ lO^. In both
preparations the virus is thermostable and remains transmissible following
exposure to a temperature of 85°C/30 minutes. Although we earlier demon-
strated that the virus passes through millipore filters of 450nm and 220nm
we have only recently observed neuropathological lesions of kuru disease in
the brain of a chimpanzee inoculated with the filtrate of a lOOnm filter.
In addition to heat stability, kuru virus can be lyophilized, partially
purified by high speed centrifugation and stored for several years without
appreciable loss in infectivity. As previously reported, numerous attempts
to demonstrate specific NT, CF, HI, or FA antibody in the sera from affected
humans and animals have proven unsuccessful. Further, attempts to demon-
strate a serological relationship of kuru to one or more conventional viruses
have not yielded evidence of an etiological relationship. _In vitro growth
of brain and visceral tissues from kuru affected animals have resulted in the
successful isolation of over 200 strains of viruses representing 6-9 major
virus groups. (See Sub-Project VII of this report.)

Significance: Serial passage of kuru virus in chimpanzees confirms the
infectious nature of this human brain disease. The successful transmission
of the disease from human patients to chimpanzees, squirrel monkeys, spider
monkeys and capuchin monkeys and the successful serial passage of the virus
in these new-world monkeys provides a more economically feasable host in
which to further elicit kuru virus properties. These later findings may well
serve to break additional species barriers. The properties of the virus thus-
far elicited with the exception of the size by filtration are remarkably
similar to the properties of scrapie virus and suggest the viruses of these
two diseases are similar.

Proposed Course: Continued serial passage of the virus in chimpanzees,
spider monkeys, and other new-world monkeys in an effort to elicit additional
properties in the nature of kuru; expanded efforts to adapt the virus to more
conventional laboratory hosts; and, attempts to purify and more completely
identify kuru virus.

24s

Serial No. NDS (CF)-62 OAD 969

SUB- PROJECT III: Characterization and pathogenesis of Creutzfeldt-Jakob
disease virus

Principal Investigators: Clarence J. Gibbs, Jr., Ph.D., and
D. Carleton Gajdusek, M,D.

Other Investigators: Raymond Roos, M<,D», John Hooks, Ph.D., Paul Brown, M.D, ,
Nancy Rogers, M,So, David Asher, M.D. , Vincent Zigas, M.D.,
Robert Cornelius, D.V.M. , Mint Basnight, M.S.,
Ronald DiGiacomo, D.V.M,, and Larry Fry, Ph.D.

Cooperating Investigators: William Greer, D.V.M., Carlos Espana, Ph.D.,
Peter Lampert, M.D., Morris Schaeffer, M.D.,
Gerald Ward, D.V.M., Peter Daniels, M.D. , Elisabeth Beck,
Kenneth Earle, M.D., R. Heffner, M.D. , Charles Chany, M.D. ,
Francoise Cathala, M.D., Louis Horta-Barbosa, D.V.M.,
John Sever, M.D., and Jacob Brody, M.D.

Project Description:

Objectives: To elucidate the morphological, biological, chemical,
immunological and physical properties of the virus causing Creutzfeldt-Jakob
disease by serial transmissions to chimpanzees; to broaden the experimental
host range of the virus; to determine the nature of the virus and the patho-
genesis of the disease.

Methods Employed: As described for kuru. (See Sub-Project II of this
report.)

Major Findings: Employing the techniques we have developed in our studies
on kuru we have successfully transmitted a second subacute degenerative dis-
ease of the human brain, Creutzfeldt-Jakob disease, from 10 patients to 12
chimpanzees on primary passage. More recently we have been successful in
transmitting this disease from two patients to two species of new-world mon-
keys, the spider monkey and the squirrel monkey. Further, successful serial
passages of the virus from humans to chimpanzees and from chimpanzees to
squirrel monkeys and the woolly monkey have been accomplished. Although the
asymptomatic incubation periods observed in new-world monkeys (9-25 months)
are somewhat more variable and longer than those observed in chimpanzees
(11-14 months) , these less expensive, more readily available and more easily
housed and handled animals are providing greater facility for expanded studies
of this disease. During the past year independent confirmation of trans-
missibility of this disease was obtained with the development of a fatal dis-
ease in a chimpanzee inoculated in the laboratories of Drs. Charles Chany and
Francoise Cathala in Paris, France. The virus is stable at —70 C and is de-
tectable in the supernatant fluids of brain suspensions that have undergone a
centrifugation of 5000rpm/30 minutes. Complement- fixation and hemagglutina-
tion-inhibition techniques were employed to test sera from humans dying with
the disease as well as to test pre- inoculation and serially collected post
inoculation sera from chimpanzees in an attempt to elicit serological evidence
of relationship of antibody in Creutzfeldt-Jakob disease infected hosts with
known viral antigens. No specific patterns suggestive of an etiological re-
lationship were observed when these sera were tested with the following

25s

Serial No. NDS (CF)-62 OAD 969

antigens: influenza types A,B,C; para- influenza types 1, 2, and 3; measles;
mumps; respiratory syncytial virus; Newcastle disease virus; echo types 6,
9, 11, 16, 20; coxsackie types A2, A4, A8, A9, and Bl through B6; polio-
virus types 1 through 3; Herpes simplex; cytomegalovirus; varicella-zoster;
group adenovirus; group reovirus; vaccinia; rabies; lymphocytic choriomenin-
gitis; rubella; SV40; psittacosis; K virus; polyoma; SV5; and, 58 strains of
arboviruses representing groups A, B, Bunyamwera super group, and minor and
ungrouped isolates from Europe and the North American continent.

Significance: These studies have demonstrated that a second subacute pro-
gressive degenerative disease of the CNS of man is caused by a virus = This
strongly supports the continued investigations to attempt to demonstrate a
virus etiology for other presenile and senile dementias of man. These
studies are providing new and significant data on our understanding of sub-
acute spongiform encephalopathies of man and are opening new vistas in neuro-
virology. The collection of specimens from 68 cases of Creutzfeldt-Jakob
disease within a 2-3 year period suggest the not so rare, as previously
thought, occurrence of this disease on a world-wide distribution basis.
Successful transmissions have been accomplished with specimens from patients
residing in the United States, Canada, England and Belgium.

Proposed Course: Emphasis will continue on the characterization, purifi-
cation and identification of the virus. Continued efforts are being made to
broaden the host range. Human tissues and tissues from affected animals are
being extensively studied in an effort to rescue the viral genome and "helper"
and/or "hinderer" virus interactions in Creutzfeldt-Jakob disease. Additional
characterization of this virus as a member of the group of subacute spongiform
encephalopathies will be continued. The epidemiology of the disease is being
pursued.

26

Serial No. NDS (CF)-62 OAD 969

SUB -PROJECT IV: Studies on the characterization and nature of scrapie,
mink encephalopathy and visna viruses

Principal Investigators: Clarence J. Gibbs, Jr., Ph.D., and
D. Carleton Gajdusek, M.D.

Other Investigators: John Hooks, Ph.D., Raymond Roos , M.D. , Paul Brown, M.D. ,
Nancy Rogers, M.S., Mint Basnight, M.S., and
David Asher, M.D.

Cooperating Investigators: J. A. Morris, Ph.D., DBS; Jacob Brody, M.D., NINDS;
Paul Albrecht, M.D., DBS; Michael Stewart, M.D.,
Hilton Levy, M.D., Larry Sturman, M.D. , William Hadlow,
D.V.M. , NIAID; Leslie Weiner, M.D,, Richard Johnson, M.D. ,
and Robert Herndon, M. D., School of Medicine, Johns Hopkins
University; Neal Nathanson, M.D. , Gerald Cole, Ph.D., and
Don Gilden, M.D. , Johns Hopkins School of Public Health and
Hygiene; James Hourrigan, D.V.M. , and A. Klingsporn, D.V.M.,
USDA; John Hotchin, M.D. , Albany; C. Chany, M.D., and
F. Cathala, M.D. , Paris; H. Koprowski, Wistar Institute;
D. Porter, M.D., University of California; Peter Lampert,
M.D. , University of California; M. Oldstone, M.D. ,
Scripps Clinic and Research Foundation

Project Description:

Objectives: To study scrapie, and mink encephalopathy as animal models
of subacute spongiform virus encephalopathies; to determine the pathogenesis
of these diseases in laboratory animals in an effort to elicit the mechanisms
of infection, virus-cell-relationships, nature of the virus per se and the
role of these viruses in inducing an aging process in infected animals. To
study visna, a demyelinating disease of sheep, as a model in the study of
demyelinating diseases of man, particularly, multiple sclerosis.

Methods: As previously described in earlier Annual Reports.

Major Findings:

Scrapie and mink encephalopathy: The recent results of our studies on
these tw/o diseases places them in the group of viruses which we now call
subacute spongiform virus encephalopathies primarily affecting the gray
matter of the brain: two of man: kuru and Creutzfeldt-Jakob disease; and, two
of animals: scrapie and mink encephalopathy. This classification is based
on (1) each is transmissible to an animal host though not necessarily the
same host, (2) each is associated with a long incubation period of from
several months to several years; (3) each induces a clinical syndrome con-
sisting of progressive cerebellar ataxia, tremors and postural instability
which is always fatal in termination; and, (4) each manifest histopatho-
logical lesions only in the gray matter of the brain consisting of severe
intraneuronal vacuolation and dropout, astrocytosis , fibrous gliosis and
moderate to marked status spongiosis without signs of acute inflammatory
response.

During the period covered by this report we have continued our studies
on the characterization of scrapie and mink encephalopathy. Wa have con-

27s

Serial No. NDS (CF)-62 OAD 969

tinued to study the effects of altering the immune mechanisms of the host by-
surgical spleenectomy, thymectomy and through the use of anti-lyraphocytic
serum, cyclophosphamide, and exposure to X-irradiation. None of the pro-
cedures employed had an effect on incubation periods, development or duration
of clinical disease or fatal termination of the process. In a series of ex-
tensive experiments we were again unable to demonstrate antigen-antibody
reactions by complement-fixation procedures utilizing crude or sucrose ex-
tracted suspensions of brain, kidney and spleen taken from mice at 30 days
intervals after IC inoculation of scrapie. Sera employed in these studies
were mouse, hyperim.munized rabbits and roosters, and naturally infected
sheep. During the course of these studies we observed that following inocu-
lation of scrapie IC into mice, virus remains detectable in the brain, spreads
rapidly to the spleen and to a lesser concentration is detectable in the
kidney. Virus in the kidney is associated with a pre-zoning effect. Whether
this is due to the presence of specific neutralizing gamma globulin deposits
on the basement membrane of glomeruli or whether the effect is due to non-
specific interference is under study. Our studies suggest that the reaction
is probably not due to the production of interferon since no increased levels
of interferon have been detected in scrapie infected animals. Further, pro-
phylactic treatment of mice with high concentrations of potent mouse inter-
feron given before and at intervals after exposure to scrapie have shown no
effect on the subsequent disease. Our studies of scrapie and mink encephalo-
pathy have further confirmed the unusual properties of thermostability,
resistance to ultraviolet irradiation, formalin and proteolytic enzymes. We
have successfully repeated our studies on the size of scrapie as determined
by filtration and our findings of 20nm-30nm has been independently confirmed
by scientists in NIAID and at Corapton, England. Administration of poly I-
poly C given in large doses at a variety of times had no effect on incubation
period, clinical signs or fatal termination of the disease.

Visna: During the period covered by this report we have developed direct
and indirect methods of demonstrating visna virus in vitro and antibody in
vivo by fluorescent microscopy. We have developed highly specific techniques
for demonstrating neutralizing antibody to visna virus in sheep serum in
tests conducted in sheep choroid plexus, lamb kidney cells and sheep testis
cell cultures. Consistently negative results have been obtained in studies
to adapt visna virus to mice, rats, rabbits, guinea pigs and hamsters by
inoculation of high concentrations of virus followed by serial blind passages.
The use of immunosuppressants did not alter the non-reactivity of animals to
the virus. Multiple inoculations of high concentrations into mice and
rabbits have failed to induce detectable levels of NT or FA antibodies.

It is of significance that disease has not occurred in chimpanzees,
rhesus monkey, cynomolgus monkeys or African green monkeys in the more than
5 years since they were inoculated intracerebrally only or by multiple routes
peripherally with scrapie, mink encephalopathy and visna virus.

Progressive pneumonia of sheep (Montana sheep disease): We had earlier
postulated that progressive pneumonia of sheep in Montana was caused by a
virus closely related to maedi and visna viruses [ In 1967 we obtained brain
tissue from a sheep and lung tissue from a second sheep both of which had
clinically diagnosed progressive pneumonia. Viruses were isolated from both
specimens submitted. We have now demonstrated chat both strains of virus are

28 1

Serial No, NDS (CF)-62 OAD 969

antigenically related to each other as well as to visna virus by cross
neutralization and cross fluorescent antibody tests. This work has been
confirmed by investigators in NIAID, NIH and at RML, in Montana. These same
investigators have extended these findings to show that the viruses are BINA
viruses associated with an RNA.-DNA dependent polymerase. This plus their
morphological similarity to the RNA oncogenic group of viruses as determined
by EM warrants investigation into their potential oncogenic and tumorigenic
capacities .

Significance: Studies on scrapie, mink encephalopathy, visna and the two
strains of viruses isolated from sheep with progressive pneumonia provide
optimal models for the ±n vivo and ±n vitro mechanisms that are involved in
slow infections of the nervous system. Data can be extrapolated to assist in
the design of experiments and interpretation of results of studies of human
neurological diseases processes. The self fusing properties of visna virus,
even when inactivated, offer a possible mechanism for interpreting the in
vivo process of demyelination, and the relationship of the visna-Montana
sheep disease virus complex to the oncogenic group of RNA viruses.

Proposed Course: Continued studies into the mechanism of infection, the
pathogenesis of disease and the immune mechanisms that may influence slow
infections due to scrapie, mink encephalopathy, visna virus, and the virus of
progressive pneumonia. To expand these latter studies to include a search for
naturally occurring tumors in progressive pneumonia of sheep as well as
alveolar carcinoma of man.

29s

Serial No. NDS (CF) -62 OAD 969

SUB-PROJECT V: Studies on Australian antigen and antibody in chimpan-
zees , monkeys and primate handlers

Principal Investigators: Robert Cornelius, D.V.M. , D. Carleton Gajdusek,
M.D, , and Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: Lewellyn Barker, M.D. , and Michael Peterson, M.D. , DBS;
John Hooks, Ph.D., and Nancy Rogers, M.S.

Project Description:

Objectives: Australian antigen was first associated with hepatitis in
1964, and it was first found in the chimpanzee in 1969. In an attempt to de-
fine the possible role of the nonhuman primate as a source of human infection,
and to explore the carrier state, if any, in the nonhuman primate, this study
was undertaken.

Methods: The NIH laboratory at the Patuxent Wildlife Research Center has
approximately 80 chimpanzees and 350 smaller nonhuman primates. These animals
are housed in two buildings, both of which contain new and old-world monkeys,
and several species of each.

In 1969 serum samples from 95 chimpanzees and approximately 50 other pri-
mates were tested for Australian antigen and antibody. Serum samples from 59
chimpanzees and 6 other primates were also tested in 1970. Serum was tested
for Australian antigen by the Ochterlony micro technique for ppt. and by
complement-fixation, and for antibody using the Ochterlony micro technique.

Those animals possessing antigen were then bled at monthly intervals for
6 months and that serum tested for antigen and antibody, and blood chemis-
tries for liver function. Cagemates of any positive animals were subjected
to the same schedule. To attempt to ascertain when the infection occurred,
serum stored at — 70°C from previous serial bleedings, coTimencing soon after
arrival, were also pooled and tested.

All laboratory personnel who came in contact with the animals, their
tissues or blood, were placed on a bimonthly bleeding schedule. This serum
was screened for antigen and antibody and liver functions.

Major Findings: Ninety-five chimpanzees were sampled in the first series.
Two animals showed 4+ antigen by CF or agar gel ppt. These two animals A28
and A62 were still in the colony when the current study was undertaken.

Eighty-eight serum samples from 59 chimpanzees, and 7 serum samples from
6 other primates, were tested more than a year later. Two chimpanzees ex-
hibited antigen titers by agar gel ppt. or CF, and one chimpanzee serum con-
tained antibody. In addition, one stumptail monkey showed antibody titer of
1:20 by CF.

The two chimpanzees that had antigen in 1970, were the two animals that
previously had shown antigen in 1969, and were still in the colony (A28 and
A62) . Chimpanzee A51 that showed antibody, was shown to have had antigen of
1:20 by ppt. in 1968, by the testing of stored serum„

The testing of stored serum from A28 and A62 . taken soon after arrival in
the colony showed that they had carried the antigen for some time. While the
titer was decreasing, A28 had been infected for at least 4 years, and A62 for
at least 3.5 years.

While A28 was housed singly, A62 was caged with A51. While this is of

30 s

Serial No. NDS (CF)-62 OAD 969

interest, it could not be established how long these two animals had been in
close contact.

Eleven laboratory personnel were bled 5 times in 7 months. Antigen was
not detected by agar gel ppt. or CF in any sample, nor were there any liver
enzyme elevations of significance.

Signif icance: Two findings were considered to be of value in this study.
The first was the demonstration that chimpanzees can show infection with
Australian antigen for at least 4 years, and still not have detectable anti-
body. And the second that transmission between cagemates appears to be
possible, or has occurred.

Proposed Course; It is planned to continue with the monitoring procedure
to detect infection, titer, persistence and carrier/shedding state. Immuni-
zation of primates for the production of antibody will be attempted. The
purpose of these procedures will be to study the feasability of the large
scale production of antibody for use in screening of donors and blood products
as well as to prepare purified gamma globulin for immunoprophylactic pro-
cedures.

31

Serial No. NDS (CF)-62 OAD 969

SUB- PROJECT VI: Fluorescent antibody studies on the intracellular

localization and identification of viral antigens in vivo
and in vitro in tissues from patients with subacute dis-
ease"s~ot the CNS

Principal Investigators: D. Carleton Gajdusek, M.D. , and
Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: John Hooks, Ph.D., Raymond Roos, M.D. , Paul Brown, M.D. ,
Nancy Rogers, M.S., Mint Basnight, M.S., and
David Asher, M.D.

Project Description:

All phases of this project remain essentially as reported for FY1969.
Notable exceptions are the preparation of several new conjugates, adaptation
of FA technique to the in vivo and in vitro study of viral strains isolated
from explant cultures and utilization of this technique in studies to detect
viral antigens and antibodies in patients with multiple sclerosis, kuru,
Creutzfeldt-Jakob disease and other degenerative diseases of man primarily
those which cause spongiform changes in the gray matter of the brain.

32 s

Serial No. NDS (CF)-62 OAD 969

SUB-PROJECT VII: Tissue and cell culture ^^ XiE££ ^'^'^^^^^ °^ viral
induced slow infections of man and animals

Principal Investigators: D. Carleton Gajdusek, M.D.,

Clarence J. Gibbs , Jr., Ph.D., and Nancy Rogers, M.S.

Other Investigators: John Hooks, Ph.D., Raymond Roos , M.D., Paul Brown, M.D.,
Mint Basnight, M.S., Helena Gilbert, David Asher, M.D. ,
and Robert Cornelius, D.V.M.

Cooperating Investigators: Harish Chopra, M.D. , NCI; S. Chou, M.D. , Univer-
sity of West Virginia; Peter Lampert, M.D., University of
California; V. ter Muelen, M.D,, Gottengen, Germany;
C. Chany, M.D. and F. Cathala, M.D,, Paris, France;
S. Baron, M.D. , NIAID

Project Description:

Objectives: To grow and maintain in vitro tissue and cell culture lines
prepared with specimens of brain, visceral, lymphatic, muscle and blood
tissues from human patients and animals succumbing to persistent, chronic,
subacute progressive degenerative diseases particularly of the central ner-
vous system, for the purpose of isolating viruses and demonstrating an
etiological relationship to the diseases under study.

Methods: Brain, visceral, lymphatic, muscle and blood tissues obtained
at surgical biopsy or early autopsy are grown in vitro as explanted organ
type cultures, trypsinized dispersed monolayer cultures, and as polykaryons
following cocultivation and Sendai virus medicated fused cultures with per-
missive cell lines for the rescue of viral genome from donor tissues. All
cultures are serially sub-cultured and maintained _in vitro over long periods
during which time they are (1) observed for spontaneously developing CPE,
(2) transformation, (3) detection of viral antigens by FA, hemadsorption,
CF, and, HA tests, (4) examined by EM for morphology and viral particles.
They are blind passaged into a wide variety of permissive cell lines and
tested for detectable viral antigens. Attempts to isolate viruses are also
being made by classical techniques of inoculating suspensions of various
tissues and organ suspensions from humans and animals into a wide variety of
primary and stable cell lines. Finally, attempts are being made to incorpor-
ate kuru and scrapie into mouse tissue culture cells during the process of
cell fusion. Two strains of cell lines are currently being employed:
C 11 D(TK) and A9(HGPRT-) which were chosen because they allow through their
enzyme deficiencies the selection of hybrid cells which grow in the presence
of amniopterin. Hybrid cells are also being investigated for the appearance
of new antigens after treatment with virus. Such treated cells combined with
Freund's complete adjuvant are used to immunize C3H/Hen mice which are sub-
sequently tested for antibody by means of immune hemadsorption test.

Major Findings: To date, over 1500 explants of brain, visceral, Ijrmphatic
and muscle tissues from humans and animals dying with subacute degenerative
CNS diseases have been grown and maintained in vitro as primary explants and
serial cultures. Over 225 viral isolates have been recovered, the vast major-
ity coming from explant cultures of chimpanzee tissues. Of these isolates

33 s

Serial No. NDS (CF)-62 OAD 969

approximately 827o are strains of new foamy viruses Pan 1 and Pan 2, described
in Sub-Project VIII. TL are strains of adenoviruses Pan 5, Pan 6, Pan 7 and
Pan 9 described in Sub-Project IX; two have been strains of reovirus and
three remain unidentified viruses from chimpanzees. One of these has been
designated Pan 8. The very slow appearance of CPE is necessitating a search
for a more satisfactory cell line to pursue characterization and identifica-
tion studies. It is significant that virus isolations have now been made
from the brains of spider monkeys (foamy- like virus) , squirrel monkeys
(herpes-like virus) and in one instance a squirrel monkey had both herpes-like
and foamy- like virus in his brains. Human tissues have yielded fewer isolates.
We have demonstrated measles virus specific antigen intracellularly in explant
cultures of brain from a case of SSPE; a single, as yet unidentified virus-
like agent has been recovered from brain explant cultures prepared from a
patient dying with ALS. An adenovirus, closely related to type 32 by neutral-
ization tests and to type 27 by hemagglutination-inhibition tests, has been
isolated from the brain of a human that died of lymphosarcoma with a subacute
encephalitis. Virions morphologically resembling adenovirus were observed by
EM in the patient's brain as well as in infected human embryo kidney cell
cultures. This is only the second reported instance that adenovirus has been
isolated from brain and the first reported instance that adenovirus can cause
a subacute encephalitis.

Proposed Course: Continued emphasis will be placed on in^ vitro growth of
human and animal tissues in attempts to isolate latent, persistent, chronic,
masked viruses and viruses which share an endosjTnbiotic relationship with the
donor hosts. Studies are being continued to elicit the etiological signifi-
cance of viral isolates to human and animal diseases. The effects of latent
viruses as they relate to presenile and senile dementias and to aging pro-
cesses in cells is being further studied.

34:

Serial No. NDS (CF)-62 OAD 969

SUB-PROJECT VIII: Characterization and distribution of two new foamy

viruses isolated from chimpanzees t issues grown in vitro
and their relationship to the RNA oncogenic group~oT
viruses

Principal Investigators: John Hooks, Ph.D., Nancy Rogers, M.S., D. Carleton
Gajdusek, M.D. , and Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: Mint Basnight, M.S., Robert Cornelius, D.V.M., and
Ronald DiGiacomo, D.V.M.

Cooperating Investigators: Peter Lampert, M.D. , University of California;
Harish Chopra, M.D., NCI; H. Thormar, Ph.D., and F.H.
Lin, M.D. , Institute for Basic Research in Mental Re-
tardation, Staten Island

Project Description:

Ob iectives: The purpose of this study is to characterize two new foamy
viruses, simian foamy 6 (Pan 1) and simian foamy 7 (Pan 2), isolated from
chimpanzees; to elicit their experimental host range, and their physical,
chemical and biological properties; to determine their etiological signifi-
cance in human and animal diseases; and, to determine what etiological role
they may have in experimental kuru and Creutzfeldt-Jakob disease in chimpan-
zees and new- world monkeys. To determine the relationship of Pan 1 and Pan 2
to prototype strains of simian foamy viruses as well as to monkey mammary
tumor virus.

Methods: Pan 1 and Pan 2 viruses were isolated from brain, visceral
tissues and lymphatic tissues from several chimpanzees affected with kuru,
Creutzfeldt-Jakob disease and from chimpanzees dying with intercurrent infec-
tions. The viruses were isolated and maintained in primary HEK cultures
which were incubated at 35°C - 37°C. Characterization studies of Pan 1 and
Pan 2 were conducted using HEK cultures as the biological system of choice.
Virus purification and assay for the presence of RNA dependent DNA polymerase
were done using the procedure described by Lin and Thormar (Journal of
Virology, 6: 1970) • Cross neutralization tests employing antibody specific
to simian foamy viruses 1 through 7 and mouse mammary tumor virus in HEK cul-
tures were performed to determine antigenic relationships between these
viruses .

Major Findings: These studies have shown that Pan 1 and Pan 2 viruses
isolated from chimpanzees are myxo-like RNA viruses which induce a foamy,
syncytia without inclusion bodies in HEK cultures. The viruses do not share
common neutralizing or fluorescent antibody antigens and neither is related
tc any of the known simian foamy viruses or to monkey mammary tumor virus.
They are morphologically indistinguishable from each other by EM and are
125nm in diameter. The virions consist of a central core 45nm, surrounded by
an inner capsule of 80nm. Virions are observed only in the cytoplasm and at
maturation obtain their outer envelope by budding into intracellular vacuoles
or at the plasma membrane. The morphology of these viruses is strikingly
similar to prototype simian foamy viruses and those viruses belonging to the
oncogenic RNA viruses e.g. mouse mammary tumor, murine leukemia, bovine syn-

35s

Serial No. NDS (CF)-62 OAD 969

cytia and monkey mammary tumor virus. Both viruses are ether, chloroform
and pH-sensitive and are inactivated by exposure to 56°C/30 minutes. They
pass through 220nm millipore filters but not through lOOnm. Homologous
antibody occurs in the serum of donor chimpanzees. Antibodies to one or both
viruses are also found in chimpanzees not inoculated with experimental kuru
or Creutzfeldt- Jakob disease. Following purification procedures two main
areas of protein and nucleic acid were demonstrable by absorbance of gradient
fraction at 260nm and 280nm. Infected materials show a larger peak at frac-
tions 5 through 15. The density of the area ranges from 1.08g/ml to 1.12g/ml.
Enzyme activity, determined by uptake of H thjonidine, was observed in frac-
tions 5-15 and to a lesser extent in other samples tested. Preliminary
studies on the acid-insoluble product show reaction at 37°C and resistance to
RNase. Cross neutralization studies showed that Pan 1 and Pan 2 viruses were
not antigenically related to each other nor is either one related to simian
foamy viruses types 1-5; monkey mammary tumor virus, visna virus, bovine syn-
cytial virus or the virus which causes progressive pneumonia in sheep in
Montana. Finally, although Pan 1 and Pan 2 share the property of RNA-DNA
dependent polymerase with monkey mammary tumor virus they differ from this
virus in their morphology.

Significance: Identification and characterization of two new foamy
viruses from multiple tissues of a high percentage of chimpanzees tested
suggest wide distribution of these viruses in nature. The CPE they induce in
HEK cultures is strikingly similar to the intracytoplasmic vacuolation of
neurons and astroglia observed in the brains of humans and animals affected
with kuru and Creutzfeldt-Jakob diseases.

Proposed Course: (1) Continuation of seroepidemiological survey to de-
termine the distribution of these viruses in man and animals; (2) increased
emphasis on the pathogenesis of these two viruses in chimpanzees to determine
their etiological significance in experimentally induced diseases of the
brain, (3) increased emphasis to determine the oncogenic and tumor inducing
properties of these viruses and (4) expanded studies to determine the re-
lationships between these viruses and other viruses now classified as onco-
genic RNA viruses.

36 s

Serial No. NDS (CF)-62 OAD 969

SUB- PROJECT IX: Characterization of newly identified adenoviruses
isolated from chimpanzee tissues grown in vitro

Principal Investigators: Mint Basnight, M.S., Nancy Rogers, M.S., and
D. Carleton Gajdusek, M.D.

Other Investigators: Clarence J. Gibbs, Jr., Ph.D., Raymond Roos , M.D. ,
John Hooks, Ph.D., and Paul Brown, M.D.

Cooperating Investigator: Wallace Rowe, M.D. , NIAID

Project Description:

Objectives: Identification and characterization of heretofore unidenti-
fied adenoviruses isolated from chimpanzee tissues explants grown in vitro.

Methods: Standard classical techniques for the isolation and identifi-
cation of viruses utilizing tissue and cell culture systems.

Major Findings: Four antigenically distinct viruses designated Pan 5,
Pan 6, Pan 7 and Pan 9 were isolated in primary HEK inoculated with fluids
from explant cultures of lymphoid tissues obtained from three chimpanzees.
In HEK and primary African green monkey kidney cells each of the viruses
induces a CPE consisting of rounding-up of cells and the formation of grape-
like clusters characteristic of adenoviruses. All four are DNA, chloroform
resistant, heat sensitive viruses which are stable at pH 3.0. Each passes
through a lOOnra millipore filter. None of the four viruses is antigenically
related to known human or simian adenoviruses by neutralization or complement-
fixation procedures. Homologous antibody occurs in the serum of each chim-
panzee from whose tissue the virus has been isolated.

Proposed Course: Seroepidemiological studies to determine the distribu-
tion of these four newly identified viruses in man and animals. Studies are
being conducted to determine the significance of these viruses in animals
developing experimentally induced kuru and Creutzfeldt-Jakob disease.

37:

Serial No. NDS (CF)-62 OAD 969

SUB- PROJECT X: Attempts to demonstrate a viral etiology for chronic
encephalitis with focal epilepsy

Principal Investigators: D. Carleton Gajdusek, M.D, , Clarence J. Gibbs , Jr.,
Ph.D., and David Asher, M.D.

Other Investigators: John Hooks, Ph.D., Nancy Rogers, M.S., Paul Brown, M.D. ,
Ra^TTiond Roos, M.D. , Larry Fry, Ph.D., Mint Basnight, M.S.,
Robert Cornelius, D.V.M., and Ronald DiGiacomo, D.V.M.

Cooperating Investigators: T. Rasmussen, M.D. and S. Carpenter, M.D.,

Montreal Neurological Institute, Canada; A. A. Smorodintsev,
M.D, and V.I. Il'yenko, M.D., Leningrad, U.S.S.R.

Project Description:

Occasional patients with chronic focal epilepsy are found at craniotomy
to have active inflammation of the brain months or years after the onset of
illness. In the USSR this condition, called Kozhevnikov 's epilepsy, seems to
follow acute tick-borne encephalitis (TBE) . There has been one published re-
port of the isolation of TBE virus from the brain tissues of two such patients,
but this has never been confirmed. Il'yenko and colleagues in Leningrad have
discovered a promising animal model for this condition; several strains of TBE
when inoculated into rhesus monkeys by the intracerebral route produce only
mild encephalitis, followed by a month or so later by the appearance of focal
choreoathetotic tremors, along with histological evidence of chronic active
inflammation of the brain, from which virus has been recovered up to a year
after inoculation. We have attempted to confirm Dr. Il'yenko's finding using
one of her strains of TBE virus. Four monkeys have been observed for three
months without evidence of movement disorder, and eight more have been inocu-
lated more recently. If the Soviet findings are confirmed we will investigate
the mechanisms of virus persistence by immunosuppression, and by Jji vitro
cultures of brain cells. Dr. Il'yenko has been invited to participate in our
studies through the U.S. -U.S.S.R. Health Exchange Agreement,

Rasmussen and co-workers have observed focal epilepsy with chronic enceph-
alitis in 23 patients, most of them from the United States and Canada. None
had preceding acute encephalitis, although many had a variety of other febrile
illnesses. In our laboratories there have been several attempts to isolate
viruses from the brains of these patients by inoculation of mice, primates and
tissue cultures, without success. Brain tissue from a recent patient has been
grown in vitro by trypsinization and explant; tissue is now in serial passage
and will be fused with several indicator cell lines in an attempt to detect
latent viruses.

38

Series No. NDS (CF) -62 OAD 969

SUB-PROJECT XI: Isolation, epidemiology and pathogenesis of mourniag
dove pox

Principal Investigators: David Asher, M.D, , D. Carleton Gajdusek, M.D. , and
Clarence J. Gibbs, Jr., Ph.D.

Cooperating Investigators: E. Dustman, Ph.D., C. Herman, Sc.D., and
L. N. Locke, D.V.M. , Patuxent Wildlife Research
Center, Department of Interior, Laurel, Maryland

Project Description:

Troublesome outbreaks of pox disease have occurred among wild mourning
doves (Zenaidura macroura) in many areas of the United States, including
Maryland. The agent of this disease was isolated in the chorioallantoic
membranes of hens' eggs. Of five orders of birds tested only doves and
pigeons were susceptible. The domestic pigeon (Columba livia) developed
only a mild form of disease. Dove-pox virus protected domestic pigeons
but not mourning doves against challenge with pigeon pox virus. A serologi-
cal study of the antigenic relationship of dove-pox virus to pigeon-pox virus
is in progress.

39 s

Serial No. NDS (CF)-62 OA.D 969

SUB-PROJECT XIJ. : lafectious and contagious disease management in a
primate colony

Principal Investigators: Robert Cornelius, D.V.M. , Ronald DiGiacomo, D.V.M. ,

D, Carleton Gajdusek, M.D, , and Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: David Asher, M.D., Paul Brown, M.D,, Raymond Roos, M.D. ,
John Hooks, Ph.D., Nancy Rogers, M.S., Mint Basnight, M.S.,
Luis Melendez, D.V.M., and Keerti Shah, M.D.

Technical Assistants: Alfred Bacote, Michael Sulima, Monica Lewis, Paul
Horst, Lloyd Horst, Paul Martin, Amos Fox, James Noll,
Galen Miller, James Webster, and Albert Bontrager

Cooperating Institutions: Animal Resources Branch, NIH; Armed Forces Insti-
tute of Pathology, Departments of Neuropathology and
Veterinary Pathology; Department of Neuropathology, Uni-
versity of California; Department of Neuropathology, The
Maudsley Institute; Department of Pathology, University
of Western Australia School of Medicine; Gulf South
Research Institute, National Center for Primate Biology;
Otisville Laboratories of the Public Health Research
Institute of the City of New York; Department of Patho-
biology, Johns Hopkins University School of Public Health
and Hygiene; New England Regional Primate Center

Project Description:

Objectives: To systematically study normal behavior, breeding habits,
clinical chemistry and hematological values, clinical and sub-clinical inter-
current infections in program and control animals employed in the study of
slow, latent and temperate viruses of the nervous system; to establish a
registry of normal and abnormal values observed in a well conditioned and
well maintained colony.

Major Findings: 1) Herpes simiae: during the later part of 1970 over
907o of the rhesus monkey population in this program was bled and examined for
NT antibody to Herpes simiae. A total of 15 of 97 rhesus tested had NT
antibody with titers of 1:100 or greater. 87 of the monkeys were imported
from India and 19% of these had antibody. There was no apparent correlation
of Ab with age, sex or length of stay in the colon}'^. 8 of 13 monkeys that
had Ab were or still are cagemates. 16 of the 97 Ab positive monkeys were
colony reared.

2) Spontaneous disease in program animals: None of the program disease
problems have been observed in a variety of animal species being employed
as experimental hosts. In primates pulmonary vascular sclerosis, pregnancy
toxemia, mycotic pneumonitis, gastric ulcer, polynephritis , meningitis and
Herpesvirus-T infection have been a problem. During the year newly imported
mink developed clinical signs of Aleutian mink disease within 5 days after
being received into the quarantine unit. This was later confirmed by histo-
logical study. In the ferret colony we have observed spontaneous occurrence
of liver tumors of vascular origin and bronchial asthma due to filarids.

40 s

Serial No. NDS (CF)-62 OAD 969

3) Collection, tabulation and program analysis of data on normative or-
gan weights and hematological parameters in the chimpanzee is currently under
study.

Significance: 1) Herpes simiae infection in a primate colony is of
importance because of the high mortality rate this virus causes in man. The
ongoing study we have initiated will serve to assess the degree of communica-
bility and exposure hazard for animal caretakers. 2) A general diagnostic
pathological workup is being performed on each animal (control and inoculated)
in our research program that dies with an intercurrent infection. Such a
surveillance provides information on diseases occurring within the colony and
provides information for successful management of a large colony which is
made up of old world and new-world monkeys. 3) Normative data on organ
weights, hematological, CSF, clinical chemistries and serological parameters
in the chimpanzee and smaller primates will assist in the rapid diagnosis of
disease.

Proposed Course: Continuation of this long-term study and publication of
a compendium on normal and diseased animal values.

418

Serial No. NDS (CF)-62 OAD 969

SUB- PROJECT XIII: Slow Virus S^nrnposia, Vlth International Congress of
Neuropathology, Paris, France

Principal Investigators: Clarence J. Gibbs, Jr., Ph.D., and
E. Osetowska, M.D.

Other Investigators: D. Carleton Gajdusek, M.D.

Cooperating Investigators: Hilary Koprowski, M.D., Wistar Institute, Phila-
delphia; Jean Lapresle, M.D., and Francoise Cathala, M.D.,
Hopital de la Salpetrifere, Paris; Richard Johnson, M.D. ,
Johns Hopkins University, Baltimore

Project Description:

The development, organization and promulgation of a major theme on
"Pathogenesis of Slow Virus Diseases of the Central Nervous System" as part
of the Vlth International Congress of Neuropathology, Paris, France, August
31 through September 4, 1970.

Results: Publication of Proceedings of Vlth International Congress of
Neuropathology, Masson & Cie, Paris, 1970.

E. Osetowska and C.J. Gibbs, Jr., Theme IV, "Pathogenesis of Slow Virus
Diseases of the Central Nervous System", Proceedings of Vlth International
Congress of Neuropathology, Masson & Cie, Paris, 1970:

Johnson, R.T. : Virus-Host relationships in acute and chronic encephal-
opathies, pp. 761-778.

Gibbs, C.J.jJr. and Gajdusek, D.C.: Characterization and nature of
viruses causing subacute spongiform encephalopathies, pp. 779-801.

Hunter, G.D. : The biochemical properties and nature of the scrapie
agent, pp. 802-817.

Oldstone, M.B.A.: Pathogenesis of cellular injury associated with per-
sistent viral infection, pp. 818-824.

Simons, M.J., Fitzgerald, M.G. , and Alpers, M.P. : Lymphocytic function
in kuru. pp. 825-826.

Brody, J. A, and Nemo, G.J.: Response of peripheral blood lymphocytes
from multiple sclerosis patients to guinea pig basic protein, and multi-
ple sclerosis patients' cerebrospinal fluid and brain, pp. 827-828.

Hotchin, J.: Immunological aspects of slow virus disease: LCM virus-
induced anti-brain antibodies as a cause of neurological damage,
pp. 829-830.

Petursson, G.: Studies on viral antibodies in visna. pp. 831-832.

42 s

Serial No. NDS (CF)-62 OAD 969

Albrecht, P.: Immune status of the organism during experimental scrapie
infection, pp. 833-834.

ter Meulen, V., Muller, D„, and Katz, M. : Immunohistological and histo-
chemical studies in subacute sclerosing panencephalitis: An example of an
analysis of a slow virus infection of the CNS. pp. 835-836.

Koestner, A., Long, J.F. , Jacoby, R.O. , Olsen, R.G. , and Shadduck, JoA. :
Canine distemper as a model of a parainfectious demyelinating encephal-
opathy, pp. 837-838.

Parry, H.B,, and Vince, A, A.: Scrapie disease of sheep: the roles of
gene and "slow virus" in pathogenesis, pp. 839-840.

Dickinson, A.G. : Classification of scrapie agents based on histological
and incubation period criteria in mice. pp. 841-842.

Barlow, R.M. , and Rennie, J.C«: Experience with mink encephalopathy in
various experimental animals, pp. 843-844.

Zeman, W.: Subacute sclerosing panencephalitis, a slow measles virus
infection, pp. 845-849.

Parker, J.C., Jr., Klintworth, G.Ko, and Graham, D.G.: Myxovirus -para-
myxovirus within lesions of the CNS of two cases following vaccination
with attenuated measles virus, pp. 850-851.

Baringer, J.R. , and Griffith, J.F.: Experimental measles virus infec-
tions of the nervous system, pp. 852-853.

Rorke, L.B., Katz, M. , Masland, W.S„ , and Koprowski, H. : Experimental
subacute sclerosing panencephalitis in ferrets. An animal model system
for study of the human disease, pp. 854-855.

Haig, D.A. : Propagation of the scrapie agent in cell culture, pp. 856-857.

Beck, E., Daniel, P.M., Gajdusek, D.C. , and Gibbs, C.J. , Jr.: Subacute
degenerations of the brain transmissible to experimental animals: a
neuropathological evaluation, pp. 858-873.

Anderson, R. McD.: Histopathology of the brain in kuru. pp. 874-875.

Nathanson, N. , Cole, GoA. , Weiner, L.P., Gilden, D.H. , and Johnson, R.T.:
Diversity of pathological lesions produced by acute virus infections of
the nervous system, pp. 876-891.

Hirano, A.: Further studies on amyotrophic lateral sclerosis and parkin-
sonism dementia complex on Guam. pp. 892-893.

43'

Serial No. NDS (CF)-62 OA.D 969

van Bogaert, L. , and Osetowska, E.: Etude comparfie de la maladie de
Carre et des encephalites de la rougeole. pp. 894-896.

Fraser, H. : Comparative morphology of ageing and scrapie, pp. 897-898.

Pattison, I.H. : Detection of the scrapie agent in the tissues of normal
mice. pp. 899-900.

Zlotnik, I.: The pathogenesis of scrapie, pp. 901-915.

Lampert, P.W. , Earie, K.M. , Gibbs, C.J., Jr., and Gajdusek, D.C.: Elec-
tron microscopic studies on experimental spongiform encephalopathies
(kuru and Creutzfeldt- Jakob disease) in chimpanzees, pp. 916-930.

Sever, J.L., Horta-Barbosa, L., Vernon, M.L., Fuccillo, D.A. , Plum, F.,
and Baringer, J.R. : Creutzfeldt- Jakob disease: Virus-like particles in
brain biopsies and tissue cultures, pp. 931-932.

Bignami, A., and Parry, H.Bo: Electron microscopic observations in slow
virus infections of the CNS. pp. 933-934.

Field, E.J.: Slow virus infection of the nervous system, pp. 935-936.

Ovary, E. , Benko, Ch., and Gombi, R. : So-called Gonatas-particles.
pp. 937-938.

Zu Rhein, G.M. , and Eckroade, R.J. : Experimental transmissible mink
encephalopathy (TME) . An ultrastructural study, pp. 939-940.

Raine, C.S., and Sheppard, R.D. : Ultrastructural observations of measles
virus in nervous tissue, pp. 941-942.

Masters, C.L. , Kakulas, B.Ao , Gajdusek, D,C. , and Gibbs, C.J., Jr.: The
significance of the recent experimental observations on slow virus infec-
tions to neuropathology, pp. 943-951.

Kurtzke, J.F, : Multiple sclerosis as a latent infection of the nervous
system, pp. 952-957.

Sever, J.L., Kurtzke, J.F. , Alter, M. , Schumacher, G.A. , Gilkeson, M.R. ,
Ellenberg, J„H. , and Brody, J„A.: Virus antibodies and multiple scler-
osis, pp. 958-959.

Benko, Ch, , Ovary, E. , and Gombi, R. : Morphological methods in the
cerebrospinal fluid (CSF) testing of subacute sclerosing panencephalitis
(SSPE). pp. 960-961.

44 3

Serial No. NDS (CF)-62 OAD 969

SUB- PROJECT XIV: Studies on the ecology, epidemiology, and pathogenesis
of arbovirus infections of man and animals

Principal Investigators: D. Carleton Gajdusek, M.D„, and
Clarence J. Gibbs, Jr., Ph.D.

Other Investigators: Nancy Rogers, M.S., John Hooks, Ph.D., Paul Brown, M,D„ ,
Mint Basnight, M.S., and David Asher, M.D„

Cooperating Investigators: Jacob Brody, NINDS ; K. Shah, M.D. , and

F. Bang, M.D,, Johns Hopkins School of Public Health,

Baltimore; A. Smorodintsev, M.D,, Institute of Influenza,

Leningrad, USSR; A. Shubladze and V. Zhdanov, Ivanovsky

Institute, USSR; B. Gavrilyuk, Institute of Biophysics,

Serpukhovsky Region, USSR; V. Il'yenko, Research Institute

of Influenza, Leningrad, USSR; J. Casals, M,D. ,

R. Shope, M,D. , and W. Downs, M,D, , Yale University, New

Haven; C. Wisseman, M,D. , University of Maryland;

R. Hornabrook, M,D„, Kuru Research Office, Okapa, New

Guinea; F. Schofield, M.D. , Nairobi; J. Sever, M,D., NINDS

Project Description:

Studies are continuing on the following areas:

1. Epidemic hemorrhagic fevers - chronic and persistent infections

2. Seroepidemiology of arbovirus infections in ecologically
isolated primitive indigenous populations:

a) Seroepidemiology of Alaskan populations

b) Seroepidemiology of the populations of the Caribbean and

Central and South American countries with particular
reference to Puerto Rico, Bolivia and Paraguay

c) Seroepidemiology of Australasian populations

3. Japanese B encephalitis studies on Guam

4. Persistence of arbovirus infections in man and animals

5. Isolation and characterization of the virus causing nephro-nephritis
hemorrhagic fever syndrome in Southeast Asia

Significance and Course: These are long-term (5 years and more) studies
being conducted on a continuing basis. Seroepidemiological and viral ecology
studies are being conducted in primitive cultures and specific patterns of
disease determined. These studies will continue until statistically sound
data are collected for publication.

45^

Serial No. NDS (CF)-62 OAD 969

Publications:

Asher, D.M, : Focal neurological disease with chronic encephalitis in
children and in an experimental primate mode. Proceedings XIII Interna-
tional Congress of Pediatrics, Vienna, 1971.

Asher, D.M. , Gibbs, C.J., Jr., and Gajdusek, D.C.: Experimental kuru in
the chimpanzee: physical findings and clinical laboratory studies.
Brain, 1971.

Asher, D.M., Locke, L.N. , and Gibbs, C.J., Jr.: Isolation of the agent of
mourning dove pox. Bacteriological Proceedings, 71:190:vl36, 1971.

Basnight, M. , Rogers, N. , Gibbs, C.J. , Jr., and Gajdusek, D.C.: Character-
ization of previously undescribed adenoviruses isolated from chimpanzee
tissue explants. Bacteriological Proceedings, 70:178:vl59, 1970.

Basnight, M. , Rogers, N.G. , Gibbs, C.J., Jr., and Gajdusek, D.C.: Charac-
terization of previously undescribed adenoviruses isolated from chimpan-
zee tissue explants. Amer. J. Epidemiology, (in press), 1971.

Beck, E., Daniel, P.M., Gajdusek, D.C., and Gibbs, C. J. , Jr.: Subacute
degenerations of the brain transmissible to experimental animals: a
neuropathological evaluation. Vlth International Congress of Neuropath-
ology, Masson and Cie, Paris, pp. 858-873, 1970.

Brown, P., Cathala, F. , Gajdusek, D.C., and Gibbs, C.J., Jr.: Measles
antibodies in the cerebrospinal fluid of patients with multiple scler-
osis. Proc. Soc. Exp. Biol. Med. (in press), 1971.

Brown, P., Hooks, J., Roos , R. , Gajdusek, D<,C. , and Gibbs, C.J., Jr.:
Creutzfeldt-Jakob disease: attempts to identify the agent by CF antibody
relationship to known viruses. Nature (in press), 1971.

Brown, P., Roos, R. , Hooks, J., Gajdusek, D.C., Gibbs, C.J. , Jr.
and Dowdle, W.R.: Viral antibodies in humans and chimpanzees with
Creutzfeldt-Jakob disease. Amer. Acad. Neurol, (in press), 1971.

Gajdusek, D.C: Slow virus infections and activation of latent virus
infection in aging. Advances in Gerontology, 3: 1971.

Gajdusek, D.C: Slow viruses in autoimmune disease. Am. J. Clinical
Path, (in press) , 1971.

Gajdusek, D.C. and Gibbs, C.J., Jr.: Transmission of two subacute spongi-
form encephalopathies of man (kuru and Creutzfeldt-Jakob disease) to
new-world monkeys. Nature, 230:5296, 588-591, 1971.

46s

Serial No. NDS (CF)-62 OkT) 969

Gajdusek, D.C, and Gibbs , C. J. , Jr.: Degenerative neurological diseases
of viral etiology: scrapie, kuru and Creutzfeldt-Jakob disease. In:
Atypical Virus Infections - Possible Relevance to Animal Models and
Rheumatic Disease. The Arthritis Foundation (in press), 1971.

Gajdusek, D.C, Gibbs, C.J. , Jr., and Lira, K.A. : Prospects for the con-
trol of chronic degenerative diseases with vaccines. In: Proceedings
of the Second International Conference on Application of Vaccines Against
Viral, Rickettsial, and Bacterial Diseases of Man (in press), 1971.

Gajdusek, D.C. and Gibbs, C.J. , Jr.: Persistent, defective and slow
virus infections of the nervous system of children. Proceedings XIII
International Congress of Pediatrics, Vienna, 1971.

Gibbs, C.J., Jr. and Gajdusek, D.C: Characterization and nature of
viruses causing subacute spongiform encephalopathies. In: Vlth Interna-
tional Congress of Neuropathology, Masson and Cie, Paris, pp. 779-801,1970.

Gibbs, C.J., Jr. and Gajdusek, DoC: Transmission and characterization of
the agents of spongiform virus encephalopathies: kuru, Creutzfeldt-Jakob
disease, scrapie and mink encephalopathy. In: Immunological Disorders
of the Nervous System, Res. Publ. Assn. Nerv. Ment. Dis., 49:XXIV:
Williams and Wilkins, Baltimore, 1971.

Gibbs, C.J. , Jr. and Gajdusek, D.C: Virological studies of agents iso-
lated from subacute and chronic neurological diseases. Proceedings XIII
International Congress of Pediatrics, Vienna, 1971.

Hooks, J.: Characterization and distribution of two new foamy viruses
isolated from chimpanzees. Ph.D. Thesis, The Catholic University of
America, Washington, D.C, 1970.

Hooks, J., Gibbs, CJ., Jr., Cutchins, E.C, Rogers, N.G. , Lampert, P.W. ,
and Gajdusek, D.C: Characterization and distribution of two new foamy
viruses isolated from chimpanzees (in press), 1971.

Hooks, J., Rogers, N. , Lampert, P., Gibbs, CJ., Jr., and Gajdusek, D.C:
Foaray viruses of chimpanzees: electron microscopy and fluorescent anti-
body staining. Bacteriological Proceedings, 70: 180-181, 1970.

Lampert, P.W. , Gibbs, CJ., Jr., and Gajdusek, D.C: Experimental spongi-
form encephalopathy (Creutzfeldt-Jakob disease) in chimpanzees. Journal
of Neuropath, and Exp. Neurol. (in press), 1971.

Lampert, P.W. , Earle, K.M. , Gibbs, CJ., Jr., and Gajdusek, D.C: Elec-
tron microscopic studies on experimental spongiform encephalopathies
(kuru and Creutzfeldt-Jakob disease) in chimpanzees. In: Vlth Interna-
tional Congress of Neuropathology, Masson and Cie, Paris, pp. 916-930,1970.

47

Serial No. NDS (CF)-62 OAD 969

Masters, C.L., Kakulas, B.A. , Gajdusek, D.C., and Gibbs, C.J., Jr.: The
significance of the recent experimental observations on slow virus infec-
tions to neuropathology. In: Vlth International Congress of Neuropath-
ology, Masson and Cie, Paris, pp. 843-951.

Rogers, N.G. , Gibbs, C.J„, Jr., Gajdusek, D.C., Andersen, S.W., and
Basnight, M. : Visna-like agents from brain and lung of sheep with Montana
sheep disease. Bacteriological Proceedings, 71: 180: v72, 1971.

Roos, R. , Rogers, N.G. , Basnight, M. , Chou, S. , and Gajdusek, D.C.: Isola-
tion of adenovirus 32 from human brain in a case of chronic encephalitis.
Bacteriological Proceedings, 71: 194: vl55, 1971.

Roos, R. , Gajdusek, D.C., and Gibbs, C.J. , Jr.: Liver disease in Creutz-
feldt-Jakob disease (subacute spongiform encephalopathy). Amer. Acad.
Neurol. 1971.

48 s

AMUAL REPORT

JULY 1, 1970 THROUGH JUNE 30, 1971

EPIDEMIOLOGY BRANCH

COLLABORATIVE AND FIELD RESEARCH

NATIONAL INSTITUTE OF NEUROLOGICAL

DISEASES AND STROKE

Introduction

The major areas of activity of the Epidemiology Branch during the

period of this report have been:

I. epidemiologic studies of neurologic diseases;

II. laboratory studies related to the epidemiology and immunology of
neurologic and perinatal diseases;

III. the continued activities on Guam and the Trust Territories;

IV. genetic studies of neurologic diseases;

The following professional personnel have joined the Epidemiology
Branch: Dr. Charles E. Morris, Associate Professor of Neurology at the
University of North Carolina, has been our Of f icer-in-Charge on Guam.
Dr. John Stanhope, an epidemiologist who was with my staff here in Bethesda
was transferred to Guam for the current fiscal year. Dr. Roger Bobowick, who
completed his residency in neurology, joined my staff in Bethesda. Dr. Jeffrey
Allen, who completed his internship in Seattle at King County, is in the
Genetics Section. Kathy O'Meara, Biologist, is also in the Genetics Section,
and Marjorie Matthews,' R. N. , is working with the Epidemiology Branch. Mr. Otis
Turner, Statistician, joined our staff for 6 months in Epidemiology, but
subsequently accepted a promotion to Associate Director, Division of Maternal &
Child Health Services, and Gary Cooper, Medical Technician, is currently
working in the Laboratory. Dr. A.V. Bird, a neurologist from South Africa
has joined the Branch for 6 months as a Visiting Scientist to pursue studies
in the epidemiology of multiple sclerosis.

The following people have left the Branch: Dr. Roger Detels left to
accept an Associate Professorship in Epidemiology at U.C.L.A. , Renee Owens,
Biologist, left to join her husband in Texas, Jane McNew, R.N., left to get
anM.P.H. degree at the Johns Hopkins School of Hygiene and Public Health.

In terms of organization and planning for next year, we are seriously
hampered because the position of Deputy Branch Chief is vacant. Dr. E.
Michael Holden, who is finishing his second year of neurology residency at
the Boston City Hospital, will go to Guam as Of f icer-in-Charge for two years.
Dr. Robert Stern, who is finishing his internship at Mt. Sinai, New York
City, will be joining the Branch in Bethesda.

It

We have pursued the mission of the Epidemiology Branch in studying the
epidemiologic aspects of neurologic disease in which, through our approaches
we can contribute significantly to the understanding of specific aspects of
neurologic disease, which will be of immediate value to physicians and patients,
We believe that through the technique of specific hypothesis testing in
defined populations rather than through large descriptive population studies
we can perform this function most effectively. Our major contributions
during this year have been: As an offshoot of our Guam studies we have
demonstrated that CNS dopamine metabolism is diminished in amyotrophic
lateral sclerosis patients in the United States. This is the first demon-
strated chemical abnormality in the CNS of ALS patients. Through studies of
SSPE we have shown that this disease apparently results from the combination
of an abnormal infection with measles and a subsequent zoonotic trigger.
Epidemiologic studies of MS in California and Washington State have produced
provocative information suggesting that a protective factor exists among
people born in low risk areas and further that the risk of developing MS can
be altered by migration after puberty. We also found that rates for Japanese
and Chinese for MS are consistently low in California and Washington State.
Serologic studies of MS continue to implicate the measles virus and a
possible familial factor. Several difficult studies have been established
and are proceeding well. These include studies of the natural history of
Parkinson^ disease and the effect of L-Dopa, twin studies on multiple
sclerosis, studies of ALS among veterans and a case/control study of Jakob-
Creutzfeldt disease. We have made significant advances in diagnosis and
treatment of acoustic neuroma and studies relating to I.Q and retinoblastoma
and torsion dystonia continue to bear promise.

I . Epidemiologic studies of neurologic disease.

Multiple Sclerosis

We have completed the first phase of our studies of multiple sclerosis
in California and Washington State and have made the following observations.
Orientals who have an apparently low rate of multiple sclerosis relative to
their latitude of birth have low rates of multiple sclerosis after migration
to California and Washington. Further, second generation Orientals persist
in having a low rate of MS suggesting a possible genetic factor. People born
in Washington State had higher rates of MS than those born in California
which would be expected according to the north-south differential of this
disease. People migrating from northern states to the West Coast had higher
rates when they migrated to Washington than to California, implying that they
either acquired protection in California or the causative factors are less
common in California than in Washington. The implication of this is that the
causative factors persist after puberty, which is contrary to current belief.
Those who migrate from low risk southern areas did not develop a high rate of
MS either in California or Washington (small numbers). This implies that
by the time of migration they have protection from multiple sclerosis. If
protection can be acquired as these data would seem to imply, there is some
hope of developing a preventive mechanism for MS.

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In collaboration with Dr. John Sever, Head, Section on Infectious
Diseases, Perinatal Research Branch, C&FR, NINDS, we have pursued our studies
of measles serology in patients and family members. Recent data suggest
that measles antibody is slightly but consistently elevated in MS patients
when compared to controls. This elevation is not consistently encountered
among siblings of the same sex of the MS patients, suggesting a possible
familial factor in this disease. We are still hoping to extend our studies
to the Shetland and Orkney Islands where an ideal population for study exists.
A brief trip to these islands confirmed the observation that the rates of MS
in these small populations are three times higher than the highest rate
reported anjnA/here else.

We have developed several new studies of the epidemiology of multiple
sclerosis. We are investigating some 15 or 20 twins from the Veterans Twin
Registry in which one of the twins has MS, in order to determine if there are
distinct variations in exposure or disease history prior to onset of MS among
the twins. We are also conducting patient, sibling studies of a small group
of MS patients in which the mother is alive and available. We hope to
detect possible differences in early infections such as we encountered with
SSPE or in other factors which have been suggested as having possible
relevance in the etiology of MS. Further, through the contract mechanism we
are involved in a study of 6,000 veterans with MS in order to further
elucidate migratory patterns and predisposing factors in this population and
selected controls.

Amyotrophic Lateral Sclerosis (stateside studies)

Following our observation on Guam that ALS patients had diminished CNS
dopamine metabolism, we extended our studies to stateside ALS patients and
again encountered diminished CNS dopamine metabolism. This finding has now
been confirmed by others at NIH, at Columbia, at Harvard and at Montreal.
Initial treatment trials of ALS patients with L-Dopa have not yielded
impressive results. The observation of a chemical abnormality in the CNS of
ALS patients is to our knowledge the first substantial chemical abnormality
in the CNS of these patients, and hopefully will lead to greater insights in
abnormal neurometabolite patterns among patients with motor neuron disease.

We continue to follow 12,000 statesiders who worked for more than one
year on Guam between 1945 and 1955. The final information on approximately
207o of the 2500 people in this group who have died continue to trickle in.
We have now encountered two patients who died of ALS which does not seem
excessive for a population of adult males.

We are pursuing our epidemiologic study to test the hypothesis that
pancreatic insufficiency is causally related to ALS which has been suggested
by several investigators. We continue to follow some 3,000 veterans who had
duodenal ulcer between 1948 and 1958. In one group only vagotomy and
pyloroplasty was performed, while in another group gastrectomy with Billroth
II anastomosis was conducted and hence the patients had induced pancreatic
insufficiency. The clinical phase of this study has ended with the suggestion
that an excess of non-specific neurologic disease occurred in those receiving
the Billroth II procedure as opposed to those receiving vagotomy and
pyloroplasty. We have no evidence of ALS in these groups. We are now

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attempting to follow-up through the Veterans Administration on causes of
death in this unique population. We are having difficulty in accumulating
the final data but there is a suggestion of an excess of several neurologic
diseases including Parkinson's disease and multiple sclerosis.

We have developed a protocol with Dr. Gilbert Beebe, Director, Follow-
Up Agency, National Research Council of the National Academy of Sciences and
Dr. John Kurtzke, Chief, of the Neurology Service, Vererans Administration,
Washington, D.C., to conduct studies of ALS veterans patterned generally on
the initial MS study among veterans. We have also developed and field
tested a detailed questionnaire to be administered to 200 ALS patients and
200 patients with brain tumor in VA hospitals in order to detect possible
predisposing factors to ALS. We will also analyze exposure to Guam as a
possible predisposing factor to this disease.

In conjunction with our studies of multiple sclerosis in California and
Washington State we have analyzed patterns of ALS among patients bom in
California and Washington and among migrants. In striking contrast to our
findings with MS we have detected no geographic patterns in the incidence
of ALS in these populations.

Parkinson's Disease.

In collaboration with the National Parkinson's Foundation, Inc., in
Miami, Florida, we are studying the effects on long term administration of
L-Dopa and the influence of L-Dopa on the natural history of Parkinson's
disease. We are following a cohort of up to 500 Parkinson's disease patients
who developed their disease from 1962 to 1965 (pre- L-Dopa) and will match
these individuals with a similar number who developed their disease after
1968 and are receiving L-Dopa. Our initial findings suggested a possible
cohort phenomenon with patients in the L~Dopa treatment group being 5 to 6
years older than those in the pre-L-Dopa group. We have reviewed several
other series of patients and have not been able to confirm this impression.
It is important however that an adequate understanding of this observation is
achieved because if there is a cohort phenomenon, it would imply the gradual
disappearance of Parkinson's disease as has been suggested by some investiga-
tors.

We are continuing our study of identical schizophrenic twins receiving
phenothiazines in order to determine if phenothiazine- induced extrapyramidal
disease is the result of a genetic inability to handle these drugs. Analysis
of our first six sets of twins suggests that there is no genetic control over
this phenomenon. Further, we have been studying the appearance of Parkinson's
disease among blacks and whites and have the impression that the disease
occurs less frequently among blacks. In our studies of drug- induced Parkin-
son's disease however, the rate of disease is similar in blacks and whites.
In collaboration with Dr. Thomas Chase, Chief, Unit on Neurology, Laboratory
of Clinical Sciences, NIMH, we have shown that schizophrenics who receive
phenothiazine derivatives and who do not develop extrapyramidal signs have
significantly elevated dopamine and seratonin end products in their CSF.
Those who do develop extrapyramidal signs have significantly depressed levels,
particularly of dopamine end products in the CSF. Thus, a possible mechanism

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can be postulated that over- stimulation of dopaminergic cells from pheno-
thiazine utilization resulted in exhaustion of these cells in a certain number
of cases and subsequent development of parkinsonian features.

Subacute Sclerosing Panencephalitis.

It is extablished that measles in some way is causally related to SSPE.
As a result of our case/control studies we have determined that SSPE develops
in individuals with very unusual measles histories. In up to 2/3 of the
patients (P=.001) there was either no history of measles, measles under age
one, or measles approximately 2 months after exposure to chicken pox.
Further, we have confirmed the observation that SSPE occurs with a strong male
preponderance and essentially in rural areas. Since measles even under age
one does not follow this male, rural pattern, we believe some form of zoonotic
triggering mechanism, perhaps an animal virus, is also involved in the
pathogenesis of SSPE. Our patients had a significantly higher rate of
exposure to sick animals including dogs and fowl, but not cats, than our
controls.

Serum samples collected from patients and their families and controls
and their families are now being tested for measles antibody and will be
available for studies of other appropriate agents.

Stroke

As the result of a field trip to Panama, a potentially valuable study
of stroke among blacks has been developed and awaits funding. We wish to
explore the relative effect of genetic factors and environmental factors on
the extraordinarily high rate of essential hypertension among blacks.

Jakob-Creutzfeldt Disease

We are conducting a case/control study of 40 patients with J-C disease.
Approximately 15 patients and control have been examined and interviewed.
Analysis of these data are not yet available.

II. Studies on Cellular Immunity in MS

Our studies on lymphocyte function in MS have continued in an attempt
to further define the role of cellular immunity in the pathogenesis of
demyelinating diseases. These have consistently failed to show any
significant differences between MS and normal persons. However, our results
demonstrate that suspensions of brain material (especially white matter)
are definitely stimulatory to lymphocytes in culture. This has been true
regardless of the health of the donor. The nature of this effect is uncertain
but must be considered in all investigations on the response of lymphocyte
from patients with neurologic illnesses to CNS antigens.

During the year we continued to investigate the effects on circulating
lymphocytes following measles vaccine. Results have been inconsistent; in
some experiments a depression in lymphocyte transformation was seen while
In others no effect was apparent. Attempts to demonstrate a lymphocyte-

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depressing serum factor following vaccination have been equally negative with
multiple sclerosis patients and normal control persons. These findings,
although involving limited numbers of patients, are in contradistinction to
reports by other investigators of an ant i- lymphocyte factor in the serum of
patients with MS.

Studies on Cellular Immunity in Neurological Diseases other Than
Multiple Sclerosis

In order to investigate the possible role of lymphocyte-mediated
immunity in the Guam ALS and the PD syndromes, we initiated a cooperative
effort with our staff on Guam. The lymphocyte transformation technique
was carried out with the lymphocytes from selected patients on Guam; at
the end of the tissue culture period, the lymphocytes were "harvested"
and sent to our Bethesda laboratory where the remainder of the DNA extraction
procedure was done. The results of these experiments indicate that such
studies are feasible despite the limited facilities and laboratory personnel
on Guam. Definitive studies are now underway.

Basic Studies on the Lymphocyte Transformation Phenomenon and
Related Tests of Cell-Mediated Immunity

Liquid scintillation quantitation of tritium incorporation by dividing
cells has been adopted as the best measure of lymphocyte transformation.
Using this technique we have completed a series of experiments on the effects
of a synthetic double-stranded RNA (poly I-C) on vaccinia virus induced
transformation. The results of these experiments have substantiated and
extended the observations described in last year's report. Complementary
studies using a double-stranded RM. virus (Reo-1) have not shown any effect
on lymphocyte transformation. Other laboratory studies carried out by
Dr. Jeffrey Allen have extended our knowledge on the effects of storage of
lymphocytes for hours or days prior to initiation of culture; we anticipate
that these will prove useful in future investigations using cells from patients
with diseases such as subacute sclerosing panencephalitis (SSPE) and Jakob-
Creutzfeldt disease. In addition. Dr. Allen has initiated a series of
experiments to study the effect of the measles virus on lymphocyte transforma-
tion induced by non-specific mitogens and specific antigenic stimulants.

Because of reports that cellular immunity to CNS antigens in MS patients
can be demonstrated by a different technique (human macrophage migration
inhibition), we are now initiating studies using this method to complement
our lymphocyte transformation investigations. To learn this technique.
Dr. Nemo spent one week in Dr. Bartfeld's laboratory at New York University
and is currently setting up the method in our laboratory.

Studies on Chronic and Congenital Viral Infections in
Experimental Animals

Approximately 50 mice whose mothers received injections of the minute
virus of mice (MVM) late in gestation have been observed for one year. The
only abnormalities observed to date has been the occurrence of solid tumors
in two animals. The first such animal was lost; the second was found to

6t

have a large tumor in the area of the right uterine horn with metastasis to
the liver. Preliminary results suggest the presence of MVM in both the
tumor and the liver and raise important questions as to the role of chronic,
congenital MVM infection in the etiology of solid tumors of the mouse.
Further studies are in progress to evaluate the oncogenicity of MVM and cell-
free extracts of the uterine tumor.

Other experiments with MVM have suggested that maternal infection
early in gestation may result in reabsorption of the infected embryo. This
problem will be studied further during the next year.

Our studies on the epidemiology of SSPE led us to hypothesize that a
fundamental step in the etiology of that disease may be an initial exposure
to measles virus at a time when persistent maternal antibodies might interfere
with the normal development of cellular immunity and hence might allow for
an unusual persistence of the viral genome in certain long-lived cells such
as neurones. Dr. Nemo and Mr. Cooper are currently attempting to develop an
animal model for such a system and are investigating the course of infection
with measles virus in young mice whose mothers were inoculated with measles
a few weeks prior to mating. No results are available at this time.

Studies on the Minute Virus of Mice (MVM)

This agent was selected for use in the development of a model system
for the study of chronic and congenital viral infections. It is hoped that
an elucidation of mechanisms involved with establishment of chronic MVM
infections will contribute to our understanding of certain chronic neurologic
diseases whose etiologies may also involve a persistent, latent or
recrudescent virus infection. MVM has been reported to cause cerebellar
hypoplasia and congenital ataxia but has been the subject of limited inves-
tigation because of technical difficulties involved with its detection,
quantitation and propagation in high concentration. The efforts of Dr. White,
Mrs. Sutton and Dr. Nemo during the past year seem to have resolved these
difficulties: they have developed methods of propagation yielding virus at
concentration 100 to 1000 times higher than previously possible, and have
improved their infectivity quantitation techniques from a six week rather
unreliable test to a one week procedure with sharp endpoints. In addition,
they have demonstrated the growth of MVM in 4 different continuous cell lines,
although reports from other laboratories had been confined to only 2 primary
cell types. Of particular interest is the development by Mrs. Sutton of
chronic MVM infection of one of these cell lines. This system is proving
useful as a continuous source of large amounts of virus and may advance our
understanding of chronic infections in vivo.

Experiments in two cell types have substantiated the impression that
MVM replication requires that the infected cell divide, even though the virus
can infect and persist in a non-dividing cell. This observation may be
relevant to the viral latency which may be a prerequisite to the pathogenesis
of SSPE, J-C disease, and progressive multifocal leukoencephalopathy (PML) .
Our working h5rpothesis is that MVM infection, like certain papova virus (SV-40) ,
polyoma and perhaps the papova-like agents involved in PML is associated with
an integration of viral DNA into chromosomal DNA., and that this relates to

7 t

the tendency for MVM to persist in non-dividing cells. Experiments are
underway to test this hypothesis by defining the sequence of molecular events
which occur in the course of MVM infections in vitro.

III. The continued activities on Guam and the Trust Territories

The epidemiologic patterns for ALS and PD on Guam have been updated
to include the past five years. A slight but apparently not significant
decline in rates was noted. The pattern of age-specific attack rates,
however, has not changed. This suggests that we are not observing a cohort
phenomenon which would have occurred as a result of a massive common exposure
at some time in the past and implies that the cause of causes of ALS and PD
on Guam are still operative. L-Dopa trials on PD patients are in their
15th month with some observable benefit in the extrapyramidal features of
this disease. Drug trials in ALS patients with L-Dopa, isoprinosine and
placebo are in their second month. The studies are "blind" and no dramatic
improvement or untoward responses have been observed. We have further
confirmatory evidence that CNS dopamine production in PD patients is markedly
depressed while CNS dopamine production in ALS patients of Guam and in the
continental United States are significantly comprised but to a lesser degree
than encountered in PD or classical paralysis agitans.

We are not attempting to develop an assay of impaired CNS dopamine
metabolism in which we used expired air and urine rather than CSF to conduct
our measurements. The technique is now being used on patients and controls.
If is is feasible we will conduct studies of CNS dopamine metabolism in
family members of ALS and PD patients and also in geographic areas where the
diseases seem to concentrate.

We conducted a follow-up study of the congenital blindness among the
Pingelapese population on the Eastern Caroline Islands. Six patients have
been seen at NINDS and the diagnosis is now believed to be achromatopsia
with the single reservation that the high myopia encountered in the affected
patients is perhaps a separate entity. The unusual age distribution of
patients observed in our previous trip report which had suggested a possible
extrinsic factor was not encountered in our most recent survey.

IV . Genetic studies of neurologic diseases

The two main interests of the Genetic Section continue to be movement
disorders, especially the torsion dystonias, and hereditary tumors of the
nervous system. In addition the Section is embarking on a study of a highly
inbred group of Irish Tinkers living in the Southeastern United States who
are thought to have several unusual genetic traits.

Our efforts regarding the dystonias are directed toward determining
the basic neurochemical defect in the autosomal i;ecessive and autosomal
dominant forms. Ten of our patients have participated in a study of
catecholamine metabolism on the research ward of Dr. Thomas Chase, NIMH.
Results to date, although not pinpointing a specific abnormality do suggest
we are dealing with the abnormal pathway. Evaluation of various therapeutic
regimens using L-Dopa, peripheral dopa decarboxylase inhibitors and other

8t

drugs is being conducted by collaborators with patients from our series. No
single regimen seems effective for all patients so that tailoring of drug and
dose for each patient is still necessary. Finally, we continue to see new
patients and their families with unusual movement problems. One interesting
group which has emerged consists of professional musicians troubled by a mild
movement abnormality which, however, is sufficient to impair proper performance,
This may be a distinct new entity or else may represent the heterozygous state
of autosomal recessive dystonia.

Acoustic neuroma remains our chief interest in the area of hereditary
neoplasms. On the basis of our study of the large Pennsylvania kindred with
acoustic neuroma and review of the literature we concluded that this seemingly
rare trait might actually be fairly common if the appropriate population were
sampled. Therefore, we looked for the presence of bilateral disease and
positive family history in individuals with onset of sjmiptoms of acoustic
neuroma at a young age. To date we have information on over 2100 individuals
in 51 families and have found at least 4 with positive family history of
acoustic neuroma. Since only 4 families have been reported in the literature
we have been able to double the number of reported families by this approach.
The interesting question of relationship between neurofibromatosis and
acoustic neuroma is being investigated through clinical study, cell culture
techniques and chromosome analysis.

The retinoblastoma study, which deals with perception of affected by
sighted individuals, is moving into its final stages. Preliminary results
show no significant difference in performance between patients and unaffected
sibs used as controls. This is at variance with the two reported studies on
this question. Chromosome studies in familial cases show no abnormalities
unlike sporadic atypical cases.

Our interest in genetic factors in ALS and PD as seen on Guam continues
through study of select families on Guam and among migrants living in
California.

Groundwork is being laid for a comprehensive genetic, demograph and
medical study of the Irish Tinkers of Murphy Village, South Carolina. Since
this group is understandably wary of strangers, great care is being paid to
establishing rapport with elders and other informants within the community.
We are enthusiastic about this project since it is through study of such
isolates that many new recessively inherited traits have been described.

A canvass is being made of isolated communities along the coast of
Maine for similarly inbred groups.

^

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CONTRACT NARRATIVE

Epidemiology Branch, C&FR, NINDS

Fiscal Year 1971

NATIONAL RESEARCH COUNCIL, FOLLOW-UP AGENCY (PH43-64-44)

Title : New Epidemiologic Study of Multiple Sclerosis in U.S. Military
Veteran Population

Contractor's Project Director: Gilbert Beebe, M.D.

John F. Kurtzke, M.D.

Current Annual Level: $34,700

Objectives : The contractor will perform an extensive survey of multiple
sclerosis among veterans of the Second World War and the Korean War.
This will be an update of an initial survey which included 600 patients
and will include approximately 6,000 patients. Patients will be matched
with controls to determine geographic patterns, socio-economic status,
urban-rural localization and numerous other variables which have been
tested for multiple sclerosis. Parallel information will be available
for Negro MS patients and controls and white female MS patients and
controls. In addition an extensive investigation of migrant and non-
migrant populations to and from high and low risk areas for multiple
sclerosis will be conducted. Emphasis will also be placed on the
relationship between the communicable diseases experienced in childhood
and subsequent multiple sclerosis.

Major Findings : The project has just commenced and data is still being
accumulated .

Significance to NINDS Program and Biomedical Research: Multiple
sclerosis is a major neurologic disease in the United States. Previous
epidemiologic studies have indicated that there is in the Northern
Hemisphere a north-south gradient in the rates of multiple sclerosis.
From a previous study, certain interesting correlations were noted
among military personnel who developed multiple sclerosis, such as an
excess of people with higher I.Q.s, with urban residence, with higher
socio-economic status and with defective eyes. The recent emphasis on
risk of multiple sclerosis among migratory populations is of crucial
importance since it must be determined if the causative factors of this
disease are more prevalent in northern areas or if there are protective
factors or mechanisms operating in southern areas . If clear patterns
can be delineated by this study, the results could suggest in which
populations we must concentrate in the search for the etiology and
prevention of multiple sclerosis.

Proposed Course of Project: This project was initiated in March 1971
and will run for three years .

lot

CONTRACT NARRATIVE

Epidemiology Branch, C&FR, NINDS

Fiscal Year 1971

THE JOHNS HOPKINS UNIVERSITY (PH43-67-1347)

Title: The Epidemiology of Parkinson's Disease

Contractor's Project Director: Abraham M. Lilienfeld, M.D.

Irving I. Kessler, M.D.

Current Annual Level : 0

Objectives ; The contractor will: (a) Select a group of hospitals to
provide sufficiently large groups of patients with Parkinson's disease;
(b) Assemble groups of patients with parkinsonism, Parkinson's disease
and similar neurological deficits who have been hospitalized for any
reason or seen in any hospital clinic; (c) Assemble matching groups of
patients without parkinsonism symptoms who have been hospitalized at
the same institutions and who are similar to the cases in age, sex,
race, and other pertinent demographic characteristics; (d) Select a
probability sample of physicians (neurologists, general practitioners,
general surgeons, and internal medicine practitioners) and from them
secure a cohort of patients with parkinsonian symptoms who have not
been hospitalized; (e) Compare the patients and their matched controls
with regard to epidemiologic characteristics of possible relevance to
the incidence of or morbidity from Parkinson's disease, including
familial mortality experience and smoking histories; (f) compare,
within the patient groups, the epidemiologic characteristics of those
with specific parkinsonian symptom complexes; (g) Compare the hospital-
ized and nonhospltalized parkinsonian patients in order to test whether
there are methodologic biases in studies restricted to hospitalized
patients; (h) Assemble a large group of patients known to have had
Parkinson's disease, parkinsonism or similar neurological deficits as
the basis for an analysis of the overall and cause specific mortality
of the decedents; (i) Reach conclusions as to the adaptability of the
case-control method proves to be inadequate, design other more suitable
methods for the epidemiological study of Parkinson's disease.

Major Findings; The first phase of the analysis of this study concentrated
on previously reported lower rates of Parkinson's disease among smokers
than non-smokers. In the population sampled in the present study, this
difference was not encountered and in general, patients were less likely
to have ever smoked, and smokers tended to smoke less than a comparable
group of patients without Parkinson's disease. Further, analysis of
the data are currently underway.

Significance to NINDS Program and Biomedical Research: At present,
understanding of risk factors and natural history in parkinsonism is
limited to a few studies primarily in clinic populations . The
extension using a case-control matching could provide important
information for this extremely important neurologic condition.

lit

Contract (PH43-67-1347)

Proposed Course of Project: The project has run for two years. The
second year was funded at a reduced rate. Therefore, analysis has been
more time-consuming and the final report is not yet available.

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CONTRACT NARRATIVE

Epidemiology Branch, C&FR, NINDS

Fiscal Year 1971

THE UNIVERSITY OF CALIFORNIA-BERKELEY (PH43-68-36)

Title: Health Survey of Stateside Guamanians

Contractor's Project Director: Reuel Stallones, M.D.

Dwayne M. Reed, M.D.

Current Annual Level : 0

Objectives : The contractor will: (a) Analyze mortality experience of
Guamanian residents of California for the past ten years in comparison
to that of California residents generally; (b) Contact adult Guamanians
residing in California and enlist the voluntary participation in the
survey of approximately 1,000 subjects; (c) Secure family and individual
health histories from recruited subjects, and administer a screening
examination comprised of: neurologic examination, psychological
examination to test for dementia and motivational aspects, EKG, BP,
blood tests for cholesterol, triglycerides, uric acid, and glucose;
(d) Conduct similar examinations of a matched group of Guamanians
residing on Rota Island, Saipan Island, and Guam; (e) Code and process
data gathered and analyze results, demonstrating whether or not disease
patterns vary between Guamanian residents in the U.S. and the general
population, and between Guamanian residents in the U.S. and Guamanian
residents in the Pacific.

Major Findings: The investigators documented an unusually high rate of
hyperuricemia and hyperglycemia among Chamorros living on Guam, in
California, or on Rota. The patterns of diabetes and gout differed to
some extent. The major conclusion was that there were no important
differences in disease patterns as a result of westernization.

Significance to NINDS Program and Biomedical Research: Establishing
a baseline on Guam for metabolic disease and cardiovascular disease
as well as some knowledge of psychological patterns and patterns of
neurological disease will be invaluable to our studies on Guam. They
vri.ll also provide important information concerning patterns of disease
among migrating populations.

Proposed Course of Project: The project as originally proposed has
terminated and a report received. A portion of the funds were unspent
and the investigators were authorized to pursue their study in another
Micronesian population on Palau. The field work is complete and we
have received the final report.

13t

CONTRACT NARRATIVE

Epidemiology Branch, C&FR, NINDS

Fiscal Year 1971

THE MASSACHUSETTS GENERAL HOSPITAL (PH43-68-982)

Title: Screening of Blood and Urine for Abnormal Amino Acid Patterns

Contractor's Project Director: Vivian E. Shih, M.D.

Current Annual Level: 0

Objectives : The contractor will perform cromatographic screening of
approximately 1,000 samples each of blood and urine for detection of
disorders of amino acid metabolism among residents of Guam and the
Trust Territories. Testing will be performed by routine methods
developed and utilized in the contractor's laboratory conducting
repeat screening on tests of blood or urine samples as needed in
order to confirm or nullify the significance of unusual patterns.

Major Findings: No major abnormalities in infants, retarded children
or ALS or PD patients were encountered in the survey of blood or
urine of 435 patients with various diseases and 574 normal infants
and 20 normal adults in Guam and other islands of Micronesia. One
girl, apparently healthy but with a history of seizure disorder
(apparently a febril convulsion), many years ago, was found to excrete
an unknown sulphur amino acid. Other non-specific differences in
amino acid patterns were encountered in different populations and were
apparently the result of dietary factors in the given communities.

Significance to NINDS Program and Biomedical Research: As the study
continues on Gxoam we become more convinced that metabolic diseases
play an important role in causing amyotrophic lateral sclerosis and
parkins onism-dementia. In addition, on many islands in the Trust
Territories we notice abnormal genetic patterns of disease which
suggest that certain inborn errors of metabolism may exist. Should
we identify these specific metabolic errors on Guam or in the other
islands we may gain important insights into the cause of the neurologic
diseases in Guam and by using population isolates gain information
concerning metabolic pathways in a given population which would provide
information on metabolic processes in human populations anywhere.

Proposed Course of Project: A final report has been received and a
joint publication has been prepared for submission to a Pediatric
Journal. Further investigation on the family of the girl with the
previously undescribed sulphur amino acid is underway.

14t

CONTRACT NARRATIVE

Epidemiology Branch, C&FR, NINDS

Fiscal Year 1971

THE JOHNS HOPKINS UNIVERSITY (NIH71-2026)

Title: The Study of Regional Differences in Stroke Mortality

Contractor's Project Director: Dean M. Nefzger

Current Annual Level: $21,937

Objectives : The contractor will: (a) Analyze death certificates of
all veterans dying in 1967 in Georgia (high mortality area) and five
Rocky Mountain States (low mortality area) . From the certificates
approximately 1,000 certified CVA deaths in each area and 100 randomly
selected controls will be chosen for further analysis (2,200 cases
total). From these basic data, the frequency of reported CVA among
veterans will be compared with male populations in similar areas to
determine if the geographic variations reported for civilians occur
among veterans; (b) By review of available hospital records and when
necessary by physician or family interview, the validity of the diagnosis
will be established in order to estimate the relative frequency of
mistaken diagnosis or failure to make the diagnosis of CVA; (c) By
review of the accumulated information on veterans dying of CVA an
estimate of the relative frequency of specific types of CVA will be
compiled; (d) All verified stroke deaths and all errors in death
certification will be analyzed in terms of geography, age, race, place
of residence, marital status, from the point of view of sources of
information (competence of certifying individual) and other variables;
(e) During this investigation the complete Military and Veterans
Administration folders will be reviewed for a subgroup of 50 cases and
controls per state in order to evaluate the usefulness of these records
in subsequent studies. In addition, all other avenues of ascertainment
of a valid rate of CVA among veterans will be explored in order that a
definitive study of veterans population be conducted in the future when
the great bulk of veterans of the Second World War arrive at the age of
high risk for CVA.

Major Findings: A total of approximately 1,000 stroke deaths and 2,800
deaths from other causes have been compiled. The data on these cases
are ready for final analysis. A preliminary review of the stroke cases
in Georgia which has a high mortality rate from stroke versus the
Rocky Mountain States which have a low reported mortality from strokes
has revealed surprising and potentially Important information. It
appears that this wide discrepancy may be purely artifactual and the
result of different reporting habits by physicians in the two areas.
It is uncommon for stroke to be listed as underlying cause of death in
the Rocky Mountain States. This finding is at variance with the Johns
Hopkins University Epidemiology of Stroke study which we supported by
contract (PH43-66-920) .

15t

Contract (NIH71-2026)

Significance to NINDS Program and Biomedical Research: The
epidemiological patterns of stroke are poorly understood, although it
is suspected that there are regional differences throughout the U.S.
Any information confirming these differences and indicating a cause
for these differences could lead to a better understanding of causation
and prevention in this important cause of morbidity and mortality.

Proposed Course of Project: The contract has expired, but final
processing of data is not complete. The investigators have received
a contract of $21,937 to complete this work. We are awaiting a final
report .

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Project Title:

Serial No. NDS (CF) - 55 E 201

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Studies on amyotrophic lateral sclerosis/parkinsonism-
dementia complex of Guam (ALS-PD)

Previous Serial Number: Same

Principal Investigators :

Jacob A. Brody, M.D.
Charles E. Morris

NINDS Research Center
John M. Stanhope, M.D.

NINDS Research Center

Other Investigators;

Jose Torres

NINDS Research Center
Francisco Leon Guerrero

NINDS Research Center
Manuel T. Cruz

NINDS Research Center
Olivia Cruz, M.D.

NINDS Research Center
Roswell Eldridge, M.D.

Consultants :

Kwang-Ming Chen, M.D.

National Taiwan University, Taipei, Taiwan
Yoshiro Yase, M.D.

Wakayama Medical College, Japan
Leonard T. Kurland, M.D.

The Mayo Clinic, Rochester, Minnesota
Donald W. Mulder, M.D.

The Mayo Clinic, Rochester, Minnesota
Haruo Okazaki, M.D.

The Mayo Clinic, Rochester, Minnesota

Cooperating Units ;

NINDS Research Center, Agana, Guam

Special Chronic Disease Studies, C&FR, NINDS

Laboratory of Slow, Latent, and Temperate Viruses,

C&FR, NINDS
Department of Epidemiology, School of Public Health,

University of California, Berkeley
The Mayo Clinic, Rochester, Minnesota
Department of Pathology, Massachusetts General Hospital,

Bos ton
Amino Acid Laboratory, Massachusetts General Hospital,

Bos ton

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Serial No. NDS (CF) - 55 E 201

Department of Neurology, Wakayama Medical College,

Wakayama, Japan
Trust Territory Health Office
University of California, Los Angeles

School of Public Health, University of Hawaii, Honolulu
Unit on Neurology, NIMH

Section on Neurology, University of Montreal
Department of Neuropathology, Albert Einstein College

of Medicine, New York
Department of Neurology, Neurological Institute of

Columbia University, New York
Neuropathology Branch, Armed Forces Institute of

Pathology, Washington, D.C.

Man Years

Total:

Professional;

Other:

3/4
1/4

Project Description:

Objectives: To determine the cause of ALS and PD, and to determine the
epidemiological, clinical, neuropathological and physiological significance
of these diseases and to develop therapeutic approaches to the diseases.

Methods employed: Routine methods for epidemiological, clinical,
neuropathological, and neurochemical and therapeutic investigations.

Major findings: As of February 1, 1971, we were following a total of
90 patients. Of these there were 30 confirmed ALS, 13 with sxispect ALS,
12 patients with definite PD and 35 with suspect PD . During the calendar
year 1970 there were 15 deaths from ALS, 3 deaths from PD, 3 deaths among
PD suspects and no deaths among ALS suspects. Of these, autopsies were
performed on 17.

We have completed the 5 year updating of the NINDS ALS and PD patients.
We have analyzed most of the data and have completed early drafts of
manuscripts for publication. The rates of ALS and PD have been declining
slightly but apparently not significantly. Age-specific death rates for
1950 - 59 and 1960 - 69 are identical indicating that no cohort phenomenon
relative to some form of early common exposure or practice is apparent from
these data. This indicates once again that the caxises of these diseases are
still present on Guam. ALS and PD each caused 10% of all deaths among
Chamorros on Guam over age 25 through 1970, Thus 1 in 5 adult Chamorros
will die of one of these diseases.

Analysis of offspring of 100 ALS and 100 PD patients and matched
controls will be completed shortly upon securing further information on
a small residual of people. We hope that analysis of this data will furnish

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Serial No. NDS (CF) - 55 E 201

familial and possibly genetic patterns of the diseases.

Once again we have noted several microfoci of ALS and PD patients in
various areas on Guam. Prior to ray next trip in August our staff will
attempt to identify these foci and map them in order that we may conduct the
appropriate field surveys during my stay.

Results of our study of patients with ALS, PD and controls to determine
the rate of CNS synthesis of dopamine by administering probenecid and
sampling the spinal fluid at appropriate intervals are available. A
manuscript has been prepared and accepted for publication.

The data clearly indicate that PD patients synthesize very little
dopamine in the CNS while in ALS patients dopamine production is compromised
but to a lesser degree. Similar findings among stateside ALS patients were
encountered. This is perhaps the most significant finding emanating from
the Guam studies. It is the first documentation of an altered CNS metabolic
pathway in ALS and may lead to a breakthrough in the understanding of the
pathogenesis of this disease.

In order to explore the apparent deficit in dopamine production in the
CNS of Guamanian PD patients, ALS patients and some controls, we are studying
CNS dopamine synthesis by a new method developed by Dr. Chase and his group
in which we sample urine and expired air rather than CSF. Results are not
yet available.

In addition to routine case finding and documentation we are continuing
trials with L-Dopa. At present of the 8 people in the original trial, 5 are
still receiving the drug after 15 months . One patient started on L-Dopa died
but we were unable to conduct an autopsy. A paper analyzing our first 6 to
8 month experience with PD patients on L-Dopa has been prepared and submitted
for publication. In general, extrapyramidal features in most of the patients
improved to some degree on L-Dopa while there was no clear evidence of
amelioration of the dementia. Some heightened awareness and interest,
however, was observed. Rigidity responded best in our series while tremor
also appeared improved by L-Dopa. Bradykenesia was not improved to the
degree which we expected, suggesting that part of this feature of the disease
may be the result of destruction in non-dopaminergic areas of the brain
(frontal lobe) . We are following all patients carefully in order to determine
if L-Dopa actually affects the natural history and life expectancy in this
degenerative fatal disease.

We are planning to initiate treatment trials in other PD patients using
a peripheral decarboxylating agent (MD 486-Merck) and much lower doses of
L-Dopa. A protocol has been developed for this trial in collaboration with
Dr. Thomas Chase, Chief, Section on Neurology, Laboratory of Clinical Sciences,
NIMH, and the Merck Pharmaceutical Company.

Significance to biomedical research and the program of the Institute:
Almost all ALS patients are now in one of our drug trials. At present 7

19t

Serial No. NDS (CF) - 55 E 201

patients have been receiving L-Dopa since late October 1970, and 12 patients
are receiving isoprinosine since December while 8 patients are receiving
placebo. The studies are double-blind and we analyzed our early results.
Dr. Chen and Yase re-evaluated all patients and scored their results and
our medical staff gave subjective evaluations of each patient's status.
Dr. Stanhope and I have the code and we compared results. It is clearly too
early to make any definitive statement. Patients are tolerating medication
extremely well and are enthusiastic and cooperative in our studies. Our
staff has worked very hard and diligently and each patient is seen at least
once a week. Because of this heightened interest and attention and our
insistence that patients eat at the time they take medication we have
introduced a rather interesting potential bias. Patients appear subjectively
improved and are eating better than before. We should be able to evaluate
this factor over time by comparing patients on drugs and on placebo. The
studies will remain double-blind and when Dr. Stanhope leaves, the code and
pill dispensing will be supervised by Mrs. Hernandez. We believe our
isoprinosine series is now sufficient and new patients will be placed in
the L-Dopa (with Mk 486 when available) drug trial.

Dr. Haruo Okazaki, Section of Experimental and Anatomic Pathology of
the Mayo Clinic continues his systematic study of the occurrence of neuro-
fibrillary changes in the brains of approximately 100 Guamanians who died
of causes other than ALS or PD . Dr. Okazaki visited Guam for 4 days in
December as a consultant and discussed relevant matters with our staff
and with the pathologists of the Giiam Memorial Hospital.

In Guam we have perhaps the highest incidence in the world of motor
neuron disease and of a primary CNS degeneration. The documentation of the
epidemiological, clinical, and neuropathological aspects of ALS and PD, a
major neuromuscular disease and an important primary CNS degeneration have
added to the world's knowledge concerning these neurologic diseases. In
fields in which there are no known causes and no known cures, data such as
these provide one of the most likely avenues for development of concepts and
facts which lead to causes and cures. We are also exploiting this unique
opportunity to test new drugs of potential benefit to patients and through
our studies we have discovered a dopamine deficiency in ALS patients on
Guam and in the U.S. that is the first promising lead in the understanding
of this disease.

Proposed Course: In addition to pursuing the above studies we are
planning the following:

Lymphocyte transformation: These studies have been interrupted because
of the increased clinical activities related to the drug studies.
Mr. George Nemo is preparing a new protocol and material v/ill be sent
to Guam to pursue our investigations of possible immune factors in ALS
and PD.

Tissue culture : Some work on tissue culture from fresh brain material
continues on Guam and in Bethesda under the supervision of Dr. C. Joseph
Gibbs, Head, Laboratory of Slow, Latent and Temperate Virus Infections,

20 t

Serial No. NDS (CF) - 55 E 201

C&FR, NINDS. We hope we will be able to pursue these studies more
actively perhaps by future visits to Guam by Dr, Gibbs and Mr. Nemo.

We are awaiting results of the chemical analysis of neurofibriles from
Dr. Michael L. Shelanski, Assistant Professor of Neuropathology, Albert
Einstein College of Medicine. He received two hemispheres of Chamorro
PD patients ' brains in whom autopsies were performed within three hours
after death and material was frozen in liquid nitrogen. Five brains were
sent to Dr. Andre Barbeau, Director, Department of Neurobiology, Clinical
Research Institute of Montreal. These include ALS and PD patients and
controls and Dr. Barbeau is conducting his to-chemical analysis in order to
determine the patterns of dopamine and other substances in various areas of
the brains . Material from general autopsies conducted previously and stored
as tissue blocks in Bethesda were returned to Dr. Loerzel and Dr. Varona for
review.

Honors and Awards : Official Citation of Commendation from Legislature of
Guam to NINDS Research Center (Resolution #381 of 10th
Guam Legislature)

Publications: Brody, J. A. and Chen, K.M. : Changing epidemiologic patterns
of amyotrophic lateral sclerosis and parkinsonism-dementia
on Guam. Motor Neuron Diseases, Grune & Stratton, 1969,
pp. 61-79.

Chen, K.M., Brody, J. A., Kurland, L.T. and Elizan, T.S.:
Patterns of neurologic diseases on Guam. II. Clinical
and genetic aspects. Neurology , 20:954-964, 1970.

Brody, J. A., Hussels , I., Brink, E. and Torres, J.M.: A
preliminary report on hereditary blindness among the
Pingelapese people of the Eastern Caroline Islands.
Lancet, 1:1253-1257, 1970.

Brody, J.A. : Phenomenal incidence of amyotrophic lateral
sclerosis and parkinsonism-dementia on Guam. Presented at
the 97th Annual Meeting of the American Public Health
Association, Philadelphia, Pa., November 1969.

Brody, J.A.: Studies of the phenomenally high incidence of
amyotrophic lateral sclerosis and parkinsonism-dementia on
Guam. Clinical Society and Commissioned Officers Association
Fifth Joint Meeting - March 31 - April 3, 1970, Washington,
D.C.

Reed, D., LaBarthe, D. and Stallones, R.: Health effects
of westernization and migration among Chamorros . Amer . J .
Epid. 92:94-112, 1970.

2it

Serial No. NDS (CF) - 55 E 201

Brody, J. A., Chase, T.N. and Gordon, E.K.: Depressed monamine
catabolite levels in cerebrospinal fluid of patients with
parkinsonism-dementia of Guam. New Eng. J. Med. 282:947-950,
1970.

Brody, J. A., Hirano, A. and Scott, R.M.: Recent neuropathologic
observations in amyotrophic lateral sclerosis and parkinsonism-
dementia of Guam. Neurology. In press.

Chase, T.N., Schnur, J. A., Brody, J. A. and Gordon, E.K.:
Parkinsonism-dementia and amyotrophic lateral sclerosis of
Guam: Effect of probenecid on monamine catabolite levels
in cerebrospinal fluid. Arch . Neurol . In press.

Schnur, J. A., Chase, T.N. and Brody, J.A. : Parkinsonism-
dementia of Guam: Treatment with L-Dopa. Neurology.
In press.

22t

Serial No. NDS (CF) - 63 E 1103

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Neurological diseases other than ALS/PD on Guam

Previous Serial Number: Same

Principal Investigators: Charles E. Morris, M.D.

NINDS Research Center
John M. Stanhope, M.D.
NINDS Research Center

Other Investigators: Kwang-ming Chen, M.D.

Mayo Clinic
Jacob A. Brody, M.D.
Leonard T. Kurland, M.D.

Mayo Clinic

Cooperating Units: NINDS Research Center, Agana, Guam
Mayo Clinic, Rochester, Minnesota

Man Years :

Total: 1/4
Professional: 3/16
Other: 1/16

Project Description:

Objectives : A survey in 1954 by Donald W. Mulder, M.D. and Leonard T.
Kurland, M.D. gave the impression that not only ALS, but also other heredo-
familial neurologic disorders seemed unusually prevalent while multiple
sclerosis and perhaps CNS tumors are uncommon. The objective of this study
is to try to determine the validity of this data and to see if it is related
to ALS and PD .

Methods employed: Since the establishment of this Center in 1956, we
have occupied a unique position on Guam. It is the only neurological
consultation service available to all ethnic groups on Guam and sees most
neurological patients at Guam Memorial Hospital and Naval Hospital. Therefore,
it is expected that most of the significant neurological cases are eventually
brought to our attention. Because of this unique position we hope to
determine the frequency of various heredo-familial neurological disorders on
the island.

Major findings: During the year 188 patients with neurologic diseases
other than ALS and PD were seen and 99 EEC's were performed. From 1960

23t

Serial No. NDS (CF) - 63 E 1103

through 1966, in conjunction with ongoing studies of amyotrophic lateral
sclerosis and parkinsonism-dementia on Guam, 1,028 Chamorro patients were
referred to our neurologic clinic. In comparison with other populations and
particularly that of Rochester, Minnesota, the residents of Guam had higher
rates of convulsive disorders, myotonic dystrophy, peroneal muscular atrophy,
and hereditary ataxias. There was no indication of an unusual incidence of
central nervous system neoplasms, and no cases of progressive muscular
dystrophy, myasthenia gravis, or indigenous multiple sclerosis were seen.
No patient with proved classic paralysis agitans was observed in the Chamorro
population. One sibship of 13 was followed in which 4 patients died of
various brain tumors and 2 of acute nyelogenic leucemia.

Significance to biomedical research and the program of the Institute;
This study adds to the general body of knowledge being collected by the
Branch regarding the island of Guam and provides information on diseases
possibly related to ALS and PD .

Proposed course: We are expanding studies of neurologic diseases on
Guam and the Trust Territories using the same techniques.

Honors and Awards : None

Publications: Chen, K.M., Brody, J. A. and Kurland, L.T.: Patterns of

neurologic diseases on Guam, Arch. Neurol. 19:573-578, 1968.

Chen, K.M., Brody, J.A., Kurland, L.T. and Elizan, T.S.:
Patterns of neurologic diseases on Guam. II. Clinical and
genetic aspects. Neurology, 20:954-964, 1970.

24t

Serial No. NDS (CF) - 66 E 1319

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: A search for automimmune mechanisms in the pathogenesis of
chronic neurological diseases by the use of peripheral
lymphocytes

Previous Serial Number: Same

Principal Investigators: Jacob A. Brody, M.D.

George Nemo, Ph.D.

Other Investigators: Minnie Toure, Biologist
Gary Cooper, Biologist

Cooperating Unit: None

Man Years :

Total : 1/4
Professional: 1/12
Other: 3/12

Project Description:

Objectives : To study the role of the small lymphocyte in the pathogenesis
of neurologic disorders suspected to be of autoimmune etiology.

Methods employed: Peripheral lymphocytes from patients with multiple
sclerosis (MS) were challenged in vitro with specific antigens. The
incorporation of tritiated thymidine into DNA during the synthetic phase
of lymphoblast transformation is used as an indicator of lymphocyte
responsiveness. In order to quantitate tritiated thymidine incorporation
more accurately, our laboratory has converted from autoradiography to
liquid scintillation spectrometry. Liquid scintillation is more sensitive,
far less time consuming and subject to a minimal degree of hximan error.

Major findings: Lymphocytes from normal patients and patients with
multiple sclerosis were challenged with brain antigens and cerebrospinal
fluid from patients with MS. Basic protein extracted from defatted guinea
pig brains was also used as a test antigen. The results show that no
significant lymphocyte transformation occurred in the samples tested.

Significance to biomedical research and the program of the Institute:
Since it is well established that the lymphocyte is the mediator of cellular
immunity, the lack of a significant lymphocyte response as demonstrated in
our study casts serious doubt on the hypothesis that MS is an autoimmune
disorder .

25t

Serial No. NDS (CF) - 66 E 1319

Proposed course: It may well be that only a small proportion of the
total lymphocyte population sampled was sensitive to the antigens tested.
The total number of reactive cells may have been too few to elicit a
measurable response. If this supposition is indeed correct, then
experimental manipulations designed to elevate the response to detectable
levels might prove fruitful.

Several reagents are currently being tested for their ability to
enhance lymphocyte transformation. Preliminary data indicate that
Polyriboinosinic-Polyribocytidylic acid (Poly I:C), a synthetic double-
stranded polynucleotide, increases the lymphocyte response to vaccinia
virus as much as 40%.

Honors and Awards: None

Publications: Brody, J. A., Harlem, M.M., Plank, C.R. and White, L.R.:

Freezing human peripheral lymphocytes and a technique for
culture in monolayers . Proc. Soc. Exp. Biol. & Med. 129 :
968-972, 1968.

Brody, J. A., Harlem, M.M., Kurtzke, J.F. and White, L.R.:
Unsuccessful attempt to induce transformation by cerebrospinal
fluid in cultured lymphocytes from multiple sclerosis patients,
New Eng. J. Med. 279:202-204, 1968.

26t

Serial No. NDS (CF) - 66 E 1320

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Stateside Guamanian study

Previous Serial Number: Same

Principal Investigator: Jacob A. Brody, M.D.

Other Investigator: None

Cooperating Units: NINDS Research Center, Agana, Guam

School of Public Health, University of California,
Berkeley

Man Years :

Total : 1/6
Professional: 1/12
Other : 1/12

Project Description:

Objectives : This study was instituted in July 1966 to determine if
ALS and PD occur with the same high frequency among Guamanians who have
left Guam. Since the bulk of the stateside Guamanians are in California,
efforts have been concentrated there.

Methods employed : A household census was completed in the fall of
1967 which included information on neurologic disease. Names of heads of
households were obtained from relatives on Guam, the office of the Guamanian
representative to Congress, local Guamanians, social organizations, an eight
year old NINDS California Guamanian registry and from other Guamanians
already living in this country. Household information was obtained by
trained Guamanian interviewers living in California and by personnel from
the Branch. Follow-up examination of suspect cases of ALS and PD was
conducted by specialist physicians.

Ma j or findings : The ALS rate in California is as high as it is on Guam.
Two patients with presumed Parkinson's disease and/or dementia have died and
autopsy studies in one are consistent with parkins onism-dementia although
the patient's age is unusually advanced and in other changes were more
compatible with paralysis agitans without dementia as seen in the United
States. These findings are being included in a report by Brody and Hirano.

27t

Serial No. NDS (CF) - 66 E 1320

Significance to biomedical research and the program of the Institute:
The results suggest that a genetic factor and/or early exposure to an
environmental factor is responsible for ALS and PD on Guam. The PD patient
in California is the only PD patient ever encountered off Guam and the
Marianas. The disease is not known to occur in non-Chamorros . Since the
rate of PD off Guam is lower than that of ALS it suggests that these
diseases are not a spectrum of CNS diseases with a single cause. The
patient with paralysis agitans is the first documentation of this disease
in a Chamorro. He lived in the United States for 8 years which may be a
clue as to the incubation period of paralysis agitans.

Proposed course: It is planned to maintain contact with this migrant
population over the years because of the valuable clues we may gain regarding
etiology of amyotrophic lateral sclerosis and parkins onism-dementia as seen
on Guam, as well as more general medical, epidemiological and social data
as the culturation proceeds . Thus far only Guam-born Chamorros in California
are old enough to develop ALS or PD . As the population ages we will attempt
to determine if these diseases occur at high rates in United States-born
Guamanians .

Honors and Awards : None

Publications: Eldridge, R., Rosario, J. and Brody, J. A.: Amyotrophic
lateral sclerosis and parkinsonism dementia in a migrant
population from Guam. (A preliminary report.) In Trans .
Amer. Neurol. Ass. 93:204-206, 1968.

Eldridge, R., Ryan, E., Rosario, J. and Brody, J. A.:
Amyotrophic lateral sclerosis and parkinsonism dementia
in a migrant population from Guam. (A full report.)
Neurology, 19:1029-1037, 1969.

Reed, D., LaBarthe, D. and Stallones, R.: Health effects
of westernization and migration among Chamorros. Amer.
J. Epid. 92:94-112, 1970.

28t

Serial No. NDS (CF) - 66 E 1321

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Japanese encephalitis on Guam

Previous Serial Number: Same

Principal Investigators: Roger Detels, M.D.

Jacob A. Brody, M.D.
C. Joseph Gibbs, Ph.D.

Laboratory of Slow, Latent and Temperate Viruses,
NINDS
Other Investigators: None

Cooperating Unit: Laboratory of Slow, Latent and Temperate Viruses, NINDS

Man Years :

Total: 1/3
Professional: 1/6
Other: 1/6

Project Description:

Objectives : A Japanese encephalitis epidemic occurred in 1947 on Guam,
but, according to serologic studies, involved only 20% of the population
before apparently disappearing from the island. It is the objective of
this study to determine if Japanese encephalitis virus (JEV) is persisting
on Guam at a low level and to determine why it has not established an
epidemic pattern as in Japan, Taiwan and Korea or an endemic pattern as
in Malaysia, despite the presence of a suitable vector and reservoir hosts.

Methods employed: Sera will be collected from Guamanians born prior
to, during and after the occurrence of the 1947 epidemic and will be
analyzed for antibody to JEV and other Group B and Group A arboviruses.
Sera will also be collected for antibody screening from animals. Mosquitoes
will be collected to determine the types of culicenes present on the island
which might act as vectors.

Major findings: Eighteen percent of sera from 498 Guamanians born
since 1900 contain hemagglutination inhibition antibodies to JEV.
Twenty-one percent born prior to 1950 and 8% born since 1950 have HI
antibody to JEV suggesting that there has been Group B arbovirus activity
on Guam since 1950. Nonetheless, only 1 of 100 pigs bled had HI antibody
to JEV . Culex tritaeniorhynchus, but not Culex annulus , has been identified
on the island.

29t

Serial No. NDS (CF) - 66 E 1321

Significance to biomedical research and the program of the Institute:
JEV was thought to have disappeared from Guam contrary to the usual pattern.
However, the finding of HI antibodies to JEV in 8% of Guamanians born since
1950 but not in pigs when all the known necessary ingredients are present
for JEV to be either endemic or epidemic invite investigation to determine
the uniqueness of Guam and thus contribute to the knowledge of factors
important to the epidemiology of JEV.

Proposed course: Tissue culture neutralization tests will be done on
all positive HI sera and an aliquot of HI negative sera using several
antigens in addition to JEV. Further sera are being collected from
Guamanians born since 1950 for neutralization tests. The C . tritaeniorhynchus
from Guam will be subclassif ied, since Barnet has proposed that only
C. tritaeniorhynchus summorosus acts as a vector for JEV.

Honors and Awards : None

Publications: None

30 1

Serial No, NDS (CF) - 67 E 1325

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: One year experience of all births on Guam with special
reference to diabetic complications

Previous Serial Number: Same

Principal Investigator: Jacob A. Brody, M.D.

Other Investigators: John M. Stanhope, M.D.

NINDS Research Center
Anne Kantor

Office of Biometry

Cooperating Units: NINDS Research Center, Agana, Guam
Office of Biometry, OD, NINDS

Man Years :

Total :

1/4

Professional:

1/12

Other:

1/6

Project Description:

Objectives : The present project was designed to expand the scope of
this study by analyzing all births for one year. During the year 1965
there were 2,523 births and we compiled data on date of birth, place of
birth, birth weight, birth order, length of gestation, birth defects, age
and race of parents, maternal complications such as diabetes, etc.

Methods employed : A study by Yen in 1963-64 revealed an unusually
high incidence of abnormal carbohydrate metabolism during pregnancy among
the native population of Guam. He also found that obesity and large babies
appeared to be a constant finding in mothers with abnormal carbohydrate
metabolism and were connected with an increased incidence of maternal and
perinatal complications .

Major findings: These data revealed an excess of low weight and high
weight babies among diabetic mothers. Differences were not statistically
significant and in general infant mortality on Guam is similar to that in
the United States.

Significance to biomedical research and the program of the Institute:
The data will be of value for public health and anthropological studies.

3Ib

Serial No. NDS (CF) - 67 E 1325

We will also conduct prospective retrospective studies on the use of birth
weight as an indication of diabetes in families and communxties .

Proposed course: This study has been terminated.

Honors and Awards: None

Publications : None

32 t

Serial No. NDS (CF) - 67 E 1485

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Analyses of abnormal urine and blood amino acids metabolism
among Guamanians

Previous Serial Number: Same

Principal Investigators: Jacob A. Brody, M.D.

Vivian Shih, M.D.

Massachusetts General Hospital

Other Investigators: Jose M. Torres

NINDS Research Center
Manuel T. Cruz

NINDS Research Center

Cooperating Units: NINDS Research Center, Agana, Guam

Amino Acid Laboratory, Massachusetts General Hospital,
Boston, Massachusetts

Man Years :

Total: 1/6
Professional: 1/12
Other: 1/12

Project Description:

Objectives: Earlier observation indicated that indigenous Guamanians
have difficulties in handling protein and carbohydrate. However, the
relationship between the observed hyperuricemia and hyperglycemia on Guam
and the neurologic manifestations is not clear.

Methods employed: A contract with the Amino Acid Laboratory of the
Massachusetts General Hospital for the broad testing for inborn errors of
metabolism was secured and blood and urine are being sent to the lab from
Guam.

Major findings: No major abnormalities in infants, retarded children
and ALS or PD patients were encountered in the survey of blood and urine
in 435 patients with various diseases and 574 normal infants and 20 normal
adults in Guam and other islands of Micronesia. One girl apparently healthy
but with a history of seizure disorder (apparently a febril convulsion) many
years ago was found to excrete an unknown sulfur amino acid. We are
reviewing her family clinically and collecting appropriate urine samples
for confirmation of this finding. No specific changes were found in PD or

33t

Serial No, NDS (CF) - 67 E 1485

ALS patients. B-amino isobutyric aciduria was detected in 56.8% of normal
infants. Taurine excretion was prevalent in normal infants on the Caroline
Islands; it was probably related to breast-feeding. Cystathioninuria was
present in 9 normal infants . We have the impression that the infant
population in Guam and other areas have relatively low rates of abnormal
amino acids when compared with other populations . This cannot be claimed
conclusively because sampling methods differed in our series. The Department
of Public Health of the Government of Guam has routinely screened all new
boms for PKU for the past 6 years and no case has been encountered.

Significance to biomedical research and the program of the Institute;
This study contributed to knowledge of metabolic abnormalities of the
Chamorro people of Guam.

Proposed course: Collection of specimens is complete. We will follow
up on the one unusual patient referred to above.

Honors and Awards : None

Publications: Shih, V.E., Brink, E.W., Peneva, P. and Brody, J. A.: Blood

and urinary amino acid patterns in Guamanians and Micronesians .
Amer. J. Pis. Child. In press .

34 1

Serial No. NDS (CF) - 67E 1486

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Torsion Dystonia - a clinical and genetic study

Previous Serial Number: Same

Principal Investigator: Roswell Eldridge, M.D.

Other Investigators: Irving S. Cooper, M.D.

St. Barnabas Hospital
Morris B. Gross, M.D.

Hunter College in the Bronx
Wolfgang Zeman, M.D.

University of Indiana
Mary Bazelon Coleman, M.D.

Children's Hospital
Kathy O'Meara

Cooperating Units:

Department of Neurologic Surgery, St. Barnabas

Hospital, New York
Laboratory of Clinical Science, NIMH

Man Years

Total

1

Professional:

2/3

Other:

1/3

Project Description:

Objectives : Torsion dystonia (TD) comprises a heterogeneous group of
conditions characterized by disordered movement. TD may be due to either
genetic or environmental factors. The present study has already defined
further the nosology of these conditions. Further clinical family
studies may suggest the basic defect in each.

Methods employed: Initially, probands with a history of TD selected
through 180 neurologic and neurosurgical centers provided the families
for study. Recently, physicians and affected individuals themselves
have contacted us requesting help. A detailed clinical family history
is obtained. The latter stresses geographical origin of ancestral
couples. The proband and all available relatives were given physical
examinations. Patients from all areas of the U. S. treated by various
methods are seen to avoid geographic, ethnic, and therapeutic bias.

35t

Serial No. NDS (CF) - 67 E 1486

Maior findings: Clinical, genetic, psychometric and therapeutic
aspects of dystonia have been evaluated in more than 200 patients in 130
families. The results have appeared in publications indicated below.
Among the conclusions are: at least two hereditary forms of dystonia
exist; there is variation in clinical features and course of the
hereditary types of dystonia; psychotherapy has a limited role as
primary treatment; drugs reported to be helpful in the dystonias generally
have been ineffective in most patients over a long period; and recent
neurosurgical procedures offer hope.

Significance to biomedical research and the program of the Institute:
Elucidation of the fundamental defect in these forms of dystonia will be of
practical importance. In addition to suggesting specific treatment,
it should be possible to distinguish between the recessive and dominant
forms chemically. The application to genetic counselling of such a test
is obvious. As in other inborn errors of metabolism, such a study could
provide basic, new information about central nervous system physiology.

Parkinson's disease shares certain clinical features with dystonia and is
relieved by the same operative procedure so that information gained from
the dystonia study may bear on this important problem.

Proposed course: Present efforts in this project are directed toward
documentation of all abnormalities in the hereditary dystonias and search
for appropriate therapy. In the latter connection we are working with
other medical centers including the National Institute of Mental Health,
Department of Neurology at Children's Hospital, Neurological Institute of
New York City, St. Barnabas and Albert Einstein Hospitals, New York City,
University Hospital, Cleveland and University of Montreal, Montreal.

We are pleased with the ground work that has been laid in terms of under-
standing the nosology and clinical course of the dystonias.

Honors and Awards : None

Publications:

Eldridge, R, , Ryan, E. , Brody, J. A. and Cooper, 1,8.: Dystonia musculorum
deformans: Evidence for two hereditary forms. Excerpta Medica International
Congress Series No. 175, Progress in Neuro-Genetics , Vol, I of the Proceed-
ings of the Second International Congress of Neuro-Genetics and Neuro-
Ophthalmology, Montreal, September 1967, pp, 772-788, 1969,

36t

Serial No. NDS (CF) - 67 E 1486

Eldridge, R. , Harlan, A., Cooper, I.S., and Riklan, M. : The Hereditary
Torsion Dystonias (Dystonia Musculorum Deformans): Geographical
distribution and I.Q. in dominant and recessive forms. In Transactions of
the American Neurological Association, 94, 1969.

Eldridge, R. , Harlan, A., Cooper, I.S. and Riklan, M. : Superior intelligence
in recessively inherited torsion dystonia. The Lancet 1:7637, pp. 65-67,
1970.

Eldridge, R. , Edgar, A., and Cooper, I.S.: Genetics, Geography and
Intelligence in the torsion dystonias. Proceedings of the Second
Conference on the Clinical Delineation of Birth Defects, May 1969. The
National Foundation-March of Dimes (In press)

Eldridge, R. : The Torsion Dystonias: Literature Review: Genetic and
Clinical Studies. In The Torsion Dystonias (Dystonia Musculorum Deformans).
Editor, Roswell Eldridge, Neurology suppl. 20:11, Part 2, November 1970.

Eldridge, R. and Koerber, T. : The Torsion Dystonias: Some Genetic and
Psychiatric Implications. The Psychiatric Forum. April, 1971.

37t

Serial No. NDS(CF) - 67 E 1487

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Genetic analysis of family data on Guam ALS cases

Previous Serial Number: Same

Principal Investigators:

John M. Stanhope, M.D.

NINDS Research Center
Jacob A . Brody , M.D.
Charles E. Morris, M.D.

NINDS Research Center
Roswell Eldridge, M.D.

Other Investigator:

Manuel T. Cruz

NINDS Research Center

Cooperation Unit: NINDS Research Center, Agana, Guam

Man Years :

Total: 1/4
Professional: 1/6
Other: 1/12

Project Description:

Objectives: To utilize the accumulation of 20 years of experience for
an indepth genetic analysis of pedigree information of Guam ALS .

Methods employed : Pedigrees were developed for the 370 definite cases
of Guam ALS by Guamanian practitioners aware of actual biologic parents . Of
these, 70 were suitable for segregation analysis. Also examination was made
of sibs whose parents were both affected by ALS.

Ma j or findings : The initial data indicate that Guam ALS may be inherited
as a simple autosomal recessive trait. In 46 families suitable for test of
the autosomal recessive hypothesis, 64 cases were observed while 74 cases
would be expected from truncate analysis. The 95% confidence limits for
such analysis cover the range from 64 cases to 84 cases so the observed
number of cases is compatible at this level of significance although barely
so.

Two of 13 over 35 years of age whose parents both had ALS were found to
have signs compatible with early ALS. If ALS is recessive all such offspring
should eventually be affected.

39t

Serial No. NDS (CF) - 67 E 1487

In testing for the autosomal dominant hypothesis in the 16 families
suitable for this analysis, 9 cases were observed, 27 would be expected and
19 to 35 cases would be the range at a 95% limit confidence. Therefore,
autosomal dominant inheritance is possible only if one postulates the gene
is not penetrant (i.e., there is no expressing of the disease) in 50% to 70%
of those carrying it.

Troublesome to any simple mode of inheritance postulated is the 2:1 male
to female ration of ALS patients. A finding which may answer this discrepancy
and possibly shed light on the basic process of the disease is that in these
siblings there is an excess of female deaths under one year of age. In
addition, 11 of our ALS or PD patients have only one Guamanian parent. We
are also following the offspring of the first 100 ALS and PD patients and
controls referred to above.

Significance to biomedical research and the program of the Institute:
Obviously establishing a genetic basis for Guam ALS and PD would have far-
reaching consequences. The elusive question of the cause of Guam ALS and
PD would be answered. A new genetic disease would be added to the expanding
catalogue of inherited neurologic diseases.

Proposed course: Complete information is being obtained on pedigrees
of other Guam ALS and PD cases so as to increase the sample studied.
Detailed study of offspring now at risk of specific mating types is underway.

Honors and Awards : None

Publications : None

40 1

Project Title:

Serial No. NDS (CF) - 67 E 1488

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individiaal Project Report

July 1, 1970 through June 30, 1971

Serological studies of common viruses in cases of multiple
sclerosis (MS) and controls

Previous Serial Number: Same

Principal Investigators;

Other Investigators:

Jacob A. Brody, M.D.
John L. Sever, M.D.

Perinatal Research Branch, NINDS
Anne H. Edgar
Jane McNew, R.N.

Mark Dyken, M.D.

Neurology Department, Indiana University Medical

Center
A. Donald Merritt, M.D.

Department of Medical Genetics, Indiana University

Medical Center

Cooperating Units: Section of Infectious Diseases, PRB, NINDS

Neurology Department, Indiana University Medical Center,

Indianapolis
Department of Medical Genetics, Indiana University

Medical Center, Indianapolis
The Wis tar Institute, Philadelphia, Pennsylvania

Man Years :

Total: 2 1/6

Professional: 1 1/6
Other: 1

Project Description:

Objectives : To test the hypothesis that MS may be caused by an unusual
response to a common virus infection. To search for possible distortions of
segregation and association among MS patients, siblings and controls.

Methods employed: MS patients known to the Neurology Department, lUMC
and to the Indiana Chapter of the National Multiple Sclerosis Society were
contacted and asked to participate. In addition, for each case several
controls with similar backgrounds and infectious disease experience were
selected. Controls are classmate friends of the patient who grew up in the
same community. Siblings of MS patients were also tested. A second sample

4lt

Serial No. NDS (CF) - 67 E 1488

of MS patients and siblings was taken from the Washington, D.C. area.
Patients and controls answered standard questions regarding infectioiis disease,
environment, course of illness and family history, and blood specimens were
taken.

Serological analysis was conducted in the Section of Infectious
Diseases, PRE, NINDS using a battery of common virus antigens by
hemagglutination and complement fixation methods. Differences between the
patient, his sibling and his controls were analyzed.

A portion of frozen serum is being banked to test promising hypotheses
in the future.

Major findings: In the Indiana series we found that MS patients have
higher titers than matched controls for measles, mumps, influenza C,
parainfluenza type 3, varicilla and herpes virus hominus . In no case,
however, were titers of MS patients higher than their siblings of the same
sex. In the Washington series, female siblings had titers as high as
female patients to measles while male siblings titer were lower than those
of the patients .

Significance to biomedical research and the program of the Institute:
The observation that MS patients do have consistently higher titers against
many viruses than controls supports an infectious or immune mechanism as
being involved in the etiology of MS. The finding that higher titers also
occur in siblings suggests that the phenomenon may be related to a common
familial exposure or a familial immunologic defect.

Proposed course: We are now testing gamma globulin levels of these
sera in collaboration with Dr. Oldrich Kolar, Department of Neurology,
Indiana University Medical Center, and testing the rabies titers in
collaboration with Dr. Hilary Koprowski, Director, The Wistar Institute.
We hope to extend our studies to the Shetland and Orkney Islands where MS
occurs at a rate three times higher than elsewhere in the world.

Honors and Awards : None

Publications: Henson, I.E., Brody, J. A. and Sever, J.L.: Elevated measles
antibodies in patients with multiple sclerosis and in their
siblings. Presented at the 97th Annual Meeting of the
American Public Health Association, Philadelphia, Pa.,
November 1969 .

Henson, T.E., Brody, J. A., Sever, J.L., Dyken, M.L. and
Cannon, J.M.: Measles antibodies in patients with multiple

sclerosis and their siblings and controls. JAMA, 211:1985,
1970.

Brody, J. A.: Virus antibody titers in multiple sclerosis
patients, siblings and controls. (An abstract.) (Presented

42 1

Serial No. NDS (CF) - 67 E 1488

at the American Epidemiological Society Meeting in Seattle,
Washington, April 1970.)

Brody, J. A., Sever, J.L. and Henson, T.E.: Virus antibodies
in the serum of multiple sclerosis patients and matched
controls. Neurology, 20:389, 1970. (Presented at the
American Academy of Neurology, May 1970. Proceedings to
be published. )

Brody, J. A., Sever, J.L. and Henson, T.E.: Virus antibodies
in MS patients, siblings and controls. JAMA. In Press.

43t

Serial No. NDS (CF) - 67 E 1489

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Neuropathological studies in veterans dying of ALS who
served on Guam

Previous Serial Number: Same

Principal Investigators: Jacob A. Brody, M.D.

R. Michael Scott, M.D.

Other Investigators: Kenneth Earle, M.D.

Armed Forces Institute of Pathology
Asao Hirano, M.D.

Montefiore Hospital
Joseph Seggora, M.D.

Veterans Administration Hospital
F.A. Quadfasel, M.D.

Veterans Administration Central Office

Cooperating Units: Neuropathology Branch, Armed Forces Institute of

Pathology, Washington, D.C.
Department of Neuropathology, Montefiore Hospital

New York
Veterans Administration Hospital, Boston
Neurology Section, Veterans Administration Central

Office, Washington, D.C.

Years :

Total:

1/3

Professional:

1/3

Other:

0

Project Description:

Objectives : To determine if ALS in veterans who served on Guam is an
acquired disease.

Methods employed: Dr. Hirano has reported characteristic neurofibrillary
changes in Guamanian ALS patients, but these changes are not seen in classical
stateside ALS. Since a large number of U.S. servicemen were stationed on
Guam during World War II, it is possible to examine the CNS of U.S. veterans
who died of ALS who spent considerable time on Guam. The question to be
answered is whether these men show the characteristic Guam-type neuro-
pathological changes or the classical stateside ALS changes.

45t

Serial No. NDS (CF) - 67 E 1489

Major findings: Brains from three veterans serving on Guam have been
collected. In two, there were no neurofibrillary changes, while in one
these changes were present, but not in the distribution observed in Guam
ALS patients .

Significance to biomedical research and the program of the Institute:
These findings are evidence that Guamanian ALS does not result from short
term exposure to an environmental agent. However, the brain of a Filipino
dying of ALS after being on Guam for many years has shown typical
neurofibrillary changes, suggesting that length of exposure to an environ-
mental agent may be an important factor in subsequent development of ALS.

Proposed course: This study is terminated and the data have been
written up and accepted for publication.

Honors and Awards : None

Publications: Brody, J. A., Hirano, A. and Scott, R.M.: Recent neuropatho logic
observations in amyotrophic lateral sclerosis and parkinsonism-
dementia of Guam. Neurology. In Press .

46t

Serial No. NDS (CF) - 67 E 1490

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: The application of fluorescent antibody methods to the
study of chronic neurological disorders

Previous Serial Number: Same

Principal Investigators: Jacob A. Brody, M.D.

Minnie Toure, Biologist
George Nemo, Ph.D.

Other Investigator: None

Cooperating Unit: None

Man Years :

Total: 1/3
Professional: 1/4
Other: 1/12

Project Description:

Objectives : To employ fluorescent antibody techniques using frozen
sections of CNS tissue for detection of viral antibody.

Methods employed : The two main staining techniques employed in
fluorescent microscopy, the direct and indirect methods will be employed.
Frozen CNS tissue sections 4-5u will be prepared using a microtome in a
refrigerated cryostat.

Major findings: None as yet.

Significance to biomedical research and the program of the Institute:
Fluorescent antibody methods are applicable to the study of chronic
neurological disorders thought to be of autoantibodies as well as sites
of delayed-type hypersensitivity reactions. A search for viral antigens
and sites of viral replication in CNS tissue is also made possible using
this technique.

Proposed course: To be continued.

Honors and Awards : None

Publications: None

47t

Serial No. NDS (CF) - 67 E 1496

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Sequelae of CNS diseases in childhood

Previous Serial Number : Same

Principal Investigator: Jacob A. Brody, M.D.

Other Investigators: Estelle Kornhauser, R.N.
Otis D. Turner

Cooperating Units: Office of Biometry, OD, NINDS

Children's Hospital, Washington, D.C.

Man Years :

Total: 2/3
Professional: 1/3
Other: 1/3

Project Description:

Objectives: To determine if infection with viruses capable of
penetrating the CNS cause permanent neurologic sequelae, particularly in
those cases in which infection occurred under the age of two years.

Methods employed : As the result of previoios studies we have decided
to improve our methods and techniques by investigating populations in which
known infections from encephalogenic viruses occurred.

Ma j or findings : Although we have attempted to secure the necessary
populations of children under age 1 in New York, Panama, St. Louis and
Chicago, we have not yet encountered a situation suitable for testing our
hypothesis .

Significance to biomedical research and the program of the Institute:
Although it is widely believed that infections of the CNS early in childhood
produce brain damage, there are no definite patterns of brain damage
or specific diseases which commonly are associated with brain damage.
Documentation of specific viral-tropisms to learning and performance would
be a major contribution to understanding and preventing minimal and major
brain damage.

Proposed course; Well documented arbovirus outbreaks have been observed
by MARU, NIAID and several hundred measles patients who were infected under

49t

Serial No. NDS (CF) - 67 E 1496

age 1 are known in Chicago (Kenrad E. Nelson, M.D., Assistant Professor of
Preventive Medicine, University of Illinois College of Medicine, Municipal
Contagious Disease Hospital, Chicago). We plan to study these occurrences
systematically .

Honors and Awards : None

Publications: Brody, J. A. and Wilner, E. : Measles, minor neurologic signs
and intelligence. Developmental Med, and Child. Neurol. 11;
449-454, 1969.

50 1

Serial No. NDS (CF) - 67 E 1497
i 1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Guillain-Barre (GB) - Bell's palsy (BP) study

Previous Serial Number: Same

Principal Investigator: Paul M. Hoffman, M.D.

Other Investigators: Lon R. White, M.D.

Jacob A. Brody, M.D.
George Nemo, Ph.D.

Cooperating Unit: Perinatal Branch, NINDS

Man Years :

Total:

1/4

Professional:

1/4

Other:

0

Project Description:

Objectives : To determine if GB and BP are caused by abnormal
immunological host responses to common viruses. Since during the immune
response the virus may not be recoverable from the patient we are attempting
to isolate the virus from materials collected from household contacts.

Methods employed : Personal contact was made with the neurology
residents or senior medical residents in a number of local hospitals and
letters were sent to the practicing neurologists and neurosurgeons in the
local area, so that prompt notification might be obtained whenever a case
of GB or BP appeared. When notification of a case was obtained, we contacted
the patient to learn of any contacts (preferably children) in the household.
If there were contacts we proceeded to collect blood, throat swabs, and
rectal swabs from the patient and his contacts. Blood specimens were
centrifuged and serum stored at -20 °C. Throat swabs were placed in Hank's
medium and PPLO medium; rectal swabs were placed in Hank's medium and vials
were stored at -70 °C.

When sufficient numbers of specimens had been collected it was planned
to forward them for serological studies and attempts at virus isolation.
Records were kept of patient's history and neurological status and of
contact's exposure to infections. Lymphocyte transformation studies were
also performed on the subject. The significance of serum factors depressing
lymphocyte transformation and the interaction of the isolated virus with
the patients lymphocytes as well as the reaction of the lymphocytes to

51t

Serial No. NDS (CF) - 67 E 1497

peripheral nerve will be evaluated. In addition careful case-control
studies of epidemiologic factors will be conducted in hope of encountering
precipitating factors in these diseases.

Ma.jor findings: None

Significance to biomedical research and the program of the Institute:
This disease has been associated with several abnormalities that suggest
auto-immunity as an etiology. Our work with the hypothesis of a viral
infection triggering an abnormal immune response is in keeping with our
knowledge of 3SPE and possibly MS.

Proposed course: This project will continue.

Honors and Awards : None

Publications: None

52t

Serial No. NDS (CF) - 68 E 1594

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Phenothiazine-induced neurological effects: A study among
twins

Previous Serial Number : Same

Principal Investigators: James A. Schnur, M,D.

Jacob A. Brody, M.D.
Dean F. Young, M.D.

Other Investigators: John D. Rainer, M.D.

New York State Psychiatric Institute

Cooperating Units: National Institute of Child Health and Human Development,
Children's Diagnostic and Study Branch
National Institute of Mental Health

Section on Twin & Sibling Studies, Adult Psychiatry
Branch
National Academy of Science, National Research Council
Columbia University, New York State Psychiatric Institute
Spring Grove State Hospital, Catonsville, Maryland

Man Years:

Total: 1/2
Professional: 1/3
Other : 1/6

Project Description:

Objectives : The objective of this study is to assess whether the
specific types of neurological side reaction induced by phenothiazine drugs
are influenced by genetic factors.

Methods employed: A preliminary study among 46 female geriatric patients
on long-term phenothiazine treatment revealed 33% had dvsklnetic reactions
and 13%, Parkinson-like reactions. The subjects for this study are twin
pairs, concordant for the same psychiatric diagnosis, who have been on
chronic phenothiazine therapy. Zygosity of the twin pairs is determined by
history, appearance, and extensive blood typing. A single neurological
examination was conducted on each pair to determine the patterns of
neurological reactions . By comparing the patterns of reactions in monozygotic
with those of fraternal twins, we can employ the usual methods of analysis to

53t

Serial No. NDS (CF) - 68 E 1594

determine the relative importance of genetic factors in the manifestation of
extrapyramidal signs resulting from phenothiazine induction.

Mai or findings : Six pairs of twin patients, four monozygotic and two |
dizygotic, on long time-high dosage phenothiazine treatment have been
examined. The results indicated that the specific type of neurological side
effect is not primarily determined by genetic factors, since identical twins
may develop distinctly different patterns of reaction.

Significance to biomedical research and the program of the Institute;
This work may help elucidate the presence or absence of a genetic contribution
to the occurrence of the important neurological side reactions to phenothiazine
drugs .

Proposed course: We propose to add other twin pairs to the series, and
analyze the data.

Honors and Awards : None

Publications: None

54t

Serial No. NDS (CF) - 68 E 1595

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individioal Project Report

July 1, 1970 through June 30, 1971

Project Title: An evaluation of the effect of successful thalamic surgery
on the progress of unilateral Parkinson's disease

Previous Serial Number: Same

Principal Investigators: R. Michael Scott, M.D.

Jacob A. Brody, M.D.
Joyce M. Cannon, R.N.

Other Investigators: Irving S. Cooper, M.D.

St. Barnabas Hospital
Robert S. Schwab, M.D.

Massachusetts General Hospital

Cooperating Units: Department of Neurosurgery, St. Barnabas Hospital,
Bronx, New York
Department of Neurology, Massachusetts General Hospital,
Boston, Massachusetts

Man Years :

Total : 1

Professional: 11/12

Other : 1/12

Project Description:

Objectives : To evaluate Dr. Cooper's hypothesis that in patients with
unilateral Parkinson's disease, thalamic surgery which succeeds in permanently
abolishing the tremor and rigidity on the involved side will either stop or
markedly delay the appearance of the symptoms on the other side of the body;
and to determine the natural history of vinilateral Parkinson's disease.

Methods employed: The charts of 1,700 consecutive thalamic surgical
cases from January 1963 through September 1964 at St. Barnabas Hospital
were reviewed to select 100 patients who came to surgery with symptoms of
tremor and rigidity confined to one side of the body. Seventy-two patients
were eventually contacted and examined. Two unoperated groups were studied.
The first of these consisted of 15 patients who presented to Dr. Cooper with
unilateral signs, who were accepted for surgery, but for various reasons were
not operated upon. The second control group consisted of 20 patients seen
by Dr. Schwab in Boston and subsequently treated medically.

55 t

Serial No. NDS (CF) - 68 E 1595

Major findings: Successful thalamic surgery does not affect the progress
of tremor and rigidity to the extremities of the opposite side of the body.
The rate and frequency of spread in unoperated and operated patients were 1
strikingly similar. Certain of these patients had a form of Parkinson's I
disease characterized by unilateral tremor and rigidity of long duration. '
The average age of onset of these patients was earlier than that of "classical"
Parkinson's disease patients, and tliey had a higher frequency of encephalitis
or severe febrile illness prior to the onset of their illness. Thalamotomy
was often extremely effective in these patients.

Significance to biomedical research and the program of the Institute:
This study further defines the role of surgery in Parkinson's disease. It
emphasizes that thalamotomy does not alter the course of progressive Parkinson's
disease, but can restore to normalcy certain patients with the benign
unilateral syndrome. In addition, it suggests ways in which patients with
the benign syndrome might be identified.

Proposed course: Piablications have been prepared and the project
terminated.

Honors and Awards : None

Publications: Cooper, I.S., and Scott, R.M. : The clinical and physiological
implications of 10 year cure of unilateral movement disorders
by thalamic surgery. In: Proceedings of the Ilird Parkinson's
Symposium. Edinburgh, Livingstone, (in press).

Scott, R.M., Brody, J. A., and Cooper, I.S.: The effect of
thalamotomy on the progress of unilateral Parkinson's disease.
J. Neurosurg. 32:286-288, 1970.

Scott, R.M., Brody, J. A., Schwab, R.S., Cooper, I.S.:
Progression of unilateral tremor and regidity in Parkinson's
disease. Neurology, 20:710-714, 1970.

Scott, R.M., and Brody, J.A. : Benign early-onset Parkinson's
disease: A syndrome distinct from classic postencephalitic
parkinsonism. (Presented at American Academy of Neurology,
May 1970). Neurology , 21:366-368, 1971

56*

Serial No. NDS (CF) - 68 E 1597

1. Collaborative & Field Research

2 . Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Neurologic diseases in the Trust Territories and other
Pacific areas

Previous Serial Number:

Same

Principal Investigators

Jacob A. Brody, M.D.
James A. Schnur, M.D.
NINDS Research Center

Other Investigators;

Manuel Cruz

mms Research Center
Jose Torres

NINDS Research Center
Francisco Leon Guerrero

NINDS Research Center

Cooperating Units:

Ophthalmology Branch, NINDS

Department of Public Health, Trijst Territory of the

Pacific Islands, Saipan, Marianas Islands
Population Genetics Laboratory, University of Hawaii,

Honolulu, Hawaii

Man Years :

Total:

Professional:
Other :

2/3
1/3
1/3

Project Description:

Objectives : To investigate neurologic illness occurring in the Trust
Territories and the Pacific area. Last year this involved the study of
leprosy patients in New Caledonia. This year it involved the study of
congenital blindness among the people of Pingelap.

Methods employed: A field trip was made to Ponape to examine the
Pingelapese people in order to document the nature of their eye disease
and their mode of inheritance and epidemiologic patterns which could
suggest both genetic and environmental factors.

Major findings: The congenital eye disease is apparently a form of
achromatopsia characterized by nystagmus, blinking, photophobia, blindness,
and impairment of color vision. The developiment of cataracts usually within

57 1

Serial No. NDS (CF) - 68 E 1597

the first 5 to 10 years of life and frequent appearance of high myopia. The
mode of inheritance appears to be recessive and no ns ex- linked. Our most
significant finding was that the apparent trend noted in 1969 that children
in age group 0-4 had a significantly lower rate of the congenital eye
disease was not sustained. In a follow-up trip in 1971 we observed that two
new patients were born with the disease since 1969 bringing the rate in the
current population age 0 - 4 up into the range of those rates for older
populations. Six patients were brought to the Ophthalmology Branch, NINDS
for study. The diagnosis appears to be achromatopsia with unexplained
high myopia.

Significance to biomedical research and the program of the Institute:
The group of diseases which are included under tapetoretinal degenerations
are poorly understood. By having a population isolate with a phenomenally
high rate (10% of the population is blind) will give a unique opportunity
for studying the full range of manifestations of this entity as the
expression of a single abnormal gene. In addition, hopefully, a mechanism
for prevention will develop.

Proposed course: We will continue to follow this population and other
populations as they become known which are of potential medical interest.

Honors and Awards : None

Publications: Brody , J. A., Hussels, I., Brink, E. and Torres, J.M.: A
preliminary report on hereditary blindness among the
Pingelapese people of the Eastern Caroline Islands.
Lancet, 1:1253-1257, 1970.

58t

Serial No. NDS (CF) - 68 E 1598

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Epilepsy on Guam

Previous Serial Number: Same

Principal Investigator: John M. Stanhope, M.D.

NINDS Research Center

Other Investigators: Jacob A. Brody, M.D.
Manuel Cruz

NINDS Research Center
Jose Torres

NINDS Research Center
Francisco Leon Guerrero

NINDS Research Center

Cooperating Unit: NINDS Research Center, Agana, Guam

Man Years :

Total: 1/2
Professional: 1/4
Other: 1/4

Project Description:

Objectives : To determine the incidence and prevalence of epilepsy on
Guam. To investigate methods for field studies of epilepsy. To determine
if previous reports of unusually high incidence of convulsive disorders on
Guam are accurate.

Methods employed: We are testing four basic approaches: (1) We are
following-up a 1962 survey of convulsive disorders in Umatac and Merizo to
determine the outcome of those children known to have had febrile convulsions
6 to 8 years ago; (2) to determine the true incidence and prevalence in
a sample population we are doing a house to house survey in the villages
of Talofofo, Merizo, and Yona; (3) as referral neurologists on Guam we are
updating all previous referrals of convulsive disorders to us and establishing
a registry. To add to this registry we are contacting all medical and
paramedical personnel on Guam to discover new cases. This registry will be
permanent and permit us to conduct studies in the Guam population; (4) to
further elaborate on methods for acquiring information we are following-up
all births on Guam in 1958 and 1963 throughout the entire island to determine
the rates of convulsive disorders in these preselected populations.

59*

Serial No. NDS (CF) - 68 E 1598

Major findings: We have already published that the rates of "true"
epilepsy and of febrile convulsions are higher on Guam than elsewhere. We
are completing the first three phases of the Epilepsy Study outlined in
previous reports . We have the information on the rate of "true" epilepsy
(the fourth part of our program) and have established sf permanent Epilepsy
Registry on Guam. There seems little doubt that the rates are high on Guam,
but the reasons are not clear. Available data are now being analyzed by
Dr. Stanhope.

Significance to biomedical research and the program of the Institute:
Further studies of epilepsy in a well-defined and accessible population
will add to the understanding of this disease and contribute new information
concerning epilepsy in a tropical environment. It will also yield important
information on different survey techniques and their relative accuracy.

Proposed course: The analysis will be completed as soon as possible and
more detailed studies of epilepsy will develop from our basic information.

Honors and Awards : None

Publications : None

60t

Serial No. NDS (CF) - 68 E 1605

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Phenothiazine-induced parkinsonism in white and Negro
patients with nonorganic psychoses

Previous Serial Number : Same

Principal Investigators: James A. Schnur, M.D.

NINDS Research Center
R. Michael Scott, M.D.

Other Investigators: Jacob A. Brody, M.D.

Cooperating Units: Spring Grove State Hospital, Catonsville, Maryland
Crownsville State Hospital, Crownsville, Maryland

Man Years :

Total: 2/3

Professional: 1/2
Other: 1/6

Project Description:

Objectives: To determine whether increased skin pigmentation is
associated with decreased prevalence of phenothiazine-induced Parkinson-like
syndromes .

Methods employed: This project was originally designed to investigate
influence of skin pigmentation on the prevalence of naturally occurring
Parkinson's disease by use of a questionnaire which was to be mailed to a
sample of American physicians. A pilot study, however, showed this approach
to be impractical, and therefore, it was abandoned. In view of the possible
relationship between drug-induced and naturally occurring parkinsonism, we
then decide to study comparable white and Negro populations who were on
treatment with phenothiazines . Thus far, we have examined approximately 75
patients in each group, samples sufficient in number for a comparative
analysis .

Major findings: Our initial results suggest that there is no difference
in the prevalence of phenothiazine-induced parkinsonism between the two
populations surveyed.

6lt

Serial No. NDS (CF) - 68 E 1605

Significance to biomedical research and the program of the Institute:
To further define the relationship between melanogenesis in the skin and in
the pigmented nuclei of the basal ganglia.

Proposed course: Analysis of data is now in progress.

Honors and Awards : None

Publications: Brody, J. A.: Genetic considerations in Parkinson's disease.
Presented at the Laurentian L-Dopa Conference, Montreal,
November 1969. In: L-Dopa and Parkinsonism, Edited by Andre
Barbeau and Fletcher McDowell, F.A, Davis Co., Phila., 1970,
pp. 27-30.

62t

Serial No. NDS (CF) - 69 E 1774

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Neurologic signs and symptoms associated with malabsorption

Previous Serial Number : Same

Principal Investigators: Paul M. Hoffman, M.D.

Jacob A, Brody, M.D.
Anne H. Edgar

Other Investigator: None

Cooperating Units: Central Veterans Administration Authority, Washington,
D.C.
Veterans Administration Hospital, Atlanta, Georgia
Veterans Administration Hospital, Long Beach, California
Veterans Administration Hospital, Durham, North Carolina
Veterans Administration Hospital, Los Angeles, California

Man Years :

Total: 1 1/2
Professional: 1 1/2
Other : 0

Project Description:

Objectives : To determine if both clinical and subclinical malabsorption
is associated with a high incidence of neurologic signs and symptoms.

Methods employed : The Cooperative Veterans Administration Retrospective
Study of surgery for peptic ulcer served as the source for patients in this
study. A review of abstracts from this study showed that a given hospital
was more likely to have done surgical procedure for the majority of its cases
then another. For this reason, patients with 3 different surgical procedures
performed at 4 hospitals were chosen. A fourth group of patients who had
simple closures of perforated ulcers were selected from all 4 hospitals .
Patients who had a subtotal gastrectomy with a Billroth II reanastomosis
were selected from the Durham Veterans Hospital, patients who had a hemi-
gastrectomy and vagotomy were selected from the Atlanta Veterans Hospital,
and patients who had a vagotomy and pyloroplasty were selected from the
Long Beach and Wadsworth Veterans Hospital. In order to insure that this
population would be in the most susceptible age group, patients between the
ages of 30 and 80 who had had surgery between 1952 and 1957 were selected
from abstracts and hospital records where available. Patients were not

63*

J

I

Serial No. NDS (CF) - 69 E 1774

with neurologic complications, any systemic disease with known neurologic
complications, or a history of neurologic disease prior to their ulcer
surgery. Patients were also excluded from the study if a revision of their
original ulcer surgery or subsequent surgery for a recurrent ulcer had been
performed. No attempt was made in this study to analyze the causes of death
or those who died since surgery. A mortality study of the 2,800 original
cases which includes this sample has also been undertaken. All patients
included in the study had letters sent to their last known address asking
them to report on one of several days to the outpatient clinic of the hospital
where their surgery was performed. A complete history with special attention
on the neurologic and gastrointestinal systems as well as a complete neurologic
examination was performed on all patients by one of us (PMH) and a random
sample of all patients as well as all patients with abnormal findings were
examined by a staff neurologist.

Major findings: No cases of motor neuron disease have been found in the
examined group. A large number of cases of peripheral neuropathy were
identified in the vagotomy and hemi-gastrectomy group. Only two cases were
seen in the vagotomy and pyloroplasty group. Through evaluation of all
patients with unexplained neurologic disorders in the Atlanta group showed
that malabsorption, poor nutrition, chronic alcohol intake, and weight loss
was all more prevalent in this group than in those without neurologic findings.
In reviewing 500 death certificates of the original study group we encountered
2 patients with multiple sclerosis, 2 with Parkinson's disease and 1 with
amyotrophic lateral sclerosis.

Significance to biomedical research and the program of the Institute;
There have been many clinical reports of cases of malabsorption who have
shown signs and symptoms of nervous system disease. There have also been
scattered reports in literature of patients who are known to have motor
neuron disease, who had a history of having had a Billroth II type of gastric
surgery performed many years prior to the onset of their disease. There has
never been, however, a matched, controlled population study of the association
of these two abnormalities . If this association is valid then further study
into the mechanism of absorption of essential nutrients and their incorporation
into nervous tissue may be a meaningful approach to the study of chronic
neurologic disease.

Proposed course: This project will continue with emphasis on causes of
death within this population.

Honors and Awards : None

Publications : None

64^

I

Serial No. NDS (CF) - 69 E 1775

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Comparison of MS among Irish and Italian immigrants in
Bos ton

Previous Serial Number: Same

Principal Investigators: Roger Detels, M.D.

Jacob A. Brody, M.D.

Other Investigator: None

Cooperating Unit : None

Man Years :

Total: 11/12
Professional: 1/6
Other: 3/4

Project Description:

Objectives : To determine if people migrating from an area of low
multiple sclerosis incidence to an area of high multiple sclerosis incidence
retain the low rate of the country of origin or acquire the high rate of
the area to which they migrated.

Methods employed: Boston, a city of 2 1/2 million, has a large Irish
and Italian population, the majority of which migrated about the turn of
the century. Thus, individuals in these groups dying of multiple sclerosis
would be expected to have died between 1920 and 1940. During these years
an estimated 5,000 deaths occurred each year among the Irish and the Italians
in Boston. Since these groups would use the same medical facilities it is
possible to do a comparative study of the rates of multiple sclerosis between
them. Italy is an area of low multiple sclerosis incidence while Ireland and
Boston have high rates. Fatality rates for Irish and Italian immigrants and
first generation Americans of Italian and Irish background can be determined
by a review of death certificates during this period. As a further control
the ratio of multiple sclerosis to amyotrophic lateral sclerosis deaths can
be compared since amyotrophic lateral sclerosis rates are considered to be
independent of latitude.

Death certificates for the period 1920 to 1940 from the city of Boston
will be reviewed for all deaths due to multiple sclerosis and amyotrophic
lateral sclerosis and for all deaths occuinring among Italian and Irish

est

Serial No. NDS (CF) - 69 E 1775

immigrants and first generation Americans of these descents. Information
will also be obtained for years of residence in the United States and in the
Boston area.

Major findings: Preliminary analysis revealed that the Boston population
is apparently not suitable for this study.

Significance to biomedical research and the program of the Institute:
It is known that populations migrating from high incidence areas of multiple
sclerosis to low incidence areas, retain the high rate of the country of
origin suggesting that events early in youth (possible infectious) cause
multiple sclerosis. This study will provide the corollary data about
migration from low risk areas to high risk areas and about multiple sclerosis
rates among first generation migrants.

Proposed course: This study will be terminated.

Honors and Awards : None

Publications: None

66t

Serial No. NDS (CF) - 69 E 1776

1. Collaborative & Field Research

2. Epidenilology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1969 through June 30, 1970

Project Title: Cerebrospinal fluid amines in drug-induced extrapyramidal
parkinsonism-like disorders .

Previous Serial Number: Same

Principal Investigator: James A, Schnur, M.D.

Other Investigator: Thomas N. Chase, M.D.
NIMH

Cooperating Units: Unit on Neurology, NIMH, Spring Grove State Hospital,
Catonsville, Maryland
Crownsville State Hospital, Crownsville, Maryland

Man Years :

Total: 2/3
Professional: 1/2
Other: 1/6

Project Description:

Objectives : To study central nervous system amine metabolism in
patients with drug-induced extrapyramidal disorders .

Methods employed : Spinal fluids of clinically defined cases of drug-
induced extrapyramidal disorders are analyzed by conventional biochemical
methods for amine metabolites.

Major findings: Levels of HVA and 5-HIAA in lumbar CSF were measured
in 20 female psychiatric patients. Those receiving long-term treatment with
antipsychotic drugs who remained free of extrapyramidal dysfunction had
substantially higher concentrations of these monoamine catabolites than
patients not taking these drugs. Individuals who developed parkinsonian
or dyskinetic signs while receiving antipsychotic agents appeared to have
significantly lower concentrations of both HVA and 5-HIAA than patients not
manifesting these disorders despite similar drug exposure.

Significance to biomedical research and the program of the Institute:
The foregoing observations support the hypothesis that the compensatory
acceleration of cerebral monoamine metabolism induced by antipsychotic drugs
may be impaired in patients who develop extrapyramidal dysfunction and this
is the mechanism of acquisition of this side affect.

I

67 t

Serial No. NDS (CF) - 69 E 1776

Propos ed cours e : This phase of the study is completed and project
terminated .

Honors and Awards: None

Publications: Chase, T.N. , Schnur, J. A. and Gordon, E.K.: Cerebrospinal
fluid monoamine catabolites in drug-induced extrapyramidal
disorders. Neuropharmacology , 9:265-268, 1970.

68*

Serial No. NDS (CF) - 69 E 1777

1. Collaborative & Field Research

2. Epidemiology Branch

3. Beth es da, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: ALS among non-Chamorros after residence on Guam

Previous Serial Number: Same

Principal Investigator: Jacob A. Brody, M.D.

Other Investigator: Estelle Kornhauser, R.N.

Cooperating Unit: Bureau of Data Processing and Accounts, Social Security
Administration, DHEW

Man Years :

Total: 1/2
Professional: 5/12
Others : 1/2

Project Description:

Objectives : This project was developed to determine if prolonged
exposure (over one year) to the environment of Guam increases the likelihood
of the development of ALS among statesiders.

Methods employed : Through various workers in the Department of Defense
we were put in contact with several construction companies which had
maintained large staffs of statesiders on the island of Guam after World
War II. These companies were asked to supply us with the names, birthdates,
and social security numbers of all personnel employed on Guam and from these
lists we selected only those workers who had spent more than one year on
Guam.. This was a group of approximately 12,000 individuals. The Social
Security Administration searched its records to determine which of these
workers had died and where they had died. We are contacting the individual
states to obtain the death certificates of the deceased workers and
determine the cause of their death.

Major findings: Of the approximately 12,000 cards submitted to the
Social Security Administration, 450 were found to contain incorrect
information making follow-up Impossible. 7,730 were considered as
representing individuals still alive, and the remainder (4,000) were
identified as to place of death. The names were submitted to the individual
states and death certificates requested. About 80% of the requested
certificates have been returned to date. Preliminary analysis indicates that
there was no excess of ALS in this group.

69t

Serial No. NDS (CF) - 69 E 1777

Significance to biomedical research and the program of the Institute;
Although results on this study are far from complete, the initial trend is
that the rate of ALS among statesiders who have spent considerable time on
Guam remains the same as that for statesiders who have never spent time in
that environment. If this trend is borne out by subsequent findings, it
would suggest that the environmental factors on Guam are less likely to be
responsible for high rate of ALS among its native population.

Proposed course: The study will be completed by submitting to Social
Security approximately 600 punch cards in order to search for death
certificates we were unable to locate through the individual states. These
cards will then be coded and searched for possible ALS deaths.

Honors and Awards : None

Publications : None

70t

Serial No. NDS (CF) - 69 E 1778

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Familial cortical cerebellar degeneration

Previous Serial Number: Same

Principal Investigators: Paul M. Hoffman, M.D.

William H. Stuart, M.D.
Kenneth Earle, M.D.

Other Investigators: Joyce M. Cannon, R.N.
Anne Harlan Edgar

Cooperating Unit: Armed Forces Institute of Pathology

Man Years :

Total: 1/2

Professional: 1/3 ,

Other : 1/6

Project Description:

Objectives : To determine the genetic pattern and the incidence of
cortical cerebellar degeneration in a family in the northern Georgia section
of the Blue Ridge Mountains .

Methods employed: A complete family history and a family tree were
obtained from the proband and his family who are now living in Hiawasee,
Georgia. Affected members were examined as well as those who were at risk
but who have not shown the signs and symptoms of the disease. Hospital
records and autopsy material, when available, was collected. An autopsy
was performed on the index case.

Major findings: The proband is an 80-year-old white male who demonstrated
abnormal finger to nose and heel to shin tests as well as inability to maintain
his balance, and persistent truncal titubation. He also exhibited scanning-
type of cerebellar speech for the last two years. There was no evidence of
long-track involvement or of abnormalities in position or vibratory sensation.
The reflexes were described as normal. Further study of other affected
members showed that the symptoms of ataxia, scanning spastic speech, and mild
upper extremity involvement were constant throughout the family. Six affected
members in two generations were examined. The neuropathology consisted of
atrophy of the superior and anterior cerebellar cortex with secondary olivary
degeneration. The disease was transmitted as an autosomal dominant.

L

7lt

Serial No. NDS (CF) - 69 E 1778

Significance to biomedical research and the program of the Institute:
The hereditary cerebellar and spinocerebellar degeneration have been described
as unique syndromes. Presently, however, it has been observed that symptoms
within a family have been very variable. This family has similar symptoms in
all affected members. Interest has now been shown in treating various types
of spinocerebellar and cerebellar disorders with L-Dopa . The well-defined
hereditary syndromes offer the greatest hope of finding an enzyme deficiency
that might result in disordered biogenic amine metabolism.

Proposed course: A report has been accepted for publication and this
study was terminated.

Honors and Awards : None

Publications: Hoffman, P.M., Stuart, W.H., Earle, K.W.,and Brody, J.A. :
Hereditary cerebello-olivary degeneration of late onset.
Neurology, 20:400, April 1970. (Presented at the American
Academy of Neurology, May 1, 1970.)

72^

Serial No. NDS (CF) - 69 E 1779

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Ihe epidemiology of motor neuron disease in the United
States

Previous Serial Number: Same

Principal Investigators: Paul M. Hoffman, M.D.

Jacob A. Brody, M.D.

Other Investigator: Joyce M. Cannon, R.N.

Cooperating Units: Bureau of Disability Insurance, Social Security
Adminis tration
North Carolina State Department of Public Health
University of North Carolina School of Medicine
Duke University School of Medicine
Charlotte Memorial Hospital

Man Years :

Total: 11/12
Professional: 10/12
Other: 1/12

Project Description:

Obj ec tives : To determine if the diagnoses on death certificates are
an adequate reflection of the incidence of motor neuron disease in the
United States.

Methods employed : Hospital records were reviewed on those patients
who had a diagnosis of motor neuron disease in years 1958 through 1962 at
the three North Carolina Hospitals. Death certificates were then obtained
on those patients who had died in the state of North Carolina. Those
patients who are still living were followed-up through the County Public
Health Departments within the state of North Carolina and information was
obtained on these patients.

Major findings: Of the death certificates obtained, the diagnosis of
motor neuron disease appeared on 72 percent. Of these 70% were certified
at death as having ALS . MS was certified in 10% of these patients,
suggesting a possible systematic error of overdiagnosis of MS at death
and underreporting of ALS.

73t

Serial No. NDS (CF) - 69 E 1779

Significance to biomedical research and the program of the Institute:
Most of the estimates of the prevalence of motor neuron disease are based
on mortality reporting. Our study cast doubt as to the validity of this
technique.

Proposed course: A report has been prepared and accepted for
publication. The project was terminated.

Honors and Awards : None

Publications: Hoffman, P.M. and Brody, J. A.: The accuracy of mortality

statistics in clinically proven amyotrophic lateral sclerosis.
Trans. Amer . Neurol. Ass. 95:261-263, 1970.

Hoffman, P.M. and Brody, J. A.: The reliability of mortality
statistics for amyotrophic lateral sclerosis. J. Chron. Pis.
23, 1971.

74t

Serial No. ITOS (CF) 69 E I78O

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title: Familial bilateral acoustic neuroma

Previous Serial Number: Same

Principal Investigator: Roswell Eldridge, M. D.

Other Investigators:

Cooperating Units:

Dean F. Young, M.D.

New York, New York
Henry Hood, M.D.

Danville , Pennsylvania
W. J. Gardner, M.D.

Cleveland, Ohio
George T. Nager, M.D.

Johns Hopkins Hospital
Frank H. DeLand, M.D.

Johns Hopkins Hospital

Department of Otolaryngology,
Department of Radiology,
Department of Neurosurgery,
Geisinger Medical Center

Danville , Pennsylvania
Johns Hopkins Hospital
Baltimore , Maryland

Man Years :

Total : 2

Professional: 1
Other : 1

Project Description:

Objectives: To perform genetic, clinical and physiologic studies of a
large family with hereditary bilateral acoustic neuroma.

To clarify the relationship of this trait to other disorders with acoustic
neuromas such as neurofibromatosis.

Methods employed: Field studies were conducted in evaluating family
members. On those seen personally, physical examinations were performed,
stressing neurological and skin examinations. In addition, audiometric
examinations including air and bone conduction and caloric examinations were
conducted in the field.

75t

Serial No. NDG (CF) 69 E I78O

Genealogic information was obtained from family members, family records,
D.A.R. records, state and military records, census recordings. Medical
history was obtained from family members, hospital and physician records,
occasionally from school records or military records.

On select patients extensive outpatient studies have included audiometric
and vestibular testing, complete EIW and neurologic examinations, skull
x-rays and brain scans using radioactive techniques. These were undertaken
in cooperation with the Departments of Neurology, Radiology and Otolaryn-
gology of Johns Hopkins Hospital.

Major findings: The study of this, the largest kindred with hereditary
neoplasm yet reported, has generated information which has been particularly
useful to neurosurgeons and otolaryngologists since it casts considerably
different light on prognosis and genetic risks to those individuals who
develop such a disease at a yoiing age.

Significance to biomedical research and the program of the Institute:
The mode of inheritance to this trait has been confirmed and the place of
this syndrome among those associated with neural sheath proliferation is
clearer. Of primary importance is the establishment of appropriate diag-
nostic techniques for early cases and treatment of these cases.

Proposed course : Affected individuals are being followed to evaluate
various forms of treatment. Relatives not known to be affected but at
risk are being examined periodically to evaluate various diagnostic techniques,

The initial phase of this project has been completed and resulted in the
publications listed below.

Honors and Awards : None

Publications :

Young, D.P., McNew, J. and Eldridge, R. : Hereditary Acoustic Neuroma -
Clinical and Genetic Aspects. In Transactions of the American Neurological
Association. 9i|, I969.

Young, D.F., Eldridge, R., Na^er, G.T., DeLand, F.H., and McNew, J.:
Hereditary bilateral acoustic neuroma (central neurofibromatosis). Pro-
ceedings of the Second Conference on The Clinical Delineating of Birth
Defects, May I969. The National Foundation -March of Dimes (in Press)

Young, D., Eldridge, R., and Gardner, ¥. J. : Bilateral Acoustic Neuroma in
a Large Kindred. JAMA. 21^1-, October 1970.

76t

Serial No. NDS (CF) - 69 E 1781

1. Collaborative Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Twin Studies in Parkinson's Disease

Previous Serial Number: Same

Principal Investigators: Roswell Eldridge, M.D.

Zdenek Hrubeck, M.D.

National Academy of Sciences

Other Investigator: Kathy O'Meara

Cooperating Unit: The National Academy of Sciences - National Research
Council Twins Registry, Washington, D. C.

U. S. Veterans Administration
Washington, D. C.

Man Years:

Total: 1/4

Professional: 1/8
Other: 1/8

Project Description:

Objectives: Parkinsonism is not a single entity but rather a symptom
complex which may be idiopathic, or may be due to cerebral arteriosclerosis
or may follow encephalitis. Genetic factors have been considered important
by a number of authors but it is not yet established how important such
factors are in any one form of Parkinsonism or if there is a form which has
a simple genetic basis independent of acquired neurologic disease. The aim
of the present proposal would be to use the twin method to evaluate genetic
factors in various forms of Parkinsonism and/or to distinguish genetically
determined Parkinsonism feom other forms.

Methods employed: Survey of the VA twin registry in 1968 has revealed
eight cases of Parkinsonism in one of a Veteran twin pair. Each of the
pair were apparently discordant for Parkinsonism. Two twin pairs were
monozygotic, five were dizygotic and in one the zygosity was unknown.
We would contact each of these individuals and his co-twin, and others with

77t

Serial No. NDS (CF) - 69 E 1781

che diagnosis who may be ascertained through updating of the registry, and
arrange for an appointment with the individual in his home at a time when
available relatives could also be present. During the family interview
a pertinent medical history would be obtained and physician examination
would be performed. Permission for review of hospital records would be
secured and arrangements might be made for additional neurologic studies.
To define zygosity, photographs would be taken of the twins, blood would be
drawn for genotyping and dermatoglyphics might be recorded. (Personal
examination of both twins and available relatives is important in order that
mild cases not be missed).

Major Findings: Interest continues in this project although there has
been no additional ascertainment of cases since the last report. The
National Science Foundation have not yet agree to our contacting patients on
their registry but we expect twin pairs will be available to us in the
future.

Significance to biomedical research and the program of the Institute:
In a chronic condition with late onset it is often difficult to determine
the role of genetic factors in causation. The twin method provides a
relatively simple method involving small numbers to answer this question.

The nosology of Parkinson's disease is especially important now that the
drug L-Dopa has been shown to help some with Parkinsonism. Is this drug
most helpful in a specific form of the disease? Is it effective in
hereditary Parkinsonism?

Proposed course: See "Methods employed".

Honors and Awards: None

Publications: None

78t

I

Serial No. KDS (CF) 69 E I782

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Twin Studies in Torticollis

Previous Serial Wumher: Same

Principal Investigators: Roswell Eldridge, M. D.

Zdenek Hruhec, M. D.

National Academy of Sciences

Other Investigators: Kathy O'Meara

Cooperating Unit: The National Academy of Sciences-National Research
Council Twins' Registry, Washington, D. C.

U. S. Veterans Administration
Washington, D. C.

Man Years :

Total: l/U
Professional: I/8
Other : 1/8

Project Description:

Objectives: Torticollis may be broadly divided into infantile and post-
infantile fonns. The former may be congenital or appear several weeks after
birth but in either the cause appears due to events preceding birth. Post-
infantile torticollis consists of a heterogeneous group of disorders which
have been ascribed to a number of causes including trauma or inflammation of
the cervical spine, myositis of nuchal musculature, functional illness, and
disease of the peripheral or central nervous system. In addition torticollis
on a hereditary basis, either as an isolated symptom or in association with
other movement disorders such as torsion dystonia, has been the subject of
numerous reports. The aim of the proposed study is to weigh the genetic
factors in the post-infantile forms of torticollis and, if possible, to
distinguish between discrete hereditary types.

Methods employed: In I968 a review of the Veterans Administration twin
registry disclosed 11 individuals with a diagnosis of torticollis. Each of
the 11 pair were said to be discordant. Five were monozygotic, two were
dizygotic and in the four the zygosity could not be established. We would
contact each of these individuals and his co-twin, arrange for an appoint-
ment with the individual in his home at a time when available relatives could

79t

Serial No, HDS (CF) 69 E I782

also be present. (We would hope also to ascertain new cases by assisting
in the up-dating of the twin registry). During the family interview the
pertinent medical history would be obtained for all relatives and physical
examination performed on those present. Pei-mission for review of hospital
records would be secured and arrangements might be made for additional neurol-
ogic studies. To establish zygosity^ photographs would be taken of the twins,
blood drawn for genotyping, and dermatoglyphics might be recorded.

Major findings: Interest continues in this project although there has
been made no additional ascertainment of cases since the last report. The
National Science Foundation has not yet agreed to our contacting patients
on their registry but we expect twin pairs will be available to us in the
future .

Significance to biomedical research and the program of the Institute:
Torticollis may be hereditary or acquired but under each of these headings
there appear to be a number of discrete entities. The twin method presents
a relatively simple means to distinguish genetically determined fonns.
Concentration on torticollis which is simply inherited is worthwhile since
such disorders should have a discrete biochemical basis which might be
revealed by study with the neurochemical techniques now available.

Proposed course : See "Methods Employed".

Honors and Awards : None

Publications: None

80t

Serial No. NDS (CF) - 70 E 1832

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Measurement of immune response after antigen stimulation

Previous Serial Number: None

Principal Investigators: George Nemo, Ph.D.

Lon R. White, M.D.

Other Investigators: Harrie Anne Sutton, Biologist
Minnie Toure, Biologist
Gary Cooper, Biologist

Cooperating Unit: Unit on Congenital and Chronic Viral Infections, DB, NICHD

Man Years :

Total:

5/6

Professional:

1/3

Other:

1/2

Project Description:

Ob j ec tives : To investigate the changes in mltogenic, cytotoxic and
anti-viral capacities of lymphocytes in relation to the acquisition of
specific immunity.

Methods employed : Studies will involve both humans (immunized to
vaccinia virus as part of routine health care practices) and experimental
animals (mice, rats, guinea pigs). The antigens with which the animals
will be inoculated include mycobacteria protein, vaccinia, sindbis, reovirus
I, and the minute virus of mice. Lymphocyte transformation studies will
be done with spleen, blood and lymph node cells. The effects of immune
lymphocytes on the course of virus infection in vitro will be studies using
monolayer tissue culture cells isogenic with the lymphocyte donor. Cyto-
toxicity will be assayed visually and by Cr^l release. Virvises will be
titered using standard techniques. Macrophage-migration inhibition will
also be employed as an indicator of cellular immunity.

Major findings: None as yet .

Significance to biomedical research and the program of the Institute:
It is widely thought that "autoaggressive" lymphocytes are involved in the
etiology of chronic demyelinating diseases. It is further believed that
lymphocytes may be important in recovery from viral infection. Experimental
animal systems have suggested the possibility that certain chronic viral

81t

Serial No. NDS (CF) - 70 E 1832

infections may owe their chronicity to impaired lymphocyte competence and
that as this competence begins to be restored, host cells which bear viral
antigen determinants on their surfaces may be injured, giving the appearance
of an autoimmune disease. Such theories must be based and examined on the
results of investigations such as those described.

Proposed course: To be continued.

Honors and Awards : None

Publications : None

82±

Serial No. NDS (CF) - 70 E 1833

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Immune mechanisms in chronic and congenital viral infections

Previous Serial Number: Same

Principle Investigator: Lon R. White, M.D., DB, NICHD

Other Investigators: George Nemo, Ph.D.

Jacob A. Brody, M.D.

Harrie Anne Sutton, biologist, DB, NICHD

(HD DB - 5)

Cooperating Units :

None

Man Years :

Total:

0.6

Professional:

0.3

Other:

0.3

Project Description:

Objectives : 1. To establish one or more models of chronic and/or
congenital viral infection in experimental animals. 2. To define the
development of cellular and humoral immunity to the same and to heterologous
antigens .

Methods employed : Neonatal and pregnant mice are injected with either
a reovirus type 1 or the minute virus of mice (MVM) . The persistence of
virus in animals is determined by isolation and flourescent and immuno-
flourescent techniques. Serum levels of hemagglutination inhibition
antibodies to both agents are followed. Lymphocyte transformation and
cytotoxic activity is studied in spleen cell cultures in the presence of
phytohemagglutinin on specific antigens . Any tumors which may develop are
studied by light and electron microscopy. Extracts of MVM-containing tumor
cells will be injected into newborn mice in addition to being studied in
tissue culture.

f Major findings; Attempts to infect embryonic animals by maternal infection

in the first week of pregnancy have failed, presumably because infected embryos
are reabsorbed. Late gestation maternal infection of 8 animals has resulted
in the live birth of 53 animals, 39 of which survived to adult life. All
progeny have appeared to be normal, most having been observed for 1 year. Two
such animals have died of solid tumors; one of these was lost to study and the
other was found to have a tumor in the area of the right uterine horn with

L

^3t

Serial No. NDS (CF) - 70 E 1833

hepatic metastases. Extracts of both the tumor and liver yielded a hemagglu-
tinating material presumed to be MVM antigen, suggesting that the animal was
either reinfected late in life or had been chronically infected since fetal
life. Cell-free materials from these tissues are now being studied both
in vivo and in vitro in an attempt to define the possible role of MVM, either
directly or indirectly, in the etiology of the tumor.

Significance to biomedical research and the program of the Institute:
Infection of the human embryo or fetus with rubella or cytomegalovirus is
associated not only with developmental abnormalities and mental retardation,
but also with infection persisting for months or years after, despite the
presence of neutralizing antibody in the serum. There are several examples
of related phenomena in experimental animals infected during prenatal or
neonatal life. Previous studies have suggested that an impairment of the
immune function of lymphocytes may be associated with such persistent
infection. These phenomena provide insight into the question of how viral
infection is normally terminated, and represent a challenge to older ideas
on the role of specific cellular immunity in the natural history of virus
infections. The investigation described represents a direct experimental
approach to elucidating the cause and course of chronic infection following
initial exposure to the agent during immunologic immaturity. The results of
this investigation will be of immediate relevance to our understanding of
other types of illness known or suspected to be associated with chronic viral
infection. In addition, they may suggest new approaches to research in the
role of congenital viral infection as a cause of diseases of unknown etiology
such as prematurity, idiopathic growth failure, malignancy, mental retardation,
developmental malformation, and autoimmune diseases.

Proposed course: To be confined at present level of effort.

Honors and Awards : None

Publications : None

at

I

Serial No, NDS (CF) - 70 E 183A

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Immune mechanisms in experimental allergic encephalomyelitis
(EAE)

Previous Serial Number: Same

Principal Investigator: Lon R. White, M.D.

Other Investigators: George Nemo, Ph.D.

Marian Kies , Ph.D., Section on Myelin Chemistry, NIMH

Cooperating Unit: Section on Myelin Chemistry, Laboratory of Cerebral
Metabolism, DBBR, NIMH (HD DB - 6)

Man Years :

Total: 0.20
Professional: 0.15
Otiier: 0.05

Project Description:

Objectives: To demonstrate in vitro "activation" of lymphocytes from
guinea pigs with EAE by a myelin derived basic protein.

Methods employed ; Techniques of lymphocyte cytotoxicity and blastogenic
transformation have been employed.

Major findings: No cytotoxicity has been demonstrated. Transformation
has been observed in some, but not all, experiments. These results seem to
relate to the following variables: a) the "batch" of basic protein used,

b) allotypic identity or dissimilarity between lymphocytes and target cells,

c) strain of guinea pig utilized, d) interval between inoculation on animals
and testing of lymph node lymphocytes.

Significance to biomedical research and the program of the Institute:
EAE is generally held to be pathogenically similar to certain human demyeli-
nating diseases, particularly the post-infections encephalitides and multiple
sclerosis, and may involve lymphocyte-mediated autoimmunity. The in vitro
demonstration of a basic protein "activatable" lymphocyte would be of great
value to a more complete londers tanding of the experimental disease and would
suggest new approach to the human diseases. The knowledge gained relating
to lymphocyte function would be of immediate relevance to problems of

85t

Serial No. NDS (CF) - 70 E 1834

autoimmunity and chronic infection in certain diseases definitely or possibly
due to viral infection during prenatal and neonatal life.

Proposed course: No further experiments planned at this time.

Honors and Awards : None

Publications : None

86 t

Serial No. NDS (CF) - 70 E 1835

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

Project Title:

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Epidemiologic and immunologic study of families of subacute
sclerosing panencephalitis patients and families of matched
controls

Previous Serial Number : Same

Principal Investigators

Roger Detels, M.D.
John M. Stanhope, M.D,
Jacob A. Brody, M.D.
Jane McNew, R.N.
Anne H. Edgar

0 th er Inves t igato rs :

John Sever, M.D.

Head, Section on Infectious Diseases, Perinatal

Research Branch, NINDS
Richard L. Parker, D.V.M.

Assistant Chief, Office of Veterinary Public Health,

Center for Diseases Control, Atlanta

Cooperating Units: Section on Infectious Diseases, Perinatal Research
Branch, NINDS
Veterinary Branch, National Communicable Disease Center,
Atlanta

Man Years :

Total:

Professional:
Other :

1 2/3

1 1/2

1/6

Project Description:

Objectives : To determine, through the use of a matched control study,
factors related to the etiology of subacute sclerosing panencephalitis.

Methods employed : Cases of SSPE were selected from the areas around
St. Louis, Missouri, North Carolina, and California in order to evaluate
the occurrence in distinctly different environments. 49 families with
matched controls and an additional 8 families for whom a suitable control
could not be obtained have been interviewed. Controls were selected on the
basis of close friendship and similar background to the proband. A detailed
history of events possibly related to the etiology of SSPE including episodes
of measles, unusual illnesses, exposure to sick animals, etc. was asked of

87 1

Serial No, NDS (CF) - 70 E 1835

probands, controls and their families. A 20cc sample of blood was drawn
from each individual in the proband and control families .

Major findings: Preliminary analysis of the 40 initial cases indicates
that the median age of measles among probands was 18 1/2 months, 2 years
younger than the median age of measles among controls . One-quarter of the
probands, but none of the controls, had measles under one year of age and
two of the probands were diagnosed by their physicians as having had measles
twice. One-quarter of the probands had no history of measles. All of the
patients came from middle or lower income families, and only one of the
probands came from a truly urban environment. History of warts, herpes
virus infection, allergies and neurologic disease were similar in proband
and control families. All but 3 of the 40 probands had pets and 2 of these
had had intimate exposure with animals. A history of exposure to sick
animals was more than twice as frequent among probands (68%) as among
controls .

Significance to biomedical research and the program of the Institute:
We have shown that the measles infection in cases was unusual and occurred
frequently during a time when passive immunity was present. Further SSFE
occurs in an unusual epidemiologic pattern with rates among males far
exceeding those of females and rates in non-urban areas vastly exceeding
those in urban areas much as would be expected with a zoonotic disease.
Thus we have postulated that SSPE is caused by an unusual measles infection
and a subsequent triggering event which is probably a zoonotic virus.

Proposed course: Sera from 57 proband families and 49 control families
will be analyzed for presence and titers of measles antibodies. Additional
testing will be made as indicated by the results of the analysis of the
questionnaires. Final analysis of the histories obtained from the proband
and control families will be completed. Intensive search for the possible
"zoonotic trigger" will be carried out.

Honors and Awards : None

Publications: Brody, J. A. and Detels, R.: Subacute sclerosing
panencephalitis: A zoonosis following aberrant
measles. Lancet, 11:500-501, 1970.

Brody, J. A., Detels, R. and McNew, J.: Evidence that
subacute sclerosing panencephalitis is caused by an
aberrant measles infection followed by a zoonosis.
Presented at Annual Meeting of the American Academy of
Neurology, April 1971. To be published.

Serial No. NDS (CF) - 70 E 1836

1. Collaborative & Field Research

2 . Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Epidemiologic factors in Jakob-Creutzfeldt 's disease

Previous Serial Number: Same

Principal Investigators: Jacob A. Brody, M.D,

A. Roger Bobowick, M.D.
Raymond P. Roos , M.D.
C&FR, NINDS

Other Investigator: Marjorie Matthews, R.N.

Cooperating Unit: Laboratory of Slow, Latent and Temperate Virus
Infections, C&FR, NINDS

Man Years :

Total: 1/4
Professional: 1/6
Other: 1/12

Project Description:

Objectives : Jakob-Creutzfeldt 's disease is one of the spongiform
encephalopathies which has been transmitted to chimpanzees. Preliminary
investigations suggest that two virus particles occur in the brains of
patients. There are many forms of Jakob Creutzfeldt 's disease which are
distinct clinically and to some degree neuropathologically . Nothing is
known of the epidemiology of this syndrome. We wish to determine if there
are common factors among patients who develop Jakob-Creutzfeldt 's disease
and if there are specific factors related to distinct forms of this syndrome.

Methods employed: Letters were sent to neuropathologists soliciting
pathologically proven cases. With the cooperation of the referring physicians
the families of the patients are contacted and asked to participate in the
epidemiologic interview. At the time of the interview session a detailed
questionnaire is completed on the relative who is the interviewee, on the
patient, and on an age and sex matched friend of the patient who serves as a
control. Blood samples are also taken for future seriologic studies.

Ma j or findings : From the series of approximately 50 patients, we have
interviewed, thus far, 12 families. The methods outlined above have proved
workable. The families have been extraordinarily cooperative and in fact
grateful.

Serial No. NDS (CF) - 70 E 1836

Significance to biomedical research and the program of the Institute:
This neurologic disease is caused by a virus or a combination of viruses.
Transmissible diseases must be studied epidemiologically if predisposing
factors are to be elicited. The documentation of a predisposing factor to
a virus-induced degenerative disease would be a major contribution to the
understanding of the disease, its treatment and prevention.

Proposed course: This series will be developed to include between
30 and 50 cases and analysis of the data will begin.

Honors and Awards : None

Publications : None

90 1

Serial No. NDS (CF) - 70 E 1837

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Multiple sclerosis among native-born and migrants to
California and Washington States

Previous Serial Number: Same

Principal Investigators: Roger Detels, M.D.

Jacob A. Brody, M.D.

Other Investigators: None

Cooperating Units : None

Man Years :

Total: 2/3
Professional: 1/3
Other: 1/3

Project Description:

Objectives : To determine whether rates of multiple sclerosis among
native-born in Washington and California are related to latitude, and to
determine if migrants from high and low risk areas within and without the
United States retain the rate from place of origin or acquire the rate of
the place to which they migrated.

Methods employed: Washington and California have a combined population
of approximately 30 million of whom about 20 million are migrants. Decedents
with multiple sclerosis and amyotrophic lateral sclerosis between 1954 and
1964 can be analyzed by birthplace, race, sex, age and duration of life in
California. ALS patients can be used as a partial control, since the ALS
rate is thought to be stable regardless of latitude. Death rates can also
be ascertained for both MS and ALS using 1960 census data as a baseline.

Major findings: Death rates from MS were studied among 7 million
native-born and 8 million migrant white, Japanese and Chinese in California
and Washington. The death rate among native-born Washingtonians was higher
than among native-born Calif ornians . Low death rates were found among
American-born and foreign-born Japanese and Chinese in both states suggesting
that racial factors may influence susceptibility to MS. Migrants to both
states from areas reported to have low rates of MS had low rates suggesting
that they had acquired protection before migration. Migrants to Washington
from areas reported to have high rates had higher rates than similar groups

91 1

Serial No. NDS (CF) - 70 E 1837

migrating to California. These findings suggest that causative factors may
still be operative in the third and fourth decades of life in high risk
areas and that a protective factor may also be involved in the etiology
of MS.

Significance to biomedical research and the program of the Institute:
If these findings are substantiated they would lead to major insights in MS.
This is the first large study of people moving from low risk to high risk
areas and we have new evidence of a protective factor in MS. Data on possible
changes in risk among those migrating from high risk areas suggests that
causative factors persist after adolescence. The concept of separate
protective and precipitating factors in MS may provide the clue in
understanding the genesis of the disease.

Proposed course: This study will be expanded through prevalence
studies focusing on various migrant groups.

Honors and Awards : None

Publications: Detels , R., Brody, J. A. and Edgar, A.H.: Multiple

sclerosis among American, Japanese and Chinese migrants
to California and Washington. New Eng . J . Med .
In press.

92 1

Serial No. NDS (CF) - 70 E 1838

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Study of the incidence of Parkinson's disease among offspring
of Parkinsonians

Previous Serial Number: Same

Principal Investigators: Roger Detels, M.D.

Jacob A. Brody, M.D.

Other Investigators : None

Cooperating Units: Church of Jesus Christ of Latter-day Saints,

Salt Lake City, Utah

The Genealogic Society,

Salt Lake City, Utah

Man Years :

Total :

Professional:

Other:

Project Description:

Objectives : To determine if genetic factors play a part in the etiology
of Parkinson's disease by examining the incidence of disease among offspring
of probands .

Methods employed : Fifty members of the Church of Jesus Christ of
Latter-day Saints who died with the diagnosis of Parkinson's disease will
be selected for review of death certificates in Utah between 1930 and 1940
and an additional 50 members who had a hospital diagnosis of Parkinson's
disease between 1930 and 1940 selected from a review of hospital records from
the major hospitals in the Salt Lake City area. A matched index control will
be selected for each proband by taking the next death certificate or hospital
record of an individual of the same sex whose age was within 5 years of the
proband at the time of death or illness. Offspring of probands and index
controls will be identified, a review of hospital records, of obituary
notices , and a review of the records of the Genealogic Society and of the
Church files of the Church of Jesus Christ of Latter-day Saints. Addresses
of offspring will be obtained from the Church and questionnaires will be sent
out to the offspring. Cases of Parkinson's disease will be confirmed when
possible by physical examination or in the case of death through available
medical records .

93t

Serial No. NDS (CF) - 70 E 1838
Major findings: None

Significance to biomedical research and the program of the Institute;
In a chronic condition with late onset, such as Parkinson's disease, it is
often difficult to determine the role of genetic factors. Members of the
Church of Jesus Christ of Latter-day Saints maintain accurate, up-to-date
genealogies . Thus , offspring of Parkinsonians can be located and the
incidence of Parkinson's disease among them determined, providing a relatively-
simple method for determining the genetic factor in the etiology of
Parkinson's disease.

Proposed course: See "Methods employed . "

Honors and Awards : None

Publications : None

9h^

Serial No. NDS (CF) - 70 E 1839

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

Project Title:

PHS-NIH

Individiial Project Report

July 1, 1970 through June 30, 1971

Studies on the relationship of chromosomal abnormalities and
certain viral infections in mice. (Alternate title: Effects
of viruses on mitosis and meiosis in the mouse.)

Previous Serial Number: Same

Principal Investigators: Beverly J. White, M.D,, LEP, NIAMD

Lon R. White, M.D., DB, NICHD

Other Investigators: George Nemo, Ph.D.

Jacob A. Brody, M.D.

Cooperating Units: Laboratory of Experimental Pathology, NIAMD (LEP/NIAMD-17)
Developmental Biology Branch (Unit on Chronic and
Congenital Viral Infections), NICHD

Man Years :

Total: 1.5

Professional: 0.9
Other: 0.6

Project Description:

Objectives : a) . To localize within dividing mouse spleen cells in vitro,
particularly in relation to chromosomes, the site of residence of the
infecting viral genome (of the minute virus of mice - MVM) and the MVM
template DNA. b) . To search for evidence of injury by virus to chromosomes
of spermatogonia and somatic cells, c). To determine whether perinatal
infection results in infection of the germ cells and, if so, to determine
the duration of infection as well as the chromosomal and reproductive
consequences of such infection.

Methods employed: The viruses under study are two strains of MVM and
two strains of reovirus type 1. The animal used is the Cumberland View
Farms C57BL/6 mouse. The viruses are "grown" and quantitated in tissue
culture. Intracellular MVM genome (infectious or template DNA) is localized
by autoradiography using tritium "tagged" MVM DNA. Chromosome studies are
carried out on short term spleen cell cultures as well as on cytologic
preparations made directly from the spleen, testes, and bone marrow of
infected and control mice. Viral antigen is localized using immuno fluorescent
and immunoh is to chemical methods.

95t

Serial No. NDS (CF) - 70 E 1839

Major findings: a). A "fate" study in mouse spleen cells using tritium-
labeled virus preparations showed no difference in autoradiographic grain
localization between control and virus infected cultures. These were carried
out with "tagged" viruses of relatively low infectivity and specific activity
(radioactivity), b). Meiotic preparations from the testes of adult mice were
studied 4 days, 1 week, 3, 6, and 12 weeks following MVM infection. The
only notable observation was of a relative decrease in number of certain cell
types, suggesting an effect on the course of sp ermio genes is .

Major improvements in methods involved with the propagation and
quantitation of MVM have been accomplished. These have been used to
produce viral "reagents" to be used in additional experiments.

Significance to biomedical research and the program of the Institute:
The role of viruses in the etiologies of chromosomal and developmental
diseases is widely discussed, but negligibly investigated. The interaction
of noncytolytic , non-oncogenic viruses with chromosomes and the spindle is
also essentially undefined. The potential of these investigations is vast;
basic information may be gained important to an understanding of human disease
pathogenesis as well as to current concepts on the evolution of viruses and
the basic nature of their interactions with dividing, differentiating cells.

Proposed course: To be continued at same. level of effort.

Honors and Awards : None

Publications : None

96t

Serial No. NDS (CF) - 70 E 1840

1. Collaborative &. Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Retinoblastoma, Clinical, Genetic and Psychometric Aspects

Previous Serial Number: Same

Principal Investigator: Roswell Eldridge, M.D.

Other Investigators: Kathy O'Meara

Jeffrey C. Allen, M.D.
David Kitchen, M.D.

Cooperating Units: Johns Hopkins Hospital, Baltimore, Maryland

Children's Hospital of D. C, Washington, D. C.
Department of Psychology, Downstate Medical Center,
New York

Man Years :

Total: 4
Professional: 3
Other: 1

Project Description:

Objectives: Only a few traits are said to be associated with
increased intelligence. Such a correlation has been documented for torsion
dystonia, the recessive type. Retinoblastoma is another condition for
which such an association has been reported. Because we have reservations
about choice of controls and value of data obtained in a handicapped
population, we wish to evaluate this association in retinoblastoma
ourselves.

Methods employed: Efforts will be concentrated on testing the
psychometric performance of affected individuals with family history who
are sighted. The unaffected sibs will form the control group.

Major findings: To date over 20 affected individuals and 20 controls
in 12 families have been evaluated by us. In addition, retrospective data
from school groups testing is being obtained.

Significance to biomedical research and the program of the Institute;
This work is of great potential significance. If there is an association

97 1

Serial No. NDS (CF) - 70 E 1840

between the gene for retinoblastoma and higher intelligence, then

finding the underlying chemical abnormality should add to our knowledge of

intellectual development as well as suggest the cause of neoplasm.

Honors and Awards: None

Publications: None

98t

Serial No. NDS (CF) - 70 E 1842

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Mortality among Japanese- Americans in relocation camps

Previous Serial Number: Same

Principal Investigator: Roger Detels, M.D.

Other Investigator: None

Cooperating Units : None

Man Years :

Total : 1/2

Professional: 1/6
Other: 1/3

Project Description:

Objectives : To determine death rates among American and foreign-born
Japanese in relocation camps in 1943 and 1944 from selected neurologic,
cardio -vascular and malignant diseases.

Methods employed : During the period 1942-45 there were over 120,000
Japanese-Americans in the U.S., all of whom were under the authority of
the War Relocation Board. The majority were in relocation camps. Copies
of the death certificates of persons dying in these relocation camps are on
file at the Bancroft Library of the University of California at Berkeley.
Records, including medical charts for all internees, are on file at the
National Records Center in Suitland, Maryland. Thus, it is possible not
only to establish death rates from varioxis caxjses, but also to determine
the accuracy of the diagnosis and the course of the disease by referring
to the medical charts. Baseline statistics of Japanese-Americans in the
U.S. in 1943-45 and of those in relocation camps were accurately maintained
at 6 month intervals and are available at the National Archives.

Major findings: Data were reviewed. No patients with MS or ALS were
encountered.

Significance to biomedical research and the program of the Institute:
The availability of death certificates and medical charts allowed us to
determine accurate death rates among American and foreign-bom Japanese-
Americans and to correlate these rates to length of residence in Japan
and the U.S. Thus, we should be able to comment on relative effects of

L

99 1

Serial No. NDS (CF) - 70 E 1842

environment and genetic make-up on such diseases as multiple sclerosis,
amyotrophic lateral sclerosis, stroke and other cardio-vascular and
malignant diseases.

Proposed course: This study was terminated.

Honors and Awards : None

Publications : None

loot

I

Serial No. NDS (CF) - 70 E 1843

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Causes of death among siblings of MS and ALS patients

Previous Serial Number: Same

Principal Investigator: Jacob A. Brody, M.D.

Other Investigator: Estelle Kornhauser, R.N.

Cooperating Unit: Department of Neuropathology, Montefiore Hospital,
New York

Man Years :

Total: 1/3
Professional: 1/4
Other: 1/12

Project Description:

Objectives: Recently, we have developed serologic evidence that
familial patterns of antibodies of MS patients differ significantly from
controls. This suggests a familial process may be involved etiologically
in MS. We wish to determine, therefore, if siblings of MS patients have
unusual illnesses possibly related to immunologic defect. The diseases we
are interested in include collagen diseases, thyroid diseases, and
rheumatoid arthritis.

Me th o ds empl oy ed : We will control the siblings of MS patients with
the siblings of ALS patients . We have selected 100 MS patients and 100
ALS patients from the autopsy file of Dr. Harry Zimmerman, Department of
Neuropathology, Montefiore Hospital. We will contact surviving siblings
to determine major illness in the sibships.

Major findings: At present, we have information on 60 ALS patients
and 70 MS patients in whom the diagnosis was confirmed at autopsy. Various
attempts to locate survivors has been unsuccessful.

Significance to biomedical research and the program of the Institute:
There is no firm evidence that an autoimmune mechanism or a virus is the
cause of MS. There is, however, accumulating suggestive evidence that this
is the case. There is also evidence of altered serologic responses among
siblings of MS patients. If we can document that the siblings of MS patients

k

lOlt

Serial No. NDS (CF) - 70 E 1843

have illnesses related to immunological mechanisms we would have valuable
evidence that these mechanisms are involved in the etiology of MS.

Propos ed co urs e : Attempts using other patient source will be made in
hopes of getting better access to follow-up data on siblings.

Honors and Awards : None

Publications : None

102 1

Serial No. NDS (CF) - 70 E 1844

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Amyotrophic lateral sclerosis in veterans

Previous Serial Number: Same

Principal Investigators;

Other Investigators

Cooperating Units ;

John Kurtzke, M.D.

Chief, Neurology Service, Veterans Administration

Hospital, Washington, D.C.
Gilbert Beebe, M.D.

Director, Follow-up Agency, National Research

Council of the National Academy of Sciences
Virginia C. Karl, M.A.

Social Work Service, Veterans Administration

Central Office, Washington, D.C.

Jacob A. Brody, M.D.
A. Roger Bobowick, M.D.

Veterans Administration

National Research Council of the National Academy of
Sciences

Man Years :

Total: 1/3
Professional: 1/4
Other: 1/12

Project Description:

Objectives: We will determine if there are distinctive epidemiologic
features related to amyotrophic lateral sclerosis using the veteran
population. We will also determine if service in Guam and the Marianas
Islands was a risk factor in developing ALS . Life-table estimates will be
constructed to clarify prognosis as to survival with ALS.

Methods employed : The methods paralleling those of the veterans study
of multiple sclerosis will be used to obtain the "records sample." In
addition, we will survey approximately 200 veterans with ALS and 200 controls
with brain tumors using a detailed historical questionnaire. This population
will be called the "interview sample." Finally, a prognostic study will be
constructed from Veterans Administration Hospital admissions of the 1957 -
1964 period for ALS and other motor neuron diseases.

103 1

Serial No. NDS (CF) - 70 E 1844

Major findings: The records sample should number at least 425 World
War II and Korean war veterans with ALS . With about 500 discharges annually
for ALS, the large Veterans Administration Hospital system will supply
sufficient case material to carry out the interview sample and construct
the prognostic study. Dr. Franklin 0. Meister, Director, Neurology Branch,
Veterans Administration Central Office, Washington, D.C. has endorsed the
project and suggest that only VA Hospitals with neurologic units be used.

The detailed historical questionnaire for the interview sample has been
drafted and preliminary field tests indicate that it will be quite workable.

Significance to biomedical research and the program of the Institute:
Regional differences and socio-economic differences have been commented upon
for amyotrophic lateral sclerosis in the United States. This study will
determine for a large population if there are predisposing factors to this
disease. Should we determine such factors, it would provide a valuable clue
to the understanding, treatment and prevention of this disease. Prognosis
for survival should also be clarified.

Proposed course: With our collaborators, we will collect all known
veterans with ALS and try to detect possible predisposing factors .

Honors and Awards : None

Publications: None

IQl+t

Serial No. NDS (CF) - 70 E 1845

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Natural history of Parkinson's disease and the effect
of L-Dopa

Previous Serial Number: Same

Principal Investigator: Jacob A. Brody, M.D.

Other Investigator: A. Roger Bobowick, M.D.

Cooperating Unit: National Parkinson Foundation, Inc., Miami, Florida

Man Years :

Total:

5/24

Professional:

1/8

Other:

1/12

Project Description:

Objectives : We wish to determine if L-Dopa affects the natural history
of Parkinson's disease or is merely symptomatic treatment.

Methods employed: We will study a cohort with diagnosed Parkinson's
disease who had the disease between 1958 and 1964, prior to the advent of
L-Dopa. We will then develop life tables to determine the life expectancy
and specific cause of death of patients with Parkinson's disease before
the advent of L-Dopa, We will then follow a matched series of patients who
are receiving L-Dopa to determine life expectancy and cause of death.

Major findings: Review of the records at the National Parkinson
Foundation, Inc. in Miami revealed that the necessary information had been
reliably recorded. A sample of 50 pre- and 50 post-L-Dopa era patients has
been processed. Medical record data retrival forms and follow-up forms to
physicians have been completed. The medical record data is remarkably
complete. The follow-up information will require the usual multiple
avenues of pursuit. The staff at the National Parkinson Foundation, Inc. is
now working on the medical record data retrival forms for the remaining 450
patients in the pre-L-Dopa cohort.

Preliminary analysis of the first 100 patients suggest that in the
population of Miami the mean age increased about 3 years from the early
to the late 1960 's. This is consistent with the cohort theory of parkinsonism
which supposes a common etiology of sub-clinical encephalitis lethargica

105t

Serial No. NDS (CF) - 70 E 1845

around 1920 and predicts the disappearance of parkinsonism as a major
clinical entity around 1980. Because of the profound implications, verificatior
of this phenomenon is being sou^t in age data of Parkinson patients at
other centers .

Significance to biomedical research and the program of the Institute:
It is not known whether L-Dopa provides only s3nnptomatic relief of Parkinson's
disease or whether it actually affects the pathologic process v/hich produces
this chronic illness. If indeed it does reverse the pathologic process, we
would advance the understanding of the genesis of this disease. Since L-Dopa
is a drug with known biohazards, we will be able to determine if these hazards
shorten the life expectancy of treated patients or if they die of specific
diseases such as cardiovascular disease or kidney disease which will permit
us to focus medical attention on prevention of these specific complications
among treated patients .

Proposed course: The pre-L-Dopa cohort and the cohort receiving L-Dopa
will be matched and appropriate follow-up information concerning major
illness and death will be secured.

Honors and Awards : None

Publications : None

I06t

Serial No. NDS (CF) - 71 E 1917

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Twin study of multiple sclerosis: An epidemiologic inquiry

Previous Serial Number: None

Principal Investigators: A. Roger Bobowick, M.D.

Jacob A. Brody, M.D.

Other Investigators: John F. Kurtzke, M.D.

Veterans Administration Hopsital, Washington, D.C.
Zdenek Hrubec, Sc.D.

Follow-up Agency, NAS-NRC
Marjorie Matthews, R.N.

Cooperating Units: Neurology Service, Veterans Administration Hospital,
Washington , D.C.
Follow-up Agency, National Academy of Sciences, National
Research Council

Man Years :

Total: 1/4
Professional: 1/6
Other: 1/12

Project Description:

Objectives: The abundant epidemiological literature of multiple sclerosis
has indicated beyond reasonable doubt that critical environmental factors
influence the rate of disease. Prevelance and migration studies strongly
suggest that the environmental factor(s) is operant about the time of puberty.
In order to examine the nature of the etiologic exposure in multiple sclerosis
then, it is most prudent to concentrate on events in patients and controls
with comparable early life histories in whom host factors are equalized.
Twins discordant for multiple sclerosis at an age beyond prime risk of
acquiring MS offer this opportunity for study.

Methods employed: The NAS-NRC twin registry has identified 23 pairs of
twins one or both of whom has multiple sclerosis. This group has come from
their population of 16,000 pairs of white male twins who are veterans of
military service in World War II and born during the years 1917 to 1927.
Initial contact with the twins will be a letter from the registry explaining
the study. If the twins express an interest in the study, they will be
contacted by phone by the NIH investigators to arrange an interview date.
The information collected will be: 1) pertinent medical history and

I07t

Serial No. NDS (CF) - 71 E 1917

neurological exam, 2) an indepth epidemiologic interview concentrating on
events prior to age 20, and 3) blood samples.

Ma.i or findings : Initial contact has been made with most of the twin
pairs and thus far five pairs have agreed to participate. The first twin
pair will be visited shortly.

Significance to biomedical research and the program of the Institute:
Although exhaustive studies have been conducted to invoke specific etlologic
factors in MS, no definitive precipitative circumstances have been identified
perhaps because of the subtlety of these factors or the difficulties of
suitably controlling for numerous xmrelated circumstances. This novel
application of the twin method of study offers an efficient technique for
identifying these precipitating circumstances. Resolution of the cause and
prevention of MS would be greatly enhanced by the identification of these
precipitating factors .

Proposed course: The twins will be visited over the course of the next
year.

Honors and Awards : None

Publications : None

108 1

Serial No, NDS (CF) - 71 E 1918

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Follow-up of the Cooperative Measles Vaccine Field Trial
in four communities

Previous Serial Number: None

Principal Investigators

Jacob A. Brody, M.D,
Anne H. Edgar

Other Inves tigators ;

Marion Dressier, M.D.

DeKalb County Health Department, Decatur, Georgia
Margaret I. Rathbun, M.D.

Monroe County Health Department, Rochester, New York
Edwin P. Isacson, M.D.

State University of New York School of Medicine
Russell E. Alexander, M.D.

University of Washington School of Medicine, Seattle
Philip S. Brachman, M.D..

National Communicable Disease Center, Atlanta

Cooperating Units :

DeKalb County Health Department, Decatur, Georgia
Monroe County Health Department, Rochester, New York
State University of New York School of Medicine
University of Washington School of Medicine, Seattle
National Communicable Disease Center, Atlanta

Man Years ;

Total:

Professional;
Other :

1/4
1/2'
3/12

Project Description:

Objectives : To determine through contact with the participants in the
killed measles vaccine field trial of the early 1960s, any possible sequelae
to receipt of the vaccine.

Methods employed : Review of the addresses of the study population in
four communities (DeKalb County, Rochester, Buffalo and Seattle) provided
definite addresses for 46% of the 2091 participants plus possible location
of an additional 17%. In order to determine the likelihood of success of
follow-up, questionnaires requesting history of childhood diseases, other
infections, non-allergic diseases accompanied by a rash, and hospitalizations
subsequent to receipt of the vaccine, were sent to the families of the study

logt

Serial No. NDS (CF) - 71 E 1918

population in DeKalb County. To date, 52% of the definitely located families
and 29% of the possibly located have responded. These dame methods are to be
extended to the participants in the field trial in the other three cities.

Major findings: To date the response to the study has been good but the
small number of participants contacted allows no conclusions to be drawn.

Significance to biomedical research and the program of the Institute:
The isolation of a measles-like virus from brains of individuals with
subacute sclerosing panencephalitis has suggested a relationship between
these two diseases. Epidemiologic study has further indicated that these
patients may experience measles at an unusually early age. Evidence of high
measles antibody titers in patients with multiple sclerosis has raised the
possibility that measles might play an etiologic role in this disorder. In
addition reports have been published on an atypical measles illness occurring
in children who have received killed measles virus vaccine several years
earlier. In light of the increasing interest in measles as an etiologic
factor in disease, and the specific sequela reported to occur in those who
have received the killed measles vaccine, the medical history of this study
population subsequent to vaccination is of particular interest.

Proposed course: The participants for whom possible or definite
addresses have been located in the other three communities will be contacted
by mail and asked to supply the information requested on the questionnaire.
Those not responding to the initial request will be contacted a second time
in the hope of increasing the response rate.

Honors and Awards : None

Publications : None

not

Serial No. NDS (CF) - 71 E 1919

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Viability of white blood cells used for lymphocyte
transformation

Previous Serial Number: None

Principal Investigators: Jeffrey C. Allen, M.D.

George Nemo, Ph.D.

Other Investigator: Jacob A. Brody, M.D.

Cooperating Unit : None

Man Years :

Total :

1/6

Professional:

1/12

Other:

1/12

Project Description:

Objectives : To develop a technique for studying lymphocyte transformation
on blood specimens transported over extended periods of time so that the
technique may be used in field studies.

Methods employed : White blood cells are allowed to stand at room
temperature in either minimal essential medium or autologous plasma over
a 24-48 hour period. The ability of the white blood cells to undergo
lymphocyte transformation when exposed to various antigens is studied by
the standard technique of measuring lymphocyte transformation used in this
laboratory.

Major findings: There was considerable variability in measurements of
lymphocyte transformation to vaccinia over a 24 hour period, however, there
was no reduction magnitude of response of the cells in the interval studied.
MEM was not as good as incubation mediums as autologous plasma.

Significance to biomedical research and the program of the Institute:
If this method can be perfected the technique of lymphocyte transformation
may be used in field studies and large numbers of relevent individuals may
be examined. It is also hoped that if some of the variability is removed the
technique of lymphocyte transformation may become sensitive. In
addition, if there is a preferential die-off of granulocytes over time, a
relatively pure culture of lymphocytes may be obtained.

lilt

Serial No. NDS (CF) - 71 E 1919

Proposed course: It remains to be determined how long white blood
cells can remain in autologous plasma at room temperature and still retain
their ability to undergo transformation. The variability in response is
considerable. This variability should be studied. Such techniques as
purifying the lymphocytes, drawing blood at a certain time of the day from
the same individual, and more careful standardization of the procedure may
control some of the variability.

Honors and Awards : None

Publications : None

112 1

I

Serial No. NDS (CF) - 71 E 1920

1. Collaborative & Field Research

2 . Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: A study of pregnancies among nurses; high risk pregnancies
due to transmissible viruses

Previous Serial Number: None

Principle Investigator: Estelle Kornhaioser, R.N.

Other Investigator: Jacob A. Brody, M.D.

Cooperating Unit : None

Man Years :

Total: 1/4
Professional: 1/12
Other: 3/12

Project Description:

Objectives : This project was developed to question the effects on the
fetus of maternal virus infection.

Methods employed: A questionnaire was designed using the usual backup
history and pertinent questions relating to type of nursing service, year of
pregnancy, normal or abnormal births, and all information relating to any
type of exposure to a viral type disease. The questionnaire and letter of
explanation was pre-tested in 1160 nurses registered in the Maryland and
District of Columbia area.

Major findings: Of the 1160 questionnaires sent we have received
approximately 800 replies . To date there are 1353 pregnancies with
approximately 250 miscarriages, stillbirths, and abortions resulting for
many reasons.

Significance to biomedical research and the program of the Institute:
This study will determine if pregnant women working with people with infectious
disease have an elevated risk of fetal damage.

Proposed course: We plan to follow up with a second mailing and then
continue our study to a larger group of respondents and continue to examine
the risk factors involved in the care of patients who have infectious diseases.

Honors and Awards : None
Publications : None

113t

Serial No. NDS (CF) - 71 E 1921

1. Collaborative & Field Research

2. Epidemiology Branch
/ 3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Measles infection of mice: Effect of maternal immunity

Previous Serial Number : None

Principle Investigators: Jacob A. Brody, M.D.

George Nemo , Ph . D .

Other Investigators: Lon R. White, M.D., DB, NICHD
Gary Cooper, Biologist

Cooperating Unit: None

Man Years :

Total: 1/6
Professional: 1/12
Other: 1/12

Project Description:

Objectives : To develop an experimental animal model to study the
pathogenesis of subacute sclerosing panencephalitis (SSPE) .

Methods employed: Adult female mice previoiosly immunized with measles
virus will be bred and their offspring challanged with measles at various
times after birth. Measles antibody levels will be determined periodically
and a thorough analysis of mouse tissues for measles virus and measles-
related antigens will be performed.

Major findings: None

Significance to biomedical research and the program of the Institute;
An agent which by most virological criteria resembles measles virus has been
isolated from brains of SSPE patients . A papova-like virus has been visualized
in electron micrographs of specially treated cell lines inoculated with SSPE
brain material suggesting the possibility of a dual infection. The exact
role these agents play in the pathogenicity of the disease is unclear. An
experimental animal model may aid considerably in elucidating the mechanism.

Proposed course: See "Methods employed . "
Honors and Awards : None
Publications : None

I15t

Serial No. NDS (CF) - 71 E 1922

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Search for a negative DNA strand in cells infected with the
minute virus of mice (MVM)

Previous Serial Number: None

Principle Investigator: Lon R. White, M.D., DB, NICHD

Other Investigators: Harrie Anne Sutton, biologist, DB, NICHD
George Nemo , Ph . D .
Gary Cooper, biologist
Jacob A. Brody, M.D.

Cooperating Unit : None (HD DB - 15)

Man Years :

Total: 0.7
Professional: 0.7
Other: 0.0

Project Description:

Objectives : a). To demonstrate a "template" DNA (negative strand)
complementary to the single stranded DNA (positive strand) of the MVM
virion, b) . To determine if the negative strand is associated with the
cell's chromosomal DNA. c). To investigate the kinetics of negative strand
synthesis relative to cellular DNA replication, cellular division, virus
infection, and virus production.

Methods employed: These studies will utilize H or C "tagged" viral
DNA with annealing demonstrated by: a) liquid scintillation detection of
retained isotope on a filter, b) autoradiography with virus infected cells
in metaphase. Some experiments will use purified chromosomes and chromosomal
DNA from mitotically phased cells in culture.

Major findings: None

Significance to biomedical research and the program of the Institute:
Characterization of virus-cell interactions using a single-stranded DNA virus
(parvovirus group, of which MVM is representative) is expected to advance our
understanding of the mechanisms by which a virus infection might alter the
genetic potential of the host cell (either a somatic or germ cell) as well
as of mechanisms involved with the establishment of latent and chronic viral

iiyt

Serial No. NDS (CF) - 71 E 1922

infections. These may be directly relevant to the etiologies of certain
reproductive, developmental, and chronic diseases whose causes are now
unknown .

Proposed course: To be continued at an increased level of effort.

Honors and Awards : None

Publications : None

Il8t

Serial No. NDS (CF) - 71 E 1923

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Effect of measles virus on specific and non-specific
stimulation of lymphocyte transformation

Previous Serial Number: None

Principal Investigator: Jeffrey C. Allen, M.D.

Other Investigators: George Nemo, Ph.D.

Jacob A. Brody, M.D.

Cooperating Unit : None

Man Years :

Total:

1/6

Professional:

1/12

Other:

1/12

Project Description:

Objectives: To define the effects live and UV- inactivated measles
virias have on the ability of human peripheral lymphocytes to respond to
apecific and non-specific stimulation in vitro .

Methods employed: Measles virus from both Ostereich and Edmonston
strains will be grown to high titer in tissue culture. Lymphocytes will be
obtained from healthy volunteers and prepared in the usual manner. A
battery of specific antigens will be used including vaccinia, PPD , Candida,
diptheria and mumps. PHA will be the only non-specific antigen used.

Lymphocytes will be cultured in the usual way and incubated for the
number of days appropriate to the particular antigen. Measles virus of
varying titers, either live or killed, will be added simultaneously, before
or after the particular antigen is added. Tritiated thymidine will be
added one day before the harvest and uptake will be measured by liquid
scintillation spectrotometry . Virus cultures and antibody measurements
will be made from lymphocyte cultures at the time of harvest. In certain
experiments fetal calf serum will be used to control for the effects of
varying titers of measles antibodies of volunteers.

Major findings: Preliminary experiments suggest that measles virus
(Ostereich) will inhibit non-specific lymphocyte PHA stimulation at titers
greater than or equal to 10 PFU/cc even though there is less than 1 virus
per lymphocyte in the original culture.

119 1

Serial No. NDS (CF) - 71 E 1923

Significance to biomedical research and the program of the Institute:
Measles virus has been known for some time to suppress specific cellular
immune responses in vivo. Controversy exists with regard to its effect on
lymphocytes in vitro and current reports suggest that the virus inhibits
specific but not non-specific stimulation. The mechanism of this inhibitory
effect is unknown. It is not known what effects humoral or cellular immunity
have on this inhibitory effect. Children with thymic aplasia and absent
cellular immunity are subject to aberrant and severe measles infections. In
vitro studies may suggest the role cellular immunity plays in lost defense
against viruses.

Proposed course: After the basic work is completed, measles antigen
will be broken down to its basic protein and RNA components, and the effects
of these substances will be evaluated. Other viruses will also be investigated
such as rubella and mumps. Various disease states will be investigated such
as SSPE and MS. A more complete study of the effects of measles antibody
and antigen- antibody complexes is anticipated. Investigation at the cellular
level for the site of measles operation will be studied with the use of
autoradiography or fluorescent staining.

Finally, the supernatort of the measles-lymphocyte cultures will be
investigated for the presence of inhibitory factors.

Honors and Awards : None

Publications: None

120t

Serial No. M)S (CF) 71 E I92U

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-WIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Biochemical Studies in Torsion Dystonia

Previous Serial Number: None

Principal Investigators: Roswell Eldridge, M.D.

Thomas Chase, M. D.

Other Investigator: Kathy A. O'Meara

Cooperating Unit: Laboratory of Clinical Science, NIMH

Man Years :

Total: 2
Professional: 1 l/2
Other : 1/2

Project Description:

Objectives: Torsion dystonia has been shown recently to consist of at
least two hereditary conditions as well as an acquired form. Through
appropriate biochemical investigation it should be possible to determine
the precise biochemical defect (or abnormal gene product) in the recessive
form of torsion dystonia.

Methods employed: Patients are selected from the nationwide registry
of torsion dystonia cases we have compiled and admitted to Dr. Chase's
service for one or more weeks of the intensive evaluation. In addition
to routine neurologic and chemical study, metabolism of biogenic amines is
evaluated as completely as possible. Following this, there is often a
period lasting several months in which drug trial is carried out.

Major findings: Initial results on the small series of patients suggest
that there may be an increased turnover of catecholamines in those with
the recessive form of dystonia. This in in marked contrast to the levels
seen in Parkinson's disease and Parkinson's dementia on Guam.

Significance to biomedical research and the program of the Institute:
Demonstration of the molecular lesion in one of the torsion dystonias would
be a noteworthy achievement since another disease would be added to the
small series of inborn errors of metabolism for which the precise lesion
is recognized. In addition, undoubtedly important information with the
forthcoming regarding biochemistry and physiology of movement control.

1^1 1

Serial No. NDS (CF) 71 E I92I1

In addition, because of the recognized association of increased intelligence
and the recessive form of dystonia, it is possible information regarding
the development of intelligence would also result.

Proposed course: Continued investigation of patients from the genetics
registry. Addition of more sophisticated techniques as they become
available.

Honors and Awards : None

Publications: Chase, T.N. : Biochemical and pharmacologic studies of

dystonia. In The Torsion Dystonias (Dystonia Musculorum
Deformans). Editor, R. Eldridge. Neurology suppl. 20:11,
Part 2, November 1970.

122 1

Serial Wo. WS (CF) 71 E 1925

1. CollalDoratlve & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-WIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Intelligence in Israeli Patients with Torsion Dystonia

Previous Serial Number: None

Principal Investigators: Roswell Eldridge, M.D.

Other Investigators: Morris Gross, PhD.

Richard Goodman, M. D.

Cooperating Unit : Department of Education

Herbert H. Lehman College
Bronx, New York

Man Years :

Total : 1/2

Professional: 3/8
Other : 1/8

Project Description:

Objectives; The recessive form of torsion dystonia has recently been
shown to be associated with increased intelligence. However, the series
which this information is based was small and individuals were scattered
over a large area of the northeastern part of the United States. In Israel,
where we suspect there to be over 20 patients with the recessive form of
torsion dystonia, a more homogeneous testing situation prevails so that
results should be more meaningful. If this association is true the implications
are dramatic so that confirmation to this Israeli study is clearly in order.

Methods employed; Bona fide cases of torsion dystonia in Israel would
be selected by the principal investigator on the basis of personal examination.
Psychometric data would be obtained in a retrospective manner through the
Israeli Department of Education and similar data would be obtained for
unaffected sibs. A comparison would then be made of the performance in
these two groups.

Major findings; Although we have known for several years of the
presence of over 20 cases of torsion dystonia in Israel we have not yet been
able to arrange for visitation. Neurologists, psychologists, and educators
in Israel have been alerted and indicated their interest in participating
in this study.

I23fc

Serial Wo. EDS (CP) 71 E I925

Significance to biomedical research and the program of the Institute:
The positive association between torsion dystonia and intelligence would
suggest that the chemical abnormality producing dystonia may also enhance
intellectual development. Elucidation of this basic chemical abnormality
would suggest a method for enhancing intellectual development;, particularly
those from the retarded population.

Proposed course: Several weeks in Israel would be necessary to make
the necessary home visits and arrange for the collection of appropriate
psychometric data.

Honors and Awards : None

Publications : None

12

i^t

Serial No„ KDS (CF) 71 E 1926

1. Collaborative & Field Research

2. Epidemiology Branch
3- Bethesda, Maryland

FES-KLE

Individual Project Report

July 1, 1970 through June 30, 197I

Project Title: The Difficulty of Diagnosing Acoustic Neuroma in Young
Patients

Previous Serial Number: None

Principal Investigators: Jeffrey C. Allen, M.D.

Roswell Eldridge;, M.D.

Other Investigator: Kathy O'Meara

Cooperating Units: Department of Neurosurgery

Columbia Presbeyterian Hospital
New York

Man Years :

Total : 1
Professional: 1/2
Other: l/2

Project Description:

Objectives: To document and publicize the difficulty of diagnosis
when acoustic neuroma occurs in young patients.

During a course of clinical and genetic study of acoustic neuroma in young
individuals we have been impressed with the unusual length of time and
unusual number of specialists consulted before diagnosis of acoustic
neuroma became a consideration. This period was often one of unusual
lonnecessary stress and expense to the patient and his family and often
resulted in loss of valuable time in many instances.

Methods employed: Patients who have early onset of acoustic neuroma
were questioned in detail regarding physicians consulted and diagnostic
procedures employed.

Major findings: In general, the younger the individual when symptoms
of acoustic neuroma begin, the longer the period before diagnosis and the
greater the number of physicians consulted and the more expense to the
family.

12 5t

Serial No. WDS (CF) 71 E I926

Significance to "biomedical research and the program of the Institute:
Such documentation should impress the medical community ahout the resistance
of this condition in young individuals thereby reducing time, expense and
tuCTiioil to patient, family, and physician when a new case develops. Through
such publicity it is possible that our acoustic neuroma registry will be
expanded .

Proposed course: Documentation of similar experience in the remainder
of our acoustic neuroma patients. Publication of results.

Honors and Awards : None

Publications : None

126t

Serial No. KDS (CF) ?! E I927

1. CollalDorative & Field Research

2. Epidemiology Branch

3. Bathes da, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title : Family Studies in Young Patients with Acoustic Neuroma

Previous Serial Number : None

Principal Investigators: Jeffrey C. Allen , M. D.

Roswell Eldridge, M. D.

Other Investigators: Kathy A. O'Meara

George T. Nager, M. D.
Johns Hopkins Hospital

Cooperating Units : Department of Otolaryngology,
Johns Hopkins Hospital
Baltimore, Maryland

Man Years :

Total: 1 1/2

Professional: 1
Other: l/2

Project Description:

Objectives: To determine the nosology of acoustic neuroma by studying
clinical features, the age at onset of symptoms and presence of a family
history of the disorder in young patients with unilateral and bilateral
acoustic neuroma.

Methods employed : Field studies were conducted in evaluating family
members. On those seen personally, physical examinations were performed,
stressing neurological and skin examinations. In addition, audiometric
examinations including air and bone conduction and caloric examinations
were conducted in the field.

Patients were selected through chart review of neurosurgeons' files. All
patients selected were alive at the outset of this study and experienced
onset of symptoms before age U5.

Genealogic information and medical history was obtained from family members
and family and hospital records. (Medical history was obtained from family
members, hospital and physician records.)

127t

Serial Wo. M)S (CF) 71 E I927

On select patients extensive outpatient studies have included audiometric
and vestibular testing, complete ENT and neurologic examinations and skull
x-rays. These were undertaken in cooperation with the Departments of
Neurology, Radiology and Otolaryngology of Johns Hopkins Hospital.

Major findings: Patients with bilateral acoustic neuroma are more likely
to have earlier onset of symptoms and a positive family history of either
central, peripheral or mixed neurofibromatosis. Nine of the 5I propositi
seen so far had bilateral acoustic neuroma. Three of these had a definite
family history of the trait. Of the Ul propositi with unilateral acoustic
neuroma, a definite family history was noted in only one.

Most patients in the study commented on the length of time and number of
physicians consulted before a definite diagnosis was made. Ttie average
number of years between onset of symptoms and surgical intervention was
7.0 for the bilateral cases and ^.h for the unilateral cases. The larger
niimber of years for bilateral cases may reflect the failure of physicians
to consider this diagnosis in younger individuals.

Significance to biomedical research and the program of the Institute:
The syndrome of bilateral acoustic neuroma seems to be distinct for unilateral
acoustic neuroma using parameters such as age of onset of symptoms, likelihood
of having a positive family history, presence of stigmata of neurofibromatosis
and mode of inheritance. Unilateral acoustic neuroma on the other hand
probably is not a distinct syndrome and may represent the manifestations of
a sporadic dominant mutation or the expression of a recessive trait. Nearly
^0 percent of our patients with unilateral acoustic neuroma were of Ashkenazic
Jewish extraction. '^

Proposed course: The project will be completed after 70 kindreds have
been examined.

A large body of descriptive information has been collected on these diverse
kindreds. These kindreds could provide a fertile source for laboratory
investigation. Pathologists have not been able to differentiate between
an acoustic neuroma from a patient with unilateral or a patient with bilateral
disease. Perhaps there are differences at a biochemical level.

Patients with bilateral disease may have a generalized neoplastic diathesis
and laboratory investigation may reveal this in the patient and certain
members of their family.

Honors and Awards: Presentation at Middle Atlantic Neurosurgery Society

Publications : None

128 1

Serial No. KDS (CF) 71 E I928

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda^ Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title : Chromosome Studies in Retinoblastoma

Previous Serial TJumber : None

Principal Investigators: David Kitchen, M.D.

Roswell Eldridge, M.D.

Other Investigators : Kathy 0 'Meara

Cooperating Unit: Cytology Laboratory, Eye Institute,
Columbia Presbyterian Hospital
New York, New York

Man Years :

Total: 2
Professional: 1/2
Other: 1 I/2

Project Description:

Objectives : Retinoblastoma is a malignant tumor of infancy and
childhood which fortunately is often amenable to surgery. There appear to be
several causes for retinoblastoma. Cases are generally sporadic but in
approximately 10 percent there is a family history suggesting genetic basis.
In addition, a number of reports have now appeared of retinoblastoma
associated with gross chromosomal abnormality, generally of the D group. In
these cases there is generally an associated physical abnormality in contrast
to the other forms of retinoblastoma. Chromosome studies will be performed
on select individuals and families to add further documentation to the role
of gross chromosomal change and retinoblastoma.

Methods employed: Blood is drawn on selected patients and their
relatives during a home visit for our psychometric evaluation of retinoblastoma.
Material is sent to Dr. David Kitchen's laboratory for karyotype analysis.

Major findings: In none of the familial cases of retinoblastoma have
karyotype analysis has there been evidence of chromosomal abnormality of a
gross nature. In contrast several sporadic cases of retinoblastoma associated
with other physical defects have been found with abnormal feryotype by Dr.
Kitchen.

ia9t

Serial No. NDS (CF) 71 E I928

Significance to biomedical research and the program of the Institute:
Confirmation of the role gross chromosomal change makes to retinoblastoma
is vital in pinpointing the precise etiologic event of this form of cancer.

Proposed course : See "Methods employed".

Honors and Awards : None

Publications : None

130^

Serial Ro. WS (CF) Jl E I929

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Genetic Study of an Irish Isolate in South Carolina

Previous Serial Rixmher; None

Principal Investigator: Roswell Eldridge, M. D.

Other Investigators: Jeffrey C. Allen, M.D.
Kathy 0 'Meara
Charles Still, M.D.

Cooperating Unit : Department of Neurology,

University of South Carolina
Columbia, South Carolina

Man Years :

Total: 2
Professional: 1
Other: 1

Project Description:

Objectives: The Irish Travellers of Murphy Village, South Carolina are
a unique Catholic isolate whose exact origins are unknown. The present
population is approximately 1,100 of which U20 are under 20 years of age.
The men receive income from spraying barns and laying linoleum. They secure
their business by travelling in small trucks throughout the eastern United
States.

The Village is of particular interest to us because of the apparent high
rate of inbreeding which predisposes autosomal recessive traits.

Methods employed: At present the need is for precise demographic data
such as population break down by sex and age. Patterns and family size
would be of interest .

Genetic information may be best secured through conversation with responsible
elders in the Village. Undoubtedly the group traces from a small founding
population and precise information about this as well as information about
members added to the group in recent years would be important.

The genetic relationship of this population to the present day Irish might
be established by noting the gene frequencies with ABO, Rh and other
accessible footmarkers.

131t

Serial No. WDS (CF) 71 E I929

Medical genetic studies most certainly focus on existing recognized familial
problems such as mental retardation and skin disease. In addition, a
comprehensive medical evaluation of the community and historical account of
disorders in earlier generations would be worthwhile. Screening for newborns
looking for recognized metabolic and chromosomal abnormalities might be
productive.

Major findings: Preliminary information has already been secured
regarding the background, demography, economy, social organization,
education, medical problems and genetics. Several familial traits are
present among the Villagers suggesting that a comprehensive survey would
reveal other genetic traits.

Significance to biomedical research and the program of the Institute:
Studies of such isolates have been unusually productive in the delineation
and understanding of genetic entities. Examples include Victor McKusick's
study of the Amish and Carl Witkop's study of the Wiesorts.

Proposed course: Considerable ground work has been laid for this
project and now results await investment of time and personnel.

Honors and Awards : None

Publications : None

132^

Serial No. M)S (CF) 71 E 1930

1. Collaborative & Field Research

2. Epidemiology Branch

3. Bethesda, Maryland

PHS-WTH

Individual Project Report

July 1, 1970 through June 30, I97I

Project Title: Von Hippel-Lindau's Syndrome - Clinical^ Genetic and
Biochemical Aspects

Previous Serial Number: None

Principal Investigator: Roswell Eldridge^ M.D.

Other Investigators: Jeffrey C. Allen, M. D.
Kathy O'Meara

Cooperating Units: Department of Neurology
Johns Hopkins Hospital
Columbia Presbeyterian Medical Center

New York
Man Years :

Total : 2
Professional: 1 I/2
Other : 1/2

Project Description:

Objectives: In I9II Eugen von Hippel described a number of patients
with retinal angiomatosis. In I926 Arvid Lindau described a number of
patients, who in addition to the retinal angiomatosis, had such intracranial
lesions as cerebellar cysts and cerebellar medullary angioblastic tumors
as well as pancreatic cysts, renal cysts and adrenal tumors. The familial
nature of this syndrome has since been noted by numerous authors.

What has impressed us is the infrequency with which these tumors are
bilateral. In contrast to other hereditary traits associated with neoplasm
of paired structures the involvement is generally bilateral. Is the
tendency to unilateral involvement in the Von Hippel-Lindau's syndrome real
and, if so, does it reflect a different etiologic mechanism than seen in the
other hereditary neoplasms? Or is the unilateral involvement only apparent,
reflecting failure to scrutinize the presumably healthy member of the
paired organs?

Methods employed: The propositi with Von Hippel-Lindau's syndrome will
be ascertained through neurologic departments of selected medical centers.
Contact with individuals and their families will be made through their
personal physician, and home visits will be arranged for physical examination
and detailed history.

133^

Serial No. KTOS (CF) 71 E I93O

Major findings : None

Significance to biomedical research and program of the Institute:
The first step will be to document whether or not this syndrome tends to be
unilateral in its involvement. Later more sophisticated laboratory studies
can be undertaken to determine more closely the mechanism of abnormal gene
action which produces the neoplasias.

Proposed course : See "Methods employed".

Honors and Awards : None

Publications: None

I3^t

Serial Wo. WDS (CF) 71 E I93I

1. CollalDorative & Field Research

2. Epidemiology Branch
3- Bethesda, Maryland

FES-Wm
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Genetic study of population isolates in Maine

Previous Serial Number : Rone

Principal Investigators: Jeffrey C. Allen, M. D.

Roswell Eldridge, M. D.

Other Investigators: Morris Lambdin, M. D.

Ellsworth, Maine
Thomas Roderick, M. D.
Jackson Laboratory
Bar Harbor, Maine

Cooperating Units: Division of Public Health Nursing,

State of Maine Dept. of Health & Welfare

Maine Genetics Counselling Center
Ellsworth, Nb,ine

Maine Medical Association; Lincoln, Sagadahoc,
Washington, Waldo and Hancock Counties

lyfen Years :

Total : 1/2

Professional: l/k
Other : l/k

Project Description:

Objectives: To investigate numerous genetic subisolates along the
coast of Maine for evidence of hereditary neurological disorders.

Methods employed : A preliminary trip was made to establish contacts
and examine the feasibility of the study. Thereafter an introductory
letter and brief questionnaire was sent to every registered physician and
public health nurse in the five major coastal counties. The questionnaire
was designed to provide information on the awareness of any unusual
familial disorders; diagnosed and undiagnosed, which are prevalent in
remote areas of the state, especially on some of the offshore islands.

If feasible, demographic and historical data will be collected from State,
County and local sources to corroborate the prevalence of genetic isolation.

135'

Serial No. M)S (CF) 71 E I93I

Patients and their families will "be ■visited and an appropriate referral
will be made to local diagnostic facilities.

Major findings: So far^ (^S of II9 (58 percent) questionnaires have
been returned. Several interesting leads exist.

Significance to biomedical research and the program of the Institute:
Maine's rugged eastern coast contains innumerable islands of varying sizes.
Several of these have been inhabited for several generations and
populations vary from 50 "to 200 persons. Inbreeding is commonly practiced.
On several islands, most of the inhabitants have only two or three
different surnames » Because of the remoteness of the islands inhabitants
have not availed themselves of the usual medical facilities. They are
visited rather infrequently by a public health nurse or physician. The
likelihood of finding recessive hereditary disorders is high in these
populations .

Proposed course : Personal visits will be made to selected families.
Genetic and demographic surveys will be conducted wherever appropriate.

Honors and Awards : None

Publications : None

I

I

136^

ANNUAL REPORT

July 1, 1970 through June 30, 1971

Special Projects Branch
Collaborative and Field Research
National Institute of Neurological Diseases and Stroke
National Institutes of Health

Following the previous fiscal year's reorganization of the Special Projects
Branch, four professional personnel were added to the staff: a fully trained
neurologist and a staff associate to the Branch, a staff associate to the
Head Injury Section, and a staff associate to the Epilepsy Section. Broad
responsibilities of the staff included anticonvulsant drug program, support
of activities of the Secretary's Advisory Committee on the Epilepsies, its
sub-committees and task forces, the NINDS Ad Hoc Committee on Cerebral Death,
the NINDS Ad Hoc Committee on Anticonvulsant Drugs, and the Head Injury and
Epilepsy Information Services.

Section on Epilepsy

Meetings of the NINDS Ad Hoc Committee on Anticonvulsant Drugs were held in
October 1970 and February 1971. The Fall meeting was held in Seattle,
Washington, to enable the committee members to observe progress on the NINDS
sulthiame research contract, and the potential for other related neuropharma-
cological research there. The Committee has continued to function in a highly
efficient and useful manner providing review of research in progress, of
proposals for new research contracts, and of the pharmacology of investigational
anticonvulsant drugs. They suggested the priorities for proposed studies and
other guidance to the section. One of the most beneficial effects of the
Committee is the origination of suggestions from individual members--as the
background of each member differs, a variety of views and ideas are presented
for Institute follow-up.

During fiscal year, the pharmaceutical industry continued to cooperate with
the section. Data on drugs from seven companies was provided and discussed
by the NINDS Ad Hoc Committee on Anticonvulsant Drugs. Priorities were
established for support of clinical trials for two investigational drugs.
One will be conducted in a state hospital for patients with generalized
epilepsy; the other in university clinics for patients with absence (petit
mal) epilepsy. Preliminary studies will be employed to perfect anticonvulsant
blood level methodology and determine metabolic information prior to the
outset of full scale trials.

Research contracts for the study of ethosuximide in absence epilepsy at the
University of Virginia and The Medical College of Wisconsin were completed
in fiscal year 71. The study of ethosuximide in absence epilepsy has
examined several hypotheses. The hypothesis that ethosuximide is not
effective in control of approximately one-half of those patients who suffer
from absence (petit mal) epilepsy is apparently correct when one defines

lu

control as complete (100%) control. Ethosuximide has, however, improved
virtually every patient in whom the drug has been tolerated. The hypothesis
that failure of response to ethosuximide occurs in the presence of thera-
peutic blood levels has been demonstrated when response is defined as
complete control. The hypothesis that patients who respond to ethosuximide
will show no evidence of focal brain lesions is apparently true although some
patients with brain lesions may also respond. Another hypothesis is that
ethosuximide does not impair psychometric performance; evidence so far has
shown that indeed ethosuximide does not impair such performance when thera-
peutic blood levels are not exceeded. There is no evidence to the present
time that ethosuximide alters clinical manifestations of absence seizures;
there is also evidence that many conventional measurements of seizure
frequency do not provide a reliable assessment of seizure control.

An ancillary technique, that of EEG telemetry, has been developed during this
study, and found to be the best criterion for measurement of control of
paroxysmal abnormal discharge. The reliability of this technique is related
to the duration of the individual seizure, the longer ones being more easily
recognized. Currently, the telemetry study consists of continuous twelve-
hour EEG recordings; the information is taken from FM tapes and evaluated at
NIH.

The telemetered EEG has correlated quite well with the best clinical method
used in the ethosuximide study to date, that is, observation by a trained
observer. In almost eyery case, however, the number of seizures estimated
by the observer is less than that ascertained by telemetered recording,
strongly suggesting that the observer, regardless of experience, does not see
every seizure. This telemetry technique will be utilized in subsequent
studies. Detailed analysis of the data from the ethosuximide research contracts
will be made at the conclusion of the study. An investigational anticonvulsant
will be evaluated in this model upon the completion of the present phase.

Pursuant to a recommendation of the NINDS Ad Hoc Committee on Anticonvulsant
Drugs, a contract was awarded for the clinical evaluation of the investigational
anticonvulsant, sulthiame, in June 1970. The pilot study has been completed
and a double-blind comparison of sulthiame and di phenyl hydantoin is now
underway. Sixty patients with a diagnosis of partial epilepsy will take part
in the study. Each patient will be evaluated over a fourteen-month period.
Important features of this research are accurate reports of seizure incidence,
other drug effects, anticonvulsant blood level determinations, and measures
of psychological performance and social functioning.

The Institute will establish a pharmacology laboratory within the Section on
Epilepsy early in the coming fiscal year. Much planning has taken place to
prepare for this activity, following its endorsement by the Ad Hoc Committee
on Anticonvulsant Drugs. Harvey J. Kupferberg, Ph.D., has been recruited to
head the laboratory. The metabolism and interactions of many anticonvulsant
drugs will be studied initially.

Staff members collaborated with officials of the National Institute of Mental
Health to plan an epidemiology survey for the prevalence and incidence of

2u

convulsive disorders in seventh grade students in Washington County, Maryland.
The children are to be examined in the summer of 1971; data will be evaluated
in the coming fiscal year.

Two meetings of the Secretary's Committee on the Epilepsies were held. It
was decided to sponsor two small closed workshops to examine in depth two
neglected areas of interest to workers in epilepsy. A workshop on laboratory
methods for preparing animal models of epilepsy will be held in November 1971.
Another workshop to be held in November 1971 will examine many aspects of the
epidemiology of epilepsy. A third event jointly encouraged by the Secretary's
Committee and the Ad Hoc Committee will be a detailed examination of the
pharmacology of anti epileptic drugs. World leaders in the field will present
papers to an audience of clinicians so their treatment of patients will be
enhanced. Unfortunately, much of the researchers' efforts have not yet been
applied to the treatment of epileptic patients. A monograph will be published
following the symposium.

The Section issued two publications for workers in the epilepsy field; a
review of the literature on the effectiveness of marketed anticonvulsants,
by James J. Coatsworth, M.D., a consultant to NINDS, and a review of the
literature on anticonvulsant blood level determination methods and clinical
importance by members of Branch personnel. Editorial support was provided in
both instances by Branch personnel. Dr. Coatsworth 's report is of especial
interest, for it highlights the meager information available on anticonvulsants
in use today, and the paucity of well -documented clinical studies that have
been conducted in the past. NINDS-sponsored studies on anticonvulsants have
been designed to overcome these shortcomings and will provide useful information
on the drugs' efficacy and safety.

Epilepsy Abstracts is now in its fourth year of publication. The Excerpta
Medica Foundation has assumed all publication and distribution responsibilities
with modest support from the Institute. Epilepsy Abstracts Retrieval System
(EARS), a free text search and retrieval system based upon Epilepsy Abstracts
was proven a powerful research tool. Every word in the abstract is a key
word permitting rapid thorough searches of the literature from any of a
variety of terminals. EARS was demonstrated at the meetings of the American
Epilepsy Society, American Academy of Neurology, and American Neurological
Association. In each case, reception was most favorable. It is planned to
have this retrieval system available to research workers throughout the
country in the coming year.

A computer-based bibliography on epilepsy research from 1900 to 1959, with
some 8,000 index citations, has been developed. It will be issued in the
coming fiscal year and will compliment the published Epilepsy Abstracts 1947
to the present time.

The investigational anticonvulsant albutoin was compared with two marketed
anticonvulsants, primidone and diphenylhydantoin, in a double-blind study at
New Castle State Hospital, New Castle, Indiana, September-December 1970,
under a NINDS research contract. The design of the trial was improved,
based upon experiences of the 1969 trial, when albutoin was compared with
phenobarbital . Accurate seizure records, blood level determination, and

3u

other laboratory and ward data were recorded through the NIH WYLBUR
computer system for ease in analysis. Plans are underway for a trial of
another investigational anticonvulsant, carbamazepine, using the patient
population at New Castle State Hospital, and methodology developed during
the albutoin study.

Section on Head Injury

In response to a recommendation of the National Advisory Council, Neurological
Diseases and Stroke, the NINDS Collaborative Study of Cerebral Death, a
directed study under contract has been established. With guidance and support
from the Office of the Associate Director for Collaborative and Field Research,
the staff prepared comprehensive bibliographies and reprint collections with
translations of the literature on cerebral death. These documents were
provided to the NINDS Committee on Cerebral Death, and to prospective
principal investigators who would collaborate under the contract mechanisms.

The overall objectives of the collaborative study of cerebral death are to
verify or modify the current clinical and electroencephalographic (EEG)
criteria for cerebral death in relation to patient age and cause of coma: to
determine the minimal time that clinical and EEG criteria must be operative
to indicate cerebral death in relation to age and cause of coma; and to
assess the neurologic deficit of patients who recover after having fulfilled
all criteria for some length of time. In addition to the clinical and EEG
evaluations of patients, several contractors will conduct ancillary studies
of cerebral death; these will include computer analysis of electroencephalo-
grams, bedside EEG monitor, averaged evoked potential responses, electro-
retinograms, analysis of cerebrospinal fluid for chemical indicators of
brain death, and detailed neuro-ophthalmological examinations.

Experience dictates that prior to embarking on a large-scale collaborative
project of this type a pilot or feasibility study is needed to pretest
procedural and technical elements of the overall study, to develop the
statistical model, and to establish executive capability; specifically, its
aims include the following: to determine whether the contemplated neuro-
logical, EEG, and other appropriate examinations can be adequately handled
in a collaborative study, and to establish a basis for achieving standardiz-
ation; to determine whether, in a collaborating hospital, any cases of
suspected cerebral death would be missed, and if so, why? to develop means of
determining when a clinical and EEG picture consistent with cerebral death is
the result of sedative drug intoxication; to develop procedures for collecting,
reviewing, editing and processing data for the later or definitive study; to
evaluate means for guaranteeing uniformity in handling patients, making the
necessary observations, and recording data for analysis; to determine the
minimal significant set of clinical, EEG, laboratory and demographic observa-
tions that will be needed for each patient in the definitive study; to determine
the best statistical model for describing patients suspected of cerebral
death, so that the risks associated with an operating rule for determining
brain death can be estimated most efficiently from data gathered in the
study; to determine the sample size needed in the later study in order to
obtain desired precision of estimates, with due regard for variability in the

4u

patient population with respect to age and etiology of coma; and to create
an executive capability that could be extended to the definitive study.

To date, the project has been in a planning phase and will not become
operational until about September 1, 1971. Preliminary findings are expected
to accrue in the coming fiscal year and major findings in subsequent years.

A thorough search of available literature in the area of cerebral death has
clearly indicated that there is currently much disagreement as to which
criteria are necessary and sufficient for the diagnosis of cerebral deatti
and that so far no systematic attempt has been made to refine the existing
criteria with respect to patient age, cause of coma, or length of time that
the criteria must be satisfied. There are cogent medical, legal, and social
reasons for establishing valid and refined criteria for cerebral death, and
it is anticipated that these criteria will become widely accepted as a result
of the collaborative study. Hence, families could be spared the emotional
distress and financial burden of an unnecessary delay in the declaration of
death in appropriate circumstances and at the same time be protected against
undue haste in cessation of efforts at resuscitation. It is further possible
that potential organ donors could be more readily identified and at an
earlier time, resulting in more viable organs for transplantation.

Contract negotiations for the pilot phase of the study are scheduled for
completion in June 1971. The project will then become operational on or
about September 1, 1971, and run for approximately one year. At the end of
the pilot phase it should be possible to make a decision concerning the
advisability of continuing with the collaborative study. If a decision to
continue is made, the investigators will meet and agree to a protocol for the
remainder of the study. This later or definitive phase will proceed until
a sufficient number of patients are studied to give valid statistical results,
all data is collected and analyzed, and a final report is drafted.

Contractor's Project Director:

Dr. A. Earl Walker (University of New Mexico)

Dr. Reginald G. Bickford (University of California-San Diego)

Dr. David C. Poskanzer (Massachusetts General Hospital)

Dr. Francis E. McGee, Jr. (Medical College of Virginia)

Dr. Julius Korein (New York University Medical Center)

Dr. Benjamin Boshes (Chicago Wesley Memorial Hospital)

Dr. Wigbert C. Wiederholt (Ohio State University)

Dr. Lorenzo G. Runk, III (University of Pennsylvania Graduate Hospital)

Dr. Donald R. Bennett (University of Utah)

The head injury model construction program has been phased out. The data
collected during the past three years is being reviewed and will provide the
basis for a monograph on the mechanical properties of the head and neck.

The final report on the survey of head injured veterans of the Korean campaign
has been submitted to the Director of the Institute.

I

5u

The section is continuing its collection and classification of head injury
literature. Its recurring classified bibliographies are being shared with
more than one thousand interested investigators in the field.

6^

CONTRACT NARRATIVE

Special Projects Branch--Section on Epilepsy

July 1, 1970— June 30, 1971

NEW CASTLE STATE HOSPITAL (PH-43-68-1310)

Title: Study of the Anticonvulsant Properties of Albutoin

Contractor's Project Director; Joseph T. Brock, M.D.

Current Annual Level: $90,000

Objectives: To study the relative anticonvulsant properties of albutoin,
diphenylhydantoin, and primidone administered to patients with seizures
refractory to treatment, and to evaluate the possible side effects of the
drugs and drug blood levels.

Course of Contract: Patient trials began September 14, 1970. Forty-nine
patients began the thirteen-week Latin cube experiment. The investigational
drug albutoin was compared with the two marketed drugs, each for three week
periods with two week intervals of regular medication. Clinical data was
collected and sent to the Section on Epilepsy, NINDS , Bethesda, for review
and preparation for computer-aided analysis.

Major Findings: Detailed analysis is now underway. A preliminary review of
the clinical data shows when compared to patients' ordinary medications,
single drugs are less effective in preventing seizures. There was a greater
incidence of seizures and side effects with the investigational drug than
with either diphenylhydantoin or primidone.

Significance to NINDS program and Biomedical Research: The pharmaceutical
industry has demonstrated a little interest in developing new anticonvulsant
agents. Aside from the economic factors involved, one of the industry's major
problems is to obtain satisfactory clinical studies of anticonvulsant drugs.
Through this contract and others, NINDS has supported clinical studies of
anticonvulsant drugs. Well controlled studies will be significant indicators
of therapeutic merit of new anticonvulsant drugs. It may encourage industry
to develop its promising new agents for clinical trial. It is anticipated
that through NINDS sponsored studies anticonvulsant drugs will reach the
market more readily.

Proposed Course of Contract; The contract will be amended without additional
funds to allow performance in FY72 . Additional anticonvulsant trials are
planned .

7u

CONTRACT NARRATIVE

Special Projects Branch — Section on Epilepsy

July 1, 1970--June 30, 1971

MEDICAL COLLEGE OF WISCONSIN (NIH-69-2169)

UNIVERSITY OF VIRGINIA SCHOOL OF MEDICINE (NIH-69-2196)

Title: Study of Anticonvulsant Properties of Ethosuximide

Contract Project Directors; Philip T. White, M.D., (MCW)

Fritz E. Dreifuss, M.D., (UV)

Current Annual Level: $ 65,462 (Medical College of Wisconsin)

$ 53,474 (University of Virginia School of Medicine)

Objectives : To study the effect of ethosuximide on the frequency and inten-
sity of petit mal epileptic seizures in patients previously untreated for
this disease; to evaluate the effect of ethosuximide therapy on physiologic,
psychometric, and other functions in human subjects.

Course of the Contract: Due to the difficulty in acquiring previously un-
treated petit mal patients, the contracts were extended to June 30, 1971.
Each center will be successful, or nearly so, in reaching its goal of 20
patients. Data is collected for each patient during the initial hospita-
lization period, during an outpatient and during the final hospitalization
period, and sent to the Section on Epilepsy, NINDS , Bethesda, for review
and entry for computer-aided analysis.

Major Findings: The procedures and protocol established during the colla-
borative study of epilepsy were proven valuable in conducting an evaluation
of an anticonvulsant agent. Detailed analysis of the clinical results will
be made at the end of the study. EEG telemetry has been developed as an
ancillary technique.

Proposed Course: The study will be extended to permit comparison of etho-
suximide with an investigational anticonvulsant.

9u

CONTRACT NARRATIVE

Special Projects Branch--Section on Epilepsy

July 1, 1970— June 30, 1971

UNIVERSITY OF MINNESOTA (NIH 70-2269)

Title: Development of a Gas-Liquid Chromatographic Analysis of Blood

Contract Project Director; Harvey J. Kupferberg, Ph.D.

Current Annual Level: $7,819

Objectives : To develop a specific GLC method for analysis of blood and
CSF for levels of sulthiame, and for levels of carbamazepine.

Course of the Contract: The contractor has completed the project, pro-
viding methodology for blood level determinations of the anticonvulsants
which are or will be studied in research contracts for clinical evaluation
of sulthiame and carbamazepine.

Major Findings; The GLC methodology was readily adapted from, the contrac-
tor's animal studies to preliminary patient studies. Information on
absorption characteristics and biological half-lives of the drugs was also
obtained.

Proposed Course: The contract has been completed.

llu

w^

CONTRACT NARRATIVE
Special Projects Branch — Section on Epilepsy
July 1, 1970— June 30, 1971

UNIVERSITY OF WASHINGTON (NIH 70-2281)

Title: Study of Sulthiame in the Treatment of Partial Epilepsy

Contractor's Project Director: John R. Green, M.D.

Current Annual Level: $106,597

Objectives: To study the relative anticonvulsant properties in partial
epilepsy of sulthiame and diphenylhydantoin; to measure these drugs in
patients' blood, and to assess patients' psychological competence and so-
cial function.

Course of Contract: A preliminary study of hospitalized patients was com-
pleted and enabled the contractor to undertake the long term study on
October 20, 1970. More than half of the goal of 60 patients have been ac-
quisitioned; each will be evaluated over a 14-month period.

Major Findings: The contractor will review and analyze the data at the con-
clusion of this two-year double blind study. The bioavailability of the
specially prepared diphenylhydantoin was found greater than the commercial
preparation the patients had been receiving. This necessitated a revision
in dosage schedules to prevent toxicity.

Proposed Course; The contract will be amended with additional funds to
allow completion of the second year planned when the contract was awarded.

I

13'

I

CONTRACT NARRATIVE

Special Projects Branch — Section on Epilepsy

July 1, 1970— June 30, 1971

EXCERPTA MEDICA FOUNDATION (NIH-71-2018)

Title: Epilepsy Abstracts, Volume 4

Current Annual Level: $39,220

Objectives: To scan serial publications and periodicals from approximately
3000 of the world's biomedical journals, select appropriate articles to be
included in Epilepsy Abstracts in accordance with the guidance of the Project
Officer and his editorial advisors; prepare abstracts with appropriate trans-
lations into English from foreign languages, classify, index, and store the
abstracts in a computer retrievable form; and produce a 9-track computer tape
in the NIH-PRS format, to be delivered when Volume 4 is completed. The
Excerpta Medica Foundation will produce camera-ready copy for each monthly
issue of Epilepsy Abstracts, which includes an Index of subjects and authors,
and will print and distribute the journal monthly, including a cumulative
index at the end of the volume. In order to pay for the production of the
camera-ready copy, the printing, and distribution, the Excerpta Medica
Foundation will sell subscriptions to recover the cost of production of
camera-ready copy, printing, and distribution.

Course of Contract: Following award of the contract, the Excerpta Medica
Foundation promoted the subscription of Epilepsy Abstracts, and acquired
900 subscriptions at $15 each. At the close of this report period, subscrip-
tions are still being acquired. The Excerpta Medica Foundation produced
monthly issues as directed and have increased slightly the number of original
articles abstracted.

Proposed Course of Contract: It is anticipated that all the remaining issues
will be distributed as scheduled and that the computer tape will be delivered
in accordance with the contract.

15 u

Serial No. NDS (CF)-71 SP 1932

1. Collaborative and Field Research

2. Epilepsy Section

3. Bethesda, Maryland

PHS--NIH

Individual Project Report

July 1, 1970--June 30, 1971

Project Title: Long-term electroencephalographic telemetry of patients
with absence (petit mal) seizures.

Principal Investigator: J. Kiffin Penry, M.D.

Other Investigators: Roger J. Porter, M.D.

Fritz E. Dreifuss, M.D.

Cooperating Units: Department of Neurology, University of Virginia School
of Medicine, Charlottesville, Virginia.

Man Years :

Total: 1.33
Professional: 0.33
Other: 1.0

Facilities and Equipment:

The telemetry equipment and FM tape recorder were provided by the
Section on Epilepsy. An oscilloscope and the clinical research unit were
provided by the University of Virginia Hospital at Charlottesville.

Project Description:

Objectives : The purpose of this study is to evaluate patterns of
paroxysmal abnormal discharge in patients with absence (petit mal) seizures.
Another primary objective is to determine if this method can be established
as a primary mode of evaluating any absence drug.

Methods employed: Each patient has a 12 hour telemetered electroencepha-
lographic recording on the clinical research unit of University of Virginia
at Charlottesville. The telemeter equipment is placed on the patient's head
by an EEC technician who also applies the electrodes. The patient is free to
move about the ward and and the signal from the transmitters is taken to a
dipole antenna and then to two receivers where two channels of EEC are fed
to an FM tape recorder. Quality control is maintained by observation of the
incoming data on the oscilloscope. The 12-hour recordings are taken back to
the laboratory of the Epilepsy Section in Bethesda where they are played back
at four times recording speed: Each 12-hour record is then read by a physi-
cian. Patterns of discharge and effects of anti-absence drugs are evident
when the data is processed by a 360 IBM computer and the Cal Comp plotter.

17^

I

Serial No. NDS (CF)-71 SP 1932

Major Findings: The initial 12 patients with absence epilepsy have all
shown some improvement on ethosuximide . Many patients have shown complete
remission of paroxysmal activity. It was felt that this criterion of seizure
control is probably the most objective available, and it is the intent of the
Section on Epilepsy to incorporate this method in evaluation of investigational
anti-absence drugs.

On further evaluation 16 patients with absence seizures have shown a
decline in seizure duration with increasing age. While this is a confirma-
tion of previously suspected phenomena, this is the first time that such
information has been collected in other than an anecdotal way.

Significance to Biomedical Research and to the Program of the Institute:
This study has applied telemetry techniques to improve the evaluation of
clinical research in anti-absence pharmacology. The study has further sug-
gested patterns of paroxysmal discharge in patients with absence seizures and
points a way for further investigation of the mechanisms of absence epilepsy.

Honors and Awards: None

Publications :

Penry JK, Porter RJ, Dreifuss FE: Quantitation of paroxysmal abnormal
discharge in the EEGs of patients with absence (petit mal) seizures,
for evaluation of antiepileptic drugs. Epilepsia (Amst) 12: 1971.

Penry JK, Porter RJ, Dreifuss FE: Patterns of paroxysmal abnormal
discharges in twelve-hour telemetered EEGs of untreated children with
absence (petit mal) seizures. Neurology (Minneap) 21: 392, 1971.

Serial No. NDS (CF)-71 SP 1933

1. Collaborative & Field Research

2. Epilepsy Section

3. Bethesda, Maryland

PHS— NIH

Individual Project Report

July 1, 1970— June 30, 1971

Project Title: Quantitation of clinical manifestations of spike-wave
activity by a reaction time method.

Principal Investigator: Roger J. Porter, M.D.

Other Investigators: J. Kiffin Penry, M.D.

Fritz E. Dreifuss, M.D.

Cooperating Units: Department of Neurology, University of Virginia School
of Medicine, Charlottesville, Virginia.

Man Years :

Total: 0.5
Professional: 0.25
Other: 0.25

Facilities and Equipment:

Electroencephalographic equipment and space for testing were provided
by the University of Virginia Hospital at Charlottesville. The reaction time
equipment including seizure detection device and digital timer were designed
and built by the Section on Epilepsy. The video-recording apparatus was
provided by the Section on Epilepsy.

Project Description:

Objectives : The purpose of this study is to determine whether reaction
time in absence patients is or is not impaired in a gradual fashion from the
point of spike-wave initiation as has been suggested by some authors but dis-
puted by others. There is some evidence for a "trough-like" pattern decrease
of consciousness. The onset of decrease clinical functions during spike-wave
paroxysms is evaluated by the reaction time method.

Methods employed: A device is employed which gives instantaneous recog-
nition by voltage criteria that a spike-wave burst has started. This burst
is of much higher than normal background, and this factor alone is used to
electronically trigger the reaction timer. On instantaneous recognition the
reaction timer is triggered and a tone is delivered to the subject. The sub-
ject responds by turning off the high pitch tone with a telegraph key. Be-
tween paroxysms the patient is maintained in a state of alertness by a program
of approximately 10 random stimuli per minute. All the data is collected

19U

Serial No. NDS (CF)-71 SP 1933

by television, including a portion of the screen reserved for the reaction
time from the digital clock. There was no age limits in selecting patients,
but they must all have spike-wave paroxysmal discharge.

Major Findings: The study is not yet complete but preliminary findings
suggest that patients maintain some ability to respond early during the
paroxysmal burst; this responsiveness is frequently not seen 1-2 seconds
after onset. Analysis of responsiveness during short bursts suggests that
patients may retain a normal reaction time during euch paroxysms.

Significance to Biomedical Research and to the Program of the Institute;
This study has applied video recording techniques and sophisticated electronic
methods to improve the quality of clinical research. Specifically, this study
is an analysis of the relation of the patient's behavior to his EEG during
paroxysmal electroencephalographic events. An understanding of this rela-
tionship is import ant --not only as a guidepost for further research in the
mechanism of epilepsy, but also in determining the day-to-day therapeutics
of the epileptic patient.

Proposed Course; The study will be continued with additional patients
in the coming fiscal year.

Honors and 'Awards: None

Publications: None

20u

CONTRACT NARRATIVE
Special Projects Branch — Section on Head Injury
July 1, 1970— June 30, 1971

NATIONAL ACADEMY OF SCIENCES (PH 43-64-44. Task Order 11)

Title: A 15-Year Follow-Up of Head-Injured Veterans of the Korean Campaign

Contractor's Project Director: Seymour Jab Ion, M.S.

Current Annual Level: No additional funds

Objectives: 1. To provide NINDS with the current addresses of the head-
injured veterans participating in the study. 2. To select and locate
matched controls. 3. To code the Red Cross interview schedules. 4. To
key punch the Red Cross coded forms. 5. To key punch the original coded
acute data for the so-called Meirowsky cases. 6. To obtain General
Classification Test scores from retired records in St. Louis. 7. To prepare
a data tape embodying original (acute) and Red Cross Interview information.
8. To prepare tabulations and analyses from the total material, relating to
characteristics of the original wound and its treatment.

Major Findings: All 8 objectives were accomplished. A report has been
submitted to the director, NINDS.

Proposed Course of Contract: This project will be concluded in fiscal
year 1971.

21 u

i\

I

CONTRACT NARRATIVE
Special Projects Branch — Section on Head Injury
July 1, 1970— June 30, 1971

WEST VIRGINIA UNIVERSITY (PH 43-67-1137)
CASE WESTERN RESERVE UNIVERSITY (NIH-69-2201)
UNIVERSITY OF WASHINGTON (NIH-69-2232)

Title: Determination of the Physical Properties of Tissues

Contractor's Project Director: Dr. Russell R. Haynes (West Virginia Univ.)

Dr. Albert H. Burstein (Case Western

Reserve Univ.)
Dr. Colin H. Daly (Univ. of Washington)

Current Annual Level: $41,000 (West Virginia University)

$27,000 (Case Western Reserve University)
$30,000 (University of Washington)

Objectives: To determine certain physical properties of the tissues of the
head (scalp, skull, dura, brain, fluids); and to develop mathematical
solutions for various simple mechanical force problems relating to effect of
force upon the head. These objectives represent Phase I and preliminary
work on Phase IV of a program to construct and test accurate physical models
of the head. Various physical properties such as bulk modulus, shear
strength, tensile strength, compressive strength, etc., are being determined
using specimens from human cadavers, autopsies, biopsies, and similar
specimens from the Macaca mulatta.

Major Findings: This project period represents the fourth year of this
collaborative study. The first year was generally devoted to developing
test equipment, determining test parameters, establishing mechanisms for
securing test specimens, and designing standardized test procedures. The
second year saw the beginning of data collection. During the third year,
both the University of Michigan and West Virginia University completed their
determination of the relevant properties of the head and made considerable
progress in the identification of substitute materials. In order to fill
certain vital gaps in the data, two smaller contracts were let last year:
to the University of Washington to determine the dynamic material properties
of the cerebral vasculature and to Case Western Reserve University to
evaluate the mechanical properties of the head-neck junction. During the
current year preliminary data on the latter two contracts have been collected,
and a final report is due in June 1971. A final contract was awarded to the
University of West Virginia for a coordinated analysis of all the data
obtained in this program. This data analysis and review is currently being
studied by the project officer and will form the basis of a monograph on the
mechanical properties of the head and neck.

Significance to NINDS Program and Biomedical Research: The data collected
provide an essential basis for the study of cell and tissue reaction to
mechanical forces, which is essential for understanding head injury.

23U

Serial No, NDS (CF)-69 SP 1785

1. Collaborative and Field Research

2. Special Projects Branch

3. Bethesda, Maryland

PHS— NIH

Individual Project Report

July 1, 1970— June 30, 1971

Project Title: A 15-Year Follow-Up of Head Injured Veterans of the Korean
Campaign

Previous Serial Number: None

Principal Investigators: A. R. Taylor, M.B., F.R.C.S.

S, Jablon, M.S.

Other Investigators: W. F. Caveness, M.D.
A. M. Meirowsky, M.D.
A. C. Dresser, M.S.W.
R. W. Hurt, M.D.
C. Kretschmann

Cooperating Units: National Research Council Follow-Up Agency, Washington, B.C.
American National Red Cross Service to Military Families,
Washington, D.C.

Man Years:

Total: 0.4
Professional: 0.3
Other: 0.1

Projection Description:

Objectives:

1. To obtain information on employment patterns and the incidence of
posttraumatic sjrmptoms and epilepsy over the past 15 years in a group of
head-injured veterans of the Korean Campaign.

2. To relate these findings to the initial data on the severity, therapy,
and sequelae of the injuries; and to compare the status of the injured men to
that of a group of non-injured control subjects.

3. By determining the incidence of seizures in the parents, siblings
and children of the veterans, to see if a genetic factor might be involved
in predetermining epilepsy after head trauma.

4. To identify those veterans who would be willing to participate in a
comprehensive 15-year follow-up hospital evaluation.

25 ^

Serial No. NDS (CF)-69 SP 1785

Methods Employed: Acute data were supplied by Dr. Caveness and
Dr. Meirowsky. The National Research Council Follow-Up Agency took a 30
percent sample of the injured group and selected non- injured controls who
were in Korea in the same units as the injured men at the time the head
injuries were sustained. Current addresses, induction AGCT scores and pre-
induction employment of the injured and non-injured men were obtained by the
National Research Council Follow-Up Agency. The staff of the Section on Head
Injury obtained permission of the veterans for interviews. American National
Red Cross Service to Military Families workers interviewed the veterans,
members of their families, and their employers. The medical data were edited
by the staff of the Section on Head Injury. The National Research Council
Follow-Up Agency edited the employment information, coded the interview
schedules, transferred all acute and follow-up data to a seven-track tape
file, and prepared tabulations on which the final report was based.

Major Findings: The final report has been submitted to the director,
NINDS . The following deductions were made about the future treatment of
combat head injuries:

1. Investigation should be made into the possible ways of treating
memory and concentration defects in line with present methods employed in
speech therapy.

2. Positional vertigo, the common posttraumatic variety, should be
intensively studied to uncover possible lines of treatment.

3. Head injured men should be educated, from the time of their first
reception, not to fear the outcome and not to regard the brain as the "master
organ".

4. They should not be segregated from limb and trunk injuries.

5. There should be a national organization to continue education in
civilian life and be ever present to sustain the veteran when his sjmiptoms
occur or recur in response to stress.

Significance to Biomedical Research and the Program of the Institute:
This study has provided data on employment and the posttraumatic state
fifteen years following head injury.

Proposed Course: With submission of the final report, this project has
been concluded.

Honors and Awards : None

Publications: None

26^

ANNUAL REPORT

For Period July 1, 1970 through Jvine 30, 1971

Perinatal Research Branch

National Institute of Neurological

Diseases and Stroke

National Institutes of Health

TABLE OF CONTENTS

I. General Sunnnary
II. Specific Summaries

Pajge No.

A. Section on Obstetrics 9

B. Section on Pediatric Neurology 11

C. Section on Behavioral Sciences 13

D. Section on Infectious Diseases 15

E. Section on Pathology 21

F. Section on Epidemiology and Genetics 29

G. Section on Data Management and Retrieval 35
H. Section on Project Services

Medical Literature Services 37

I. Section on Speech, Language and Hearing 41

III. Contract Narratives

The Wayne State University (PH-43-68-669)

Maternal ajnino acid level as related to fetal

birth weight of the infant. 45

The Johns Hopkins University School of Medicine
(PH-43-68-710) Long-range effects on the fetus

of certain maternal infections during pregnancy. 47

The University of California (NIH 69-4)

Cytomegalovirus infections in pregnancy. 49

IV. Individual Project Reports

Section on Infectious Diseases 51

Office of the Chief 93

Section on Obstetrics 101

Section on Pediatric Neurology 117

Section on Behavioral Sciences 139

Section on Epidemiology and Genetics 157

Section on Pathology 193

Section on Speech, Language and Hearing 225

.^•.oiD\-riq ti ANNUAL REPORT

For Period Jiily 1, 1970 through June 30, 1971

Perinatal Research Branch

National Institute of Ne\irological

Diseases and Stroke

National Institutes of Health

SUMMARY OF PROFESSIONAL OR SCIENTIFIC ACC0t4FLISHMENTS

A. FINDINGS

A number of scientific findings that came to light from COLR data during
this fiscal year can be highlighted in reference to the development of
mental and motor performance in the growing child:

The proportion of variability in four-year IQ attributed to the
genetic component (heritability) is lower among Negroes than among
-•'■ whites.

Any measure of social class is an important predictor of Stanford-
-Binet IQ at age four and is a significant determinant of mental and
motor performance. Within both white and Negro children, those with
the higher socio-economic index do better on the Binet at age four
than those with the lower index. Higher IQ (Binet at age four) in
either race is associated with higher educational level of the mother.
Females have a higher IQ (Binet) at age four than males in either
- race.

The IQ in singletons seems to decline in the interval between ages
four and seven years vJaereas the IQ of twins seems to show an
improvement, suggesting that twins who usually perform more poorly
•' than singletons during early life are able to "catch up" diiring
childhood.

The offspring from interracial matings do not differ significantly
in birth weight or birth length or in mental or motor performance
•■' at eight months from the offspring of intraracial matings of either
race. However, by age four months and at one year the interracial
offspring were significantly smaller than the controls, and at age
four years, the IQ's of interracials were lower than either control.
' This suggests an adverse environmental rather than an adverse genetic
influence on the interracial offspring which is not experienced by
intraracial progeny.

Birth weight is better than gestational age in predicting mental and
motor performance at eight months of age and of IQ at four years,
independent of race, social class, or sex.

Head size at one year of age may be a good predictor of IQ at age
four. There is a 50^ chance of an IQ being less than 80 at age four
for the one year male with a head size less than ^3 cm, and for the
one year female with a head size less than k2 cm.

A number of additional studies on twins and sibs speak to the physical
development of the growing child:

Half sibs compared to full siblings show more RH inconipatibility,
seizures, congenital heart defects, mental retardation, club foot,
and Polydactyly. Evidence suggests that genetic factors play an
important role in seizures and mental retardation and environmental
(prenatal) factors in congenital heart defects and club foot.

Billington's hypothesis that sensitization of the mother during
pregnancy against paternal antigens leads to non-pathological
placental hypertrophy and increased birth weight in succeeding
pregnancies has been supported and confirmed.

Respiratory Distress Syndrome occurs more frequently in twins than
in singletons.

The relationship between type of twin placentation and zygosity was
studied in 569 sets of twins. The perinatal death rate is lk%
in either race. The frequency of congenital malformations was not
higher in twin than in singleton deaths. Malformations in mono-
chorionic deaths were multiple and lethal. Heavy infants and twins
with large intra-pair birth weight differences were common in
separate dl amniotic -dichor ionic groups; the fused di-di group had
light infants and the lowest death rates.

In comparing somatic vs. visceral growth rates, histological study
of the kidneys of 51^ neonatal deaths and stillbirths revealed that
the growth in birth weight and body length is faster in the Negro
than in the white during the early phases of pregnancy, but visceral
growth, as of the kidneys, proceeds at similar rates in both races,
independent of whether the growing fetus is small or large for dates.

A few studies focused on the blood and vas.c\ilar conditions in the growing
child:

High RH antibody titers are not as highly associated with serious
morbidity in the Negro as in the white progeny.

Neonatal polycythemia is associated with longer gestation, lower
birth and placental weights, less placental pathology, and lower
socio-economic status than controls. Polycythemic neonates do not
differ from controls in psychological scores at eight months or in
neurological findings at age one, but showed, especially among the
Negro females, a lower four year Binet IQ.

Chorangioma cases show an increased association with neonatal throm-
bocytopenic purpura, toxemia, fetal hemangioma, erythroblastosis and
single umbilical artery. The condition is commonest in whites and
females. The prematurity rate of survivors is not different from
that of single live births.

2v

I

Neonatal Retinal Hemorrhage occurs in approximately 20^ of vertex
births. The possible relationship of such hemorrhage to birth
weight, mental and motor performance at eight months, Binet IQ, at
four years and birth position is being investigated using matched
non-retinal hemorrhage cases as controls.

Generalized skin hemorrhages at birth seem to be associated with
subsequent marked hearing loss, speech and behavior problems and
possible mental retardation. This apparent association is being
investigated to assess whether it is spurious or valid.

Among 306 cases of single umbilical artery, in utero and neonatal
deaths occurred in 13.8^ of cases. Many of these deaths are due to
congenital malformations. Compared to controls matched for race,
sex, institution, birth weight, gestational age, and socio-economic
index, the single umbilical artery group had six-fold more occurrences
of velamentous and marginal insertions of the cord, slightly more
neurologic ally abnormal cases but similar mean mental and motor
scores at eight months and four year IQ scores.

The cumulative fertility rate, the perinatal death rate, birth
weight of offspring, and gestational age of mothers with sicklemia
were the same as in non-sickling mothers. The infant and child
death rates, however, were higher in the sickling group.

An important finding with reference to cerebral palsy revealed that
spastic and non-spastic prematures of equal immaturity do not differ
in their Apgar scores, bilirubin levels, or history of birth trauma.
Cerebral hemorrhage, however, is one suspected cause of spastic
diplegia. In the effort to elucidate the etiology of cerebral palsy
in the prematures, this carefully designed study lends no support
either to the genetic or the anoxic or toxic (hyperbilirubenia) or
traumatic or nutritional (intrauterine blighting) hypotheses. The
only statistically significant difference between spastic and non-
spastic prematures was a low post-natal hematocrit level in the
spastic s, and reason for this difference should be explored.

A few studies on virology and immunology pointed out that patients who
had carcinoma of the uterine cervix were found to have increased amounts
of antibody against Type II Herpes Virus two years before the clinical
emergency of cancer. The Australian Antigen was detected in the blood
of 600 cases of infectious hepatitis studied during an epidemic in the
United States.

B. STUDIES m PROGEESS

In addition, a considerable number of studies are currently in progress
and are specifically described in the Individual Project Reports. It
must be pointed out that all this research activity is separate from and
has anteceded the research activity of the Ad Hoc Task Forces that came
into being during the last fiscal year. Under the aegis of these Task
Forces, the main data analysis activity may be highlighted as follows:

3v

I. TASK FORCES

1) Labor and Delivery — This group is analyzing the characteristics
of labor and delivery and its complications in relationship to
perinatal loss, child development and neurological and intellec-
tual deficits.

2) Infectious Diseases — The main objective here is the investigation
of the role of infectious agents and particularly viruses which
occur in pregnancy and diiring the perinatal period in the fetal
deaths of subsequent child development.

3) Genetics and Congenital Malformations — The focus is the pursxdt
of genetic factors related to human development and especially
neurological and intellectual deficits. Further, this group is
assimilating information regarding congenital malformations and
its epidemiology. The unique population of twins born to mothers
in the Study will be followed to at least fifteen years of age

so that observations under mental and physical development can
be made to at least through puberty. The development of special
protocols for the yearly examination of these twins and for the
special neiirological and psychological examinations at ages
twelve and fifteen are currently under way. Furthermore, a
chromosomal study of children from five Collaborating Institutions
(Boston, Philadelphia, Buffalo, Tennessee, and Oregon) to relate
major and minor chromosomal aberrations in these children to the
outcome of pregnancy has been launched with the University of
Colorado acting as the coordinator. In addition, a comprehensive
study of sickle cell anemia to relate the occurrence of sickling
in the mother with the outcome of pregnancy and to study the
growth and development of children who are themselves sicklers
is also iinder way. A proposal by Dr. J. V. Neel of the University
of Michigan has been endorsed to establish genetic markers used
in a variety of polymorphic blood loci for correlations with
pregnancy outcomes; the necessary blood samples will be drawn
and stored to be typed later. A request by Dr. Osborne is
currently being considered by more than one NIH Institute to 1
collect dental cast impressions on the seven year olds in the
Collaborative Study with a view to assessing the timing of the
occurrence of an insult in utero and its correlation with the
emergence of specific morbidity in the child; this is part of a
larger study of morphological and developmental asymmetries of
interest to geneticists and teratologists. I

^) Drugs in Pregnancy — This group studies the large number of drugs
that are taken by women during pregnancy to explore whether these
relate to any observed differences in fetal outcome including
congenital malformations or subsequent child development.

5) One Year Task Force — The concern is the analysis of pre- and

perinatal factors related to neurological abnormalities identified
in one year old children. The one year outcome was classified in

4v

ovt o;)Ti.specific categories in a mutually exclusive hierarchical order

of disease conditions; these categories along with neurologically
normal cases will he compared for frequencies of a large array
of pre- and perinatal variables to detect associations. Instances
where strong associations exist will he subjected to more

■ • definitive analysis.

6) Four and Seven Year Task Force — The objective is the analysis of
pre- and perinatal factors related to neurological and intellectual
deficits identified from four to seven years of age. The intent

is to discover predictors of IQ at four years of age by means of
multiple regression analysis. The number of variables will be
reduced by obtaining a simple correlation of study variables
with four year IQ. Variables lacking specific correlation will
be eliminated from the regression analysis or analyzed separately.
In addition a few composite indices made up of IQ and other four
year performance tests will be subjected to regression analysis
against the whole array of antecedent factors.

7) Three and Eight Year Speech, Language and Hearing Task Force
This group is concerned with analysis of pre- and perinatal
factors related to speech, language and hearing performance as
judged in three year olds, and then again in ei^t year old
children.

8) Physical Growth — This task force is concerned with the development
of the physical growth profiles based on the physical measurements
obtained on Collaborative Study children from birth on through
age eight. Further, the group is looking at physical growth
characteristics in children who differ by socioeconomic back-
ground and other developmental characteristics.

"^'"■'f^) Toxemia — This task force is concerned with the evaluation of the
utility of the clinical signs and symptoms which are part of the
toxemia complex and its significance vis-a-vis perinatal mortality
and prematurity. The First International Workshop on Clinical
Diagnostic Criteria of Toxemia of Pregnancy scheduled in
December 1971 is sponsored by the Perinatal Research Branch sind
shall present data from the Study as well as a proposed set of
guidelines for the development of the theory of toxemia and its
implications in clinical practice.

10) Pathology Task Force — This task force is concerned with the
analysis of the brain specimens from children dying during the
perinatal period to get insight into the kind and frequency of
apparent brain injury identified from postmortem examination.

11) Basic Document — Frequency and cross tabulations of continuous,
discrete and dichotomous variables are used to reveal the
prospective relationship, if any, between selected obstetrical
variables and mortality, birth weight and one year neurological
outcome by race and institution.

5v

12) Anesthe sia-Analges ia — Gravida have been categorized into two
grotrps, "normaJ." aind those that are "not normal" as defined.
The effect of anesthesia-analgesia in various combinations, as
defined, will be tabulated in relation to selected outcomes from
these two populations, to assess the effect, if any, of selected
anesthesia-analgesia agents as administered. The protective
effect of barbiturate against lowering Apgar by anesthesia will
be investigated.

13) Epidemiological and Statistical Advisory Committee — This was
established by the Perinatal Research Committee to offer merit
review of study proposals as submitted by the various task
forces prior to final approval.

II. OTHER STUDIES

1) The relationship between weight gain during pregnancy and the weight
of the baby is to be investigated by the Primate Nutrition Study
currently being funded jointly by the National Institute of Child
Health and Human Development, the National Institute of Neurological
Diseases and Stroke, and Johns Hopkins University. The pilot phase
is to explore through experimental design the lead that came out
from COLR data, confirming previous findings, that increase in
maternal weight gain reduces the frequency of low birth weight
children.

2) The Collaborating Institutions at Minnesota and at Oregon have been
funded by the Office of Education to investigate the relationship,
if any, between elementary school performance and antecedent pre-,
peri-, and postnatal information available in the Project.

3) Dr. Rosenblith, in Providence, has established a neonatal behavioral
scale working on the Providence sample of the Project population
and is now validating it against the parameters of data collected
at various endpoints in the Study. This behavioral scale, if validated
on the population of the Project as a whole, will serve as an
additional source of identification of high risk infants, especially

in areas where more efficient or more accurate methods are needed
than the medical examinations can provide.

h) In cooperation with the Collaborating Institution at Boston and

through funds made available through the National Institute of Child
Health and Human Development, under the scope of the COLR contract for
data collection, a study of dermatoglyphics is iinder way to find out
if normal progeny can be thus distinguished from abnormal progeny.

5) In cooperation with a member of NICHD, the Branch will carry out a
multi-factor analysis of the contribution of various preciirsors to
the occurrence of minimal brain dysfunction.

OTHER FUNCTIONS

The Branch, through the Office of the Chief, continues to monitor and overview
the flow of activities in the following eight areas:

Sample maintenance, overdue forms, early monitoring of data collected,
quality control (inter- and intrainstitutional), form processing
(editing, coding, punching, storage and magnetic tape), study proposals
from the task forces as well as from individual investigators, merit
review of manuscripts prior to publication, the Project Officer's role in
contract negotiations and supervision of data collection in the
Collaborating Institutions.

The Branch is initiating and exploring various modsilities and patterns of
enlisting and involving other Institutes within NIH in design and funding of
a nimber of studies in cooperation with one or more Institutions in the
field. The Institutes thus far involved are: National Cancer Institute,
National Heart Institute, National Institute of Infectious Diseases and
Allergy, National Institute of Dental Health, National Institute of Mental
Health.

The Branch is investigating and exploring mechanisms of merit review and
funding of studies based on local data ansilysis by the various Collaborating
Institutions. This is needed after the sharp fiscal stringency that has
come about during Fiscal Year 1972.

The Branch is making contact with several organizations to carry forward
cooperative efforts. A Joint Conference on Minimal Brain Dysfunction has
been agreed upon among the CIBA Corporation, NICHD, and NINDS and has
received approval for presentation under the auspices of the New York
Academy of Science in March 1972. At this conference, the present status of
theory, research, etiology, management, and treatment will be explored and
will receive broad dissemination throiigh the Academy's annals publication.

The Branch has also been negotiating with the Office of Education to carry
out a study relating pre- and perinatal variables to learning disability in
early grades. It has been agreed by the two units involved that the actual
study be carried out during FY 1972. It is now being planned.

The Branch has held discussions with the Bureau of Indian Health, HSMHA, in
regard to research on communication research — including audiology, treatment
of the deaf, and prediction of stroke by analysis of dysarthria. Interaction
and agreement on our prospective areas of interest and capability will
continue.

The Branch has received a number of requests for reviews of grant applications
by Branch members who are specialists in the area of perinatal research.

The Branch has carried out an analysis of its own and NINDS -wide research on
the child aged 0-5, following a request by the Division of Research Grants,
which is pursuing the question for the Director of NIH in response to the
President's proposed initiative on children's health in that age group.

7 V

The Branch has received, and has either satisfied or is in process of
accommodating, a niunber of requests for different kinds of data emerging
from the Collaborative Study. Among these are The Bureau of Economdc
Research; Division of Epidemiology, Columbia University Medical School;
Faculty of Law, Tel Aviv University; Vocational Counseling Associates;
School of Public Health, North Carolina University; School of Business
Administration, University of Washington; and Downstate Medical Center,
State University of New York.

The Branch has carried out an advisory function for certain organizations.
The Branch Chief was appointed to the Advisory Liaison Committee, American
College of Obstetricians and Gynecologists, and to the World Health
Organization's Expert Committee on the Prevention of Perinatal Mortality
and Morbidity.

PROBLEMS

The imposed arbitrary cut of $2.1 million within the Perinatal Project dxiring
the coming fiscal year was at long last negotiated and absorbed. With the
help of the Perinatal Research Committee, the Branch was able to adjust to
this unwarranted cut both within its own internal operation and throuighout
the Collaborating Institutions. The collaborators showed an acute sense of
responsiveness and loyalty despite the growing sense of anxiety and low morale
amongst them. To adjust to this cut was an experience which involved
considerable effort and time, and was most pa3.nful, to say the least.

PROGRAM DEVELOPMENT

During the fiscal year, the Branch has undertaken an analysis of research
needs and capabilities for the purpose of developing a research program to
follow the termination of data collection in the Collaborative Study. A
five year plan is being prepared in this context to explore leads from the
current phase of the Perinatal Program and to pursue new and other leads in
the whole area of developmental neiirology. This plan is to be reviewed for
design and budget prior to Fiscal Year 1972. In its targeted research, the
plan utilizes laboratory as well as field studies to explore in depth the
effect of genetic, biologic, physical, and psycho-social factors on the
developing nervous system. Implementation will not be limited to any one
group of institutions or to many institutions at a time, nor will it be
necessarily limited to any one funding mechanism.

8v

A. SECTION OW OBSTETRICS
Report for the Period July 1, 1970 through June 30, 1971

I. SUMMARY OF SdEEfTIFIC OR PROEESSIOHAL ACCOMPLISHMEMTS

With the active cooperation of 9 experts in clinical research on toxemia
of pregnancy and selected members from the Collaborating Institutions,
data from the Perinatal Research Study were analyzed for the association
of toxemia of pregnancy as reported in the Study protocol with fetal and
neonatal mortality and on the reporting of edema, proteinuria and blood
pressures during pregnancy. This information was then used to design a
new study, utilising the original data on the clinical diagnostic criteria
of toxemia. A chronological data basis for specified periods of pregnancy
was established for blood pressures, proteinuria, edema, and their
combinations, in association with the Immediate and long-term outcomes of
pregnancy. These data, in turn, are being used to define "iso-risk zones"
of blood pressures, edema, proteinuria, and combinations of the same in
the gravida for specified outcomes of pregnancy: live birth, birthweight,
fetal death, neonatal death. From these findings, critical limits will
be set for identification of cases with toxemia in the Study pregnancies.
Our preliminary findings indicate that the epidemiologic approach to the
study of toxemia may contribute to the resolution of some of the complex
riddles of toxemia. The methodological problems involved in this study
were presented and discussed at the annual meeting of the Swiss Obstetric
Society. We are cooperating with Dr. E. Hughes, Chairman of the Committee
on Nomenclature of the American College of Obstetricians and Gynecologists,
on the definition and nomenclature of the toxemias.

Together with the members of the Task Force on Labor and Delivery, a
study has been designed to determine the influence of specific labor and
delivery factors on the fetus in terms of immediate outcome and later
neurologic and psychologic outcome. In this study, a methodology -"
developed by Dr. E. Friedman for the quantification of uterine activity
and its deviations in relation to the phases of labor will be used to
measure possible damage to the fetus, dependent on the dyscoordination or
changes in the duration of any one or combinations of the phases of labor.
The identification of gravidae with specified types of labor will produce
several standard cohorts that may be used by other Study sections and
task forces as variables for their respective special studies. The design
of this study was completed in three workshops of the Task Force on Labor
and Delivery.

Through the cooperation of Dr. K. Benirschke, the karyotypes of the
surviving children with Down's syndrome in the Perinatal Research Study
have been identified through skin and leucocyte cultures.

II. PROBLEMS

No problems except for the shortage of professional and clerical-
I statistical man power.

9v

Ill, PROPOSED FUTUEE OBJECTIVES

The studies on toxemia and labor and delivery will more than fully
occupy the staff of the Section during the next fiscal year.

The experience gained in the collection of obstetric data will be
employed in the planning of a comprehensive prenatal care organization.

10,

B. SECTION ON PEDIATRIC KEUROLOGY
Report for Period July 1, I97O through June 30, I97I

I. SUMMARY OF SCIENTIFIC OR PROFESSIONAL ACCOMPLISHMENTS :

A. Logistics:

Editing of data gathered from examinations of Perinatal Project
children continues to occupy most of the effort of the Section on
Pediatric Neurology. This data is provided from pediatric and neuro-
logic examinations performed at seven years of age and is recorded
on forms (PED-7^, PED-75, PED-76). In addition, diagnostic summary
forms are prepared (lDC-77). To date, 21,711 IDC-77 forms (the
final step in the data recording process) have been completed.
There is a small "backlog of incomplete forms; 19I forms are in prog-
ress and kkl are ready for final, hrief review.

Staff members serve resource and recorder functions on the Four-
and Seven-Year Outcome Task Force of the Perinatal Research Committee
(PRC). This Task Force is engaged in comprehensive analysis of
perinatal and postnatal factors as they may affect the child's
intelligence at four years of age.

Tvfo staff members are serving resource and secretarial functions on
the Physical Growth and Development Task Force of the PRC.

Members of the Section are participating in the One-Year Outcome
Task Force. This task force, originating in the Office of the
Chief, Perinatal Research Branch, will study associations between
prenatal and perinatal events and the neurologic status of the
infant at one year of age.

One staff member has been serving on the Basic Document Committee
and for other task forces of the PRC.

Attending Quality Control examinations, conducted at the Collabora-
tive Study insitutions, occupies 25^ of the time of one staff
physician.

The Section continues the task of maintaining a postcard inventory
of examinations given in order to prevent cases from being lost to
the Study,

B. Research Progress:

Accomplishments in research have been meager in the past year be-
cause priority for resources to analyze data has been assigned to
task forces of the Perinatal Research Committee, and to the Basic
Document effort.

11 V

A procedure for definitive analysis of Perinatal Research Branch
data, designed in 1969? is still being programmed in the Office of
Biometry.

The study of electroencephalograms of Project children was termi-
nated by Perinatal Research Committee action.

II. PROBLEMS :

None can be mentioned.

III. PROPOSED FUTURE OBJECTIVES:

The axialyses of the One-Year Outcome Task Force and the Four- and Seven-
Year Outcome Task Force will occupy much attention in the coming year.

A research design to study spastic diplegia of premature infants is
being developed. This study might be launched after completion of the
present commitment to analyze project data which has been accumulated.

12 V

C. SECTION ON BEHAVIORAL SCIENCES
Report for the period July 1, 1970 through June 30, 197I

SUMVIARY OF SCIENTIFIC OR PROFESSIONAL AGGOMPLISHMENTS

A? Research

I. Task Force. The major research effort has been devoted to the
planning and execution of a study relating findings dirring pregnancy,
delivery, infancy and early cliildhood to intellectual and motor per-
formance at four years of age. This research is being carried out in
collaboration with the Four and Seven Year Neurological and Psychological
Task Force, Two hundred and seventy one precxarsor variables are being
related to: (l) Stanford-Binet IQ; (2) the condition of low IQ (mental
retardation); (3) a factor score representing overall perfonnance on the
four year battery which includes tests of concept formation and fine and
gross motor abilities in addition to IQ; (^) a second score representing
overall test performance which is characteristic of "brain damaged"
children.

Completion of all analyses related to the first two outcomes is expected
by the end of this fiscal year.

Individual research efforts are continuing in the areas of (l) item
analyses of the four year battery, (2) heritability of intelligence as
estimated from the study of twins and sibs, (3) outcomes in children
from interracial and from consanguineous matings, (h) follow-up of
children rated as dysfluent at three years of age. A study of the
relationships of birthweight and gestational age to Bayley scores at
eight months and IQ at four" years has been con^ileted and is being
prepared for publication.

In the area of methodological studies, test-retest reliabilities have
been established for all sub-tests and overall ratings in the four and
seven year psychology batteries on two small random samples of children.
(Ns = lij-0 and 228 respectively) The four-year results are now coxi5)lete.
This data is collected through the procedure of Inter-institutional
Quality Control. All children are retested after an interval of
approximately three months by an examiner from another institution. All
trials (15 per year) are supervised by a psychologist from this Section.
He observes the re-testing, records the data and discusses the results
with the psychologists involved. In the four-year battery, the test-
retest reliability for the Stanford-Binet is high and the retest-observer
reliability, which reflects scoring agreement among examiners from
different institutions, is very high. For the Graham- Ernhart Block
Sort Test these reliability coefficients are moderate and very high
respectively. For the three gross motor tests many of the relatively
small n^umber of children who fail on the first test, pass on the second
test. This is also true for the fo-ur fine motor tests. For the Overail
Behavior Rating, the "suspect" category appears to be quite unstable,
(it is also small), with most of these children being rated as normal on
the second test. This same tendency is evident in the "suspect" category

13V

for the Overall Test Impression Rating.

In the seven-year battery test -ret est reliabilities for the three WISC
IQs, the Bender-Gestalt Test, the Aaditory-Vocal Association Test, the
Goodenough-Harris Drav;-A-Person Test and the three sub-tests of the WHAT
are high and the retest-observer reliabilities are very high. For the
Tactile Finger Recognition Test, the test-retest reliability is moderate
and the retest-observer reliability is very high. The same tendency
observed in the four year sair^jle for children who were rated initially
as "suspect" in Overall Behavior and in Overall Test Impression to be
rated the second time as normal appears in the seven year sample.

B. Data Collection

From the period 7-1-70 to 3-l-71j 5^7 four-year psychology examinations
have been received in the Section. Data collection with this instrument
has been completed. As of 3-1-71^ S5^ four -year examinations have been
processed (edited). The current backlog of these examinations is three.

From the period 7-1-70 to 3-1-71^ 3872 seven-year psychology examinations
have been received. Projected to 6-30-71^ 5^72 such examinations will
have been received. As of 3-1-71^ ^015 seven-year examinations have been
processed (edited). The current backlog of these examinations is 2U2.

II. Proposed Future Objectives

These are completion of the four-year study described above and planning
in collaboration with the Task Force for a second comprehensive study
using the results of the seven-year psychological test battery as end
points.

III. IVtLscellaneous

Organizational Changes

The position of research psychologist vacated by Dr. Lee Willerman has
been filled by Dr. Paul Nichols.

Ik

D. SECTION ON INFECTIOUS DISEASES

Report for pwlod July 1, 1970 through June 30, 1971

I. SOMKART OF SCIENTIFIC OR PROFESSIONAL ACHIEVaiENTS

Thft Section la organized into eight Independent but Integrated Units.
The research activities are divided Into four broad areas:

A. Large serological surveys of perinatal infections in support of the
Periaat&l Research Study.

B. Extended perinstal iiiivestigatlons, including both clinical studies
and laboratory investigations based on leads froa the Perinatal
Research Study.

C. Brain tissua culture studies of perinatal infections and chronic
neurological diseases.

D. Hutritioa and infection in aan and prlaates.

The S^tion published approxisately 38 nanuscripts and presented 42
papers during the present fiscal year.

A. Serological Survei^ of Perinatal Infections

1. Serelogical Investigations Using Co«ple«ent Fixation and
HsaagglntinatioB Methods

tephasis has been centered on selected studies of pregnancies with
abnonal outcones and Matched controls. Tests have been coapleted
on patients with abortions, stillbirths, cataracts and ■icro-
cephaly, along with aatched controls, to detemiae the possible
laflnaace of virus infections in relation to these eutcoaes.
Several of these studies have now been published; others
are in press. In each ease the Multiple senn speclnens froa
apprexlaately 100 patients and 200 natched controls* are used and
■ere than 20 virus antigens are generally es^loyed.

Sladlar studies now in progress include cancer of the cervix,
repeat abortions and stillbirths.

Studies have Included the use of Australia antigen to provide
Now inforaation on hepatitis related infections. Recent tests
have been initiated for Au antibody deteralnations.

2. Serological Tests Bnploying Tissue Cultures

Specific tests for cytomegalovirus, rubella, EB virus and other
antigens are conducted in the laboratory for tissue culture investi-
gations. This laboratory, under the direction of Dr. Fuccillo,

15 V

pcrfonu tlic •pacific neutralization taats as well as flsvraacant
aatibady determiaatioiui for SB Tlrtts and has daralopad specific
hcaagflatlnation tasts foz harpcsvims, typas 1 aad 2» as wall as
cjtf Bgel iMnrir Tha laboratory has alao bean actlvaly lovolTad
in tha Tims isolation stndias to conf Im the sarologlcal
obsarrations. This iavolvad tha attcaptad isolation of
▼irasaa froa ovar 2500 placental spacisans and an oi|«al
auBbar of throat swab spociaans as wall as tha laboratory
support for tha isolation of cytonagalovims froa the pregnant woaen
under study at the Kaiaer Hoapital is Los Angeles and the children
being atudied in laltlnore and Pr«lerick, Maryland. Ve are alao
actively involved in isolating rubella virus as well as other
viruses fron the tissue speclaens obtained fron experiaentally
infected anlnala and wenen Inannixed for rubella. Keperts of
these investigations are in press.

3. laManoglebttlia Peterninations

To help identify cengenitally Infected children in the Perinatal
Research Study, we are testing the 30,000 cord sera which are avail-
able for li^ levela. More than 2/3 of these have been tested and
the reaaining 1/3 should be conpleted in the next fiscal year. It
would appear that approzinately 1000 of the 30,000 will have elevated
1^ levels. With these identified we will proceed with nore specific
testing for viral antibody in the Iffl conponeut of the serum. This
will require the use of fluorescent antibody techniques and ultra-
centrifuge tion .

B. Extended Perinatal Inveatigations

Clinical studies have been in progress in four areas. These represent
an extension of perinatal inveatigations based on leads obtained in the
Collaborative Perinatal Research Study. Two of these investigations
are funded by contracta.

1. Cytaaegalovirus Infections in Pregnant Wonen

This is a three year study lAich is now In its third year of active
work. Serial urine apeclneaa are being collected at the Kaiser
Hospital la Loa Angeles. Pregnant weaen are studied for antibody
as well aa virus excretion throughout pregnancy and the children
are ezaained and atudied for the presence of infection at birth.
Specimens are aent to the Section where the virua isolation and
antibody detemlnatlons are actually conducted. Virus isolationa
are being aade and the study is proceeding aa expected. The
investigation la supported under contract and is in collaboration
with Dr. Margaret Jones, Professor of Pediatrics, UCLA.

16 V

2. Mycoplasiiui Infections In Pregnant Women

This investigation is now In its third year and is conducted in
collaboration «d.th the Naval Medical Center, Dr. Melvin Moseles.
Serial vaginal samples are obtained daring pregnancy and tested in
our laboratory for the presence of Mycoplasma and T-Strain infection.
In addition, throat swabs and blood samples are obtained from the
^ children at birth. Approximately 15Z of babies are found to have
Mycoplasm infection as are their mothers. The study also involves
the attempted recovery of Mycoplasma from the ovaries of women
undergoing hysterectomy. The first phase of work is being
prepared for publication.

3. Serial IgM Determinations in High Risk Infants

High risk and low birtfaweight infants at the University of Tennessee
are being studied under a contract arrangement with Dr. Sheldon
Korones. Serial determinations of IgM are being evaluated as a
means for identifying children with perinatal Infections. The
study is now in its third year. Reports from this study were
published this year.

A. Longitudinal Study of Rubella-Damaged Children

This is an ongoing study of children at the Johns Hopkins Medical
Center who are identified as having congenital rubella « The
longitudinal observations have permitted the association of early
second trimester rubella with deafness and peripheral pulmonic
stenosis in the children. Follow-up is being continued to permit
further identification of more subtle defects, such as mental
retardation in association with perinatal infection. The study la
funded under contract and the principal investigator is Dr. Janet
Bardy. Several publications from this study appeared this year.

5. Experimental Animals

Investigations using pregnant rabbits with vertically transmitted
rubella Infection have demonstrated not only the transmission of the
infection but the particular predilection of the vims for repli-
cation in the cartilaginous growing tissue of the fetus. These
studies are being extended and because of the observations particu-
lar emphasis is being placed on human clinical material to deter-
mine if there is an unusual involvement of the cartilaginous tissue
In fetal specimens of man as well as In rabbits. Recent work on
the experimental production of vaginal herpesvirus homlnis I
infection in the Cebus monkey has been published. This animal
•yatem appears to be an excellent model for the study of vaginal
and congenital herpes Infections.

6. Volunf^r Studies

Huaan Toluntcer studies are being conducted with the use of low
teapersture adapted vaccine material. The antigenic response and
shedding characteristics of this vaccine preparation are being
studiad.

The effect of aannitol on the excretion of hunan cytomegalovirus was
tested in volunteers. The drug was found to produce no increase in
viurea or virus in the nasopharynx.

7. Drug Evaluation Studies

Because of the severe damaging effects of cytomegalovirus and Herpes
simplex virus, studies have been conducted in conjunction with the
Children's Hospital of the District of Columbia - Dr. Gordon Avery on
the possible value of drugs for these diseases. These Investigations
are being reported. In addition investigations on the use of 5-IDU
for congenital herpes Infection continue in conjunction with Dr. A. J.
Nahmias. We arc planning a joint collaborative study with Dr. Nahmlas
and others.

8. Clinical and Experimental Transmission of Toxoplasmosis

Because of recent evidence on possible importance of the cat in the
transmission of toxoplasmosis, an lovestlgatlon was carried out in
Puerto Rico on the possible shedding of toxoplasmosis by children in
this incidence population. There was no shedding of T. gondii among
these children, however serological studies demonstrated the high rate
of infection which occurred primarily between one and three years of
age. This report is now in press.

9. Hepatitis

Antigen studies utilizing the Australian antigen with complement
fixation and gel diffusion tests demonstrated the high frequency of
antigen among children with mongolism at the Pacific State Hospital.
Comparative studies in other populations are now in progress as are
investigations of the experimental transmission of the virus In monkeys
and the development of improved techniques for antigen and antibody
determinations. Congenital transmission of Australia antigen has
been found to occur. An epidemic of Infectious hepatitis involving
over 300 patients has been studied. Au antibody has been tested for
in mothers of mongoloid children. Reports of these studies are in press.

10. Immune Mechanisms in Perinatal Infections

It has become increasingly apparent that specific studies on the Inmune
mechanisms of the developing fetus and newborn are needed so that we may
better understand the deficiencies which permit congenital infection and

18 V

chroalc Infection to occur in the fetus and persist In the child. Experl-
■ental studies utilizing the haaster, rat and monkey are In progress In
vfalch coRblned antibody, delayed hypersensitivity and interferon deteral-
natloQS are nade serially In Infected and non-Infected anlnals as well as
In association vlth antigenic stlaalatlon.

11. Cytoacgalovlrns - Serotypes Associated with Clinical Disease

At least three strains of cytoaegalovlrus appeared to be Identifiable.
The iaportance of the various strains In clinical disease is unknown.
Cosparatlve serological tests are now in progress in our laboratory to
try to distinguish these strains and correlate this with the clinical
findings In the patients. In addition because of the high frequency of
infection and disease with c3rtoaegalovirus in patients receiving iasrano-
suppresslve drugs and/or patients with leukemia, collaborative studies
with investigators In the National Cancer Institute are in progress in
which strains of cytomegalovirus are being isolated and compared to each
other and the clinical findings in the patients. Reports are being
prepared for publication.

C. Brain Tissue Culture Studies

With the isolation of measles virus from the brains of patients with
subacute sclerosing panencephalitis, particular eaq>hasls has been placed
on the comparison of various strains of virus isolated from these patients
and characterization of these strains in relation to wild measles virus
and vaccine virus. In addition Imswnologlcal studies have proceeded and
doMnstrate no specific lanme deficiency in the patients with the disease.
Additional reports on these topics have been published by the laboratory
this year. Epidemiologic investigations conducted in conjunction with
Dr. Jabbonr at the University of Tennessee demonstrated that approximately
one-half of the patients identified are in the southeastern part of the
United States. Additional investigations are emphasizing Creutzfeldt-
Jakob Disease, progressive multifocal lenkoencephalopathy, multiple scler-
osis and Parkinson's Disease r utilizing both biopsy and autopsy specimens.
Work being reported this April desonstrates the presence of chronic
suppressed measles infection in the Ijrmph nodes of children with SSPE.
This finding significantly changes the concept of the infection from
one localized in the central nervous system to a generalized Involvement.
T— ime suppression may be related to this disseminated infection.

II. PROBLEMS

The past year has seen a considerable turnover of personnel. Difficul-
ties arose because of the loss of technicians «uid statisticians in the
face of an increasing demand for testing.

Further, losses of technical personnel, either through resignation,
maternity leave, or leave without pay, has slowed down the effort in the
exploration of serum samples from the Collaborative Perinatal Program.

1<) 7

E. SECTION ON PATHOLOGY
Report for the Period July 1, 1970 through June 30, 1971
I. SUMMARY OF SCIENTIFIC OR PROFESSIONAL ACCOMPLISHMENTS
A, Project Activities and Material

1. Pathology Task Force

On October 7, 1970 the first meeting of the Pathology Task
Force was held in Bethesda under the co- chairmanship of
Drs. Stanley Aronson (Providence, Rhode Island) and
Lewis E. Lipkin. The following pathologists, in addition
to the co-chairmen, agreed to serve as members:

Dr. Shirley G. Driscoll (Boston, Massachusetts)

Dr. Floyd Gilles (Boston, Massachusetts)

Dr. Bernard Klionsky (Pittsburgh, Pennsylvania)

Dr. Haruo Okazaki (Rochester, Minnesota)

Dr. Edward P. Richardson, Jr. (Boston, Massachusetts)

The project neuropathologic material was considered in
considerable detail by the Task Force. Its characteristics
and the implications for data development from such specimens
were explored. It was agreed in general that, in addition to
modifications in the then current analysis and reporting
procedure, the material warranted several in-depth studies.
The latter are under exploration at this writing and will be
noted under "III. Proposed Future Objectives."

2. Case Integration and Analysis

Following the last Task Force meeting, considerable effort was
devoted to completing the procedural modifications begun last
year (c.f. I,A,1. FY 69-70). In designing these changes,
particular attention was paid to meeting the needs of the other
project sections and the contributing institutions, while at the
same time accelerating the initial review process. The danger
inherent in such partial workup of an individual case is always
the possible necessity for repeating the work at a later date.
In reformulating procedures, relaxation of previously established
standards was necessary in several areas. In particular:

a. The deliberate maintenance of ignorance of clinical aspects
by the examiner was sacrificed.

b. The usual multiple stain examination of the same structure
was deferred.

21 V

At this time only a limited number of tissue blocks are checked
and only the H&E sections examined, rather than as previously
all blocks and all four stained slides for each. A procedure
for block selection based on gross photographs has been developed
as has a new histologic protocol. The latter not only
facilitates the examination, but serves to document the scope
of the current examination, thus reducing future duplication.

3. Tissue Processing and Preservation

Almost the entire technician effort in this laboratory is
devoted for the most part to:

a. The preparation of histologic slides from the paraffin
embedded blocks.

b. The preservation of the remaining wet tissues which are
regarded as "public documents" in the same sense as project
forms or sera in other sections.

For long-term efficiency, we have been preparing a full
complement of three special stains and one unstained blank for
each block of every case. In order to meet the requirements
of the accelerated review, it has been necessary to dispense
with all blanks and special stains except in urgent instances,
and to prepare only a single H&E slide for each block. During
the first phase of this effort more than 100 cases were so
processed making nearly 3,200 H&E sections available for
examination later in the calendar year.

The remaining technician effort is largely devoted to regular
checking and filling of storage containers (specimens are
preserved in 807o alcohol when total embedding in paraffin is
not feasible). Despite diligent efforts, no fully satisfactory
bagging method is available. We therefore are in the process
of transferring all specimens to specially selected and sealable
polyethylene containers which will reduce the need for
continual checking and filling.

B. Special Studies

1. Biologic Pattern Data Processing

This project was subject to two commendatory reviews during the
past calendar year. The planned-for quantitative analysis of the
parameters of chromatolysis has been begun and data regarding
cell size and shape, quantity and dispersion characteristics
of Nissl substance, nuclear and nucleolar eccentricity,
nuclear size and shape, etc., are being determined on hundreds
of chromatolytic and contralateral control neurons which have
been automatically scanned. The simultaneous determination of

22 V

DNA content and synthesis rate in Feulgen stained radio-
autographs is proceeding in tissues of a transplanted lymphoma,
and about to be begun in embryonic neural tube. The marrow
and blood cell identification component of the grain counting
system being specified for NCI is in progress.

Extensive equipment additions and modifications have been
accomplished without preventing the scanning microscope from
being used for data acquisition on the above mentioned projects.
These are detailed in the relevant individual project report, as
are the numerous computer programs created, debugged and made
available for general image processing use. (Serial No. NDS
(CF)-65 PR/P 1278)

2. Twin Placentation in Relation to Zygosity

The relationship between type of twin placentation and zygosity
was studied in 569 sets of twins in the Collaborative Study.
Perinatal death rates, pathologic findings on deaths, birthweight,
gestational age and birthweight differences were evaluated in
relation to twin placentation. This paper has been published
and the study is completed. (Serial No. NDS (CF)-68 PR/P 1650)

3. Kidney Malformations in Fetuses of AxC Line 9935 Rats

The rate of spontaneous genito-urinary malformations in AxC
line 9935 rats is being determined. Randomly selected
pregnant rats are killed at or near term and the mothers and
fetuses were examined in detail for malformations involving
the genito-urinary system. This paper has been published and
the study is completed. (Serial No. NDS (CF)-69 PR/P 1763)

^- Placental Study of Abortion Material (Obtained by an Induced
Abortion )

A detailed histological review of more than 100 specimens of
induced abortion material was undertaken by Dr. Fujikura in
cooperation with the Department of Anatomy at Kyoto University
in Japan. The material was compared with the products of
spontaneous abortions. The paper has been accepted for
publication. (Serial No. NDS (CF)-69 PR/P 1764)

5. The Interrelationship Between Selected Congenital Malformations
and Major Pathologic Findings

The relationship between selected malformations in autopsied
deaths and major pathologic factors in the baby as well as in
the mother and placenta are being analyzed. (Serial No. NDS
(CF)-69 PR/P 1765)

23 V

6. Reproductive Ability of the American Negro with Sickling
and its Public Health Implications

The reproductive performance of 654 sicklers and 1,890
nonsicklers in the Collaborative Study was compared. The
cumulative fertility rate of mothers with sicklemia was the
same as that of nonsickling mothers. Because of evidence
that mothers with sickling have normal reproductive abilities,
the gene will continue to propagate and a plea is being made
that sickling tests be done on all Negroes. (Serial No, NDS
(CF)-69 PR/P 1766)

7 . The Significance of Chorioangiomas

A review of 81 histologically verified cases of chorioangioma
showed an increased association with neonatal thrombocytopenic
purpura, toxemia, fetal hemangioma, erythroblastosis, and
single umbilical artery. There is a distinct female preponderance
and the condition is more common in whites. The prematurity rate
in the surviving group is not different from that of single live
births in the Collaborative Study. The paper has been published
and the study is completed. (Serial No. NDS (CF)-69 PR/P 1769)

8. Pathologic Effects of Ligation of the Anterior Spinal Artery
and/or the Great Radicular Artery in Monkeys

Spinal cords of monkeys which had been subject to ischemia by
means of surgical ligation of the anterior spinal artery and/or
great radicular artery. The ligation of the anterior spinal
artery just below its anastomosis with the arteria radicularis
magna of Adamkiewicz produced clinical paraplegia and severe
necrotic changes in the spinal cord caudal to this level. The
ligature of either the arteria radicularis magna or the anterior
spinal artery above the anastomosis produced anoxic changes which
might be only temporary and were not reflected by serious clinical
deficits. These findings are significant and of practical
application in case one of these arteries must be sacrificed or
is involved in a pathologic process. The publication is in
preparation. (Serial No. NDS (CF)-69 PR/P 1772)

j

9. Thrombocytopenic Purpura and Placental Hemangioma

This combination heretofore unreported and found in two cases in
the Collaborative Study is discussed. The paper has been
published and the study is completed. (Serial No. NDS (CF)-70
PR/P 1858)

2k

I

10. Mental and Motor Development in Monozygotic Co-Twins with
Dissimilar Birthweights

Eight-month mental and motor series as well as four-year I.Q.'s
of 125 sets of identical twins in the Collaborative Study who
had unequal intrapair birthweights showed no difference in these
parameters between co-twins. Since the lighter of monozygotic
twins presumably suffers drastic nutritional setbacks in utero
compared to its co-twin, these findings suggest that the human
brain is fairly resistant to the effects of intrauterine
malnutrition. Submission of this paper for publication is
being suspended pending data analysis of the seven-year I.Q.
(Serial No. NDS (CF)-70 PR/P 1859)

11. A Follow-up of Children with Single Umbilical Artery

A follow-up study of 355 cases of single umbilical artery is
being undertaken with particular emphasis on the somatic,
mental and motor development of those who have reached four
years of age. (Serial No. NDS (CF)-71 PR/P 1911)

12. Birthweight in Relation to Renal Glomerular Development and
Gestational Age in Whites and Negroes

A histologic evaluation of the kidneys of 514 neonatal deaths
and stillbirths in the Collaborative Study was made and the
findings assessed in relation to birthweight and gestational
age. Racial differences are discussed particularly in regard
to birthweight and in infants whose birthweights do not match
gestational age. (Serial No. NDS (CF)-71 PR/P 1912)

13. The Clinical Signifiance of Generalized Petechiae at Birth

In a pilot study about a quarter of the infants diagnosed as
having had generalized skin hemorrhages at birth, had significant
changes in the form of marked hearing loss, speech and behavior
problems and, one instance of mental retardation accompanied by
distinct visual impairment. On the hypothesis that significant
skin petechiae could be accompanied by hemorrhages in internal
vital structures such as the brain, inner ear, retina, etc., a
careful evaluation of the balance of these children is being
made and findings compared with those of matched controls.
(Serial No. NDS (CF)-71 PR/P 1913)

Other Activities

There have been numerous presentations of work done within the
section. Presentations at the first Biological Congress, the
SPIE, and the New York Microscopical Society have covered various
aspects of the work in image processing. The last meeting in
May 1971 listed three individual talks by involved personnel.

25V

The Head of the Section has served as contract officer for the
collaborative program with the Artifical Intelligence Group of
the National Bureau of Standards as described in the individual
project report. The Section Head continued his working relation-
ship with the Image Processing Group, DCRT and the National Cancer
Institute.

II. PROBLEMS

A. Personnel

The basic problem of a contracting, financial and personnel base
and the concurrent desire to evaluate the overall nature of the
pathological material has made for a serious problem. The need
for additional professional personnel is undoubted, and can only
partially be met by developing work-type task forces. Additional
professional personnel will require additional filing and clerical
help since slides alone are examinable at times at faster rates
than they can be refiled even with our present relatively minisucle
professional staff.

PROPOSED FUTURE OBJECTIVES

A. The accelerated overall case review has and will continue to receive
first professional and technical priority within the section. At
this writing, the results of the first 35 microscopic examinations
completed under this revised procedure are under review, in order to
assure ourselves that no information essential for other sections or
the Collaborating Institutions is likely to be slighted.

B. Several in-depth studies are in the planning stage. These include:

1. A population study of selected obstetric variables within the
autopsy population in order to elicit suggestive structures,
lesions or patterns to be specifically searched for within the
material. Dr. Rudolf Vollman, Head, Section on Obstetrics, has
agreed to collaborate with us in this study.

2. We are hoping to interest members of the Pathology Task Force
in participating in studies of such characteristic lesions of
the newborn period as the Virchow or Banker lesions of the
periventricular white matter. These lesions are of particular
interest, and our material represents a unique opportunity for
a definitive study of such changes.

3. Collaboration with the Task Force on Genetics in an extensive
study of sickling. This will largely be under the direction of
Dr. Froehlich who has for several years pointed out the importance
of such work in the context of our placental material.

26 V

We continue our plans to apply the technics developed by the
biologic image processing project to the project neuropathologic
material. Current work on neurocellular degenerations such
as chromatolysis will provide the basis for such future
applications.

I

27 V

F. SECTION ON EPIDMIOLOGY AND GEHETICS
Report for the period Jiily 1, 1970 through June 30, 19T1

I. SUMMARY OF SCIEIiynFIC MP PROFESSIONAL ACCOMPLISHMENTS

During the past fiscal year the major effort of this Section was
directed towards organizing the task force on Genetics and Congenital
Malfoimations; implementing the comprehensive plan for analysis of
genetic and socioeconomic data which was developed hy a panel of genetic
experts last year and was adopted and extended by the task force on
Genetics and Congenital Malf oimations ; reorganizing and reorienting the
previous data analysis efforts; improving the quality of the data and
completing oux data file.

The Section has continued to receive, edit and code infoimation on the
Family Health History Review form (FHH-9). Daring this period, a-pproxi-
mately 6,000 FHH-9 forms were received and approximately 5^000 have been
processed and sent to punch. The FEIH-9 is being used to derive a new
socioeconomic index for comparison with that derived for each family
seven years earlier. For this, the family income item is being rescored
with a more realistic ceiling of $15,000 rather than $10,000 used on the
Socioeconomic Interview foim (SE-l). This will require recalling of all
schedules already processed but it is anticipated that they can be re-
scored without undue delay.

The most significant event in the operations of this Section diiring the
last fiscal year has undoubtedly been the creation of task forces to plan
euid direct all data analysis in particular areas, . The task force respon-
sible for the analysis of genetic and socioeconomic data is that of
Genetics and Congenital Malformations, which at present is composed of
Drs. Richard Osborne, Kuxt Hirschhom, Walter Nance, William J, Schull, _, ■
and Ntinos C. Myrianthopoulos, assisted by professionals from the Office
of Biometry, and the Perinatal Research Branch. The task force idea is
not new to this Section. As reported in last year's Annual Report, this
Section had already convened a pan.el of genetic experts to discuss problems
of genetic analysis and help develop a comprehensive and workable analysis
plan. That group had been most successful in developing such a plan and
the newly-created task force adopted it into, and has proceeded to im-
plement it and extend it in special areas outside the protocol.

Thus the efforts of this Section toward data analysis are vigorous and
the prospects seem good. Some projects have been completed, some are
now in progress, some have been approved for immediate implementation,
and some are now being planned. These are described in detail in the
indiATidual project reports. One part of the genetic study of intellectual
and motor perfoimance which is being done in collaboration with investi-
gators at the University of Minnesota has been completed. Among some of
the interesting preliminary results of this study are that the within
family environmental variance is larger among Negroes than among whites,
which means that the heritability of mental perfoimance is lower among

i 29v

among Negroes than among whites; and that the coiirparison of the if-year
to the T-year IQ scores shows aji improvement in twins hut a decline in
singletons which may mean that the twins who usijally perfoim poorer than
singletons during their early period of life, may he ahle to catch xjp
during childhood and adolescence. Dr. Kaylor's study with Dr. Warbiirton
on the effect of parity, matenaal age and change of mate on placental
weight and birthvreight has also heen completed and appeared in the
January issue of the American Journal of Humsui Genetics,

The task force, operating always on the premise that the major contri-
bution of genetic ajialysis \-rlll be in the area of special studies, has
approved a nijmber of new projects. Among these are, a comprehensive
study of congenital malformations, a genetic study of obstetric variables,
a study of ABO and Rh incompatibility in pregnancy wastage and infant
survival and a study of heritability of twinning in man.

The task force during their meetings and discussions took the position
that analysis of Collaborative Study data should not constitute an end
in itself but should be used to locate areas in which specific problems
lie. These problems shouJ.d then be pursued as logical extensions of the
objectives ajid scope of the Collaborative Study, In accordance with these
guidelines the task force recommended to the Perinatal Research Committee
that the unique population of twins bom to mothers in the Collaborative
Study be followed to at least 15 years of age so that observations on
their mental and physical development can be made at least through puberty.
The Perinatal Research Committee approved the proposal and the task force
is now in the process of developing special protocols for yearly examina-
tion of these twins and special neurological and psychological examinations
at ages 12 and 15 years.

The task force has approved the participation of this Section in a
chromosomal study of children from five collaborating institutions to
relate major and minor chromosomal aberrations to abnormalities found in
these children. The participating institutions are in Boston, Mass.,
Philadelphia, Pa., Birffalo, N.'Y,, Memphis, Tenn,, and Portland, Ore, Dr,
H, Lubs of the University of Colorado is acting as a coordinator for the
study. The task force has approved the participation of this Section in
a comprehensive study of sickle-cell anemia to relate the occurrence of
sickling in the mother with the outcome of pregnancy and to study the
growth and development of children who are themselves sicklers. The task
force has also endorsed a proposal by Dr, J.V,' Weel of the University of
Michigan to establish genetic markers using a variety of polymorphic blood I
loci for correlations with pregnancy outcomes. Because of difficulties
with funding of such an extensive project it was decided to draw and
store the necessary blood samples now and type them at a more convenient
time later. Since there is a reasonable probability that the effects of
these genes as well as tha-t for sickle-cell anemia may be influenced by
the degree of black ancestry the task force recommended that a simple
estimate be made of skin color as an index of this ancestiy for control
purposes.

The task force on Genetics and Congenital Malformations has been in

30 v

constant contact and collalDoration with other task forces with common
inte2re,its in data analysis. Dr. Myrianthopovilos and Dr. K. French of
the University of Oregon are collaborating with the task force on four
and seven year examinations to provide the demographic and socioeconomic
description of the population; a joint study is planned with the task
force on physical growth and development of the physical growth and de-
velopment of twins; advice and help has "been provided with the analysis
of some of the speech and hearing data to the appropriate task force;
and suggestions have been made to the task force on infectious disease
concerning new approaches for analysis of the relationship between vital
antibodies and congenital malformations.

Dr. Waylor has been instrumental in completing three files of special
genetic interest. One is the consajiguinity file whose codes have been
exhaustively hand-reviewed. All efforts are now being made to resolve
code discrepancies, especially in second and subsequent pregnancies.
Another is the record linkage file which involves over 6,000 women who
reported having close relatives participating in the Study. This has
now been completed and all the infoimation on the women and their repor-
ted relatives has been put on tape. A hand review of another file, that
of interracial matings has also been completed and this file now contains
199 cases with known outcomes.

II. PROBLEMS

The major problem which has confronted the Section during the past year
has been that of the shortage of trained professionals to direct and
participate in data analysis. As in past years, the Section has tried
to resolve this problem by inviting qualified professionals from within
and without the Collaborative Study to participate in data analysis.
This plan has for the most part been successful but is no substitute for
the constant presence of the investigator at the Section headquarters
and his immediate access to all data.

III.- FUTUKE OBJECTIVES

The future objectives of the Section are to implement the analysis of
the genetic and socioeconomic data of the Collaborative Study according
to the plan of genetic analysis which was developed with the help of
genetic experts and the task force on Genetics and Congenital Malformations,
Although the plan calls for collaboration with investigators and insti-
tutions outside the Collaborative Study, there is no doubt that the plan-
ning and direction of research must come from this Section. A second
objective, therefore, is to strengthen our professional staff in the
Section,

At the same time the Section, with the advice and guidance of its task
force is continuously trying to identify specific areas of fruitful
■ research from the first round of analysis, which, although not within
the protocol, should be pursued as logical extensions of the objectives

3IV

of the Collaborative Study. Considerable progress in this area has
already been made.

MISCELLANEOUS

A« Personnel

Tlie personnel of the Section on Epidemiology and Genetics consists at
present of the following: Professional, Dr. N.C. Myrianthopoulos, Head,
Dr. A.F.I Waylor, geneticist. Miss T.L.' Martin, statistician; one secretary,
five statistical assistants. Daring the last year this Section lost two
positions, that of Miss A. Baszynski, fieldworker, and that of one sec-
retary. The Section is now recruiting for a fieldworker whose duties
vrould be to assist in the follow-up of the t-(-7ins to age 15 years.

B. Activities of the Section Head and the Professional Personnel

The Head of the Section, Dr. Myrianthopoulos, in addition to his foimal
duties has continued his independent investigations in the genetics of
neurological disorders, especially the lipidoses, and has contributed
chapters in several textbooks on these topics. Dr. Myrianthopoulos has
maintained his affiliation with George Washington University School of
Medicine as a Clinical Associate Professor of Neurology and Director of
the Genetic Counseling and Research Center. He is a member of the Medical
Advisory Board of the Huntington's Chorea Foundation, a private orgajii-
zation which supports research in Huntington's chorea, and of the Commit-
tee upon Huntington's Chorea of the V/orld Federation of Neurology. Dr.
Myrianthopoulos has been elected a member of the Academy of Medicine of
Washington, D.C

In September, 1970, Dr. Myrianthopoulos attended the Fourth Annual Work-
shop of the World Federation of Neurology Eesearch Group on Huntington's
Chorea in Munich, Geimany, where he chaired the session on Genetics and
Epidemiology and discussed the current status and future prospects of
research in Huntington's chorea. In January, 19T1, Dr. Myrianthopoulos
also attended a meeting of a special committee of this research group in
Louvain, Belgium, during which plans were made for the Huntington Centen-
nial Symposium in Columbus, Ohio for April, 1972 and for the preparation
of a complete bibliography on Huntington's chorea. In addition. Dr.
Myrianthopoulos helped to set up a special study of Huntington's chorea
in the low countries and the north of Fi-ance. Dr. Myrianthopoulos also
attended the annual meeting of the American Society of Human Genetics in
Indianapolis in October, 1970, where he presented a paper on respiratory
distress syndrome in twins.

Dr. Naylor participated in all of the meetings and discussions of the
task force on Genetics and Congenital Malformations and has been instru-
mental in designing many of the ongoing studies, often doing the program-
ming himself. As mentioned earlier, he also designed and completed three
data files of special genetic interest. Dr. Naylor attended the annual
meeting of the American Society of Human Genetics in Indianapolis in
October, 1970, where he presented a paper based on Collaborative Study

32 V

data adducing evidence for a powerful parity effect in spontaneous
a.bortion.

Bibliogi-aphy

Myrianthopoulos, N.C,', Naylor, A.tF.I and Aronson, SoMJ: Tay-Sachs disease
is probatly not increasing. Nature 22^:60^, 19T0»

Myrianthopoulos, W.'tiJ: A survey of twins in the population of the
Collaborative Study. Acta Genet. Med. Gemellol. 19:15-23, 19T0o

Mjrrianthopo-ulos, W.C,'': Review of "Genetic Counseling. A Guide for the
Practicing Physician" "by W, Fuhrmann and F. Vogel. Social Biology
17:244-246, 1970.

Myrianthopoulos, W.C.': Genetic aspects of multiple sclerosis. Handbook
of Clinical Neurology Vol, 9, Ch, 5^ Amsterdam, Worth -Holland
Publishing Co,, 1970,

Aronson, S,M,* and Myrianthopoulos, N.C: Epidemiology and Genetics of
the Sphingolipidoses, Handbook of Clinical Neurology Vol. 10, Ch. 24,
Amsterdam, North -Holland Publishing Co., 1970.

Willeiman, L,, Naylor, A.F,' and Myrianthopoulos, N.C: Intellectual
development of children from interracial matings. Science 170:1329-
1331, 1970.

Myrianthopo-ulos, N.CJ: An epidemiologic survey of twins in a large,
prospectively studied population. Am. J. Hum. Genet.. 22:611-629,
1970.

Warburton, D. and Naylor, A.F.^: Effect of parity on placental weight
and birthweight: An immunological phenomenon? A report of the
Collaborative Study of Cerebral Palsy. Am. J. Hum. Genet. 23:4l-54,
1971.

Myrianthopoulos, N.C,^: Respiratoary distress syndrome in twins. Acta
Genet. Med. Gemellol. in press.

33 V

G. SECTION ON DATA MANAGEMEIW AND RETRIEVAL
Report for Period July 1, I97O through June 30, I97I

I. SUMMARY OF ACCOMPLISHMEMS

This Section is responsible for recei-\n.ng, coding, filing and storing
forms in accordance with a system designed to facilitate form and/or
data retrieval. The Acting Head of this Section has continued in this
assignment from the Office of Biometry umtil June 1, 19?!^ at which
time Dr. B. H. Fox Asst. to the Chief, Perinatal Research Branch
became the Acting Head. He also supervises the systems analysts and
programmers providing services to PRE from the Office of Biometry to
aid in data analysis. The current data file consists of 58,806
registrants -- of this number 5^,177 were core study cases. Of these
core study cases 53^837 have delivered.

Approximately 3'^ million forms will have been submitted by June 30,
1971' The available data from approximately 3-1 million of these forms
has been processed into over 5*9 million piinch cards in I5I card
formats and converted into active computer tape files, h-9,631 births
have been reviewed at 1 year of age, ^3^888 at k years of age and
32,194 at 7 years of age.

Personnel staffing, with the expected decrease in volume of study forms
received, decreased from 33 "to 27 . Personnel have been encouraged to
take advantage of training being offered through career counselling and
self -improvement requests in an effort to permit a continued smooth
transition.

During the year, work was continued in extending the development and
use of complete program packages to provide an extended Variable File
System with update and editing in conjunction with PRB operated master
files and data banks, thus reducing future cost of operations. In
addition several standard programming packages have been utilized
during this fiscal year period for both studies and statistical analysis
of data greatly facilitating this type of work previously done by other
methods. This included the use of Express III System, Data Check System,
Table Mak.er 2, Inquiry Response System, and Remote Terminal programs.
Personnel have been trained in remote terminal operation for providing
appropriate services.

The research activities of PRB were supported with special infonnation
retrieval for case selection, tabulations, and creation of subfiles of
the Variable File System. Also a major effort has been accomplished
with the production of quantities of tabulations for large-scale studies
involving considerable amounts of data, utilization of dedicated
equipment, extensive systems analysis and complex data processing. A
major project was the production of Monograph II in addition to major
assignments received from the Research Task Forces organized last year.
•Supplementary production of tables for the Basic Doc\jment was also
produced during the year.

35^

The Forms Accountability Program for review of study forms was
continued with the objective of maximizing of all recoverable data for
inclusion in the PRE master files and data banks. A quality review
system of file folders has continued with review of coding, organizing
and binding of forms in the file folders which facilitate their use
while preventing loss or misfiling of individual study forms. This
has been extended by the initiation of file clerk review of folders
for organization of forms in a standard order and identification and
refiling of misfiled forms. An inventory control card system is
completed summarizing the status of each file folder and included in
the file.

II. PROBLEMS

The Section, which contains a large number of non-professional staff
has continued shifts of assignment of personnel including retraining
where required to meet program needs. Continued efforts for
retraining and placement of personnel will help in problems that can
be expected to arise with a reduced workload in data collection.

III. PROPOSED FUnmE OBJECTIVES

Continuation in the development of sophisticated information retrieval-
systems to supply research needs more rapidly is planned with the
integrated use of dedicated computer equipment and maximation of remote
terminal operation. The conversion and retraining of staff will
continue so that present personnel can be reassigned to functions as
required. New systems development are planned to be continued which
will fijTther effect reduced turnaround time on study requests as well
as economy of operation. For example, improved file structure systems
tailored to groups of studies with increased flexibility of use are
planned.

36

H. SECTION ON PROJECT SH^VICES
Report for the period July 1, 1970 through June 30, 1971

SUMMARY OF ACCOMPLISHMENTS

The Section on Project Services, Medical Literature Unit, is responsible
for the acquisition of selected medical and statistical textbooks,
abstracts, scientific directories, atlases, annual publications of books,
and periodicals, and the organizing and indexing of published literature
on all medical- scientific subjects, which in any way contribute to the
various studies undertaken by the Perinatal Research Branch. The present
book collection in the PRB Reading Room numbers 1082. Subscriptions to
leading journals and abstracting periodicals pertinent to the program now
total 102. In the interest of economy, a number of journals not being
utilized to the optimum were discontinued during the past year. Up-to-date
listings of journals and new book acquisitions are now in the process of
compilation and will soon be circulated to Branch personnel. In addition,
our locator file, containing a listing of books by author and title, now
numbers 1109, and controls the books housed in the various sections of the
Branch, readily retrievable upon request.

Approximately 1200 visits were made to our Reading Room this past year.
Its facilities were utilized by PRB personnel, consultants, task forces,
and others.

The activities of the Unit are wide and varied and encompass the following
functions:

Provides a Reading Room and maintains a lending library.

Processed books and/or journals borrowed from PRB Reading Room collection;
processed book, journal and photocopy requests, through NIH-NIM Library,^
procuring for Branch personnel, items not available in PRB Reading Room;
xeroxed from journals and/or books, articles of interest pertinent to the
program, and circulated these to the staff; provided a quick service for
acquisition of literature, urgently needed and not available in-house,
from NIH Library. In most instances material was procured the same day.

Xeroxing on the premises of approximately 25,100 pages of material,
requested by PRB Staff.

Dissemination of material and information pertinent to the Program and in
answer to a variety of inquiries and requests from the scientific and lay
community. This was accomplished by correspondence, by telephone and by
mail.

Periodic mailing of NINDS publications and reprints relevant to the Study
and of possible interest to PRB Staff, Project Directors, Consultants,
Perinatal Research Committee and Task Forces.

37v

Annual distribution of prepared lists regarding new acquisitions of
books, journals and abstracts, etc. to those utilizing our facilities.

Literature searches for specific articles, subjects or authors as
requested by investigator.

Compilation and maintenance of mailing list for annual distribution of
Bibliography of Collaborative Perinatal Project Publications.

Coordination and compilation of Annual Report in proper format for
submission to NINDS Director.

Book ordering for PRE Reading Room.

Compilation of Bibliography of Collaborative Perinatal Project Publica-
tions. This is updated annually at the end of each fiscal year and
includes publications based on pooled core data, local core data, and
non-project studies of special interest to authors at PRB and those
from collaborating institutions who are paid in part or in toto from
Project funds. During the past year a Quarterly Bibliography of all
COLR publications, with summaries, was initiated to enhance the visibility
of our research effort on a more concurrent basis than is possible with
the Annual Bibliography.

Collection of publications by authors from the collaborating institutions i
prior to 1965 which fall into the following categories: publications
using Project population, publications of personnel supported in part
or in toto by Project funds, and ancillary studies.

Indexing reprint collection. These are reprints in the perinatal area

of critical interest to our scientists. They are retrievable on demand

by author, subject and number. Subject headings parallel those used in
Index Medicus.

Semi-annual preparation of bibliography of Branch personnel publications
for inclusion in Scientific Directory and Annual Bibliography, NIH.

Clearinghouse for scheduling of meetings and conferences in the PRB
Reading Room. During the past year 84 meetings were hosted and on
occasion information was gathered and material xeroxed and collated for
use at a specific meeting.

Reorganized our Collaborative Project reprint file for instant accessi-
bility and retrieval of material.

Arranged tours of National Library of Medicine and Clinical Center for
those foreign visitors to the Branch who were interested and also
collected literature of interest for them; arranged for the loan of
equipment through NIH Audio Visual Unit for use by PRB Staff members, etc.

38

II . PROBLEMS

Backlog is the big issue and continues in the following areas:

Subject indexing of reprints for Reprint Retrieval File

Cutting, annotating and filing of reprints

Collection of COLR reprints published prior to 1965 from

collaborating institutions in our continuing effort

to update the file.

III. PROPOSED FUTURE OBJECTIVES

Will continue to concentrate on giving service and meeting the needs of
the Branch in literature retrieval, special reference searches,
compilation of bibliographies, etc., as requested.

I

39 V

I. SECTION ON SPEECH, LMGUAGE AMD HEARING
Report for the Period July 1, 1970 through June 30, 1971

I. SUMMARY OF ACTIVITIES AND DEVELOPI/JENTS

A. Routine

1. Processing of 3-year Speech, Language and Hearing (SLH) forms. Status:
essentially completed (15I forms received last year).

2. Collection of 8-year SLH data. Status: continuing at six
collaborating instititions.

3. Processing of 8-year SLH forms. Status: continiiing (l3,711 forms
edited to 2-28-71).

k. Supervision of inter-institutional quality control. Status:
continuing on trimester basis.

5. Project site visits by Section Head as Project Officer. Status:
continuing.

6. Collection of data for Language Organization Scales. Status:
continuing at q.uality control visits.

B. New

1. Development of forms and method for machine tabiilation and analysis
of quality control results. Status: essentially completed.

2. Organization of SLH Task Force for data analysis. Status: completed
and in operation.

a. Section Head f\mctions as coordinator. Speech Pathologist as
secretary for SLH Task Force.

3. Development of data analysis plan. Status: first steps taken.

h. Analysis of concordance in case identification by exams at 8-years,
7-years and 3-years. Status: corripleted.

5. Tabulation of first 5000 8-year exams by test, institution, race, sex,
birthweight, I.Q. and education of gravida. Status: completed.

6. Tabulation of cases with dysfluency, preliminary to devising study
proposal. Status: completed.

7. Preliminary look at cases with significant petechiae and. abnormal
speech, language or hearing findings. Status: in operation.

I

i4-lv

C. Discontinued

1. Eight-year SLH exams at 6 institutions (New York Medical College,
Columbia-Presbyterian Medical Canter, Medical College of Virginia,
Philadelphia Children's Hospital, Brown University, Charity Hospital
of New Orleans). Reason: recommendation by the Perinatal Research
Committee.

2. Updata of 8-year SLH manual and forms. Reason: completed.

D. Non-Project

1. Consultant and training activities by Section Head serving the National
Center for Health Statistics regarding hearing tests in the National
Health Survey.

2. Active participation by the Speech Pathologist as Chairman and member
of the NIWDS EEO Advisoiy Committee, and member of the EEC Task Force
whose task is to organize and implement an Affirmative Action Plan
for NINDS.

3. Participation by the Speech Pathologist as panel member at the annual
convention of the American Speech and Hearing Association in New York,
November l8, 1970.

k. Appointment of Section Head as NINDS member of the Committee on Hearing,
Bioacoustics, and Biomechanics (CHAEA) of the National Research
Council-National Academy of Sciences and attendance at its annual
meeting at Cape Canaveral, March-April, 1971*

5. Project Head serves as resource person in Speech, Language and Hearing
areas for NINDS.

II. PROBLEMS

A, Personnel needs.

1. Language Specialist (Speech Pathology or Psychology)

2. Secretary

3. Statistical assistant (one statistical assistant was transferred to
another branch to achieve promotion; this section now is comprised

of one statistical assistant, one Speech Pathologist, one Audiologist-
Section Head).

III. OBJECTIVES

A. Development of an effective plan for data analysis, leading to the
disclosure of relationships between:

1. Perinatal conditions sind communicative disorders:

42 V

2. Test results at 3 -years and at 8-years;

3. Inter-instutional disparities and causative factors;
h. Communicative disorders and learning disabilities.

43 V

CONTRACT KAEEATIVE
Perinatal Research Branch. — Section on Pediatric Neurology
July 1; 1970 through June 30, 1971

WA^IE STATE UNIVERSITY (PH U3-68-669)

Title: Maternal Amino Acid Level as Related to Fetal Birthweight of the Infant

Contractor's Project Director; Kamram S. Moghissi, M.D.

Current Annual Level; $18,000

Objectives;

To investigate relationships in pregnancy between dietary intake of protein,
blood amino acid, protein and globulin fraction and weight gain of gravida
and dimensions of newborns and Bay ley test.

Course of Contract:

The last contract year of the study has ended as of January 25, 1971» I^e
workscope of the project was more than accomplished. There remains only to
complete developmental testing of babies as they reach 8 months of age; and analy-
sis of all individual amino acids on those cases tested at 8 months as they are
completed.

A new finding of particular interest is an association between branch chain
amino acids and the 8-month Bay ley test scores.

Major Findings:

The following publications have resulted from the study:

1. Churchill, J.A., Moghissi, K.S., Evans, T.W. and Frohman, C. :
Relationships of Maternal Amino Acid Blood Levels to Fetal
Development. Obstetrics and Gynecology. 33; kS2-k9^, I969

2. Moghissi, K.S., Churchill, J.A., and Frohman, C. ; Relationships of
Maternal Amino Acid Blood Levels to Fetal Development. In Perinatal
Factors Affecting Human Development. Proceedings of the Special
Session held during the Eighth Meeting of the PABO Advisory Committee
on Medical Research, Washington, D.C., June 10, I969, Washington, D.C.,
Pan American Health Organization, Sci. Publ. No. I85, I969, pp. 16-I9.

Significance ;

The study may furnish a means by which to identify gra-vlda's providing sub-
optimum amounts of nutrients to the developing infant. Nutrient supply may
improve the outcome of these pregnancies.

Proposed Course of Contract;
Study is in last contract year.

i+5v

i

CONTRACT NABRATIVE

Perinatal Resaarch Branch - Section on Infectious Diseases

July 1, 1970 — June 30, 1971

JOHNS HOPKIMS PMIVERSITY SCHOOL OF MEDICINE (PH-4 3-68- 710)

Title; Long-range effects on the fetus of certain aaternal Infections
during pregnancy

Contractor's Project Director; Janet B. Hardy, M. D.

Current Annual Level; $22,207

Objectives; The project Is designed to study the long-range effects of
certain maternal Infections during pregnancy on fetal outcome with observa-
tion of the identified children for a period of at least seven years.
Serologic speciaens and pertinent clinical information relating to perinatal
infections and pediatric clinical findings are being obtained longitudinally
from approximately 2100 children. Handprints tor dermatogl3rphlcs are
available from approximately 800 children with particular eiphasis on
children with congenital infections. Serial serum specimens are available
from approximately 300 children with definite or suspect congenital infections
and complete data and specimens are available from the Johns Hopkins
Comprehensive Care and the Frederick County Study of cytomegalovirus,
rubella and toxoplasmosis. Clinical data and longitudinal follow-up Is
available for approximately 200 non-Collaborative Study children with
congenital rubella.

Major Findings; A list of the publications assisted at least in part from
this contract Includes a number of papers. Of particular importance is the
data published on cord Immunoglobulin levels from 2750 cases. Additional
important data has been generated concerning age specific distribution of
certain infections in the Inter-dty population as compared to the Frederick
Cevaty population. This study Included one phase of analysis of buccal
smears for sex chromatin. The rubella studies have been extremely pro-
ductive.

Significance te HIHBS Program and Biomedical Research; The present study
of the long-range effects of certain maternal Infections on the fetus
provides the longitudinal observations necessary to define the complete
spectrum of fetal damage caused by these agents. Combined clinical and
laboratory observations permit the analysis of the long-term follow-up
data.

Proposed Course of Contract; The total contract period is designed to
complete the collection of clinical and laboratory data pertaining to
cord immnnoglobullns ; age specific distribution of certain infections
(cytoaegalovirus, rubella and toxoplasmosis); effect of perinatal toxo-
plasmoals; dermatoglyphlcs and Barr Body studies; and longitudinal studies
of children with congenital rubella.

GONTSACT NASKATIVE

P«rlAatal R«stt«rch Branch - Section on lafoctloos Dlsoasos

July 1, 1970 — JttBO 30, 1971

OWIVKRSITT OP CALIPOBMIA (MIH 69-4)

Tltlo: CytOBOgnloTlrao lnfoc£ions In prognnncy

Contractor *■ Projoct Director; Dr. Hargaret Jonaa

Currant Annnal Laval; $ 33 , 300

ObjactlTae; Study of watamal and contaaltal Infactlona with eytooMgalorlrua
and harpearlrua honinla utilizing aerologlc tacfanlquaa aa well aa Tlrua
laolatlon frea urine aad tlaaua apeclaena.

Major Plndlmta; During the aecond year of the contract approzlaately 1287
petlenta have been enrolled. To date 3140 urine apeclMena aad 4308 blood
apeclaana hare been collected. In addition, 7 abortion tlaane apeclaeaa
have been obtained. A total of 685 throat airab apeclaena have been collected
along with 410 cerrlcal awaba aad 159 cervical aaeara. The aera are being
teated for antibody to cytonega 1 ovlrua and herpesvlrua honlnla.

Serology apeclaena taken through Deceaber 1970 have been teated for antibody
to cytoaagalovlrua. The rate of aeroconveralona for CKV approaehea IZ In
the flrat 1500 woaen. Theae reaults are being recenf Iraed ; checked for
Herpea I and II; teated for cord Ig^; analyzed la relation to vlrua aheddlag,
tlae of pregnancy aad clinical flndlnga In the child.

Slgnlflcaace to NIMDS Prograa and Bleaedlcal Reaearch; Congenital cytoaegalo-
vlrua lafectlona can cauae aevere fetal daaage aad death. The preaeat atudy
la dealgaed to deteralne the tlae durlag pregnancy In which aatemal lafectlona
aay reault la congenital dlaeaae. The data will alao def lae for the flrat
tlae the frequency aad peralateace of aatemal aad fetal lafectlona.

Propoaed Courae of Contract; The coatract will provide the coatlaued atudy
of pregaaat woaea. We aatlclpate belag able to atudy 400 patleata durlag
the third coatract year, brlaglag the total aoaber of petlenta studied to
2000. Speclaeas of urlae, blood aad tlaaua will be forwarded, aa la the paat,
for laboratory atudy of the tlae of lafectloa during pregnancy and the tread
of coagealtal lafectloa.

49 V

Project Title:

Serial No. KDS (CF)-57 TR/TD U02 "

1. Perinatal Research Branch

2. Section on Infectious Diseases

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

Jull^ 1, 1970 through June 30, I97I

Serological and Virus Isolation Studies of Infectious
Diseases in the Collaborative Study on Cerebral Palsy,
Mental Retardation, and Other Neurological and Sensory
Disorders of Infancy and Childhood.

PreAd-Ous Serial Number: Same

Principal Investigators;

Other Investigators:

Dr.
Dr.
Dr.

Dr. John L. Sever, PRE, NINDS
Dr. David A. Puccillo, PRE, NINDS
Mrs, Anita Ley, PRE, NINDS
Mrs. Renee Traub, PRE, NINDS
Mrs. Maiy Ruth Gilkeson, PRE, NINDS

Gabriel Castellano, MBA

Janet Hardy, Baltimore, Maryland

Sheldon Korones, Memphis, Term.

Cooperating Units:

Collaborative Institutions in the Perinatal

Research Study
Laboratory of Infectious Diseases, NIAED
NICHD
Cooperating Institutions in California and Havaii

("With the Section on Infectious Diseases)
Microbiological Associates

Man Years ;

Total:

Professional:
Other :

3.5
5-5

Project Description:

Objectives: The purpose of the infectious disease investigations in the
Perinatal Research Study is to determine insofar as possible the role of
infections in the production of abnormal pregnancy outcomes. To accomplish
this, serial specimens are taken throughout pregnancy, at delivery, and at
six weeks postpartum. These sera are being tested with antigens to determine
the antibody responses of the patients during pregnancy and postpartum, and
then to relate this serological infonnation to the clinical data for the
pregnancy and the child. In addition, serum specimens from the children at
one year of age were obtained from the last 10,000 study pregnancies and

51'

Serial No. NDS (CF)-57 PR/ID U02

these are now being studied. In special cases when congenital infection was
suspected on the basis of clinical or laboratory findings, throat swabs and
blood specimens were obtained from the children.

Methods Employed: To accomplish this program, blood specimens were
obtained from pregnant women at set intervals throughout pregnancy and post-
partum. Initially, during 19^9^ "the collection of blood specimens was made
once every trimester. Late in I96O, the requirements for the collection were
increased and strengthened so that blood specimens were taken at the time of
registration, at set intervals of approximately every two months throughout
pregnancy, at delivery, and at six weeks postpartum. At the same time, a
uniform method for the collection and processing was adapted as a required
study procedure. The use of special vacutainers, sterile technique, special
sterile vials, and new shipping containers were all required. Intensive
laboratory training sessions were held at NIH by the Section on Infectious
Diseases for all technicians in the collaborating institutions. These sessions
were repeated each year and a training film was prepared and sent to collab-
orating groups on request. Careful, complete control was mainatined on all
collection and processing of serum. At the Serum Center of the Section,
personnel checked every vial of serum as received for quality and quantity.
Regular reports of this information were sent every three months to each
study institution. Telephone calls were made immediately to Project Directors
whenever there was a decrease in the quality and quantity of sera received.
Completeness of sets of specimens from each patient was also reviewed by
Serum Center personnel. To improve the collection of complete sets of sera,
a special reporting form was devised for the collaborating institutions and
monitored by the Section.

By the spring of I96I all institutions attained the minimum requirement
of 90^ satisfactory specimens for quality and quantity. Satisfactory quality
was defined as straw colored sera with no hemolysis. Satisfactory quantity
was a minimum of h vials, each with 3 iilL of serum and proper labels. At each
meeting of the Project Directors a report was given concerning the quality
and quantity of specimens submitted by each institution. This detailed in-
tensive review was conducted throughout the entire time of sampling of sera
from the mothers and continues at present with specimens being received from
children at one year of age, special study specimens, and sera from cooperating
groups .

Completeness of sets of serial specimens was determined from the sera
submitted to the Serum Center of the Section and the special reporting form
sent to the Section. Data for the first 28,386 patients showed that there
was at least one specimen submitted for 9^.2^ of the patients. The majority
of the omitted specimens occurred during the first months of 1959. A computer
analysis of specimen set completeness was prepared.

With the development of a firm base for obtaining the required specimens
and tissues, the program embarked on a large commitment for the development
of necessary antigens, new techniques, and the training of a competent
laboratory staff for the study of the specimens. Antigen development was

52v

Serial Wo. NDS (CF)-5T PR/ID il02

conducted by the personnel of the Section and experienced investigators under
contract. Professional laboratory personnel were selected for the Section's
Units on Statistics and Design^ Serology^ Virology, Experimental Animal
Research, Immunological Studies, Epidemiology, and Experimental Pathology and
Neurology. Three years ago the Unit on Cytogenetics was added. Ihis program
is jointly sponsored by the National Institute of Child Health and Human
Development and the National Institute of Neurological Diseases and Stroke.

To document the occurrence of an infection, two approaches are available:
l) Isolation of the microorganism from the patient, or 2) Detection of an anti-
body rise in serum specimens. Both approaches are being used in the present
studies. Appropriate isolation procedures for virus, bacteria, and protozoa
are being used with throat swab specimens from the children. This approach
was not used for detecting infections in the mothers since it would have
required obtaining throat swab and anal specimens at the time of each infec-
tion. When this has been tried in the past, it has been unsuccessful because
the women are usually unable to come to a laboratory for the collection of
specimens when they have minor illnesses. Furthermore, a great many of the
infections under consideration frequently do not result in significant illness
of the women. These subclinical infections go unnoticed and \inrecognized.
For these reasons, the serological approach was selected. By collecting
serial serum specimens, antibody levels for various viruses and protozoa can
be determined. The development of antibody to a microorganism in a patient
who was previously antibody negative provides indirect evidence for infection.
The presence of specific antibody indicates prior exposure to that antigen or
mi cr oorgani sm .

The serological test most frequently employed in these studies is the
complement fixation method. This basic method has been used for many years
as the Wasserman test for the diagnosis of syphilis. With the use of viral
antigens the test is very versatile, performed rapidly, and provides broad
coverage of a great many of the more than 125 viruses which are known to be

of importance to man. Antigens were prepared for most of these viruses. Tests
of specificity were conducted with animal sera. For man, considerable data is
available from our studies and those of many other laboratories to indicate
that both group and specific reactions occur with these antigens. The adeno-
virus CF (Type 2) antigen, for example, is group reactive and provides evidence
for adenovirus infections in general. To date, there are 31 adenoviruses
recognized for man. Rubella CF antigen on the other hand is very specific
and detects infection with rubella only. The sensitivity, specificity, and
persistence of the test is also known. With this type of information, it is
pissible to design serological studies for the -viruses and protozoa. Bacterial
antigens are usually not available for specific serological tests. Only direct
evidence for these infections can be used. The history of infection as
reported by the patient has proven to be quite unreliable in most cases and
is used only in general information.

The majority of initial serological studies are conducted with the use
of the complement fixation method. All tests are reproduced completely and
a minimum of 90^ agreement within twofold variation is required. All sera

i

53^

Serial No. NDS (CF)-5T PR/ID U02

showing significant change in antibody, together with any sera which did not
reproduce, are tested a third time. For more specific testing or confirmation
of these resialts the hemagglutination inhibition and neutralization methods
cs,u be used. These latter serological procedures are very specific and are
also employed for follow-up testing whenever the initial studies with the
complement fixation method suggest the need, for further investigation.

Major Findings: A total of lUo complement fixing antigens have been
developed. Approximately three-fourths of the antigens have been thoroughly
evaluated and are now being applied in routine testing of study sera. The
development and maintenance of large quantities (l,000 ml) of satisfactory
antigens for 50 viruses is an integral part of the investigation being carried
on by the study. The other antigens are receiving intensive developmental
work and 20 of these antigens are under test for specificity. Specific con-
trol antisera have been prepared for 90 microorganisms. In addition, to
provide improved safety, extensive work has been conducted on the inactivation
of the live virus antigens .

The serological studies are being conducted in accordance with three
major study designs: First is the epidemiological studies to determine the
frequency of virus experience among study populations. Specimens from
representative patients at study hospitals are being tested for evidence of
antibody. By studying these specimens, it is possible to establish the
frequency of antibody and change in antibody titer to each virus . The data
for each hospital is then analyzed in relation to other information from the
Collaborative Perinatal Study, and in relation to other infonnation from
epidemiological data concerning the seasonal occurrence of abnormal preg-
nancies and children.

The second and most active category of study involves the selection of
particular microorganisms for intensive testing. Studies of this type have
involved, for example, the testing of sera from a large number of patients for
antibody to toxoplasmosis or rubella. Intensive studies are now being con-
ducted utilizing antigens for Australia antigen influenza A, miimps, cytomegalo-
virus, herpes simplex, and rubella. Patients identified as having serological
evidence for an infection are then grouped and clinical data for these groups
and the remaining patients and their children are compared and analyzed.

Third, studies are designed to obtain maximum data concerning the virus
experience of patients with abnormal pregnancy outcomes, and "matched controls.'
The results of this type of study are then analyzed in terms of differences in
frequency of antibody and antibody change among the abnormals and matched
controls. The matching of the patients include factors which are known to
influence virus experience, such as time of the year during which the specimens
were obtained, race, age, niaiber of living children in the family, and
geographic location of the patients . These studies are conducted when a
sufficient number of abnormals of a particular type have been identified so
that statistical analysis might establish valid information. The initial
studies were directed at abnormalities which are relatively frequent, such as
abortions, stillbirths, and neonatal deaths. The less frequent abnormalities

5I1V

Serial No. NDS (CF)-57 PR/ID U02

or those which cannot be recognized in infancy or early childhood are being
studied as greater numbers of these patients are identified in the Collabora-
tive Study population. These studies have included spastic diplegia, CWS
malformation, cranio-facial abnormalities, and mongoloid infants.

Collaborating Studies: The primary deficiency of data in the Study has
long been recognized as the late registration of Study patients. Since only
20^ of the patients register during the first trimester of pregnancy, it is
impossible to document adequately the infectious diseases experience of
patients during the first trimester. To provide data on the first trimester
of pregnancy, one additional Collaborative Study was joined with the program
of the Section on Infectious Diseases.

Study of Viral Infections in Pregnancy

Dr. Margaret Jones, UCLA and Kaiser Hospital in Los Angeles, Calif.

In addition to this study, the collaboration with the Perinatal Study in
the Kaiser Hospital in Oakland with Dr. Yerushalmy has been an integral part
of the program since its initiation in 1959-

Significance to the Program of the Institute: The use of micro-serological
techniques for a large group of new viruses provides an opportunity to inves-
tigate the course of human disease caused by viruses which are either diffi-
cult to isolate or are resistant to evaluation because the clinical effects
are delayed until a long time after infection has subsided. This is particu-
larly true in the case of birth defects . The application of this tool of
analysis is providing valuable information on the epidemiological aspects of
viirus infections.

Proposed Course of the Project: The serological program will continue to
be expanded in terms of antigenic materials and the performance of tests .

As additional abnormal pregnancy outcomes are reported, these will be
added to existing studies on abortions, stillbirths, neonatal deaths, con-
genital malformations, and mongols . New studies will include congenital mal-
formations of various types and low I.Q. at four years of age.

A specific two-year commitment has been made to complete tests on U,000
abnormals and 4,000 controls using 10 antigens. The PES will Identify the
specifically abnormal children and the laboratory will match controls and
perform the necessary tests. The cord sera from 30,000 children are being
tested for IgM levels. This work will be completed next year (if materials
are provided by the PRC) . The 1,000 children with high IgM are being tested
and studied in detail.

I

55'

Serial No. NBS (CF)-57 PR/lD 402

Publications:

Matsen, J. M., Jones, M. H., Sever, J. L. , Goldenberg, E. D., Gilkeson,
M. R., and Justus, K. M. : Family rubella study in Los Angeles.
Calif. Med. 112: 14-19, 1970.

Henson, T. E., Brody, J. A., Sever, J. L. , Dyken, M. L., and Cannon, J.:
Measles antibody titers in multiple sclerosis patients, siblings and
controls. JAMA 211: 1985-1988, 1970.

Korones, S. B., Todaro, J., Roane, J. A., and Sever, J. L. : Maternal
virus infection after the first trimester of pregnancy and status of
offspring to 4 years of age in a predominantly Negro population.
J. Pediat. 77: 245-251, 1970.

Asbed, R. A., Masland, M. W., Weinberger, M. M., and Sever, J. L.:
Early case finding of children with communication problems. Part I.
Report of a community screening program. Volta Review. 72: 23-49, 1970.

Mendez-Cashion, D., Sever, J. L. , Nazario, R., and Gilkeson, M. R. :
Frequency of rubella antibody in Puerto Rican adiilts. Bol. Asoc. Med.
P. Rico. 61: 406-408, 1969.

Sever, J. L., and Terasaki, P. I.: Maternal -fetal incompatibility.
III. Central nervous system and cardiac anomalies. In Terasaki, P. I.
(Ed.): Histocompatibility Testing. Copenhagen, Denmark, Munksgaard,
1970, pp. 495-500.

Weinberger, M. M., Masland, M. W., Asbed, R. A., and Sever, J. L.:
Congenital rubella presenting as retarded language development. Amer . J .
Pis. Child. 120: 125-128, 1970.

Sever, J. L., Kurtzke, J. F., Alter, M. , Schumacher, G. A., Gilkeson, M. F
and Ellenberg, J. H. : Virus antibodies and multiple sclerosis.
Arch. Neurol. , In press.

Newman, S. J., McCallin, P. F., and Sever, J. L. : Attempts to isolate
H-1 virus from spontaneous human abortions. A negative report.
Teratology. 3: 279-281, 1970.

Sever, J. L. , Gilkeson, M. R., Chen, T. C, Ley, A. C, and Edmonds, D.:
Epidemiology of mongolism in the Collaborative Project. Ann. N.Y. Acad.
Sci. 171: 328-341, 1970.

Sever, J. L. : Persistent central nervous system infection by a virus:
Rubella. Res. Publ. Ass. Res. Nerv. Ment. Pis., In press.

56v

Serial No. NBS (CF)-57 PR/ID 402

Sever, J. L. : Virus infections and malformation. Fed. Proc. 30: 114-
117, 1971.

Sever, J. L. : Infectious agents and fetal disease. In Waisman, H. A.,
and Kerr, G. (Eds.): Fetal Growth and Development. New York, N.Y.,
McGraw-Hill Book Company, 1970, pp. 221-233.

Sever, J.L. : Vlroses e o feto, Revlsta de Atualizacao en Ginecologia
et Obstetricia. Vol. IV, Wo. 10, 1970, pp. 36-54.

I

57v

4

Serial No. NDS (CF)-61 PR/ID 835

1. Perinatal Research Branch

2. Section on Infectious Diseases

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Clinical Investigations in Human Volunteers and Other

Populations of Virus Effects and Production of Prototype
Human Antisera, Vaccines, and Other Agents.

Previous Serial Number: Same

Principal Investigators: Dr. John L. Sever, PRB, NINDS

Dr. Earl Matthew, PRB, NINDS
Dr. Donald Henson, PRB, NINDS
Dr. Dale Dietzman, PRB, NINDS

Other Investigators: Dr. David A. Fuccillo, PRB, NINDS

Cooperating Units: Bureau of Prisons, Department of Justice
(Dr. Myrl Alexander, Director)
Petersburg Federal Reformatory
(Dr. Joel Rascoff , Chief Medical Officer)

I

Man Years:

Total: 1.00
Professional: .50
Other: .50

Project Description:

Objectives: To study the efficacy of prophylactic and therapeutic
materials for the prevention and control of infectious diseases. To study
the safety, antigenicity, communicability and immunogenic ity of candidate
rubella vaccines. To determine whether intravenous mannitol causes
cytomegalovirus .

Methods Employed: Human volunteer studies are conducted in collabora-
tion with the Federal Bureau of Prisons. These studies are reviewed and approvec
by the Clinical Research Committee and the Medical Board of the National
Institutes of Health, and the Vaccine Development Board of the National
Institute of Allergy and Infectious Diseases. Intravenous mannitol was given
to human volunteers who were studied for CMV virus shedding excretion and
antibody. Vaccine studies with low temperature adapted rubella vaccine are
scheduled .

59'

i

Serial No. NDS (CF)-61 PR/ID 835

Major Findings; Volunteer studies were performed to evaluate the
effect of mannitol on excretion of CMV and antibody response. The mannitol
proved to be safe in that it did not increase excretion or antibody.

Significance to the Program of the Institute: Volunteer studies
provide the basic data necessary to evaluate potential rubella vaccines.
These studies should provide important and necessary information on the
effectiveness and safety of such preparations. It will also elucidate the
epidemiology of CMV in man.

Proposed Course of the Project: Additional studies will be
performed when necessary or appropriate.

Honors and Awards : None

Publications: None

60 V

i

S«rl«l H«. n>S (CF)-62 PR/ID 972

1. P«rlaatal lascarch Braoch

2. Section on Infectious Dlseaaes

3. Bethesdn, Haryland

FHS-4IIH

ladlvldvBl Project leport

July 1, 1970 through June 30, 1971

Project Title: Experlaentel Anisel Tlsaue Culture, Hlstopethologlcel and

Serological Inreatlgatlona of the Role of Ylruaea and Other
Microorgaalsna in the Perinatal Period.

Previous Serial Knaber: Saaa

Principal Investigators: Dr. VUllaa T. London, PKK, HINDS

Dr. David ▲. Fueclllo, PRB, HINDS

Dr. Johm L. Sever, P&B, HINDS

Mr. Villiaa Helas, OB, HINDS

Other Investigators: Mrs. Anita C. Ley, PRB, HITOS

Mrs. Blanche Curfnan, PRB, HIHDS

Cooperating Units: Dr. Andre J. Hahnlas, Departnent of Pediatrics,
Bnory University S^iool of Medicine, Atlanta, Ga.

Dr. J. M. Rice, Bioassay Section, EPB, NCI

Dr. Donald B. Cheek, Departnent of Pediatrics

Johns Hopkins University Medical School, Baltiaore, Md.

Dr. Louis W. Catalano, Jr., Departnent of Neurology,
College of Physiciana and Surgeons, Celunbia University,
Hev York, Hev York

Man Tears:

Total: 8
Professional: 2
Other: 6

Project Description:

Objectives: To study the role of viruses and other nicroorganlsna
in the perinatal period, the infection of gravid and nengravld aninals of
several different species by parenteral routes with various viruses and
ether nicroorgaaisns to detemlne the effects of these agents on the aninals
and their fetal tissues.

Attssqpt to recover inoculated agents froa the various aninals and fetal
tlasues and the correlation of these relsolationa with tine (In gestatloc) of
inoculation, and dosage given.

6iv

,i»

Serial Mo. NDS (CF)-62 PR/ID 972

Correlate these findings with gross and hlstopsthologlcal findings.
Correlate all of this inforaatlon with serological findings.

Initiate a nutritional study of non-hoaan prlnates using pregnant
rhesus nenkeys to test the hypothesis that there Is no causal relation
between aatemal nutrition during pregnancy and certain sensory, patho-
logical, Imunologlcal and biochemical characteristics of the Infant. It
Is considered that a nonkey trial In which the concept of randoaness is
peraitted is a necessary preliminary to a human trial.

Methods Employed t An investigation of the role of vimses and other
microorganisms in the perinatal period by the continual use of experimental
animals; tissue culture techniques; histopathological studies; and sere-
logical testing.

Pregnant mice, rabbits and monkeys are being Inoculated by various
routes with viruses and other microorganisms. These animals are being
observed and checked for evidence of disease and/or effects on fetal tissmes.

Virus isolation investigations utilizing tissue cultnre to recover
viruses from tissues and fluorescent antibody technique to study the location
of virus infection produced in experimental animals.

Histopathological and gross anatomical studies are conducted en ^
specimens obtained from the experimental animal studies.

Extensive serological studies are conducted with the many viral antigens
developed for the Collaborative Study and new antigens with materials being
studied previously mentioned.

In the non-human primate nutritional study, pregnant rhesus menke3rs
will be maintained throughout pregnancy en one of four diets restricted la
either calories or protein. One diet will be deficient in both. The
pregnant animal will be delivered at 158 days of gestation by cesarean
section and the Infant's tissues will be processed for biochemical analysis
by the Hopkins Unit. The natritionally deprived female monkeys will be
continued on their respective diets and studies for Immunological responses
to various antigens such as tuberculin, rubella and mumps virus will be
performed.

Major Findings; The Inoculation of rabbits with low passage wild
rubella virus strains and vaccine strains gave variations In antibody
response and virus shedding. These variations can be useful as markers of
strain differences. The lew passage wild virus strains produce high anti-
body levels and variable amounts of virus shedding. The vaccine strains
resulted in little or no antibody and no virus shedding.

62 V

Serial No. RDS (CF)-62 PR/ID 972

Genital Infection vna readily eatabllehed In 10 feaale Cebua Bonkeye
when Inoculated Intrayaglnally with type 2 Herpearlrus hoslnla. Infection
«aa protren by vlrtts laolatlone and aerologlcal atvdlea and the denonatratlon
of herpetic Tealdea and/or ulcers on the Tul^a and ^raglna vith noderate
cerrldtls. Relnoculatlon with HVH type 2 was perfomed In all 10 anlaals
with resulting vaginal reinfection being established In 3 aonkeys despite
the presence of serua aeetrallzlng antibodies. The lesions produced In the
Cebus aonkey appear to parallel closely the disease observed In huaan genital
Infections.

Polylnoslnlc - polycytldyllc ribonucleic acid (Poly I:C) coaplexed
with poly-D-lyslne was studied as a prophylactic and therapeutic naterlal
In nice Inoculated with herpes simplex virus. With a treatnent reglaen
over a period of several days alee treated with Poly I:C > poly-D-lyslne
had significantly less Mortality than anlaals treated with Poly I:C alone
or saline only. This enhanced protection was still detectable If treat-
aent was begun as late as 4 days following Inoculation of HSV, at which
tlae an occasional aouse was showing signs of early CHS illness.

A pilot nutrition study using 38 rhesus aonkeys has been started.
To date 8 anlaals have becoae pregnant and at least one anlaal is on each
of the four diets. It appears that the anlaals will eat the diets as
prepared and aalntain pregnancy.

Significance to the Prograa of the Institute; A prograa using ezperi-
aental anlaals, tissue culture techniques, and hlstopathologlcal investiga-
tions coapleaents the strict serological approach being used on huaan sera
obtained froa the Collaborative Study and thus balances the Investigations
of the role of viruses and other nicroorganisas in the perinatal period. It
presents the direct neans of Investigation of these agents «rhich may contribute
to perinatal pathology.

Proposed Course of the Project; Further studies using tissue froa
patients with subacute sclerosing panencephalitis, Creutzfeldt-Jakob disease,
aayotrophlc lateral sclerosis and progressive aultifocal leucoencephalitis ,
Inoculated intracerebrally into the fetus of pregnant rhesus aonkeys are
now In progress.

The Cebus herpes aonkey aodel provides an experlaental approach for the
study of congenital infection and possible prevention or control of congenital
disease. We propose a pilot study, first, to deteraine if congenital infec-
tion can be produced and, second, to explore aethods of prevention or treat-
aent if this pilot is successful.

If the above studies were successful In producing congenital herpes
type 2 Infection in the Cebus, we would propose to proceed with a study of
chemotherapy using cytoslne arablnoslde, 5-IDU, Poly I:C, and Poly Lysine
for clearing the aatemal vaginal infection prior to delivery of the baby.

L

63.

Serial Ho. MDS (CF)-62 PR/ID 972

Secoad, ve would Inroatigaeo Innmlzatloa with herpes type 2 antigen for
Its efficacy la ellailnatlat vaginal Infections and, third, we would study
cesarean section aa a «eans of hy-passlng the Infected vaginal area and
preventing congenital Infection.

At the ceapletlon of the pilot priaate nutrition study, the data will
he analyzed and any correction In aethods and diets will be aade. The aaln
phase will then be started ualng 136 rhesus nonkeys.

Honors and Awards: None

Publications:

Nabaalas, A. J., London, W. T., Catalano, L. W., Fucclllo, D. A., and Sever,
J. L.: Genital Herpesvirus hoalnis type 2 Infection: An cxperiaental nodal
in Cebus aonkeys. Science 171: 297-298, 1971.

London, W. T., Catalano, L. W. , Hahaias, A. J., Fucclllo, D. A., and Sever,
J. L.: Genital Herpesvirus homlals type 2 infection of aonkeys. Obstct. &
Gynec. 37: 1971. (In press).

London, W. T., Fucclllo, D. A., Andersen, B. , and Sever, J. L.: Concentration
of rubella virus antigen in chondrocytes of congenltally Infected rabbits.
Nature 226: 172-173, 1970.

London, V. T., Fucclllo, D. A., Ley, A., and Sever, J. L.: Antibody response
of yarious strains of rubella rirus when inoculated into rabbits. Proc. Soc.
Exper. Biol. Med. (In press).

Catalano, L. W. , London, W. T., Rice, J. M. and Sever, J. L.: Treataent of
herpesvirus encephalitis with Poly I:C - Poly-D-Lyslne complexes. Obstet. t
Gynec. (In press).

6k

Serial No. WDS (CF)-65 PR/ID 1270

1. Perinatal Research Branch

2. Section on Infectious Diseases

3. Bethesda^ Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Toxoplasmosis: Serological and Clinical Studies.

Previous Serial Number: Same

Principal Investigator: Dr. John L. Sever^ PRE;, NIKDS

Other Investigators: Dr. Joseph S. Drage, PRE;, NINDS

Cooperating Units: Section on Pediatric Neurology^ PRE, NIKDS

Man Years:

Total: .2
Professional: .2
Other: .2

Project Description:

Objectives: This study relates rises in antibody titer to abnormal
pregnancy outcomes.

Methods Employed: Computer analysis of multiple variables relating to
injection with toxoplasma and pregnancy outcome.

Major Findings: Within the group of 47 patients with titer elevations
of greater than 409 b^ or significant increases in antibody titer, five were
found to have definite toxoplasmosis and ten were suspected of having
toxoplasmosis. The ten included six with motor retardation, two pregnancies
resulting in stillbirths, and two in neonatal deaths. The sera from ten of
the remaining 32 apparently normal children were tested for antibody to
toxoplasmosis and one was found to have a high titer.

Proposed Course of the Project: Publication of findings.

Honors and Awards : None

Publications : None

65x

Serial Wo. NDS.(CF)-6t PR/ID I503

1. Perinatal Research Branch

2. Section on Infectious Diseases

3. Bethesda^ Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 19 T^

Project Htle: Epidemiologic Studies of Perinatal Infections

Previous Serial Number: Same

Principal Investigators: Dr. John L. Sever, PRE, NINDS

Mrs. Dorothy M. Edmonds, R.N., PRE, NllffiS

Other Investigators: None

Cooperating

Units :

None

Man Years:

Total:

1.50

Profess

sional:

1.50

Other :

0

Project Description:

Objectives: To utilize information from the Collaborative Perinatal
Research Study and other cooperative studies to identify pregnancies compli-
cated by maternal infections or infections in childhood; to further delineate
these cases by serologic testing of the stored sera; to utilize Collaborative
Study and related data to determine outcome of these pregnancies in relation
to the outcomes of matched controls and the general study population in order
to gain information on the frequency of maternal infections during pregnancy
and their effects on the developing fetus. Studies include: Microcephaly,
Hydrocephaly, Abortions, Cataxacts, and Bacterial and Protozoal Infections.

Methods Employed: Primary material utilized for these studies comes
from the Perinatal Research Study and other cooperative studies . For this
reason the serologic data developed by the Section on Infectious Diseases is
correlated with the clinical information available either from print-outs or
direct hand review of the charts stored in the Perinatal Research Branch.

Major Findings: The frequency of some virus antibodies has been found to
be increased in several of the abnormal patient groups . These findings are
being followed with additional testing of similar groups of patients.

Significance to the Program of the Institute : The development of the
serologic data requires the further analysis in relation to the epidemiology
of infections in the Perinatal Research Study as well as the epidemiology of
perinatal infections with other collaborating groups. This data then provides

67 V

Serial No. NDS(CF)-67 PR/ID 1503

the basis for correlating serologic and clinical information. Special studies
are initiated in populations where high frequencies of infection or abnormal
pregnancy outcomes have been noted.

Proposed Course of the Project: Special emphasis will be placed on the
possible association of neoplastic diseases and other tumors with herpes
simplex infections. We will analyze the clinical data for the 5500 reported
infections in the Perinatal Research Study. Detailed epidemiological studies
will be conducted on stillbirths, congenital malformations of various types,
and children with low IQ's at 4 years of age.

Honors and Awards: None

Publications :

Sever, J.L, : Viruses and embryos. In Fraser, F.C. and McKusick, V.A.
(Eds.): Congenital Malformations , Proceedings of the Third International
Conference, The Hague, The Netherlands, September 7-13, 1969, Amsterdam,
Excerpta Medica Foundation, International Congress Series No. 204, 1970,
pp. 180-186.

Sever, J.L. : Viral Teratogens: A Status Report. Hospital Practice.
5: 75-83, 1970.

Sever, J.L. : Viruses and The Fetus. Int. J. Gynaec. Obstet. 8: 763-769,
1970.

6a V

Serial He. MDS (CF)-67 PR/ID 1506

1. Perinatal Reaearch Branch

2. Section on Infectloes Dlaeaaea

3. Betheada, Maryland

PHS-NIH

Individual Project Report

Jvlj U 1970 through June 30, 1971

Project Title: Maternal Infection and Pregnancy Outcoae

Prerloua Serial Noaber: Saae

Principal Investlgatora : Dr. David A. Fucclllo, PRB, HIHDS

Dr. John L. Sever, PRB, HINDS

Other Inveatlgatora: Dr. Wllllaa T. Leaden, PRB, HIHDS
Mra. Ranee Trauh, PRB, HIHDS
Kra. Mary Rath Cilkeaen, PRB, HIHDS
Kra. Plera Hader, PRB, HIHDS

Cooperating Units: DnlTersity of California (Dr. Margaret H. Jonea)

Hagaaii Peraaaente Medical Croup (Dr. Paul F. McCallla)
Univeraity of Puerto Rico, School of Tropical Medicine

(Dr. Dolerea Mendez-Cashlon)
Peanaylvania Hospital (Dr. Coraon and Dr. Belogneae)

Man Tears:

Total: 4.5
Prof eaaional : 1.0
Other: 3.5

Project Deacrlptioa;

Objectivea: To utilise varioua virological technlquea in an iatenslTe
study of Tiruses to deteralne their role in the production of birth defects
and related abnoraalitiea. To develop Tirologlcal technlquea neceasary for
the iJiTeatigatioa of the natural courae of the diaeaae aa cauaed by the
infectloua ageata.

Methods Eaployed: Hea ▼Irus Isolation techniques and aerua neutralisa-
tion teata are uaed for large acale teating in the study of pregnant uoaen and
their children. The derelopaent of new technlquea, such as the HAI teat for
Herpea hoainia Typea I and II and a new test for cytoaegalorlruses will now
peralt the deteralnation of the frequency of Tlrua experience aaong atudy
populations along with the presence of antibody and change in antibody titer
to the Tirus. These tests are being uaed to eatablish the reliability of
other teata and to deteralne their aenaitivity and apeciflclty. A aerologic
atudy utilizing theae tests on sera collected on the Collaborative Study

69 V

Serial H«. HDS (CF)-67 PR/ID 1506

population was conductad to dataralne the fraquancy of aatlbody chansaa daring
pregnancy and tha effact of these Infections on the developing aad>ryo. Wa
have coapletad the testing of 2500 placentas for rubella vims for the
purpose of Identifying children with congenital rubella by the presence of
rubella In the placental material. These reports are being prepared for
publication. The use of a fluoreacent, specific antl-lgM teat la proving
to be a valuable method for ldentlf3rlng congenital Infections.

Vaccina for rubella la being given to pregnant women (at tha Pennsyl-
vania Hospital) who are subsequently being aborted. This will help determine
the effect of vaccine on the fatua.

Major Plndlnga; Three new tests were developed for specific virus
serology. These were hemadaorptlon tests for Harpea I» Herpes II and
cytomegalovlrua. Tha tests now provide highly specific, reliable, rapid,
mlcromathods for virus serology with these aganta. This Is of particular
value becauae prevloua matboda were particularly laborloua and expensive or
not specific. Also, since these are hemagglutination tests, the anti-
complementary effects found In many Perinatal Kasearch Study sera can be
avoided and tha sera are now usable.

Vlrua Isolation atndlaa of woman for cytomegalovirus show transient
as well as persistent Infections. Studies of the children of these pregnancies
are now In progress.

Tha fetal abortion studies, after rubella vaccinations, have been
completed for about 75 caaaa and results will be reported at a later date.

Rubella vaccina atudlaa are continuing with the temperature adapted
vaccine material.

Significance to the Program of tha Institute; The results from these
studies help determine what effect virus infection has on abnormal pregnancy
outcomes and alao provide valuable Information on the epidemiological aspects
of virus Infections.

Proposed Course of the Project: Studies are now in progress on
cytomegalovlrua infactiona during pregnancy and In acveral population
groupa in Maryland. Additional atudlaa are being conducted on Herpea Typea
I and II infections in women. Drug evaluation of Cytoslne Arabinoside for
CMV and 5-IDU for Harpea heminia la being conducted in monkeys.

Honors and Awarda; Nona

Pttbllcationa :

Puccillo, D. A., Modar, P. L., Traub, R. G., Hanaen, S. and Sever, J. L.:
Micro Indirect Hamagglutlnatlon Teat for Cytomegalovlrua. Appl. Microbiol.
21: 104-107, 1971.

70 V

8«rl«l ■•. »8 (eF)*67 PK/ID 1506

CmtMlMa», L. W., FkcUIq, 9. ▲., Tr««b, K. C, ami t«r«r, J. L.:
laolatiM ef l«b«ll« Tlrw fraa Plaewtas ami Thrwt CsltWM •£ lafaats.
i—r. J. Ob«f t. ftr— c. la prMs.

71 V

Serial No. NDS (CF)-69 PR/ID 1729

1. Perinatal Research Branch

2. Section on Infections Diseases

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 throttgh June 30, 1971

Project Title: Stndy of the Possible Transalsslon of Toxoplasaosls In
Huaans and Ma— als

Previous Serial Noaber: Saae

Principal Investigators: Dr. John L. Sever, PRE, NINDS

Dr. Stephen J. NcvBan, PRB, HINDS
Dr. David A. Fucclllo, PRB, HINDS
Dr. Dolores Mendez-Cashlon, Puerto Rico
Dr. Wllllaa T. London, PRB, HIHDS

Other Investigators: Hone

Cooperating Units: Departneat of Pediatrics
CeBtro-4fedlco
San Joan, Puerto Rico

Study has been coapleted.

Honors and Awards: Hone

Publications:

Hevaan, S. J., FuccUlo, D. A., Sever, J. L., London, W. T. and Mendez-
Cashlon, D.: Tozeplasnosls In Puerto Rico. II. A serological and
epidemiological study In Puerto Rlcan children. Boletln de la Asoclaclon
Medlca de Puerto Rico, In press.

73v

Serial Ho. HDS (CF)-69 PR/ID 1731

1. Pcrliuital Research Branch

2. Section on Infections Diseases

3. Bethesda, Maryland

PHS-HIH

Individual Project Report

Jnly 1, 1970 throngh Jane 30, 1971

Project Title: Experimental in vitro and In vivo techniques for Isolation
of Infectious agents fron chronic diseases. Use of zonal
centrlfugatlon and Isopycnlc ultracentrlfugatlon for
purification of the suppressed form of neasles virus fron
SSPE hralns and FML papovavlrus fron progressive nultlfocal
lenkoencephalopathy brain speclaens. Study of the role of
Interferon In chronic Infection. Csaparatlve study of the
role of delayed hypersensitivity, hunoral lanunlty and
Interferon In neurotropic neasles Infections In rats.
Study of the fetal lanmologlcal cenpetence In rhesus
■onkeys. Sevelopnent of Inaunologlc methodology In
evaluating Intra-uterlne Infections and CHS Infections.

Previous Serial Huniber: Same

Principal Investigators: Dr. Lulz Herta-Barhosa, PRB, HIHDS

Dr. Dale Dletsman, PRB, HIHDS
Dr. David A. FuccUlo, PRB, HIHDS
Dr. William T. London, PRB, HIHDS
Dr. John L. Sever, PRB, HIHDS

Other Investigators: Mrs. Rebecca Hamilton, PRB, HIHDS

Mrs. Barbara Wit tig, PRB, HIHDS

Miss Helen Krebs, PRB, HIHDS

Mrs. Anita Ley, PRB, HIHDS

Cooperating Units: University of Tennessee (Dr. J. T. Jabbour and Dr.

Charles Cape)
University of Vermont (Dr. George Schumacher)
Indiana University Medical Center (Dr. Wolfgang Zeaan)
University of Calif emia L.A. (Dr. Gary Gltnlck)
Wilmington General Hoapital (Dr. George Bolnes)

Man Teara:

Total: 5
Professional: 2
Other: 3

Project Description:

Objectives; To establish ufaether persistent or tolerant viral
infections are associated with chronic diseases such as polymlositis ,

75 V

Serial He. NDS (CF)-69 PR/ID 1731

ulcerative enterocolltla, aajro trophic lateral sclereela, Creutzf eld t~ Jakob
dlaeaee, pregreaslTe anltlfocal leekoeacephalepathy, and Multiple sclerosis.

To purify and concentrate PML and SSPE ▼Iruses and examine these
agents electron alcroscoplcally, blochenlcally, and antlgenlcally In
parallel with the conventional formm of these viruses.

To determine Interferon levels In patients with ulcerative entero-
colitis, hepatitis, and SSPE, as well as to establish whether their
peripheral white cells are capable of producing Interferon to normal levels
when compared with cells from normal subjects.

To evaluate the role of cellular laannlty, humoral lamunlty, and
Interferon In protecting experimental animals against neurotropic measles
virus.

To elucidate the maturation of the lomune system In sobryonlc life
and correlate this process with Intra-uterlne Infections.

To separate IgM ab^ IgG from cord sera from children with congenital
diseases and determine the serological specificity of the 19S Immunoglobulin
using fluorescent antibody assays In an attempt to develop a diagnostic test
for fetal Infections.

Methods Employed; Brain specimens from well-documented cases of MS,
FML, ALS, SSPE, etc, were homogenized to a lOZ suspension and Inoculated
Intracranlally In groups of 5 rhesus monkey fetuses during the first third
of gestation when the animals are expected to be inmnologlcally immature
and hence more susceptible to Infectious agents. These fetuses were carried
to term and, immediately after birth, one animal was killed and examined
hlstopathologlcally for CNS lesions. The remaining animals were carefully
observed for clinical symptoms, abnormal behavior, and antibody pattern. A
2-year follow-up has been scheduled.

Brain tissue provenlent from patients with encephalopathies, intes-
tinal mucosa from patients with ulcerative enterocolitis, muscle from
patients with polymiositls , and lymph nodes from Individuals with SSPE were
examined by electron microscopy, fluorescent microscopy and, whenever
possible, cultured In vitro . These tissue cultures were submitted to
vlrologic and serologic assays, inoculated into experimental animals, and
co-cultivated with human diploid and heteroploid cell lines in efforts to
isolate and identify intracellular agents.

Brain autopsies from patients with FML and known to contain intra-
nuclear virus were frozen and thawed three times, the nuclei extracted and
disrupted by physical means. Nuclear extracts were isopycnically banded in
CsCl and fractions examined under the electron microscope. Fractions
containing the papovavlrus were used to IflBsunize guinea pigs in order to

jGv

Serial No. NDS (CF)-69 PR/ ID 1731

produce aonospeclflc antisera Intended to Identify the virus. Slallar
approach, but using zonal centrlfugatlon to purify the virus, was planned
for SSPE and Is awaiting approval of the necessary contract.

Interferon levels In serua of hepatitis and enterocolitis patients
were obtained by Vk Inhibition test using Slndbls virus as the challenge
virus. SSPE patients' white cells were grown in vitro, infected with
different viruses (including aeasles), and Interferon on supematants
measured by the HA Inhibition method. Adequate controls were utilized.

Levis* rats were Inomnized with eeasles vaccine conblned with
Freund's adjuvant and after antibodies developed and delayed hypersensitivity
becaae ■aaifest as measured by skin tests with PPD, these animals served
as serum and lymphocyte donors to groups of histocompatlble, non-Immunized
animals. Interferon was produced by ^ vitro grown macrophages and 40,000
units were inoculated Into other groups of rats. Passively Immunized animals,
i.e., those rats receiving pre-determlned amounts of antibody, competent
lymphocytes and interferon were challenged with 10 U>so neurotropic measles
virus and protection conferred measured by comparing death rates with those
of control groups.

Mumps and chyknagunia viruses were inoculated into groups of rhesus
monkey fetuses at first, second, and third periods of gestation. At
appropriate Intervals the fetuses were removed and blood collected for
Immoaological evaluation. Antibody curve was determined by CF test;
lymphocyte transformation was measured by uptake of tritlated thymidine in
the presence of mumps antigoi; Interferon production was established by HA
inhibition assays. Concomitantly, the pathogenesis of «imps virus was
studied in some fetuses to determine the fetal susceptibility in terms of
stage of gestation and to correlate these observations with the immuno-
logical data.

lamBnoglobttlins from cord sera provenlent from cases of congenital
infections were precipitated by the ammonium sulfate method and concen-
trated 10 times in saline. These preparations were ultraccntrlfuged in
linear gradients of sucrose and 7S gamma globulins separated from 19S
molecules. The latter fractions were serologically evaluated against
different antigens (Toxoplasma, rubella, QfV) to determine antibody
specificity and thus the identity of the causative agent.

Sera and spinal fluid from patients with SSPE were compared with
specimens from patients with other neurological diseases using the immuno-
diffusion technique in gel plates. Highly concentrated measles virus
antigens were utilized in the test.

Major Findings; Virologlcal studies with SSPE lymph node biopsies
resulted in the isolation of measles virus and in the demonstration of an
unidentified bullet-shaped virion in the cytoplasm of several stroma cells.
The measles virus was isolated in mixed cultures with HeLa cells. This

77V

SarlAl Ho. RD8 (eP)-69 PK/ID 1731

finding attggaata that SSPE la a ayataoalc Infection with poaalble apaclf Ic
defoctlyenaaa of the cellolar Ijanmlty.

A alBple dlagnoatlc teat for SSPE «aa developed by the Ovchterlony
technique uaing the patient 'a aplnal flnld and concentrated meaalea antigen.
The aaaay proved highly apeclflc and hence of conalderable dlagnoatlc value.

A herpea-llke vlrua waa found aaaoclated with caaea of ulcerative
enterocolltla. The vlrua partldea were vlauallzed by electron mlcroacopy
and tlasue culturea of nucoaa and aub-mucoaa apeclaena ahoved acattered
Intracellular vlrua when aaaayed by lamunofluoreacence.

Circulating Interferon waa not found In patlenta with chronic or
acute hepatltla nor In patlenta with enterocolltla. Serua and aplnal fluid
froa patlenta with SSPE alao failed to ahow detectable levela of Interferon.
However, the peripheral white cella from theae patlenta reaponded to In
vitro Infection by producing noraal aaounta of Interferon, Indicating tiiat
the IF ayaten la not Impaired In theae condltlona.

Plconavlma-llke cryatala were aeen by electron alcroacopy In aeveral
■nade blopalea froa patlenta with polyaloaltla . tUxed culturea of auade
cella and alalan cella were prepared In atteapta to laolate vlruaea. A
papovavlrua waa purified froa a FML brain autopay and aubaequently uaed to
Inoculate laboratory anlaala. The purification waa achieved by banding the
vlrua In CaCl (1.6 gra/al). Spectrophotoaetry and electron alcroacopy
monitoring resulted In a rather pure vlrua preparation of approximately 10'
Intact partlclea/5 graas of tlaaue.

Significance to the Prograa of the Inatltute; The developaent of
alzed cell culturea to unnaak latent Infectlona provldea an excellent
methodology for the atudy of chronic dlaeaaea of poaalble viral etlologlea.

The auccesaful development of a almple dlagnoatlc teat for SSPE
warrants further Immunological atudlea with cerebrospinal fluid from patlenta
with other neurological dlaeaaea In an effort to define antibody pattema
of dlagnoatlc value.

The preaence of a latent. Intracellular measles virus In the lymph
node of patlenta with SSPE auggeata a tolerant Infection with deficiency of
the delayed hypersensitivity. This finding should lead to detailed examina-
tion of the Immune competence of SSPE patlenta aa well as that of Indlvldnala
with other CNS Illnesses.

The understanding of alow vlrua infectlona of the CRS will depend upon
purification of the auppr eased form of these agenta followed by careful
biochemical and blophyalcal analyala. To accompllah thla goal, the uae of
zonal centrlfttgatlon for cellular fractionation ahould be oiphaalzed.

78^

Serial H*. HDS (CF)-69 PR/ID 1731

Ffpo— J C»Mf of th« Proj«ct; Special ai^hasls will be placed epen
a brala caltvre reaearch prograa. lanreatlgatlea of the aeehaalaa ef patlM>-
genesla aad poaalble Ibmom def Iclenclea In patlenta vlth nenrologlcal diseases
will be conducted. Our selection of patients with aoltlple scleroels,
Parkinson's Disease, progresslre wtltlfecal leukoencephalopatby, and aayo-
trophlc lateral sclerosis Is supported by the existing data which suggest
possible Tlral etiologies for each of these diseases. Tissue speclnens and
blood from patients will be provided through collaborative-contract arrange-
■ents with Investigators throughout the country.

Utilising the nlzed culture technique «e hope to detenlne If It Is
possible to release suppressed virus from these chronic neurologic diseases
and to gain a further understanding of the pathogenesis of latent Infections
of the CMS. Antibody levels and conpctence of lynphocytes fron patients will
be exsnlned using the standard technlquea.

The SSPE strain ef aoasles virus will be sti|dled In laboratory anlnals
and efforts will be directed at the developnent of an anlaal aodel systen for
this disease.

Honors and Awards: Hone

Publications:

Horta-Barbosa , L., Fuccllle, D. A., Haailton, R., Traub, R. , Ley, A., and
Sever, J. L.: Sene characteristics of SSPE ncasles virus. Proc. Sec. Exper.
Biol, h Med. 134: 17-21, 1970.

Vernon, M. L., Horta-Barbosa, L., Fucclllo, D. A., Sever, J. L., Barlnger,
J. R. , and Blmbaun, G.: Virus-like particles and nucleoproteln-type
fllsnents In brain tissue fron two patients with Creutzfeldt- Jakob Disease.
Lancet 1: 964-967, 1970.

Horta-Barbosa, L., Krebs, H., L^, A., Chen, T. C, Gllkeson, M. R., and
Sever, J. L.: Progressive Increase In cerebrospinal fluid measles antibody
levels in subacute sclerosing panencephalitis. Pediatrics in press.

Horta-Barbosa, L., Fucclllo, D. A., and Sever, J. L.: Viral and protozoan
infections of the newborn. In Abramson, H. (Ed.): Resuscitation of the
Newborn Infant. St. Louis, Mo., C. V. Mosby Co., In press.

79V

i

Serial He. MDS (CF)-69 PR/ID 1732

1. Perlnetel Research Branch

2. Section on Infections Diseases

3. Bethesda, Maryland

PHS-NIH

Indlvldoal Project Report

July 1, 1970 through June 30, 1971

Project Title: Investigation of the Role of Mycoplasma spp, and Other
Microerganisas in the Perinatal Period.

Previous Sorial Huaber: Same

Principal Inrestigators ; Dr. David L. Madden, PRB, KINDS

Dr. Williaa T. London, PRB, MINDS
Dr. John L. Sever, PRB, NINDS
Dr. Earl B. Itatthew, PRB, NINDS
Dr. Dale Dietnuin, PRB, HINDS

Other Investigators: Dr. Melvin Museles, NNMH

Mr. Kenneth Meats, PRB, NINDS

Cooperating Units: National Naval Medical Hospital, Bethesda, Md.

Man Tears:

Total:

1.60

Professional:

.60

Other:

1

Project Description:

Objectives: To study the role of Mycoplasna spp. in perinatal diseases
of aan. To develop aniaal aodels to detemine the pathogenesis of tiiese
diseases. To develop nev test systems for the rapid diagnosis of perinatal
diseases.

Methods Employed: Yaginal and oral swabs are obtained from pregnant
vomen and monkeys at the time of labor or C-section. Swabs are obtained from
infants and mothers. These specimens are cultured in standard media. Isolated
cultures will be identified by the metabolic-inhibition (M.I.) test. Serum
samples obtained prior to delivery, at time of delivery and post-delivery are
studied for presence of antibodies by the M.I. test, indirect iDmunofluorescence
and agglutination tests. Pregnant monkeys found to be free of Mycoplawna
infections will be Inoculated with various strains of Mycoplasma to detemdne
the effect of these strains upon mother and infant. The use of newer techniques
for determining antibodies in body fluids will be used in an effort to develop
quicker and more accurate tests for detection of viral antigens and antibodies.
He will utilize new virus strains and tissues in an attempt to grow higher
tltered virus antigens. He will use new techniques such as counter-lnmnmo-
electrophoresis and radioinsmnoprecipitation tests.

8iv

Serial Ho. HDS (CF)-69 PR/ID 1732

Major Fiad<«B*i Santa froa 100 aothar-baby pairs, whose Mycoplaaaa
flora and antibody tltara for Mycopla— a had been deterained, were atvdied
for abnoraal aaoants of IgG and Ii^ antibody in an effort to deteralne if
in utero infection occurred. None aaa found. Post-natal follow-up on
children known to have been infected vith Myeoplasaa at birth has indicated
that they vere not adversely affected as jndged by birth veight, weight
gain or increased susceptibility to disease.

Inoculation of M. hoainis into the vagina of pregnant aonkeys did
not cause fetal infections. The pregnant aonkeys soon eliainated this
agent froa their bodies. In non-pregnant aonkeys, a severe vaginitis
occurred and the organisas have persisted in the vaginal tract for a long
period of tiae. It was also observed that soae of the serological character-
istics of this organisa were altered. Concurrent with this study, a natural
spread of M. orale II in the aonkey colony was observed.

An SSPE virus antigen was grown to high titers in spinner cultures
and then concentrated 100 tiaes in a Spinco centrifuge. A aethod to
concentrate spinal fluid was developed by utilizing evaporation of fluid
through cellulose dialysis tubing. This concentrated antigen and spinal
fluid in a gel diffusion or counter-iaaunoelectrophoresis test was of
coaparable sensitivity to the C? or HI test on uaconcent rated spinal fluid
and was auch less coaplicated to perfora.

Significance to the Prograa of the Institute; A prograa devoted to
studying the effect of Mycoplawm in perinatal infections coapleaents the
virological studies currently being dene. This study and the support given
to other investigators aay aore accurately define the role of Myeoplasaa in
disease. The developaent of additional techniques for the serological
diagnosis of perinatal diseases which are as sensitive or aore sensitive
than current tests being used, and which by-pass the probleas of anti-
coapleaentary sera, will increase the usefulness of the stored perinatal

Proposed Course of the Project; Further studies are being done on
the association of Myeoplasaa in noraal deliveries. Study of the pa biogene-
sis of Myeoplasaa in aonkeys will be continued to see if an aniaal aodel can
be developed for septic and non-septic abortions and other perinatal infec-
tions. Atteapts to define the association of Myeoplasaa in neurological
diseases will be aade. Continued effort will be aade to apply these tech-
niques to other antigen-antibody coabinations in order to develop additional
serological methods to detect perinatal and neurological diseases.

Honors and Awards: None

82a

S«rlAl Ho. HDS (CF)-69 PK/ID 1732

PvblleatloBs:

Itaddea, D. L., Barton, R. E. and MeCullough, N. B.: SpontanooiM infection
in Ex-gomf roft guinea plga due to Cloatrldlan porf rlngena . Lab Aninal Care
20: 45^455, 1970.

Kaddan, D. L., Hlldarbxaadt, R. J., Monlf, 6. R. 6., London, V. T., Sorer,
J. L. and McCnllongh, H. B.: The laelatlon and Identification of Mycoplaana
fron Macaca aulatta. Lab Anlnal Care 20: 467-470, 1970.

Madden, D. L., HUderbrandt, R. J., Monlf, 6. R. 6., London, W. T.,
McCullottgh, N. B. and Sever, J. L.: The laolatlon and Identification of
Mycoplaaaa fron Cercoplthecoa aethlopa. Lab Anlnal Care 20: 471-473, 1970.

MBtthev, B. B., Dletnan, D. E., Madden, D. L., Sever, J. L., Mattl, A. and
Dodaen, W. E.: The poaalble abaence of Atiatralla antigen In 6/6 trana-
locatlon type of Down 'a a3mdrone. Lancet. (In preaa)

i

83V

Serial Wo. NDS (CF)-69 PR/id 1733

1. Perinatal Research Branch

2. -Section on Infectious Diseases

3. Bethesda, Maryland

PHS-WIH

Individual Project Report

July 1, 1970 through June 30, I97I

Project Title: Viral Diseases of the Nervous System

Previous Serial Number: Same

Principal Investigator: Dr. David A. Fuccillo

Other Investigators: Dr. J. L. Sever, PRB, NINDS
Dr. S. Baron, LVD, NXAID
Dr. W. T. London, PRB, NINDS

Cooperating Units: Laboratory of Viral Disease, NXAID

Georgetown University School of Medicine,

Washington, D.C., Dr. J. A. Bellanti
Harvard Medical School, Boston, Massachusetts,
Dr. L. Johnson

Man Years:

Total: 2.0
Professional: 1.0
Other: 1.0

Project Description:

Objectives: The main objective of this project is to establish the
clinical and biological significance of the two different strains of hearpes
simplex viioas (Type I, "oral" and Type 2, "genital") in the causation of
human disease including carcinoma of the cervix.

Methods Employed: The principal methods employed are: (l) the quanta!
cross -mi croneutralization test, by which type specific herpesvirus antibody is
identified; (2) mass serological surveys of materials from selected patients
with specific disease entities; (3) virus isolation, titration, and charac-
terization procedures; (h) interferon titration via plaque reduction assays.

Major Finding: Patients with carcinoma in situ of the uterine cervix
were demonstrated to have increased amounts of antibody to type 2 herpes-virus .
Antibody was also elevated in these patients two years before they developed
cancer (collaboration -with Dr. Johnson).

Significance to Biomedical Research and the Program of the Institute:
These investigations attempt to elucidate the pathogenesis of AnLral infections
of the adult and fetus using immunological and v:.rological techniques. Herpes

85 V

Serial No. NDS (CF)-69 PR/ID 1733

simplex virus has been one agent which has received particllar attention in
these studies, since it has significant neurotropic capabilities in tenns of
newborn and adiolt encephalitides. There is considerable spe ciiilation that
this virus may have latent, "slow" virus potential in relationship to chronic
diseases of humans, including carcinoma and central nervous system infection.
Investigation of the clinical and biological properties of the two strains of
herpes simplex virus have permitted more definitive establishment of such
capabilities. Furthermore, the therapeutic potential of artificial interferon
inducers as anti-viral agents was investigated with herpesviarus in animals and
one human, particularly in relationship to central nervous system Infections.

Proposed Course of Project: Expanded study of antibody and isolation of virus
from women with carcinoma in situ and carcinoma of cervix in the Collaborative
Study .

Honors and Awards : None

Publications:

Catalano, L. W. , Jr., Fuccillo, D. A. and Sever, J. L.: Piggy-Back Micro-
transfer Technique. Appl. Microbiol. l8: 109ij-1095, I969.

Catalano, L. W. Jr. and Sever, J. L.: Role of Viruses as Causes of Con-
genital Defects. Ann. Rev. Microbiol., In Press.

Catalano, L. W. Jr., Fuccillo, D. A., Traub, R., and Sever, J. L.:

Isolation of Rubella Virus from Placentas and Throat Cultures of Infants

in a Prospective Study Following the 196^-65 Epidemic, J. Pedlat.. In Press,

Fuccillo, D. A., Moder, F., Traub, R., Hensen, S. and Sever, J. L.:

Micro Indirect Hemagglutination Test for Cytomegalovirus. Appl. Microbiol.

21: 104-107, 1971.

86v

Serial No. NDS (CF)-70 PR/ID 1848

1. Parlaatal Rsaearch Branch

2. Section on Infection* Diseases

3. Bethesda, Maryland

PHS-RIH

Individual Project Report

July 1, 1970 through June 30 » 1971

Project Title: Delayed Hypersensitivity In Chronic Viral Diseases

Previous Serial Nunber: Sane

Principal Investigator: Dr. Earl B. Matthew

Other lavestlgators: Mrs. Mary Krasay
Dr. Donald Hanson
Dr. David A. FuecUlo

Cooperating Units: Hone

Man Tears:

Total: 1
Professional: 1/2
Other: 1/2

Project Description:

Objectives: To detenlne the role of delayed fajrpersensltlvlty
(cellular Inmnlty) In chronic viral Infections.

Methods Biployed: The nacrophage migration Inhibition test (MI) ,
lynphocyte transfonatlon and lynphotozln assay vere used In tiie study.

Major Findings: The nacrophage migration Inhibition test» lynphocytlc
transfomatlon and lynphotoxln assay vere all attenpted In studying the
nottse cytomegalovirus Infection. Hone vere found to give reliable results
and no correlation could be made. This vns due In most part to the type of
animal used, the mouse not developing good delayed hypersensitivity unless
an abnormal stimulus Is utilized such as Freund's adjuvant. Unfortunately,
this distorts the natural disease picture. The mouse CMV system Is vorth-
vhlle for more refined study vhen other techniques become available.

Development of the lymphocyte transformation test as a marker of
cellular Immunity in human virus infections has been a major effort In the
past year using various viral antigens. The studies of these In subacute
sclerosing panencephalitis and multiple sclerosis are In the last stages of

L

87^

S«rl«l Ho. IDS (CF)-7e PR/ID 1848

caaplatloA. Th* 9tmij of theso In patients with acnto lyaphocytlc lottkeala
is coBpleto.

Slgniflcnncc to tho Progr«« of the Inotltute; Prior to the develop-
■ent of the above testa, no good tests for aeasurlng cellular looimlty In
himans to rlral antigens existed. Antibody teats to viral antlgena have
been developed and used In this and other laboratories for nany years.
This represents a test for only part of the body's 1— iim response. The
developaeat of lyaphocyte transfonatlon Into a clinically uaable test for
virus Infection now allows both fons of the body's protective laensne
■echanlsns, fauaoral and cellular, to be teated alnultaneously .

Proposed Course of the Project; To complete the above mentioned
specific projects prior to July 1971 lAen the principal Investigator Is
tendnatlng his stay In NIHDS.

Honors and Awards: Hone

Publications: Heme.

S«rlal No. NDS (Cr)-70 PR/ID 1849

1. Perinatal Kcsearch Branch

2. Section en Infectious Diseases

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Chronic Infection with Cytoaegalovlruses In Man and Anlnals

Prerleus Serial Nuaber: Same

Principal latest Iga tor : Dr. Donald Benson, PRE, MINDS

Other Investigators: Mr. Leonard Moore, PRB, NINDS

Dr. John L. Sever, PRB, NINDS

Dr. David A. Fucclllo, PRB, NINDS

Dr. Earl B. Matthew, PRB, NINDS

Cooperating Units: NCI, Dr. Edward Henderson, Dr. Ronald Yankee,
Dr. Stuart Slegel, and Dr. Arthur Levine
Araed Forces Institute of Pathology, Dr. A. J. Strano

Man Tears:

Total: 2
Professional : 1
Other: 1

Project Description:

Objectives: Study chronic cytonegalovlrus infection, antibody levels,
virus excretion, lymphocyte response, disease in nan and alee. Relate
specific pathologic processes to antibody levels and virus excretion patterns.

Methods Enployed: Children with congenital CMV and patients with
leukemia with CMV infections are being followed for virus excretion, antibody
response and lynphocyte response. Techniques for CMV isolation have been
perfected. Antibody response is being followed with CF, HAI and neutralization
■ethods. Lymphocyte transformation is determined with trltiated thymidine.

Major Findings; Chronic infection of the tissues of the mouse have
been demonstrated. Results have shown that the response of the host to
infection depends on the anatomy of the organs Infected.

Eighty-seven children with acute lymphocytic or acute myelocytic
leukemia have been longitudinally studied for 3 to 18 months. Results
Indicate a correlation between clinical symptoms and rises In antibody titers
to CMV. There is no association between antibody titers and frequency of

Serial Ho. NDS (Cr)-70 PR/ ID 18A9

virus Isolation froa urine or throat or langth of virus excretion In these
children. Basic problea now Is to develop tests that discriminate between
CH7 rilssealnatlon and focal chronic Infection.

Significance to the Program of the Institute; Cytomegalovirus causes
congenital disease and death as well as significant Infection In patients
with nallgnandes. An understanding of this chronic Infection and the
laatune responses of the host should be of great value In the prevention and
treatment of these diseases.

Proposed Course of the Project: This Is a new study. We will
continue along the lines outlined above.

Honors and Awards: Hone

Publications:

Henson, D. and Sever, J. L.: Effects of Viruses on the Fetus. In Marcus, S.
L. and Marcus, C. C. (Ed.): Advances In Obstetrics and Gynecology.
Baltimore, Md., Williams and WUklns Co., Vol. 2, 1971. In press.

Stflal Ho. HDS (CF)-71 PR/ID 1903

1. Parlnatal lessarch Branch

2. Section on Infoctlooo Dlaeascs

3. Bothesda, Maryland

PHS-NIH

Individual Project Report

Jttly 1, 1970 throttgh Jane 30, 1971

Project Title: Inveatlgatlon of the Etiology and Effect of Serun and
Infectious Hei»atltls In the Perinatal Period.

Previous Serial Nunber: None

Principal Investigators: Dr. David L. Madden, PRB, HINDS

Dr. John L. Sever, PRB, HINDS
Dr. Dale E. Dletznan, PRB, NIHDS
Dr. Earl B. Matthev, PRB, HINDS

Other Investigators: Dr. Benedict Hagler

Cooperating Units: Lynchburg Training School and Hospital

Man Tears:

Total: 1.75
Professional: 1.75
Other: 0

Project Description:

Objectives: To detemlne the etiology of Australia antigen associated
(serua) and infectious hepatitis. To detemlne the relationship of hepatitis/
congenital jaundice and postnatal jaundice. To develop aninal Bodels and new
diagnostic tests for these diseases.

Methods E>n>loyed: A large epidenic of infectious hepatitis (600 cases)
occurred in the Lynchburg Training School and Hospital during the suaner of
1970. Over 5000 blood and 400 fecal sanples were collected during a 4-aonth
period. These sanples represent serial speclnens obtained prior to the
developnent of disease, at tine of acute disease and post-infection. A 6-
nonth follow-up has now been carried out. Australia antigen deteminations
were perfomed on 1200 patients. A controlled study of the effect of ganaa
globulin on this strain of infectious hepatitis was initiated. Serun and
fecal sanples will be cultured in a variety of tissue culture systens and
inoculated into experinental anlnals. Material fron patients with hepatitis
hospitalized at Stdmrban Hospital has been collected and is being utilized
in coaparison studies.

I

91V

S«rlAl Ho. HDS (CF)-71 PR/ID 1903

MajT Fladl«K«; TIm rasults of the opldoalologleal atudy uadortokoa
at tho LyncUbsrg TralBlag Scliool aad Hoopltal ladlcoto that the dlaoaao
waa not aaaoclatad with Aaatralla antlgan. It waa foand that the dlaeaac
vaa tranaBlttod fnm vard to ward by infected patient workera or through
contact vlth the patlenta. It appeara that thia outbreak la the largeat
nen-ceaawn •ovree of Infoctiewi preaently known. In thla outbreak, the
uae of ganaM globulin waa not effective unleaa given 14-21 daya before the
firat dlnlcnl cane. Thla waa partly related to the occurrence of aub-
cllnlcnl cnaea which una followed by waaa contaalaatlon of the ward. The
occurrence of clrcnlntlng Au antigen In chronic carrlcra did not alter the
courae of the Infectloua hepatltla. Prellnlnary reaulta auggeat that the
incidence of Au antigen in trlaoiqr 21 karyotype patlenta waa wnch higher
than in patlantn with Down 'a ayndroae dne to other karyotypea or in patlenta
with noraal karywtypea.

Significance to the Progran of the Inetltnte; The finding of an
etlologlcnl agent or an infectloua hepatltla aaaoclated antigen would be
an iaportant atep in the pranrentlon of thla dlaoaae which affecta aatveral
thowaand ialiwiduala each year. The naterlal collected fron the Lynchburg
outbreak la probably the largest accunulntion of pre-jaundlce» acute, and
convnlaeeent natenals erer collected fron a aingle outbreak. It will be
aactraoMly valuable for future reference.

Propoaed Courae of the Project; Attcnpta to define the role and
cause of aeruw and infections hepatitis will be continued. The tlaaue
culture atndy has Just been initiated. Attcnpta to detect specific antigen
and antibody in these feces and seruas will be nade.

Honora and Awarda: None

Publications : None

92 V

Project Title:

Serial No. NBS (CF)-63 PR/OC 1144

1. Perinatal Research Branch

2. Office of the Chief

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

An Instrument For The Conduct Of A Retrospective Study Of
Seizures, Cerebral Palsy, Mental Retardation and Other
Neurological and Sensory Disorders of Infancy and Childhood.

Previous Serial Number: Same

Principal Investigators: Z.A. Shakhashiri, M.D., PRE, NINES

Leonard V. Phelps, Clearwater, Florida
Glen S. Bartlett, M.D., Case Western Reserve Univ.,

Cleveland, Ohio

Other Investigators: Lenore Bajda, M.D., PRB, NINDS

John R. Day, M.D., Chevy Chase, Maryland

Blanche L. Vincent, S.N.O., Greensboro, North Carolina

Zula C. Meekham, B.S.N. , PRB, NINDS

Rose R. Tortorella, PRB, NINDS

Cooperating Units ;

Georgetown University Hospital, Retarded Children's Clinic,
Selected Maternity Hospitals and Physicians in Metropolitan
Washington.

Man Years:

Total:

Professional:

Other:

.35
.30
.05

Project Description:

Objectives: Design an instrument for the conduct of a retrospective study of
seizures, cerebral palsy, mental retardation and other neurological and sen-
sory disorders of infancy and childhood in order to test certain basic and
important hypotheses concerning the occurrence of neurological damage.

Methods employed: Recognized damaged outcomes of pregnancy, such as seizures,
diplegias, hemiplegias and choreoathetoids are to be studied and related to
defined perinatal or postnatal events. These outcomes were selected because
they were construed to be related to or manifestations of or involved in the
biological or psycho-sociological mechanism underlying the following hypoth-
eses: (1) anoxia, (2) toxic influences on the brain, (3) metabolic influences.
(4) trauma to the head, (5) infection of the brain, (6) dehydration of the
child, (7) genetic or familial patterns, and (8) socioeconomic status.

I

93V

Serial No. NDS (CF)-63 PR/OC U44

Proposed Course of the Pro.ject; The abstraction of the maternity and nursery
records and interviewing of the mothers have been completed. The physicians
of sib controls have been contacted cind information attesting to the health
status of these sibs has been obtained. All forms have been edited and coded,
and IBM cards have been keypimched. Partial analysis of the data has been
carried out. Comparison of findings in this project with findings in the
Collaborative Study will be carried out at a later time.

Preparation of the manuscript has been delayed until the end of FY '72.

Honors and Awards: None

Publications: None

94v

I

Serial No. NDS (CF)-63 PR/OC 1146

1. Perinatal Research Branch

2. Office of the Chief

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Public Health Implications Study of Perinatal Mortality in

the Collaborative Study and in the Collaborative Study Cities,

Previous Title: Revision and Expansion of Previous Project Entitled A

Commentary On The Appropriateness Of The Use Of Certain
Tabular Data, For Formulating Generalizations Concerning
Popvilations In The Same Cities As Those In Which The
Collaborative Study On Cerebral Palsy, Mental Retardation
And Other Neurological And Sensory Disorders Of Infancy
And Childhood Is Being Conducted.

Previous Serial Number: Same

Principal Investigators: Z.A. Shakhashiri, M.D., FRB, NINDS

Leonard V. Phelps, Clearwater, Florida
Glen S. Bartlett, M.D., Case Western Reserve Univ.,

Cleveland, Ohio

Other Investigators: None

Cooperating Units: The Census Bureau and the National Center for Health
Statistics cooperated in the furnishing of necessary
statistical infoimation for the United States and cities.
The respective state or city health departments are
providing natality and mortality data for the Project
cities.

Man Years:

Total: 0.50

Professional: 0.30

Other: 0.20

Project Description:

Obj ectives : To evaluate fetal and infant mortality of the Collaborative
Study population and of the cities from which that population is drawn, with
the aim of comparing the two popiolations, city by city, and institution by
institution, on mortality characteristics.

Methods employed: In addition to the data previously available from the
National Center for Health Statistics for perinatal events, detailed data on
natality and perinatal mortality are being sought for the study cities from

95v

Serial No. NBS (CF)-63 PR/OC 1146

either the state or city health departments, whichever has jurisdiction for
these records. The data include figures for livebirths, stillbirths, and
deaths under 24 hours, 1 day to 7 days, 8 days to 28 days and 1 month to
12 months, evaluated by birthweight, length of gestation, race and sex, and
plurality for the years 1959 through 1966. Corresponding data will be com-
piled by institution for the PRB study population. The state or city health
departments have been asked to furnish either completed tabulations or raw
data to be tabiilated (arrangements finalized) by the PRB Section on Data
Management and Retrieval.

Considerable effort has been and is being expended, in connection with the
Section on Data Management and Retrieval, PRB, and the Office of Biometry,
NIKDS, to create a usable data file of the external data being obtained in
connection with this study. The aim is to provide a file with more general
utility than the limited scope of this study. When such a file is created,
the information necessary to make use of the file will be made available to
interested persons in PRB.

Ma.jor Findings: Evaluation of birthweight and length of gestation data for
all core live births (first and subsequent pregnancies) reveals differences
between races and between sexes that are generally persistent among all the
institutions. Birthweights are lighter among non-whites than among whites,
and among females than among males. With vdiite males the heaviest, the order
of decline is next white female, then, at about the same weight, non-white
males, then non-white females. There is a particular excess of non-whites at
low birth weights (2500 grams or less). Length of gestation is shorter among
non-white than among whites by about 1 week, with an excess of both short-
gestation and long-gestation deliveries among non-whites. Length of gestation
is slightly shorter among males than among females in both races, but less
consistently so among whites.

Perinatal mortality has declined in the study population since its first year,
with a transient elevation in 1962. Group I mortality (fetal deaths 1001
grams and over plus deaths under 7 days of age per 1000 total births) declined
from 28.5 in 1959 to 17.6 in 1966 (mean 21.7) among whites and from 33.2 to
21.8 (mean 28.4) among non-whites. Group II mortality (fetal deaths 501 grams
and over plus deaths under 28 days per 100 total births) declined from 34.9
in 1959 to 19.1 in 1966 (mean 26.0) among whites and from 39.8 to 25.5 (mean
33.3) among non-whites. These trends tended to persist from institution to
institution, though to varying degrees.

Previous deadline could not be met because of delays in receipt of data from
the cities and in preparing these data from their various sources into a single
program for the computer.

It is anticipated that the file will be completed and the present phase of the
study reported by the end of June 1972.

Honors and Awards : None
Publications: None

Serial No. NDS (CF)-66 PR/OC 1378

1. Perinatal Research Branch

2. Office of the Chief

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Maternal Factors Affecting Birth Weight.

Previous Title: The Prediction of Birth Weight-Multivariate Analysis.

Previous Serial Number: Same

Principal Investigator: Heinz W. Berendes, M.B., PRB, NINDS

Other Investigators! W. Weiss, Office of Biometry, NINES

J. Deutschberger, Office of Biometry, NINDS*
E. Jackson, Office of Biometry, NINDS

Cooperating Units: Perinatal Research Branch, NINDS ■

Man Years:

Total: 0

Professional: 0

Other: 0

This study has been completed.

Honors and Awards: None

Publications: Weiss, W., and Jackson, E.G.: Maternal factors affecting

birth weight. In Perinatal Factors Affecting Himian Development.
Proceedings of the Special Session held during the Eight Meeting
of the PAHO Advisory Committee on Medical Research, Washington,
D.C., June 10, 1969. Washington, D.C., Pan American Health
Organization, Sci. Publ. No. 185, 1969, pp. 54-59.

* Deceased

97 V

Serial No. NBS (CF)-68 PR/OC 1618

1. Perinatal Research Branch

2. Office of the Chief

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

Jioly 1, 1970 through June 30, 1971

Project Title: The Effect of Labor on the Outcome of the Child.

Previous Serial Number: Same

Principal Investigators: Z.A. Shakhashiri, M.D., PRE, NINDS

W. Lawrence Holley, M.D., Children's Hosp., Akron,

Ohio
A. A. Lilien, M.D., Somerville, New Jersey

Other Investigators: None

Cooperating Units: Perinatal Research Branch, NINDS

Man Years:

Total: .08

Professional: .06

Other: .02

Project Description;

In order to single out the effect of labor, if any, on fetal growth and
development, a study is made of outcomes of normal pregnancies terminated
spontaneously, compared to outcomes of similar pregnancies terminated by
elective induction of labor and to outcomes of similar pregnancies terminated
by elective cesarean section.

Methods Employed: Project infants of normal pregnancies terminated as
described above will be compared for selected outcome variables such as
mortality, Apgar, bilirubin, mental and motor scores at eight months and I.Q.
at four years. All three groups will be controlled for race, birth weight
and gestation length.

Ma j or Findings : A search is under way to ascertain whether cases are available
in adequate numbers to make the design feasible. Additional non-Project cases
might be needed.

The scope of this Project has been referred to and is being undertaken by the
Ad Hoc Task Force on Labor and Delivery. See Serial No. NDS (CF)-71 PR/OB I90U.

Honors and Awards : None

Publications: None

99^

(

I

Serial No. NDS (CF) -66 PE/oB 1331

1. Perinatal Research Branch

2. Section on Obstetrics
3- Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 19T0 through June 30, 1971

Project Title: Obstetric Factors in Twin Pregnancies.

Previous Serial Number: Same

Principal Investigator: Rudolf F. Vollman, M. D. , PRB, WINDS

Other Investigators: Jose G. Marmol, M.D. , PRE, NIWDS
Irene B. Ross, PRE, HINDS

Cooperating Units: All institutions participating in the Collaborative Study

Man Years

Total: 0.2
Professional: 0.1
Others : 0.1

Project Description:

Objectives: The early diagnosis of a twin pregnancy remains still an
important problem on which depend the prenatal care and the management of
the labor and delivery. The study has two objectives:

1. To study the outcome of twin pregnancies in relation to the time
the diagnosis was first established.

2. To make a comparison of the obstetric problems presented by the first
versus the second twin and their effect upon the fetal outcome.

Methods-: With the help of a computer printout, an established case and card
file, and an additional revie-w of a current file on abortions and fetal deaths,
the twins delivered in the Collaborative Project through December 31, 1965,
have been identified. All case records were reviewed and additional informa-
tion or clarification was solicited from the collaborating hospitals as needed.
The mothers' medical, family and reproductive history, together with informa-
tion on the course of the study pregnancy, intercurrent diseases, drugs,
obstetric complications, labor and delivery, and outcome of pregnancy were
abstracted. These data have been used to prepare a set of tabulations for the
study. of the variables specified above. This study has been completed and
the variables tabulated. The preparation of the manuscript has been delayed

I

101 V

Serial No. WDS (CF)- 66 PR/OB 1331

due to a shortage of manpower.

Honors and Awards: None
Publications: None

102 V

Serial No. WDS (CF) -66 PR/oB 1333

1. Perinatal Research Branch

2. Section on Obstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Distribution of Abortions by Chronologic and Gynecologic Age
of the Gravida.

Previous Serial Number: Same

Principal Investigator: Rudolf F. Vollman, M.D. , PRB, NINDS

Other Investigators: Jose G. Marmol, M.D., PRE, NINDS
Irene B. Ross, PRB, NINDS

Cooperating Units: All institutions participating in the Collaborative Study

Man Years

Total:

0.3

Professional:

0.1

Others:

0.2

Project Description:

Objectives: Information is accumulating which demonstrates that endocrine
and morphologic conditions for optimal reproductive performance are reached
only several years after menarche. The conventional association of pregnancy
outcome by chronologic maternal age will be compared with the mother's
gynecologic age, based on the age at menarche.

Lfethods : For study pregnancies terminating in abortion, a number of important
maternal variables have been abstracted from the study record: race,
chronologic and gynecologic age, marital status, gravidity and parity, length
of the menstrual cycle, medical and obstetric complications of the study
pregnancy, duration and outcome of the study pregnancy. These variables
serve as controls in the analysis of chronologic versus gynecologic maternal
age.

Based on the preliminary findings, this study has been extended to include all
fetal deaths in the Perinatal Research program. Work is progressing very
slowly due to a shortage of manpower.

103 V

Serial Wo. NDS (CF) -66 PR/oB 1333

Honors and Awards: None
Publications: None

104^

Serial Wo. NDS (CF)-68 PR/oB 1623

1. Perinatal Research Branch

2. Section on Obstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Prenatal Drugs.

Previous Serial Number: Same

Principal Investigator: Alan L. Scriggins, M. D., University of Vermont

Hospitals

Other Investigators: Rudolf F. Vollman, M.D., PRE, WINDS

Seymour Katsh, Ph.D., University of Colorado Medical

Center
S. Barbara Katz, Office of Biometry, WINDS

Cooperating Units: All institutions participating in the Collaborative Study

The study has been completed.
Honors and Awards: None
Publications : None

105

Serial No. NDS (CF)-68 PR/oB 1625

1. Perinatal Research Branch

2. Section on Obstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Menstruation and Ovulation in the Monkey.

Previous Serial Number: Same

Principal Investigator: Rudolf F. Vollman, M.D. , PRE, NINDS

Other Investigators: Irene B. Ross, PRE, NINDS

Cooperating Units: Department of Embryology, Carnegie Institution of
Washington

The study has been discontinued.

^

107V

Serial No. NDS (CF)-TO PR/oB I85O

1. Perinatal Research Branch

2. Section on Obstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Toxemia in Pregnancy and Its Relationship to the Outcome of
Pregnancy.

Previous Serial Number: Same

Principal Investigator: Rudolf F. Vollman, M.D. , PRB, NINDS

Other Investigators: Leon C. Chesley, M.D., Downstate Medical Center,

Brooklyn, New York
Russell R. de Alvarez, M.D., Temple University School

of Medicine, Philadelphia, Pa.
R. Gordon Douglas, M.D. , Providence Lying- In Hospital,

Providence, R.I.
Emanuel A. Friedman, M.D. , Harvard University Medical

School, Boston, Mass.
Adolph H. Sellmann, M.D., Charity Hospital, New Orleans,

La.
Gilbert J. Vosburgh, M.D., Columbia University, College

of Physicians and Surgeons, New York, N.Y.

Cooperating Units: All institutions participating in the Collaborative
Project

Man Years

Total: 1.6
Professional: 0.8
Others: 0.8

Project Description:

The purpose of this study on toxemia of pregnancy is threefold:

1. To establish quantitative gradients for defined areas of the individual
signs of toxemia versus outcome of pregnancy.

2. To measure the relative weights of the clinical signs of toxemia --
hypertension, proteinuria, edema -- singly and combined, in their
contribution to perinatal mortality and birthweight.

109 V

Serial No. NTS (CF)-TO PR/oB 185O

3. To define a cohort of cases based on 1 and 2 as "pregnancies

complicated "by toxemia" in the Perinatal Research Study for further
research in association with the long-term development of the children.

Methods: It was demonstrated in a preliminary study of the clinical reports
of toxemia in the Study that the rates of toxemia vary widely between the
participating institutions and the 2 different Study forms used, even when the
populations of the gravidae were controlled for race, age, and parity. It was
concluded that this large variability in the incidence of toxemia must be
related to the use of different diagnostic criteria by the individual
collaborating hospitals. It was therefore decided to use the original (raw)
data of the clinical symptoms of toxemia and to analyze them individually and
in combination to establish a uniform cohort of cases with toxemia for the
study. The study will be carried out in cooperation with a special ad hoc
Task Force on Toxemia and will absorb all of the professional and none
professional time available.

Some preliminary findings were presented at the International Symposium on
Gestosis in Basel, Switzerland, April 25-26, 1969.

Honors and Awards: None

Publications: Vollman, R.F. : Rates of toxemia by age and parity. In
Rippmann, E.T. (Ed.): Die Spaetgestose (EPH-Gestose) .
Basel, Switzerland, Schwabe & Co. Verlag, 1970, pp. 338-3ij-2.

110 V

Serial No. WDS CCF)-TO PR/OB 185I

1. Perinatal Research Branch

2. Section on Ohstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1910 through June 30, I97I

Project Title: Low Eclampsia Rate in the Collaborative Project.

Previous Serial Number: Same

Principal Investigator: Jose G. Marmol, M.D. , PRE, NINDS

Other Investigators: None

Cooperating Units: None

The study has been completed.

Honors and Awards: None

Publications: Marmol, J.G. : Cases of eclampsia in the Collaborative Project.
Schweiz. Z. Gynaek. Geburtsh. 1: 169-I80, 1970

f

1

I

lliv

Serial No. WDS (CF)-71 PR/OB 190U

1. Perinatal Research Branch

2. Section on Obstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 3O;, 197I

Project Title: The Study of Labor and Delivery

Previous Serial Number: None

Principal Investigator: Rudolf F. Vollman, M.D., PRE, NINDS

Other Investigators: Emanuel A. Friedman, M.D. , Harvard Iftiiversity Medical

School, Boston, M^ss.
Luke Gillespie, M.D., Boston Lying-in Hospital, Boston,

Mass.
lyfe-rvin Green, M.D., Nev York Medical College, New York,

N.Y.
Esther Jackson, Office of Biometry, NINDS
Schuyler G. Kohl, M.D., State University of New York,

Downstate Medical Center, Brooklyn, N.Y.
Kenneth R. Niswander, M.D. , University of California at

Davis, Calif.
David Rubinstein, Office of Biometry, NINDS
Vincent Tricomi, M.D., Brooklyn- Cumberland Medical Center,

Brooklyn, N.Y.

Cooperating Units: All institutions participating in the Collaborative Project

Man Years

Total: 0.3
Professional: 0.2
Others: 0.1

Project Description:

This study has two objectives:

A. To deteiroine the influence of specific labor and delivery factors on
the fetus in terms of immediate outcome and later neurologic and
psychologic development .

B. To develop a reference standard of actuarial, obstetric, and labor and
delivery features that will result in optimal outcome.

113 V

Serial No. NDS (OF) -71 PR/OB 190U

Labor is usually reported in quantitative units of hours and minutes and is
conventionally divided into short, normal, and long labor by arbitrarily
selected time intervals. These intervals, in turn, have been associated with
certain complications of labor. FKEEDMAU has demonstrated in a long series
of publications that these associations do not necessarily reflect the under-
lying physiology or pathology of uterine activity. He devised a methodology
to quantify uterine activity and its deviations in relation to the phases of
labor, which are based upon the observed progress of labor in time. Possible
damage to the fetus is not necessarily dependent upon the total duration of
labor, but on the dyscoordination or changes in the du3ra.tion of any one or
combinations of the phases of labor.

The identification of gravidae with specified types of labor will produce
several standard cohorts that may be used by other Study sections and task
forces (Pediatric Neurology, Pathology, Physical Growth and Development etc.)
as variables for their respective special studies.

Msthodology:

A reference cohort of gravidae with "normal labors" is defined, for which the
specified outcome will be tabulated. This reference cohort will be compared
with cohorts of specified types of dysfunctional labor and their respective

outcomes.

Those gravidae with incomplete data on labor and delivery in the Study record
will be identified and their general characteristics (race, age, parity, sex
of fetus) and outcomes will be tabulated separately.

Honors and Awards: None
Publications: None

114 V

Serial No. NDS (CF)-Tl PR/OB 1905

1. Perinatal Research Branch

2. Section on Obstetrics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

Jxily 1, 1970 through June 30, 1971

Project Title: Reference Bibliography on Human Teratology.

Previous Serial Number: None

Principal Investigator: Rudolf F. Vollman, M.D. , PRB, NINDS

Other Investigators: None

Cooperating IBiits: Reference and Interlibrary Loan Sections of the NIH
Library
Reference Section of the National Library of Medicine

ysan Years

Total: 0.3
Professional: 0.2
Others: 0.1

Project Description:

The scientific publications on human teratology during the past 170 years
have been reviewed and approximately 4000 items have been selected, analyzed
gind coded by content. The criteria for selection were:

1. originality of observation reported

2. morphological classification of malformations

3. morphogenesis of malformations in association with embryological stages
k. hypothesis on etiology of malformations in time and teratogenic factors

5. experimental teratology

6. review articles.

Findings : The study of teratology was initially stimulated by the curiosity
to explore the range of the lusus naturae from which very early concepts on the
embryological potential were deducted. It thus contributed to the development
of systematic research in normal human embryology. From experimental embryology,
critical times, effective factors, their quantity and quality of interaction
with the formative process were Identified and have been extrapolated to explain
possible mechanisms in human teratology.

The manuscript is ready for publication.

1115 V

Serial No. WDS (CF)-Tl PE/oB I905

Honors and Awards: None
Publications: None

116 V

Serial No. NBS (CF)-63 PR/PN 1163

1. Perinatal Research Branch, NINES

2. Section on Pediatric Neurology

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
Jvily 1, 1970 through June 30, 1971

Project Title: An Investigation into the Relationship Between Congenital
Heart and Great Vessel Anomalies and Selected Factors as
Recorded in the Collaborative Perinatal Research Project.

Previous Serial Number: Same

Principal Investigator: Lenore Bajda, M.D., PRB, NINDS

Other Investigators: Heinz W. Berendes, M.D,, PRB, NINDS
John L. Sever, M.D., PRB, NINDS

Cooperating Units: Office of the Director, NHI

Man Years:

Total: 0

Professional: 0

Other: 0

Project Description; The primary objective of this study is to assess
relationships between certain maternal variables and congenital heart-great
vessel anomalies.

Additional objectives include investigating relationships between early
signs of abnormality and the existence of definitive congenital heart lesions
and determining the existence of congenital heart-vessel anomaly in conjunction
with mental retardation as recorded in the eight-^nonth psychological examina-
tion, the one-year summary records, four-year psychological examination, and
seven-year neurological and psychological examinations.

To date, maternal parameters analyzed included age of gravida, parity, prior
pregnancy outcome, prior and current health status, ABO blood group, current
smoking pattern, and viral antibody status.

Study data was obtained from Collaborative Study records received by PRB
from the onset of the Study (January 1959) through December 1964. These
records provided 112 live and stillbirth cardiac cases for study out of a
population pool of approximately 38,000. Analysis of an expanded Study
cohort through 1965, with a population pool of approximately 55,000 providing
additional cases, is underway in anticipation that the additional cases will
support earlier findings and perhaps provide further clues for identifying
etiological relationships.

I

117 V

Serial. No. NDS (Cr)-63 PR/PN 1163

There was a definite preponderance of mothers over 30 in the C-V Study group.
Controling for race, and removing cases with chromosomal aberrations, there
were more white mothers in the 30 and over age group than expected at the
.05 level. This trend is also noted among Negroes. There was a greater than
expected number of gravida with systemic disease complications and prior
pregnancy loss among the mothers of the cardiacs. A breakdown of these
factors for greater specificity is pending.

Because the number of patients with each specific cardiac abnormality was
small, specific associations between serological findings and clinical
observations were not possible, although several interesting trends were
noted. Analysis on the larger cohort is nearing completion.

A preliminary report on the 1964 cohort study was presented at the 1966
Annual Meeting of the Teratology Society.

The urgent priority of processing the entire Collaborative Project study data
has temporarily delayed further work on this study.

Honors and Awards: None

Publications: None

118 V

Serial No. NDS (CF)-63 PR/PN ll6k

1. Perinatal Research Branch, HINDS

2. Section on Pediatric Neurology

3. Bethesda, tferyland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Early Signs as Predictors of Death and Neurological Abnor-
mality Among Premature Infants Weighing 1000-2000 Grams

Previous Serial Number: Same and incorporating Serial No. NDS (CF)-69

PR/PN YJUO

Principal Investigator: Joseph S. Drage, M.D., PRE, NINDS

Other Investigators: Karin B. Nelson, M.D., PRE, NINDS
Heinz Berendes, M.D., PRE, NINDS

Cooperating Units: None

Wfen Years:

Total: .00

Professional: .00

Other: .00

Project Description: A group of I365 single livehom premature infants with
"birth-weights of 1000-2000 grams has been prospectively studied relating the
presence of specific neurological signs during the nursery period to neuro-
logical status at one year of age. Of the I365 infants, 917 were examined
at one year, 27^ died during the first year, and 173 were lost to follow-up.
Ihere were 201 neurologlcally abnormal Infants among the 917 examined at
one year. The first fovir PED-2's (Neonatal Pediatric Examinations) were
reviewed for the presence or absence of early signs, A positive early sign
was defined as the occurrence, on at least one examination of the specific
early sign being studied. Specific signs studied included cry, suck, palmar
grasp, traction response, Moro reflex, eye movement, muscle tone, local
convulsions, general convulsions, highest serum bilirubin. Coombs* test,
procedures of resuscitation, etc. Each of these signs was considered
separately, and those showing significant association with death and abnormal
outcome were then combined. When three signs were considered in combination,
the. more that were positive the greater the association of neurological
abnormality and death, and of neurological abnormality alone.

A preliminary manuscript has been prepared. Because of Task Force study
assignments, this project is in abeyance.

Honors and Awards: None

Publications : None

119 V

Serial No. EDS (CF)-66 PR/PN 1335

1. Perinatal Research Branch, NIHDS

2. Section on Pediatric Neurology

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I971

Project Title: Mortality and Morbidity Among Infants Weighing 1000-2000 Grams

Previous Serial Number: Same

Principal Investigator: Joseph S. Drage, M.D., PRE, NINDS

Other Investigators: Karin B. Nelson, M.D., PRE, NINDS
B.H. Williams, M.D., PRE, NINDS

Cooperating Units: None

Man Years:

Total:

.00

Professional:

.00

Other:

.00

Project Description: A group

of 1

A group of I36U liveborn infants, with birthweights
ranging from 1000-2000 grams, has been studied regarding outcome within the
first year of life. This group of I36U infants represents approximately
2^ of the group of 55^000 single live births from which they were drawn.

Within the group of I36U infants, 9IT were examined at one year of age, ajid
of these 201 were considered to have definite neurological abnormality. There
were 27^ deaths during the first year of life. Thus, U75 of the infants either
died during the first year of life or were considered neurologically abnormal
by examination at one year. There were 173 infants lost to follow-up and
this represents 12^ of the original I36U cases. Over 50^ of the deaths
occurred during the first 2k hours and over 90^ occurred during the first
28 days.

The 201 infants neurologically abnormal at one year were classified in the
following way: isolated motor retardation, diplegia, hemiplegia, quadri-
plegia, monoplegia, athetosis, motor retardation with neurological signs
insufficient for other diagnosis, and other (infants with neurological signs,
but not fitting a specific diagnostic category). Congenital malformations
were also monitored for this group.

Outcome at one year (death, abnormal, normal, and lost) was then tabulated
within each 100-gram birthweight interval. In general for I36U infants,
as the birthweight increased, the percent of deaths decreased. For infants

121V

Serial Noi NDS (CF)-66 PR/PN 1335

in jeopardy (dead or abnormal at one year), the same relationship held. The
percent of lost cases within each 100-gram birthwelght interval varied
slightly. Data has been obtained and is undergoing analysis

Because of Task Force study assignments, this project is in abeyance.

Honors and Awards: None

Publications: None

:it2a

Serial No. KDS (CF)-66 Pr/pN 1338

1. Perinatal Research Branch, KIKDS

2. Section on Pediatric Neurology

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title: The Association of Mental Sub normality with Head Circumfer-
ence, Congenital IVfelformations, and Other Conditions of the
Newborn Term Infant

Previous Serial Number: Same

Principal Investigator: Lenore Bajda, M.D., PRB, NINDS

Other Investigators: Karin B. Nelson, M.D., PRE, NINDS

Cooperating Units: Office of Biometry

Man Years:

Total: 0
Professional: 0
Other : 0

Project Description: The objective of this study was to determine the
relationship between head size and certain other physical features of the
Collaborative Study child noted shortly after birth, at the one-year examina-
tion and at k year upon completion of the psychological examination. The
project was in abeyance pending updating of the data file. Meanwhile
Dr. Nelson and Mr. Deutschberger have completed their study on "Head Size at
One Year as a Predictor of Four- Year IQ" (PR/PN I5O9) using a sample of the
Collaborative Study population including a partially selected pediatric group
of 9} 379 children. They concluded that there is approximately a ^O'fo chance
of an IQ of less than 80 at k years of age for the one-year male with a head
size less than k3 cm. and a one-year female with a head size of less than h2 cm.
Plans are under way to examine in detail the low and high head measure sample
of the Nelson-Deutschberger study for other factors which might account for the
correlation between head size and the four-year IQ values. Depending on the
results of this analysis, the original study proposal will then move either
forward with a larger sample, or terminate.

The -urgent priority of processing the entire Collaborative Project study data
has temporarily delayed further work on this study.

Honors and Awards: None

Publications : None

I

123

Serial No. EDS (CF)-68 PR/pN I628

1. Perinatal Research Branch, NIMDS

2. Section on Pediatric Neurology

3. Bethesda, IVfe-ryland

PHS - NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Effects of Prenatal Protein Deprivation on Behavior and
Brain Structure of- Mice

Previous Serial Number: Same

Principal Investigators: John Ao Churchill, M.D., PRB, NINDS

J. H. Carleton, M.D., Miami, Florida
W. lawrence Holley, M.D., Children's Hosp., Akron,

Ohio
Other Investigators: None

Cooperating Units: None

Man Years:

Total: 0
Professional: 0
Other 0

Ten^jorarily discontinued pending a time -when brain specimens can he processed
and examined. The brains are now in celloidin blocks. Dr. Jack Carleton,
Co- investigator, has left the Branch.

Honors and Awards: None

Publications : None

i

125

Serial Wo. HDS (CF)-68 PR/pn 1633

1. Perinatal Research Branch, KINDS

2. Section on Pediatric Neurology

3. Bethesda, tferyland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Neuropsychologic Outcome of Children with Retinal
Hemorrhages at Birth

Previous Serial Number: Same

Principal Investigators: Arthur L. Rosenhaum, M.D. , U.C.L.A., Los Angeles, Cal.

John A. Caiurchill, M.D., PRB, NINDS

Other Investigators: None

Cooperating Units: None

Man Years:

Total: .05

Professional: .05

Other: .00

Project Description: Retinal hemorrhages in the newborn occur in approximately
20^0 vertex births. Much work has been done to elucidate the etiology of
these hemorrhages, but little is known about their significance in terms of
long term follow-up. This study was designed to study the neuropsychologic
outcome of children with retinal hemorrhages, and also the relationship
between laterality of the hemorrhage and birth position.

Approximately 200 cases of retinal hemorrhage in the newborn were studied,
and matched with "non-hemorrhage" controls. These matched pairs were then
compared in rel^itionship to the following outcome variables: a) Birthweight,
b) Bay ley mental and motor scores given at 8 months of age, and c) The Binet
IQ given at k years of age.

The retinal hemorrhage cases were also reviewed in an attempt to analyze the
possibility of a relationship between birth position and the eye in which
the hemorrhage occurred.

This material is still in the process of being prepared for submission for
publication.

Honors and Awards: None

Publications: None

I

127 V

Serial Wo. UDS (crF)-69 Er/PW 17^+8

1. Perinatal Research Branch, MIKDS

2. Section on Pediatric Wetirology

3. Bethesda, lyfe-ryland

PHS-MH
Individiial Project Report
JvCLy 1, 1970 through Jime 30, I97I

Project Title: Neonatal Polycythemia: I. A Manifestation of Chronic
Injury During Distress

Previous Serial Number: Same

Principal Investigator: Miles M. Weinberger, M.D., Nat*l, Jewish Hosp.

Denver Colorado

Other Investigators: Arthur Oleinick, M.D., EPID, NCI

John A. Churchill, M.D., PRE, EIITOS

Cooperating Units: None

Ifen Years:

Total: .05

Professional: .05

Other: .00

Project Description: The objectives of the study were to study demographic
and maternal factors associated with neonatal polycythemia.

As part of the protocol dtiring the Collaborative Study on Cerebral Palsy,
capillary hematocrits were obtained as near to k8 hours as possible (generally
between 36 and 60 hours) on hhj6&3 newborns, or SGfo of all 77 and over were
identified, and controls, matched for institution and year of birth, race
sex, socioeconomic index, and presence or absence of U-year follow-up examina-
tion were selected randomly from all infants with i+6-hour hematocrits 50
through 65. Various demographic and maternal factors were identified on both
subjects and controls, and differences were statistically evaluated.

The subject cases (those with polycythemia manifested by hematocrits 77 and
over) were found to have been the product of longer gestation, but were
smaller in weight than the control population. There was an increase in
incidence of placental pathology and the placenta of the subject cases was
significantly lighter than the weights of the controls. One-minute and five-
minute Apgar scores were both lower in the subject cases and there was a
greater incidence of dysmaturity diagnosed in the subject cases. When compared
with the whole Collaborative Study population, infajits with polycythemia were
noted to come from lower socioeconomic groups. Data suggest that neonatal
polycythemia may be manifestation of chronic intrauterine distress.

L

129^

Serial No. KDS (CF)-69 PR/pn rjkS

This material was presented at a Perinatal Research Branch (nIKDS) seminar
m April 1970. Considerable progress has been made toward completing this
study for submission for publication. However, no additional work will be
done on it until after June 1971.

Honors and Awards: None

Publications : None

130 V

Serial No. EDS (CF)-69 PR/PN 17^9

1. Perinatal Research Branch, NIKDS

2. Section on Pediatric Neurology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Neonatal Polycythemia: II. Outcome

Previous Serial Number: Same

Principal Investigators: Miles M. Weinberger, M.D., Nat'l. Jewish Hosp.,

Denver, Colorado, John A. Churchill, M.D., PRE, NINDS

Other Investigators: Arthur Oleinick, M.D., EPID, NCI

Cooperating Units: None

Man Years:

Total: .05 ■
Professional: .05
Other: .00

Project Description: The objectives of the study were to determine polycythemia
in the neonatal period as an adverse effect on neuropsychological outcome.

See project report on Neonatal Polycythemia: I. A Manifestation of Chronic
Injury During Distress covering demographic and maternal factors. In addition,
the neuropsychological status of these infants was examined at various
intervals through four years of age.

Examining only the outcome of those infants without recognized congenital
malformation and gestational ages of 36 weeks and greater, it was found that
while these infants tended to be small for gestation age and have lower Apgar
scores, there was no significant difference in other findings in the newborn
period. There was also no difference in psychological scores at eight months.
At one year, there was no difference in abnormal neiorological findings between
subjects and control cases. At four years, however, the subjects did manifest
a statistically- significant lower score on the U-year Stanford-Binet examina-
tions than the controls. This difference was greatest among Negroes, especially
among the Negro females. White males did not manifest any difference in k-yeax
IQ.

Significance is not so much that some differences in U-year IQ were found among
some of the subgroups of the polycythemic Infants, but that contrary to the
expectations from the literatiore the differences between polycythemic infants
and non-polycythemic controls were so small.

131 V

Serial No. NDS (CF)-69 PR/PN 17^9

This material is still in the process of being prepared for submission for
publication. However, no additional work will be done on it until after
June, 1971.

Honors and Awards : None

Publications : None

132 V

Serial Wo. NDS (CF)-69 PR/PN 1750

1. Perinatal Re search Branch, KLWS

2. Section on Pediatric Neurology

3. Bethesda, Iferyland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title: Congenital IVferrow Dysfunction in Down's Syndrome

Previous Project Title: Abnormal Hematopoiesis in Newborns with Down's

Syndrome

Previous Serial Number: Same

Principal Investigators: Miles M. Weinberger, M.D., Nat'l Jewish Hosp.

Denver, Colorado
Arthur Oleinick, M.D., EPID, NCI

Other Investigators: None

Cooperating Units: None

Man Years:

Total: .05

Professional: .05

Other: .00

Study has been completed.

Honors and Awards : None

Publications : Weinberger, M.M. and Oleinick, A. : Congenital marrow

dysfunction in Down's syndrome. J. Pediat. 77: 273-279, 1970

133 V

Serial No. NDS (CF)-70 PR/PN I853

1. Perinatal Research Branchy KIKDS

2. Section on Pediatric Neiirology

3. Bethesda, tfe-ryland

PHS-KEH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: The Effects of Protein Malnutrition on Ontogeny of the Brain

Previous Serial Number: Same

Principal Investigator: John A. ChTirchill, M.D., PRE, NINDS

Other Investigators: None

Cooperating Units : None

Man Years:

Total: 0
Professional: 0
Other : 0

Project Description; A review was made of the literature pertaining to the
effect of protein-calorie malnutrition of the mother during pregnancy on
the nervous system structure and function of the offspring. From this
review, a critique was prepared and presented at the meeting of the American
Association for the Advancement of Science, held in Boston, Massachusetts
on December 26-31, 1969* Study has been completed.

Honors and Awards: None

Publications : None

I

135 V

Serial No. NDS (CF)-70 PR/PN 1854

1. Perinatal Research Branch, NINES

2. Section on Pediatric Neurology

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: The Etiology of Cerebral Palsy in Prematures.

Previous Serial Number: Same

Principal Investigator: John A. Churchill, M.D., PRE, NINES

Other Investigators: None

Cooperating Units: None

Man Years:

Total: .30

Professional: .25

Other: .05

Project Description: Theories of the etiology of cerebral palsy were studied
in a group of 1364 singleton liveborn infants weighing 2.0 Kg. or less at
birth, the group comprising all such small babies in the Collaborative Project
totalling 54,370 births. Subjects diagnosed as having cerebral palsy at
1 year of age numbered 45.

No support for the genetic theory was obtained by a study of siblings of the
spastics.

The anoxia theory gained no support in that Apgar and other indirect measures
of asphyxia differed insignificantly between the spastic and equally premature
non-spastic groups.

Hyperbilirubinemia did not appear to cause spastic diplegia since bilirubin
levels differed very little between spastics and non-spastics. Of 18 cases
with erythroblastosis, none had cerebral palsy.

Intrauterine blighting of the fetus was considered unlikely. Intrauterine
disturbances often produce growth retardation. None of the small-for-dates
infants had spastic diplegia. Furthennore, 5 spastics were born by "elective"
caesarean section, one being remarkably free from abnormal prenatal events.

Evidences of cranial injury or of factors conducive to birth trauma were no
more frequent in spastic than in other prematures. The spastic cases born
by caesarean section, where travmia should be minimized, weighed against the
traimia theory.

Cerebral hemorrhage as the cause of premature spastic diplegia was found to

137 V

Serial No. NDS (CF)-70 PR/PN 1854

desei*ve attention. Hematocrits obtained in the first few postpartim days
were significantly lower in spastic than in non-spastic prematures.
HemodJlution did not explain the observation. Others have shown that low
hematocrits occur in prematures who have cerebral hemorrhage.

Compelling and direct evidence that cerebral hemorrhage causes spastic
diplegia of prematurity cannot be derived from this study. However, the
results make clear a need to investigate hemorrhagic mechanisms in prematures.

This has been submitted for publication.

Honors and Awards: None

Publications: None

138 V

Serial No. NDS (GF)-68 PR/bS l6kO

1. Perinatal Research Branch

2. Section on Behavioral Sciences

3. Bethesda, Iferyland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: The relationship of demographic, perinatal and other

developmental characteristics to intellectual and motor
performance, of pre- school children.

Previous Serial Number: Same

Principal Investigator: Sarah H. Broman, Ph.D., PRB, NINDS

Other Investigators: Jaswant Khanna, Ph.D., University of Tennessee

Joseph Weber, formerly Office of Biometry, NINDS

Cooperating Units: University of Tennessee

Office of Biometry, NINDS

Man Years:

Total: .25
Professional: .20
Other: .05

Project Description:

30 characteristics of some 13,000 study children have been related to their
pass-fail performance on individual items in the psychology battery admin-
istered at four years of age. Preliminary analyses relating four demographic
variables to performance on the Stanford-Binet show that: (l) differences
among white, Negro and Puerto Rican children in IQ, socioeconomic index,
educational level of mother and proportion of males to females are highly
significant. Whites exceed the other two groups in mean IQ and socioeconomic
index. Differences among the groups in mean number of years of mother's
education are more evenly spaced with whites ^ Negroes p' Puerto Ricans.
Sex ratios in these groups follow a different order with Puerto Ricans :?'
whites 7 Negroes; (ll) Highly significant differences in percent pass by
ethnic group occur on all 28 of the Stanford Binet items examined. These
items cover the test levels from 2 years 6 months through 6 years. Among
whites percent pass is significantly higher than among the total group on
27 items. Among Negroes percent pass is significantly lower than the total
group on 26 items. Among Puerto-Ricans, percent pass is significantly lower
than the total group on I6 items, does not differ from the total group on
nine items and is higher on three items: (ill) Within both the white and
Negro groups, children who pass have a significantly higher socioeconomic
index than those who fail on all of the 28 Binet items. Among Puerto-
Ricans however these differences occur on only 10 items; (iV) Similarly,
among both whites and Negroes, educational level of mother is significantly

139 V

Serial No. KDS (CF)-68 PR/bS l6kO

higher for passers than for those who fail on all 28 items. However for
Puerto Ricans this occiirs on only five items; (v) Among whites, se:x;
differences in performance occur on 13 of the 28 Binet items with
proportionally more males than females failing these items. Among Negroes
sex differences in favor of femaJ.es occur on 15 items and in favor of males
on one item. Among Puerto Ricans sex differences are significant on only
five items, again with more males than females failing these items.

The content of the items on which all of the above differences occur is of
course highly relevant and is now being categorized. The majority of test
items administered at this age are verbal. However the following trends in
ethnic group differences have been noted. Using McNemar's classification
of items, verbal item differences are largest between white and Puerto
Ricans; memory item differences are largest between whites and Negroes, non-
verbal item differences are relatively small among all groups. Using five
factors identified by Valett, whites are consistently higher than both
Negroes and Puerto Ricans on groups of items representing the three factors
of general comprehension, vocabulary and verbal fluency, and judgement and
reasoning. For the factor of visual motor ability, whites consistently
exceed Negroes but only slightly exceed Puerto Ricans. For the last factor
considered, memory and concentration, whites are higher than Negroes and
slightly higher than Puerto Ricans. When Negro-Puerto Rican differences
are examined, the two groups differ very little on the factors of general
con^jrehension and memory and concentration. Puerto Ricans are higher on
the visual motor items and Negroes are higher on vocabulary and verbal
fluency and to a lesser extent on judgement and reasoning items.

Subseq^uent analyses will explore the relationship of perinatal, neur-ologi cal
and other psychological variables to performance at four years of age.

Honors and Awards: None

Publications: None

1^40 V

Serial No. M)S (GF)-69 PR/bS 1752

1. Perinatal Research Branch

2. Section on Behavioral Sciences

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title: Duration of Membrane Rupture and Psychological Outcome

Previous Serial Number: Same

Principal Investigator: Dr. Lee Willerman, PRB^NINDS

Other Investigator: Dr. John A. Churchill, PRB^NINDS

Cooperating Unit: Section on Pediatric Neirrology, PRB,NINDS

This project has been discontinued.

l4iv

Serial No. EDS (GF)-69 PR/BS 1753

1. Perinatal Research. Branch

2. Section on Behavioral Sciences

3 . Bethe sda, Maryland

PHS-NIH
Individual Project Report
July 1^ 1970 through June 30, 1971

Project Title: Growth and Intellectual Development of Children from
Consanguineous Matings

Previous Serial Number: Same

Principal Investigator: Dr. Lee Willerman, PRB_, NINDS

Other Investigators: Dr. Ntinos C, ]\fyrianthopoulos, PRE, NINDS

Dr. Alfred F. Naylor, PRB, NINDS

Cooperating Unit: Section on Epidemiology and Genetics, PRE, NINDS

Man Years:

Total: .10
Professional: ,05
Other: .05

Project Description:

Objectives; Consanguineous matings have been reported to be associated
with a higher Incidence of fetal and neonatal mortality as well as mental
retardation in the offspring. The present study will examine outcomes in
the offspring of l48 sets of parents in the Collaborative Study who are
second cousins or closer. Cases coded as consanguineous are now being
reviewed for validity of such codes in the Section on Epidemiology and
Genetics. Following this review, analysis of the outcome variables is
expected to proceed quickly.

Honors and Awards: None

Publications: None

143 V

Serial No. KDS (CF) -69 PR/BS YJ^k

1, Perinatal Research Branch.

2, Section on Behavioral Sciences

3, Bethesda, Maryland

PHS-WIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Growth and Intellectual Development of Children From
Interracial IVlatings

Previous Serial Number: Same

Principal Investigator: Dr. Lee Willerman, PRB, NINDS

Other Investigators: Dr. Alfred R, Naylor, PRB, NINDS

Dr. Ntinos G. jy^rrianthopoulos, PRE, NINDS
Dr. John A. GhurchiU., PRB, NINDS

Cooperating Units: Section on Epidemiology and Genetics, PRE, NINDS

Section on Pediatric Neurology, PRE, NINDS

Man Years:

Total: .40
Professional: .35
Other: .05

Project Description:

Objectives: Offspring from Negro-white matings (n=17l) were individually
matched to children from white-white and Negro-Negro matings on hospitaJ.
of birth, socioeconomic index, and marital status. Though not significantly
different from the controls in either length or weight at birth, by four
months of age the interracials were significantly smaller than the controls.
At one year the interracials were still smaller, but the magnitude of the
differences had diminished considerably. On psychological test performance
at eight months, no differences were observed. At four years the IQs of the
interracial children were significantly lower than the white controls, but
not significantly lower than the Negro controls. Birthweights and lengths
of the interracial children were intermediate to the larger white and
smaller Negro children regardless of the race of the interracial mother.
These results suggest a genetic basis to the findings that Negro children
weigh less and are shorter than white children at birth.

Honors and Awards: None

Publications:

Willerman, L, , Naylor, A. F., ly^nrianthopoulos, N. C. : Intellectual
Development of Children from Interracial Matings. Science, I97O,
Vol. 170, pp. 1329-1331.

145 V

Serial No. NDS (CF)-69 PR/BS I756

1. Perinatal Research. Branch.

2. Section on Behavioral Sciences

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Preschool Stuttering and Early Maternal Attitudes

Previous Serial Number: Same

Principal Investigator: Raymond H. Holden, Ed.D. , Brown University

Other Investigator: Paul J. LaBenz, Sc.D., PRB, NINDS .

Cooperating Unit: Child Development Study, Brown University

Man Years:

Total: .35
Professional: ,30
Other: .05

Project Description:

This study evaluates the relationship of dysfluency in speech noted in
children at age three years, and attitudes of their mothers as reported on
the Parental Attitude Research Instrument (PARl) when the children were
8 months old, A sample of 1100 children in the Providence Child Development
Study was screened for speech, language and hearing problems at three years
of age. Twenty six children were identified as dysfluent. Two random
sanrples consisting of 26 cases showing no dysfluency and 2.6 cases rated as
unknown were selected from their respective groups within the sample.

No significant differences were found between the dysfluent and normal
groups with regard to total PARI scores, socioeconomic status or mean IQ
(Revised Stanford-Binet Form L-M, I960) at age \ years. The group rated
unknown was I9 points lower than both of the other groups in mean IQ.
Review of their records indicated that they could not be rated for dysfluency
because they were uncooperative, ixntestable and/or mentally retarded.
Follow-up on partial samples of the dysfluent group revealed a marked drop
in mean verbal IQ at age 7 years, and a high proportion of abnormal
articulation ratings at age 8 years. This study will be completed when
additional follow-up data is available.

Honors and Awards : None

Publications: None

1^7a

Serial No. NDS (CF)-69 PR/BS 1757

1. Perinatal Research Branch.

2. Section on Behavioral Sciences

3. Bethesda, Maryland

PHS-WIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Social Class and Outcome in the Neurologically Abnormal
Infant

Previous Serial Mumber: Same

Principal Investigator: Raymond H, Holden, Ed.D., Brown University

Providence, Rhode Island

Other Investigator: Lee Willerman, Ph.D., PRE, NINDS

Cooperating Unit: Brown University, Providence, Rhode Island

This study has been conjjleted.

Honors and Awards : None

Publications:

Holden, R. H. and Willerman, L. : Social class and outcome in the
neiirologically abnormal child. In Trapp, E. P. (ed.) Readings on the
Exceptional Child. 2nd Ed. App let on-Century- Crofts, I97I. In Press.

1^4-9 ■

Serial Wo. NDS (CF)-69 PR/BS 175 8

1. Perinatal Research Branch

2. Section on Behavioral Sciences

3. Bethesda, Maryland

PHS-WIH
Individual Project Report
July 1, 1970 through June 30, I97I

Project Title: Neonatal, Ethnic and Social Class Factors in Infant and
Pre- school Test Performance

Previous Project Title: Matiirity at Birth, Mental and Motor Performance at
Eight MDnths and Intelligence Quotient at Four Years

Previous Serial Number: Same

Principal Investigator: Sarah H. Etroman, Ph.D., PRB, WINDS

Other Investigator: Heinz W. Berendes, M.D., PRB, WINDS

Cooperating Unit: Office of the Chief, PRB, WINDS

Man Years :

Total:

.25

Professional:

.20

Other:

.05

Objectives: This study examined the effects of two measures of maturity at
birth on mental and motor development at eight months and on a subsequent
measure of intelligence at four years in two large samples of children who
are being followed longitudinally in the Collaborative Study in the National
Institute of Neurological Diseases and Stroke. Birthweight and gestational
age were related to Bayley Mental and Motor scores at eight months and to
Stanford- Binet IQs at four years within the context of ethnic, social class
and sex classifications. The relationship of Bayley scores to Binet IQ was
also examined. A multiple linear regression model was used.

The major findings were as follows:

1. Among infants tested at eight months (W=31,678) there is a highly
significant association between the group of predictor variables of ethnicity
(white and Wegro) sex, social class, (four levels of maternal education),
birthweight and gestational age and the criterion of Bayley mental score.
However only seven percent of the variance was explained in the criterion
measiire. The most "useful" predictor was birthweight, followed by gestational
age.

2. A similar pattern resulted from the analysis of Bayley motor scores. Nine
percent of the variance was explained and again birthweight, followed by
gestational age, was the best predictor.

3. Among children tested at four years (N=16,658) there is a highly signi-

151 V

Serial No. NDS ((IF)-69 PR/BS 175 8

ficaxLt association between the group of predictor variables of ethnicity,
sex, social class, birthweight, gestational age, eight month mental scores
and eight month motor scores and the criterion of Stanford- Bine t IQ. Twenty-
eight percent of the variance was explained in the criterion measure. The
most useful predictor was ethnicity followed by social class (maternal
education) .

The inplications of these findings and other derived from analyses of the
relationship of the predictors to the criterion measures within the four
separate ethnic-sex groups are discussed in the context of the characteristics
of the samples studied. This stxidy has been completed.

Honors and Awards: None

Publications : None

152V

Serial Wo. WDS (CF)-70 PR/BS 1856

1. Perinatal Research Branch.

2. Section on Behavioral Sciences

3. Bethesda, Maryland

PHS-NIH
Individual Project Report
July 1, 1970 through June 30, 1971

Project Title: Biosocial Influences on Euxnan Development

Previous Serial Number: Same

Principal Investigator: Dr. Lee Willerman, PRB, NIHDS

Other Investigator: None

Cooperating Unit: None

This paper has heen completed and was presented at the k-Jth Annual Meeting
of the American Orthopsychiatric Association, San Francisco, California,
March 23-26, 1970. The abstract has been published in the American
Journal of Orthopsychiatry, kO: 324, I97I.

Honors and Awards: None

Publications : None

153v

Serial No. NDS (CF)-70 PR/BS I857

1. Perinatal Research Branch

2. Section on Behavioral Sciences

3. Bethesda, Maryland

PHS-WIH
Individual Project Report
July 1^ 1970 through June 30, 1971

Project Title: The G-enetics of Intellectual and Motor Performance

Previous Serial Number: Same

Principal Investigator: Sarah H, Broman, Ph.D., PRB, NINDS

Other Investigators: N. C. l\fyrianthopoulos, Ph.D., PRE, NINDS

Lee Willerman, Ph.D., PRE, NINDS
V. L. Anderson, Ph.D., University of Minnesota
Paul Nichols, Ph.D., University of Minnesota

Cooperating Units: Section on Epidemiology and Genetics

The Dight Institute for Human Genetics, University
of Minnesota

Man Years:

Total: .25
Professional: .20
Other: .05

Project Description:

Objectives: The objective of the study is to assess the contribution of
genetics to the variance of behavioral measures, particularly intellectual
and motor performance, by the calculation of heritability in twins. The
results of the psychological tests given at ages eight months, four years
and seven years have been analyzed for all twins of known zygosity and for
all sibs from first and second study pregnancies. It is also planned to
analyze these data for an appropriate sample of unrelated child pairs. As
a physical parallel, the heritabilities of height, weight and head cir-
cumference at several age levels have been computed.

Some selected findings are as follows: (l) heritability of IQ at age four
does not appear to be lower than in older populations; (2) the between family
variance componant of the four-year IQ test was nearly twice as large in the
white population as in the Negro population suggesting that heritability
is lower in Negroes than in whites; (3) an analysis of varisjice of weight
and height measured at four years revealed that, unlike IQ, the variance
Goniponants were not different in whites and Negroes; (k) at seven years,
a high correlation (••86) was found between 12 subtests social class loading
and the Negro-white differences on the test, while no relationship was
found between Negro-white differences and heritabilities of the subtests

155 V

Serial No.' NDS (CF)-70 PR/BS I857

after controlling for the subtests' social class loading; (5) the
discrepancy in IQ between twins and singletons decreased in both races
from four to seven years.

Analyses of these data are continuing.

Honors and Awards: None

Publications: None

156 V

Serial No.NDS (CF)-63 PR/EG 11T4
lo Perinatal Research Branch
2o Section on Epidemiology

and Genetics
3. Bethesda^ Maryland

PHS
Individual Project Report
July 1, 1970 through June 30, 19T1

Project Title: Birthweight in Relation to Selected Socioeconomic Variables

Previous Serial Number: Same

Principal Investigator: Dr. N.C. Myrianthopoulos, PRB, imroS

Other Investigators : Dr. Joshua Lederberg, Stanford University

Cooperating Units : None

Man Years :

Total: .10
Professional: .05
Other: .05

Project Description:

Objectives: This is a continuation of a study to relate birthweight to
socioeconomic and medical variables. The results of the first part of the
study have already been published (Ann. Htm. Genet. 31:71-83, 196t)<>

Proposed course : In the second paii: the plan is to compare children in the
100th percentile of birthweight with children in the 50th percentile of
birthweight and children of diabetic mothers, in teims of several socioeconomic,
biological and medical variables. A magnetic tape with all the necessary data
has been prepared. Analysis is done at Stanford University under Dr.
Lederberg's direction. No further progress has been made in this study.

Honors and Awards: None

Publications : None

157 V

Serial No. EDS (CF)-63 PR/EG 1175

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda^ Maryland

PHS-WIH

Individual Project Report

July 1, 1970 through Jvme 30, 1971

Project Title: Detennination of the Zygosity of Twins Bom to Mothers
in the Collahorative Study

Previous Serial Number: Same

Principal Investigator: Dr. N.C. Myrianthopoulos, PKB, NINDS

Other Investigators : None

Cooperating Units : All Institutions participating in the Collaborative Study

Man Years:

Total: .15
Professional: .15
Other: ,00

Project Description:

Objectives; This is a continuing project to deteimine the zygosity and
epidemiologic characteristics of twins bom to Study mothers.

Methods employed: Twin zygosity is determined by comparison of sex,
placentation, blood groups, and finger and palm prints. This information is
forvarded by all Institutions to the Section on Epidemiology and Genetics
where it is classified and analyzed by special methods.

Major findings: In addition to the findings reported in last year's report,
an analysis has been made of respiratory distress syndrome in the twins.
Respiratory distress syndrome occurred in 77 of 1130 livebom twins (or 1 in
15) and appears to be from 5 to 9 times more frequent than in singletons.
This increase cannot be entirely accounted for by the higher prematurity rate
of the twins over singletons. In 46 twin pairs with at least one affected,
there was a significantly higher concordance rate among MZ than DZ pairs,
suggesting that genetic factors are of some etiologic importance in this disease.

An analysis of congenital malfonnations in the twins is also being made.

Proposed course: To study all twins in terms of a variety of genetic and
socioeconomic variables,

159 V

Serial No. WDS (CF)-63 PR/EG 1175

Honors and Awards: None

Publications: Myrianthopoulos, N.C.: A survey of twins in the population of
a prospective collaborative study. Acta Genet, ^fed. Gemellol.
19: 15-23, 1970.

Myrianthopoulos^ N.C.: An epidemiologic survey of twins in a
large, prospectively studied population. Amer. J» Hum. Genet.
22: 611-629, 1970.

IVtyrianthopoulos, N.C.: Respiratory distress syndrome in twins.
Acta Genet. Med. Gemellol., in press.

I6OV

Serial No. -WDS (CF)-63 PR/eG IITT

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 19T1

Project Title: Genetic and Socioeconomic Factors in Early and Late Fetal
Death

Pluvious Serial Number: Same

Principal Investigator: Dr. N.C, Myrianthopoulos, PRE, NIKDS

Other Investigators: Esther Jackson, OB, NINDS

Cooperating Units : None

Man Years:

Total: .10
Professional: .10
Other: .00

Project Description:

Objectives: To detennine what effect do genetic and socioeconomic factors
have on pregnancy wastage and if a distinction can be made etiologically
between early and late fetal deaths.

Methodology and major findings: Discriminant analysis of 1,877 early and late
fetal deaths with respect to kO medical, genetic and socioeconomic variables,
shows that toxemia, anemia and complications of pregnancy are good discriminants
in both whites and Negroes; prior pregnancies and prior pregnancy wastage in
whites only; and socioeconomic status in Negroes only. Knowledge of sex of
study child improves the power of the discriminant function.

When the sample is restricted to fetal deaths of mothers who registered in the
first trimester the results are not appreciably changed. This project is now
completed.

Honors and Awards : None

Publications: None

161 V

Serial No. WDS (CF)-63 PR/eG llSij-

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Population Dynamics of Tay-Sachs Disease and other
Sphingolipidoses

Previous Serial Number: Same

Principal Investigator: Dr. N.C, Myrianthopoulos, PRE, NINDS

Other Investigators : Dr. Stanley Aronson, Brown University

Cooperating Units : None

Man Years :

Total: .15
Professional: .15
Other: .00

Project Description:

Objectives: This is a continuation of a study to determine whether differ-
ential fertility favoring the Jewish heterozygote can acco\«it for the 100 fold
higher frequency of Tay-Sachs disease and the gene responsible for it among
the Jewish compared with non-Jewish population in the U.S. Evidence for
selective advantage of the heterozygote was found in the first part of the
study (Am. Jo Hum. Genet. 18:313-327; I966).

Proposed coiorse: The plan is to investigate whether or not such advantage
can be demonstrated in other sphingolipidoses, to study the demography of
these disorders and to deteimine whether resistance to tuberculosis or some
other infectious disease confers a selective advantage to the heterozygote.

Methodology: About ^,000 cases of Jewish immigrant patients who had TB have
been collected from the records of the American Chronic Disease Center,
Denver, Colorado. The distribution of places of origin of these patients has
been compared with that of ancestors of Tay-Sachs disease and a negative rank
correlation has been found suggesting that the frequency of TB had been low
in areas of high concentrations of Tay-Sachs disease, and vice versa. A
more sophisticated analysis is now being perfonned.

163 V

Serial No.' NDS (CF)-63 PR/EG 1184

Honors and Awards: None
Publications: None

1614-v

Serial No. NDS (CF)-65 PR/EG 12T4
1« Perinatal Research Branch

2. Section on Epidemiology

ajid Genetics

3. Be the s da, Maryland

PHS-Wm

Individual Project Report

July 1, 1970 through June 30;, 1971

Project Title: Genetic Bases of Neonatal Reflexes

Previous Serial Numher: Same

Principal Investigator: Dr. A.F, Naylor, PRB, NINDS

Other Investigators: Dr. N.C. Myrianthopoiolos, PRB, KINDS

Cooperating Units : None

Man Years :

Total: ,05
Professional: ,05
Other: .00

Project Description:

Objectives: To investigate the validity of regarding the suck, rooting and
other neonatal reflexes as genetic entities.

Major findings: An initial set of cases retrieved for absence of one or more
of these reflexes was reviewed and seemed to have high frequencies of various
kinds of trauma whose base line frequencies were unknown.

Proposed course: To place limits on the frequencies of losses of suck, root-
ing, palmar grasp, plantar grasp and Moro reflexes because of mutation or
segregation at gene loci specifically affecting manifestation of these reflexes.

The completion of the (condensed) Variable Data File makes practical the reacti-
vation of this project along proper lines. Base populations can be selected
for general health, especially neurological, and frequencies of isolated absence
or weakness of single neurological signs can be tested. Active work on this
project will be undertaken when most current tasks have been carried out.

Honors and Awards: None

Publications : None

165 V

Serial No. ' EDS (CF)-65 PR/EG 12J6
lo Perinatal Research Branch

2, Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Sequential Aspects of Occurrence of Spontaneous Abortion in
Family Histories

Previous Serial Number: Same

Principal Investigator: Dr. A.F, Naylor, PRB, NIKDS

Other Investigators: Dr. Dorothy Warburton, College of Physicians and Surgeons
of Colianbia University

Cooperating Units : None

Man Years:

Total: ,20
Professional: ,20
Other: .00

Project Description:

Objectives: To relate the risk of spontaneous abortion to maternal age and
prior reproductive experience. A special point under investigation is whether
apparent age effects are explicable by a tendency for intrinsic habitual aborters
to remain in the reproductive population longer in attempts to compensate for
unsuccessful pregnancies. Also conditional risks have been estimated.

Proposed course: Although certain portions of the data analysis support a ma-
ternal aging explanation for part of the trend in abortion risk, a reconsideration
of the original tab\ilar outputs shows that a very substantial additional parity
exists. An existing manuscript must be completely redrafted to reflect this
change in interpretation.

Honors and Awards: None

Publications : None

167 V

Serial Wo. KDS (CF)-6t PE/eG 15 10

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-NIH

Individvial Project Report

July 1, 1970 through Jvne 30, 1971

Project Title : The Study of Maternal Effects in the Production of
Congenital Malfoimations

Previous Serial WumlDer: Same

Principal Investigator: Dr. W.C, Myrianthopoulos, PRB, NUTOS

Other Investigators: None

Cooperating

Units :

None

Man Years:

Total :

.10

Professional:

.10

Other:

.00

Project Description

:

Objectives:

To deteimin(

To deteimine the extent to which maternal factors are involved
in the production of congenital malfoimations, and to single out those mal-
foimations and conditions of the newborn in which the role of maternal factors,
genetic and environmental, appears to he deciding.

Methods employed: The GEN 5-8 was used to obtain a history of outcome of
prior pregnancies, in families in which half-siblings are present. Study
pregnancies were also included.

Ma j or findings : In all, 152 cases were identified and these were screened
for occurrence of congenital malfoimations and other conditions among
children by different fathers. Six conditions were found to occur in high
frequency among half sibs: Rh trouble, convulsions, congenital heart
disease, club foot, mental retardation and Polydactyly.

Analysis of the distribution of these abnoimalitie s in first and second sib-
ships of the same family showed that in the case of seizures and mental re-
tardation the experience in the first sibship was significantly different
from that in the second, indicating a genetic etiology; in the case of congenital
heart defects and club foot the experience seemed to be the same in both
sibships, indicating that environmental maternal factors predominate.

169 V

Serial No,' NDS (CF)-6t PR/EG I5IO

Additional cases have now became available and a more complete analysis is
being made. The study is in progress.

Honors and Awards: None

Publications : None

170'

Serial No. WDS (CF)-6t PR/eG 1514

1. Perinatal Research Branch

2, Section on Epidemiology

ajid Genetics
3» Bathes da, Marylemd

PHS-NIH

Individual Project Report

July 1, 19T0 through June 30, 1971

Project Title: Record Linkage of Relatives Registered in the Collaborative
Study

Previous Serial Number: Same

Principal Investigator: Dr. A.F. Nay lor, PRB, NINDS

Other Investigators: Dr. N.C, Myrianthqpoulos, PRB, NIKDS

Cooperating Units : None

Man Years:

Total: .60
Professional: ,15
Other: .45

Project Description:

Objectives; To identify all relatives of gravidae registered in the Collaborative
Study.

Methodology: Abstracting of reports of relatives has been completed and the
infoimation key-punched into card images on magnetic tape.

Proposed course: In its current foim the file lists, case by case, the NIRDS
number of reporter and reported relatives and relationship code. The file must
be reprocessed into pairs so that the relative making the report is retrievable
when the reported relative is an index case; also transitive pairing — linkage
of two relatives who are both reported by one woman — must be established.

Honors and Awards: None

Publications: None

171 V

Serial No. M)S (cr)-6T PR/eG 1515

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-WIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Rh Hemolytic Disease in Negro and White Infants

Previous Serial Number: Same

Principal Investigator: Dr. A.F. Naylor, PRB, NINDS

Other Investigators: None

Cooperating Units : None

Man Years:

Total: .01
Professional: ,01
Other: .00

Project Description:

Objectives: To confiim a report that high Rh antibody levels have smaller
morbid effects in Negro than in White babies, althotigh this is not true for
ABO ajitibodies.

Major findings: Preliminary and indirect confiimation has been obtained, from
a small data sample under study in this Section, for reports in the literature
that high Rh ajitibody titers are not as highly associated with serious morbidity
in Negroes as in whites.

Proposed course: The existence of the Variable Data File will make possible
the easy execution of the required data processing.

Honors and Awards: None

Publications : None

173 V

Serial No. NDS (CF)-6t PR/eG 1516

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Be the s da, Maryland

PHS-WIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Size of Placenta in Relation to Mother-Fetus Antigenic
Difference

Previous Serial Wumher: Same

Principal Investigators : Dr. Dorothy Warburton, College of Physicians and

Surgeons of Columbia University
Dr. A. P.. Naylor, PRB, WHTOS

Other Investigators: Dr. G. Nicholas Rogentine, Immunology Branch, NCI

Dr. Robert Cefalo, Obstetrics, National Naval Medical Center
Mrs. Lois Dienes, PRB, NINDS
Mrs. Jean Nemore, PRB, NINDS

Cooperating Units: Obstetrics Department, National Naval Medical Center

Man Years :

Total: 1.20
Professional: .80
Other: ,kO

Project Description:

Objectives: To investigate the hypothesis published by Billington in 196k,
that sensitization of the mother during pregnancy against paternal antigens
leads to non-pathological placental hypertrophy and increased birthweight in
succeeding pregnancies.

Major findings: Analysis of a large sample of study data has given convincing
support to the hypothesis. A paper reporting the evidence has been published.

Proposed course : The arrangements for a laboratory study, described in the
19^-70 Annual Report have been active and 25 maternal sera have been tested
against paternal leucocytes. No attempt at analysis will be made until many
more cases have accumulated.

Honors and Awards : None

r.

1

175 V

Serial Wo. WS (CF)-67 PR/EG I516

PubU-cations: War-burton, D. and Naylor, A.F. : The effect of parity on
placental weight and birth weight : An immunological
phenomenon? A report of the Collaborative Study of Cerebral
Palsy. Amer. J. Hum. Genet. 23: ^1-5^^, 1971«

176 V

Serial No, NDS (CF)-68 PR/eG iGkG
1, Perinatal Research Branch
2o Section on Epidemiology

and Genetics
3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30^ 1971

Project Title: Study of the Physical Growth of Children in the Collaborative
Study

Previous Serial Number: Same

Principal Investigator: Dr. Glen S. Bartlett, Case Western Reserve University

Other Investigators: None

Cooperating Units: Office of Biometry, NINDS

This project has been transferred to the jurisdiction of the task force on
Physical Growth and Development,

177 V

Serial No. NDS (CF)-68 PR/eG l6kj

1, Perinatal Research Branch

2, Section on Epidemiology

and Genetics
3o Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 thro\:igh June 30, 1971

Project Title: Development and Evaluation of an Index of Reproductive
Perfoimance for the Puipose of Identifying High Risk
Pregnancy Suspects

Previous Serial Number: Same

Principal Investigator: Dr. Glen S, Bartlett, Case Western Reserve University

Other Investigators: None

Cooperating Units : None

This project has been temporarily discontinued and will be held in abeyance.

179 V

Serial No. EDS (cf)-68 PR/eG l6kQ

1. Peilnatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Farther Investigation of the Socioeconomic Index as a
Descriptive and Predictive Instrument

Previous Serial Wumher: Same

Principal Investigators: Dr. Glen S, Bartlett, Case Western University

Dr. W.C. Myrianthopoulos, PRE, NBTOS

Other Investigators : None

Cooperating Units : None

Man Years :

Total: .00
Professional: .00
Other: .00

Project Description:

Objectives: The socioeconomic status score or socioeconomic index, developed
by the Bureau of the Census for use with the I960 Census, has been used with
good results in this Section to describe the Collaborative Study population,
and by others to describe other populations. This study is designed to ex-
plore mathematically the three component parts of the index — education and
occupation of the head of household, and family income — in order to assess
their relative contributions to the index; to evaluate the ability of the
index to encompass other socioeconomic variables not used in deriving the
inde ; and to investigate whether or not the index, in addition to being
descidptive, can also be used as a reliable predictive instrument.

Methodology; Linear discriminant function and multiple regression analysis
will be used to deteimine appropriate weights to be applied to the component
factors, and correlation coefficients will be obtained between the index and
its components, and external variables.

Current status : A working paper proposing three models has been written.
Data from first study pregnancy and from the T-year follow-up examination will
be used to test these models. No further progress has been made in this project.

181 V

Honors and Awards : None
Publications : None

Serial No. NDS (CF)-68 PR/eg l6k8

l82^

Seri-al No. -NDS (CF)-71 PR/eG I906

1. Perinatal Research Branch

2, Section on Bpidemiology

and Genetics
3o Bethesda, Maryland

PHS-NIH

Individual Project Report

jTily 1, 19T0 through June 30, 1971

Project Title: Blood group effects in mother and offspring

Previous Serial Number: None

Principal Investigators: Dr. N.C. Myrianthopoulos, PRE, NINDS

Dr. Be mice Cohen, Johns Hopkins University

Other Investigators: None

Cooperating Units: None

Man Years :

Total: .10
Professional: ,10
Other: .00

Project Description:

Objectives : To deteimine the influence, if any, of maternal and/or infant ABO
and/or Rh types iipon maternal manifestations and fetal-infant- child survivor-
ship, morbidity and development.

Proposed course and methodology: A comprehensive investigation will be made
of the blood group relationships, including possible deleterious consequences
or beneficial effects in mothers and offspring of various maternal ABO and/or
Rh types and ABO-Rh combinations both per se, and grouped as potentially
"compatible" and "incompatible" combinations of mother and conceptus. Maternal
and offspring effects will be examined in terms of immediate and acute effects
ajid in terms of long-range effects in mother and offspring, through develop-
mental milestones at 7 years of age. Comparisons will be made by institution,
race, age of mother, previous reproductive history, complications of pregnancy,
n mnrunoglobulin levels, and socioeconomic status.

Honors and Awards: None

Publi cati ens : None

I

•183 V

Serial No. NDS (CF)-TI PR/eG I907

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Be the s da, Maryland

PHS-KIH

Individual Project Report

July 1, 1970 through June 30, 19T1

Project Title: Epidemiologic and genetic study of congenital malfoimations

Previous Serial Nixnber: None

Principal Investigator: Dr. N.C, Myrianthopoulos, PRB, NUTDS

Other Investigators: Dr. J. Drage, PRB, NIKDS

Dr. C.S. Chung, University of Hawaii

Cooperating Units : None

Man Years :

Total: .20
Professional: .15
Other: .05

Project Description;

Objectives: To study the epidemiology of all congenital malfoimations in
Collaborative Study children at birth ajid one year; to test the hypothesis
of quasi -continuous distribution as an inheidtance mechanism for most mal-
foimations; to assess the genetic load due to congenital malfoimations; to
test the etiologic significance of several medical, genetic and socioeconomic
factors in the occurrence of selected malfoimations; and to explore more fully
previo-us findings suggesting a relationship of diabetes in the mother and the
occurrence of congenital heart disease in the offspring.

Proposed course emd methodology: The plan is to draw a definitive list of con-
genital malfoimations and develop a file of all Collaborative Study children
who had a malfoimatlon between birth and one year of age. Consanguinity data
will be used to assess the genetic load and aji extension of Falconer's method
will be employed for the quasi-continuous distribution analysis. A prospective
analysis will be used to test the etiologic significance of medical, genetic
and socioeconomic factors.

Honors and Awards : None

Publications: None

185.

Serial No. KDS (CF)-TI PR/EG I908

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: A chromosomal study of children in the Collaborative Study

Previous Serial Nimiber: None

Principal Investigators: Dr. N.C. Myriajithopotilos, PRE, WHTDS

Dr. H. Lubs, University of Colorado

Other Investigators: None

Cooperating Units: Boston Children's Hospital Medical Center, Children's

Hospital University of Buffalo, University of Tennessee,
Children's Hospital of Philadelphia and University of
Oregon Medical School

Man Years :

Total: .20
Professional: «15
Other: ,05

Project Description:

Objectives: To study the epidemiology of chromosomal aberrations in approxi-
mately 10,000 Study children at age 7 years; and to relate major and minor
chromosomal deviants to growth, mental and motor development and neurological
status of these children.

Proposed course and methodology: A blood sample will be taken from children at
the five collaborating institutions, during their 7-year examination. Two cells
will be immediately analyzed; ten cells will be analyzed when chromosomal anom-
alies are found or when the children have some mental or motor anomaly or a
congenital malfonnation. All technical work will be done in accordance with
standardized techniques and new techniques such as fluorescence staining, will
be employed. All measurements and analysis will be done at Denver with the aid
of an automatic chromosome analyzer. A second phase of the study will be con-
cerned with the chromosomal-clinical correlations.

Honors and Awards: None

Publications : None

187 V

Serial No. EDS (CF)-TI PR/eG 1909

1, Perinatal Research Branch

2, Section on Epidemiology

and Genetics

3, Bethesda, Maryland

PHS-NIH

Individual Project Report

Jiily 1, 1970 through June 30, 1971

Project Title: Continuation of study of twins horn in the Collaborative Study
beyond the age of seven years

Previous Serial Number: None

Principal Investigator: Dr. N.C, Myrianthopoulos, PRB, NINDS

Other Investigators: None

Cooperating Units: All Collaborating Institutions

Man Years:

Total: .10
Professional: ,05
Other: .05

Project Description:

Objectives: To follow all living twins and their siblings bom in the Collab-
orative Study to age 15 years in order to study their physical and mental
development through puberty.

Proposed course and methodology: All living twins, including unlike-sexed twins,
will be typed with a broader spectrum of genetic markers to insure utmost ac-
cxrracy in zygosity and to obtain a distribution of these markers in twins and
their Study siblings for further studies. The twins and their Study siblings
will be given ein annual physical examination which will include physical and
anthropometric measurements, and more extensive examinations with appropidate
IQ and behavioral tests at ages 12 and 15 years. Investigations will include
growth and development of genetically identical and environmentally related
individuals; neurological evaluation, especially in regard to minimal abnor-
malities; differences in mental development; determination of the occurrence
of a third type of twin; genetic factors in induction of puberty; and host
resistance to disease.

Honors and Awards: None

Publications: None

189^

I

Serial No. NDS (CF)-Tl PR/EG 1910

1. Perinatal Research Branch

2. Section on Epidemiology

and Genetics

3. Bethesda, Maiylemd

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Genetics of Obstetric Variables eind the Role of Maternal

Factors in the Deteimination of Intelligence and Neurological
. Perfoimance

Previous Serial Number: None

Principal Investigators : Dr. A.F,' Nayldr, PRE, NINDS

Dr. N.C, MyrianthopoTilos, PRE, NINDS

Other Investigators : Dr. D. Warburton, College of Physicians and Surgeons of
Colxmbia University

Cooperating Units: Department of Human Genetics eind Development, Columbia
University

Man Years:

Total: .50
Professional: .50
Other: ,00

Project Description;

Objectives: To analyze the variation of obstetric and gynecological factors
into heia table and non-heritable components.

Proposed course: The genetic linkage file necessary for fall realization of
the objectives, is near but short of completion. Specialized programs necesssiry
for genetic analyses are being procured from the University of Hawaii but will
need modification for use on NIH canputers. An expanded version of the Variable
Data File incorporating needed additional information has been created. This
expanded file will be used immediately (l) to investigate biases in obstetric
variation among women reporting relatives in the Study compared to those not
reporting relatives (2) to investigate obstetric heritability at a "primary"
level to identify conditions which tend to recur in repeat Study pregnancies.

Honors and Awards: None

Publications : None

I9IV

Serial No. NDS (CF)-65 PR/P 1278

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Biologic Pattern Data Processing

Previous Serial Number: Same

Principal Investigators: Lewis E. Lipkin, M. D.

Russell A. Kirsch

Other Investigators: Peter F. Lemkin
Philip G. Stein
Stanley E. Shackney, M, D.

Cooperating Units: Artificial Intelligence Group
National Bureau of Standards

Office of Associate Scientific Director

Clinical Trials

National Cancer Institute

PDP-10 Group

Division of Computer Research and Technology

Man Years

Total: 8.2
Professional: 5.0
Others: 3.2

Project Description:

This project has been extensively reviewed during the past calendar year.
The first review in June 1970 was by the NCI directorate with uniformly
favorable comment. The second more formal review in December 1970 by an
external ad hoc committee, chaired by Dr. Kenneth Earle, Chief of
Neuropathology of AFIP, concluded: "The committee was unanimous in its
support of this important project..."

1. Substantive Projects: There have been three major biologic projects

begun during the reporting period. These are totally dependent on both
the concepts and facilities of the emerging image processing facility
in the Section on Pathology.

193 V

Serial No. NDS (CF)-65 PR/P 1278

a. The Quantitative Characterization of Chroma to lysis: Serial
histologic sections of hypoglossal neurons of rabbits rendered
chromatolytic by nerve section one or two weeks prior to sacrifice
by perfusion fixation and the corresponding contralateral controls
are selected by the observer for automatic scanning. The tapes so
generated are displayed on the PDP-lO's 340 display, where under
the biologist's visual control and by means of a Graf con stylus and
tablet cells and their components are segregated. The computer is
providing data on each cell and its components that are measures

of Nissl distance dispersion and/or loss, nuclear and nucleolar
swelling, nuclear eccentricity, change in cell size, change in cell
shape and changes in patterns of distributions of cytoplasmic RNA.
At this writing, additional subprograms are being written to enable
us to determine quantitative measures of satellitosis in terms of
such parameters as the probability of encountering a glial cell as
a function of distance from the cytoplasmic boundary of the cell
body. Since the magnetic tapes of the scanned images are preserved,
these results may be obtained without rescanning. Indeed, as
additional algorithms are developed, they may be applied to exactly
the same cell images without further microscopy.

b. Identification of Marrow and Peripheral Blood Cells in Radio-
autographs: This ability is one of the remaining requirements for
specifying the automatic grain counting device requested by the
National Cancer Institute. A library of scans is now under
construction, so that following leads such as those provided by
Mendelsohn and Prewitt, this major requirement for successful
completion may be met. Of particular interest is the very great
utility of image masks generable from multiple wavelength scans.

c. Concurrent DNA Content and Synthesis Rate Determinations in
Embryonic Neural Tube and Transplanted Lymphomas: The image
processing facility provides the unique ability to determine not
only the rate of DNA synthesis in a cell population, but also to
determine the quantity of DNA/cell in a Feulgen- stained autoradio-
graph. This ability will provide data which will provide informa-
tion which hitherto has only been available from laboriously
examined time-lapse studies and labeled mitosis counts. Material
presently being examined are transplants of rodent leukemia. In
process, however, are embryonic neural tissues with the expectation
of obtaining more quantitative information concerning mantle-
marginal relationships.

In addition to the foregoing specific projects, extensive experiments
involving three-dimensional reconstructions, automatic morphologic
analysis, etc. were performed. Some indications of their scope is
given under "4. Computer Analysis and Image Processing Results."

194 V

Serial No. NDS (CF)-65 PR/P 1278

Apparatus Design, Construction and Procurement: Significant design and
construction attention has gone into improving the scanning microscope,
while keeping it concurrently usable for productive scanning. Some of
the modifications have been discovered to be necessary as a consequence
of having used the system. For example, a new rigid table was
constructed and procurement was initiated for anti-vibration mounts
to cure a building vibration induced problem that was discovered during
the course of scanning at high magnification. In preparing to use the
scanner, photometric calibration of the photo-multiplier and associated
circuitry is necessary. To facilitate this, new gain controls were
constructed and low density filters were obtained which can be used to
simulate the optical density of biological materials to be scanned, so
as to facilitate calibration of the scanner over the full optical
density range. A standard procedure was also adopted for recording on
the magnetic tape along with an image that has been scanned, the
corresponding values of the reference beam photomultiplier, so as to
make completely available for subsequent analysis the information about
incident illumination necessary to refine the calibration of the
photometric part of the scanner. For point by point rather than raster
scanning, the scan axis drivers and associated motor controls necessary
for driving the scanner mirrors were constructed. Immediately after
scanning it is often useful to have a crude visual presentation of the
image as scanned. A beam storage tube display was obtained and
connected so as to provide both intensity versus x and y type of display
as well as the previous contouragram form of display to exhibit the
complete scan output for a single scan.

During this period the LINC-8 and the PDP-10 computers were connected
together in a flexible manner to enable the interchange not only of scan
data but of programs in both directions from the LINC-8 and hence from
the microscope to the PDP-10 and conversely. The RF08/RS08 disk system
was connected to the LINC-8 and is currently in operation. An A.B. Dick
Videojet Line Printer was connected to the LINC-8 and has made it
possible to produce full resolution displays of scanned images through
the use of about 16 gray levels of intensity encoded with suitable print
characters.

A large disk pack drive installed on the PDP-10 has a five-million word
capacity and is used exclusively by the image processing project.

A major effort has been going on in the design and construction of an
optical bench to provide the capabilities of the present scanner with
more flexibility for the addition of new scanner options. The basic
optical bench mount which had previously been obtained had several
pieces of equipment added to it . A vertical stage to take the place of
the present optical microscope stage was constructed and installed, as
was a mount for the use of the monochromator and a mount and changing
system for mounting microscope objectives on the bench. Investigation
was initiated for procurement of a new scanner to be used on the optical
bench and a microscope tube length correction system was designed.

195V

Serial No. NDS (CF)-65 PR/P 1278

Special image measuring devices for use on the optical bench were
obtained to be used in precise calibration of the optical system when
it is in operation on the optical bench. The design of a system for
distance measurement using interferometric techniques on the optical
bench was begun.

3. Programs: Many programs have been written during this period for the
LINC-8 and the PDP-10 which served to facilitate experiments using the
scanning microscope and to remove the burden from the user of having to
write detailed programs. Notable among such efforts is the construction
of successive versions of the MOSS microscope operating supervising
system which enables complex sequences of scanning and processing on
the LINC-8 to be initiated by the user with a minimal amount of
programming. Prior to obtaining special line printer equipment for
producing images, a program was written to use the NBS Stromberg-Carlson
Graphic Printer to make digitized printouts of scanned images. This
served not only the purpose of visual representation of scans, but
enabled us to gain assurance that the scan data was as intended when
the microscope was set up.

During this period the PDP-10 has become the main production device for
processing scanned data. Largely because of fine cooperation from
personnel in DCRT, the PDP-10 has proved powerful and has generally
been satisfactorily available. The total use of the PDP-10 has, at
various times, consumed over 20 hours of CPU time per month for NBS
users alone engaged in program writing and development as well as
production computing. At peak usages, NINDS and NCI users on this
project have exceeded this total. This total of 40-45 hours of CPU
time/month can probably be taken as a reasonable projected upper bound
for computer use during the next year.

During this period several specialized and some general purpose tools
have been constructed. A version of the morphological analysis procedure
for decomposing scanned images that had previously been converted from
the Q-32 computer to the PDP-10 was modified to use the full resolution
of the data obtained from the microscope scanner. Another somewhat
more general version was constructed in the LISP language to provide
the full generality over all possible scanned images of decomposition
methods. Of the generalized tools, a systematic effort enabled the
construction on the PDP-10 system of general purpose image processing
facility more powerful than the corresponding one previously built on
the Q-32 computer. This facility is written to operate in the LISP
language and hence to provide the high level language processing
capability of LISP with the corresponding efficiency of lower level code
written either in assembly language or in FORTRAN through the use of a
FORTRAN-LISP interface written by S. Bryan (DCRT). The list of major
functions available in the LISP image processing facility is as follows:

a. A routine for reading whole or partial scans from magnetic tape into
memory performing optical thresholding on the data. This routine also
allows the reference photomultiplier data optionally to be read
instead of the photometric information from the scan data.

196 V

Serial No. NDS (CF)-65 PR/P 1278

b. A routine for variously thresholding image arrays to convert them
to binary form.

c. Very high speed non-buffered input/output for swapping images from
disk to core. Because a typical image involves 16,000 words of
PDP-10 core storage, it is important to be able to swap such images
into and out of memory at high speeds to avoid unconscionably large
amounts of processing time.

d. A FORTRAN bit processing package to operate within the LISP
language .

e. A high speed assembly language program for computing the derivative
of a scanned image. The derivative data contains information about
boundaries and gradients and hence is used in morphological analysis
extensively.

f . An assembly language program to perform Boolean operations on binary
arrays considered as masks.

g. A high speed assembly language program to extract contiguous regions
(blobs) from binary arrays obtained from images.

h. A high speed assembly language program for counting the number of
elements in a binary array.

i. A FORTRAN routine for circumscribing an extracted object in an
image with a circumscribing rectangle.

j. A general purpose statistical routine for taking an image which has
been previously thresholded into binary form, and then computing
centers of gravity, mean densities, and various types of moment
computations, as well as obtaining minimum and maximum values of
intensity range for an image.

All the above programs are available at the LISP language level and
hence can be called with considerable ease by higher-level programs
without the need for the user to descend to lower level coding. The
above general and special purpose programs operate in a unique
environment involving both specialized equipment and users with
particular kinds of processing problems. Despite the specialized
nature of this environment, a systematic documentation effort is being
made to document these programs so as to describe the operations and
enable their duplication.

197 V

Serial No. NDS (CF)-65 PR/P 1278

Computer Analysis and ImaRe Processing Results: The large amount of
PDP-10 computer time used during this period is mostly attributable to
the various analyses that have been run on the computer using scan data
from various biological sources. We mention below some of these
results that have been obtained:

a. Various fluctuations in scanner illumination and line voltages
result in non-uniformities in an otherwise uniform section of a
scan. To analyze the degree of non-uniformity, a statistical
analysis was made on the values of the reference beam on the
microscope scanner. It was found that this illumination is extremely
stable, so much so that for a reasonable level of illumination,
variations in illumination level were small compared to those
attributable to quantization errors and other sources of scanning
noise.

b. In order to test the resolution of the scanner for scanning silver
grains on blood cells, the Stromberg-Carlson 4020 Display was used
to resynthesize several scanned images of labeled blood cells. In
the resynthesized image, the silver grains appeared clearly resolved
and hence the data necessary for location and recognition can be
considered to be present in the output of the scanner.

c. An analysis of a small region from one of these cells containing
multiple silver grains was made using the morphological analysis
algorithm. The result was a proper identification of the location
of silver grains and an indication that if this procedure were used
over a larger image, the silver grains would properly be identified.

d. Several experiments were performed using optical serial section data.
In the first, a specimen with small polystyrene spheres was sectioned
optically and then the image was reconstructed in three dimensions
within the computer. Since the spheres were only a few microns in
diameter, the reconstructed image, rather than being that of a
sphere, was the diffraction pattern that would be expected from such
small, largely phase, objects. It was also possible, using the
three-dimensional reconstruction inside the computer, to rotate the
image through 90 degrees and hence produce the effects of having
sectioned perpendicular to the plane of the optical slide. This
technique can be directly extended to other natural objects and in
particular can be extended to larger objects than these test
specimens.

e. A set of serial sections of two adjacent nerve cells was made which
exhibited the appropriate low depth-of-f ield for the optics that
were used and enabled in a resynthesized set of images the easy
visual identification of the vertical sectioning process.

198.

Serial No. NDS (CF)-65 PR/P 1278

f. An extensive analysis was made of several optical sections of monkey
vagal nuclei neurons. These analyses consisted of a complete
morphological decomposition using the morphological analysis algorithm
along with the determination of optical density statistics for the
decomposed objects. Using these statistics then, the original
objects were resynthesized and the computer's reconstruction was
visually compared with the original scan data. The strong
correspondence between the original and the resynthesized images
suggested that the morphological analysis decomposition procedure
preserves the morphological structure of images for purposes of
recognition of cells and their components.

g. A program was written by Bryan (DCRT) to enable manual specification
of the decomposition procedure analogously to the automatic method
used in the morphological analyzer. This program is currently being
worked on to make its operation more precise and more convenient.
Both versions are being employed particularly in the quantitative
analysis of chromatolysis.

Honors and Awards: None

Publications: Lipkin, L.E.: Resolution, scale change and information
distortion. In Lipkin, B.S., and Rosenfeld, A. (Eds.):
Picture Processing and Psychopictorics . New York, N.Y.,
Academic Press, 1970, pp. 203-215.

Lemkin, P.P.: A patch to focal-w to use the LINC-8 display.
DECUS Program Library. FOCAL8-58: 1-3, 1969.

Lemkin, P.F.: OCTMON: Octal monitor for the PDP-8. DECUS
Program Library. 8-298: 1-7, 1969.

Stein, P.G.: Image -analyzing microscopes. Anal. Chem.
42: 103A-105A, 1970.

Shapiro, H., Biryan, S., Lipkin, L.E«, Stein, P.G., and
Lemkin, P.P.: Computer aided microspectrophotometry of
biological specimens. This paper has been accepted for
publication in Exp. Cell Res.

Kirsch, R.A.: Computer determination of the constituent
structure of biological images. This paper has been accepted
for publication in Comput. Biomed. Res.

199 V

Serial No. NDS (CF)-68 PR/P 1650

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Twin Placentation in Relation to Zygosity

Previous Serial Number: Same

Principal Investigators: Toshio Fujikura, M. D.

Luz A. Froehlich, M. D.

Other Investigators: Ntinos Myrianthopoulos, M. D.

Cooperating Units: All Collaborating Institutions

Man Years

Total: 0.0
Professional: 0.0
Others: 0.0

Project Description:

Twins totalling 569 pairs were studied in relation to type of twin
placentation. Separate diamniotic-dichorionic placentas were rarely
associated with monozygosity. This and the fact that monochorionic
placentas are all monozygotic make examination of twin placentas
extremely useful in forecasting zygosity. Relatively heavy infants
as well as twins with large intrapair birth-weight differences were
common in the separate diamniotic-dichorionic group, suggesting
independent intrauterine growth of co-twins. The converse was true in
twins with fused diamniotic-dichorionic placentas, who also had the lowest
death rates. Perinatal death rate was the same in whites (14.77o) and
Negroes (14.37o), highest in male-male twin pairs, but lowest in male-female
pairs in Negroes and female-female pairs in whites. Compared to singleton
deaths, the frequency of congenital malformations was not higher in twin
deaths, but the types of malformations found in monochorionic deaths were
often multiple and lethal. This study has been completed.

Honors and Awards: None

Publications: Fujikura, T. , and Froehlich, L.A.: Twin placentation
and zygosity. Obstet. Gynec. 37: 34-43, Jan. 1971.

201 V

Serial .No. NDS (CF)-69 PR/P 1763

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Kidney Malformations in Fetuses of A x C Line 9935 Rats

Previous Serial Number: Same

Principal Investigators: Toshio Fujikura, M. D.

Other Investigators: None

Cooperating Units: None

Man Years

Total: 0.9
Professional: 0.5
Others: 0.4

Project Description:

Fifty A X C 9935 strain inbred rat litters were examined near term; 88/315
fetuses (27.97o) showed renal malformations (renal agenesis and hydroneph-
rosis). Unilateral renal agenesis occurred more often on the right side
(11.57o) than the left (3.67o), especially in males. Renal agenesis was
always associated with absence or hypoplasia of a uterine horn or vas deferens
and epididymis on the corresponding side. However, the ovaries or testes
were present and intact. Hydronephrosis was seen more often on the left
side (11.47o) than the right (3.57o). There was no significant sex difference
in the frequency of renal agenesis and hydronephrosis. In hydronephrosis
the ureteropelvic junction was patent, and hydroureter always accompanied
moderate hydronephrosis. Atresia near the vesicoureteral junction was
considered as a cause of hydronephrosis. There was a close morphogenet ic
relation between renal agenesis and hydronephrosis. This study has been
completed.

Honors and Awards: None

Publications: Fujikura, T.: Kidney malformations in fetuses of A x C
line 9935 rats. Teratology 3: 245-249, Aug. 1970.

203^

Serial No. NDS (CF)-69 PR/P 1764

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Placental Study of Abortion Material
(Obtained by an induced abortion)

Previous Serial Number: Same

Principal Investigators: Toshio Fujikura, M. D.

Other Investigators: Hideo Nishimura, M. D.
Kenichiro Ezaki, M. D.

Cooperating Units: Department of Anatomy
Kyoto University
Kyoto , Japan

Man Years

Total: 0.1
Professional: 0.1
Others: 0.0

Project Description:

The placental materials of 114 induced abortions (normal group) and
87 spontaneous abortions (abnormal group) were histologically compared.
Degenerative hydropic and necrotic villi were commonly found in the
chorion laeve of the normal group even in early gestation, but not in the
chorion frondosum. The sampling site in the chorionic sac was important
for histological diagnosis. The mean number of chorionic villi in the
abnormal group was not different from that of the normal group within each
gestational interval. This indicates that abnormal villous growth may not
be the primary factor responsible for spontaneous abortion. Up to 14 weeks
gestation, active syncytial proliferation was present but no substantial
increase of chorionic villi was found. Beyond this inactive stage the
villous number increased rapidly and conversely syncytial sprouts decreased
in numbers. The mechanism of syncytial proliferation was discussed in
relation to other prenatal conditions. Study completed.

Honors and Awards: None

Publications: Fujikura, T., Wishimura,. H., Ezaki, K. : Placental study of
abortion material (obtained by an induced abortion).
Amer. J. Obstet. Gynec, in press

205V

Serial- No. NDS (CF)-69 PR/P 1765

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: The Interrelationship Between Selected Congenital
Malformations and Major Pathologic Findings

Previous Serial Number: Same

Principal Investigators: Luz A. Froehlich, M. D.

Toshio Fujikura, M. D.

Other Investigators: None

Cooperating Units: All Collaborating Institutions

Man Years

Total: 0.05
Professional: 0.05
Others: 0.00

Project Description:

The incidences of certain variables in the baby, mother and placenta of
core deaths were compared with deaths having selected congenital malforma-
tions. Velamentous and marginal cords were more common in single umbilical
artery deaths compared to core deaths. There was surprisingly little
association with diabetes, except among the cases with agenesis of the
kidney. Meconium staining was nearly twice as high in multiple heart
malformations compared to controls. Hydramnios was common in association
with anencephaly, and to a lesser extent with spina bifida and hypoplasia
of lungs. The incidence of toxemia was more than twice as high in
anencephaly, spina bifida, and agenesis of the kidney compare to controls.
In addition, retroplacental hemorrhage was twice as high in anencephaly.
Erythroblastosis was twice as high in accessory spleen but was not found
in any of the other malformed cases. These correlations will be tested for
possible significance. The study is being readied for publication.

Honors and Awards : None

Publications: None

207 V

Serial No. NDS (CF)-69 PR/P 1766

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July I, 1970 through June 30, 1971

Project Title: Reproductive Ability of the American Negro with Sickling
and its Public Health Implications

Previous Serial Number: Same

Principal Investigators: Luz A. Froehlich, M. D.

Toshio Fujikura, M. D.

Other Investigators: None

Cooperating Units: All Collaborating Institutions

Man Years

Total: 0.4
Professional: 0.4
Others: 0,0

Project Description:

The reproductive performance of 654 sicklers and 1,890 non-sicklers in the
Collaborative Study was compared. The cumulative fertility rate of mothers
with sicklemia was the same as that of non-sickling mothers. There was no
substantial difference in perinatal death rates, birthweight and gestation
age between the sickling and non-sickling group. Only the infant and child
death rate was higher in the sickling group. Because of the normal
reproductive abilities of the sickler, the sickle cell gene may continue
to propagate in the U.S. Negroes for generations to come.

The paper has been approved by the Publications Review Board and the NIH
Review Board. Certain modifications and refinements are felt necessary prior
to submission for publication. These modifications are strongly dependent
on the performing of hemoglobin electrophoretic studies on the sicklers,
to distinguish the homozygotes from the heterozygotes. Such a plan to
allow uniform testing of all Negroes for sickling and to follow this through
with hemoglobin electrophoresis on the proven sicklers is being earnestly
pursued.

Honors and Awards : None

Publications: None

209V

Serial- No. NDS (CF)-69 PR/P 1769

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: The Significance of Chorioangiomas

Previous Serial Number: Same

Principal Investigators: Luz A. Froehlich, M. D.

Toshio Fujikura, M. D.

Other Investigators: Pearl Fisher, Ph.D., OB, NINDS

Cooperating Units: All Collaborating Institutions

Man Years

Total: 0.1
Professional: 0.1
Others: 0.0

Project Description:

Eighty-one cases of chorioangioma in the Collaborative Study were analyzed,
of which five were in twin pregnancies. Chorioangiomas were more frequent
in whites (0.337o) than in Negroes (0.207o) and in females (49 cases) more
than in males (37 cases). Two infants had neonatal thrombocytopenic
purpura unassociated with skin hemangioma. Congenital anomalies were
high in twins; in single births, the incidences of several malformations
were significantly higher than in the general Collaborative Study population.
An interesting correlation was noted among chorioangioma, skin hemangioma
and single umbilical artery. In Negroes, in particular, twin rates in
current and prior pregnancies were high as was the fact that the gravida
herself was a twin. Acute toxemia was a significant complication in both
whites and Negroes. This study has been completed.

The paper was presented at the Teratology Society Meeting in Annapolis,
Maryland on May 20-22, 1970.

Honors and Awards: None

Publications: Froehlich, L.A., Fujikura, T., and Fisher, P.: Chorioangiomas
and their clinical implications. Obstet. Gynec. 37: 51-59,
Jan. 1971.

211v

Serial No. NDS (CF)-69 PR/P 1772

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Pathologic Effects of Ligation of the Anterior Spinal
Artery and/or the Great Radicular Artery in Monkeys

Previous Serial Number: Same

Principal Investigators: Oscar Aparicio, M. D.

Other Investigators: Larry C. Fried, M. D.

Cooperating Units: Surgical Neurology Branch
Intramural Research
National Institute of Neurological Diseases and Stroke

Section on Neuroradiology

Medical Neurology Branch

Intramural Research

National Institute of Neurological Diseases and Stroke

Man Years

Total: .05
Professional: .05
Others: .00

Project Description:

Objectives: The neuropathological evaluation of spinal cords of monkeys
subjected to vascular ligations, in order to determine the practical
feasibility of sacrificing any of these vessels if necessary during the
course of a surgical procedure.

Methods Employed: The neuropathologic evaluation of the spinal cords by
means of multiple staining methods, including H&E, Luxol Blue with Cresyl
Violet, and others, in order to determine the presence, extent and
location of any lesions due to ischemia.

Proposed Course: The histopathological evaluation has been completed and
a publication is being prepared.

Honors and Awards: None

Publications: None

213^

Serial No. NDS (CF)-70 PR/P 1858

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Thrombocytopenic Purpura and Placental Hemangioma

Previous Serial Number: Same

Principal Investigators: Luz A. Froehlich, M. D.

Other Investigators: Mary Housler, R. N.

University of Buffalo

Cooperating Units:

None

Man Years

Total:

0.1

Professional:

0.1

Others:

0.0

Proiect Description

A case is presented in which neonatal thrombocytopenic purpura (TP) was
associated with hemangioma of the placenta. This combination has not been
reported in the literature, although the combination of TP and skin
hemangioma is well known. The baby had no skin hemangioma. Other known
causes of TP in the newborn such as maternal TP and sepsis were not
present. The mother took a thiazide during the last eight weeks of
pregnancy. However, the incidence of TP among those whose mothers took
thiazide (0.017o) was not higher than among those whose mothers did not
take thiazide (0.017o), indicating that thiazides and TP are not related.
Study completed.

Honors and Awards: None

Publications: Froehlich, L.A. and Housler, M. : Neonatal thrombocytopenia
and chorangioma. J. Pediat. 78: 5l6-519^ 1971.

215V

Serial No. NDS (CF)-70 PR/P 1859

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Mental and Motor Development in Monozygotic
Co-Twins with Dissimilar Birthweights

Previous Serial Number: Same

Principal Investigators: Toshio Fujikura, M. D.

Luz A. Froehlich, M. D.

Other Investigators: None

Cooperating Units: All Collaborating Institutions

Man Years

Total: 0.2
Professional: 0.2
Others: 0.0

Project Description:

Developmental measures in 125 monozygotic twin sets with unequal
birthweights between co-twins were studied. There were no significant
differences between co-twins in the Bayley mental and motor scores at
eight months nor the Stanford-Binet I.Q. at four years. A reportedly
higher I.Q. for the heavier monozygotic twins was not confirmed in this
study, even among pairs with large birthweight differences (mean
differences 26-287o). Although the effects of nutrition on the mental
development of the fetus are currently of great concern, these data suggest
that the developing human brain seems to have a strong resistance to
intrauterine deprivation.

The future of this manuscript is uncertain. Because interpretation of
the data is controversial the paper is apparently being shelved until
data on the seven year I.Q. have been similarly analyzed.

Honors and Awards: None

Publications: None

217 V

SeriaJ. No. NDS (CF)-71 PR/P 1911

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: A Follow-up of Children with Single Umbilical Artery

Previous Serial Number: None

Principal Investigators: Luz A. Froehlich, M. D.

Other Investigators: Toshio Fujikura, M. D.
John Churchill, M. D.

Pearl Fisher, Hi.D.

Cooperating Units: All Collaborating Institutions

Man Years

Total: 0.3
Professional: 0.2
Others: 0.1

Project Description;

There are 306 presently living core Study children who were born with
single umbilical artery (SUA). These are from a total of 355 core SUA
cases (identified by microscopic examination) of whom 49 (13.8%) have
died, mostly in utero or during the first week of life. Associated
congenital malformations were a significant cause of death. The surviving
cases were compared with surviving controls matched for race, sex,
institutions, birthweight, gestational age and S-E index. Velamentous
and marginal insertions of cord were about six times as frequent in SUA.
There were also slightly more neurologically abnormal cases (7.0%) than
controls (4.9%). On the whole, however, mean mental and motor and
four year I.Q. scores, and parameters of physical growth did not distinguish
between cases and controls. Statistical analysis has been temporarily
halted because of Dr. Fisher's departure, but all attempts will be made
to see the paper to completion despite this inconvenience.

Honors and Awards : None

Publications: None

219 V

Serial No. NDS (CF)-71 PR/P 1912

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: Birthweight in Relation to Renal Glomerular Development
and Gestational Age in Whites and Negroes

Previous Serial Number: None

Principal Investigators: Toshio Fujikura, M. D.

Luz A. Froehlich, M. D.

Other Investigators: None

Cooperating Units: All Collaborating Institutions

Man Years

Total:

0,

.25

Professional:

0.

.20

Others:

0,

.05

Proiect Description

Birthweight, body length and X-ray evaluation of bone deve ''^ypnient continue

to be widely used as measures of fetal maturity. Howevj^^ these are

factors of somatic growth and are not necessarily direj,^i^y related to

growth and maturity of internal viscera. On the conVj^^^-^on that histologic

examination of an organ would give a more accurate P-Jcture of the maturation

of internal viscera, a h^istologic evaluation of the Sidneys of 51^ neonatal

deaths and fresh stillbirths was undertaken. In tl early phases of

pregnancy, somatic growth appeared to proceed at r gienificantly faster pace

in the Negro, as evidenced by higher birthweight ^ . ^\^^s race compared to

whites. However, the developmental rate of vis^j, such as the kidney

seemed to proceed at similar rates in the two rj ' Racial differences

were observed in the percent of the presence o' . hroeenesis (PPN) in the

"small for dates" as well as "large for dates'" /" . f „n«-g in both races

about half of the "small for dates" infants f ^ -i i pxhibited a nephrogenic

zone compared to more than 95% of the trul" "■ t'res At the same time

two to four times as many "large for d ^ f, ^^^^^ts showed nephrogenesis

as those which were mature both for ^ir' 'weight and gestational age. In

maternal diabetes more than 807, r ^^fg^^ between 35-37 weeks gestation

showed nephrogenesis where only r, ,„, . j r^u.^ oaoer is being

J. J £ ,1- ^. b6.67o were expected, me pcifco.

readied for publication. "*

Honors and Awards: None

Publications: None J

221V

\<

I

Serial No. NDS (CF)-71 PR/P 1913

1. Perinatal Research Branch

2. Section on Pathology

3. Bethesda, Maryland

PHS-NIH

Individual Project Report

July 1, 1970 through June 30, 1971

Project Title: The Clinical Significance of Generalized Petechiae at Birth

Previous Serial Number: None

Principal Investigators: Luz A. Froehlich, M. D.

Jean G. Oliver, M.A.

Other Investigators: Toshio Fujikura, M. D.

Jean S. Nemore, R.N., B.A.

Cooperating Units: Section on Infectious Diseases, PKB, NIKDS
All Collaborating Institutions

Man Years

Total: 0.2
Professional: 0.2
Others: 0

Project Description:

In a hand review of charts of some 30 of about 120 living infants diagnosed
as having had significant skin petechiae at birth, six were found to have
significant hearing loss, an incidence far greater than expected. Speech
and behavior problems were also common. Mental retardation was found
in a mature male along with marked visual impairment.

On the hypothesis that significant skin petechiae could be accompanied
by hemorrhages in vital structures such as the brain, inner ear, retina,
etc., the review of charts of all 120 children will be continued and
findings compared with those of controls matched for institution, race,
sex, birthweight and socio-economic index.

Honors and Awards: None

Publications: None

223 V

Serial Wo. NDS (CF)-70 PR/SLH 1520

1. Perinatal Research Branch

2. Section on Speech^ Language and
Hearing

3. Bethesda^ Maryland

PHS-RIH
Individual Project Report
July 1, 1970 through June 30^ I97I

Project Title Explorative Study for the Use of a Speech and Language

Screening Examination for 3-Year Old Children in the Home
Situation

Previous Serial Number: NDS-(CF)-63 PR/BS II67

Principal Investigator: Dr. Miriam F. Fiedler

Other Investigator: Dr. Eric H. Lenneberg, Cornell University

Cooperating Units: Children's Medical Center, Boston, Massachusetts

Section on Behavioral Sciences, PRB, NINDS
Section on Pediatric-Neujrology, PRB, NIWDS

Man Years:

Total: .5

Professional: .2

Other : . 3

Project Description:

The study concerns children with non-normal speech identified at age three
years by means of interviews with the mothers of Perinatal Project children
in Boston. Findings were confirmed by subsequent speech, language and
hearing examinations. The speech data were considered in relation to
perinatal findings as an initial study, and, in a second study, with regard
to outcome at age seven years in the light of psychological and neurological
examinations. Most of the children identified as abnormal or suspect at
age three years were found to be similarly regarded by other measures at
age seven years.

Part of the study was presented by Dr. Fiedler as a paper delivered at the
Tri-City Meeting of the Obstetrical Society in Boston on May 16, I967,
\nider the title "Delayed Speech Development in Children at 3 Years of Age
Related to Peri and Postnatal Findings". The study has been completed.

Honors and Awards: None

Publications:

Fiedler, M. F. , and Lenneberg, E. H. : Explorative Study for the Use of a
Speech and Language Screening Examination for 3-Year Old Children in the Home
Situation. Pediatrics. In press.

225v

Annual Report

Associate Director's Report

July 1, 1970 through June 30, 1971

Extramural Programs

National Institute of Neurological Diseases and Stroke

A detailed summary of the NINDS extramural research grant effort and training
accomplishments in Fiscal Year 1971 are provided in the reports of the Research
Grant and Training Branches; organizational progress including program reporting
activities are included in the report of the Administrative Officer. In broad
terms, the highlights of NINDS Extramural Programs for Fiscal Year 1971 have
been characterized by:

A. A CONTINUED REDUCTION IN TRAINING ACTIVITIES: With a "hold the line"
philosophy in the training activities of the Institute, 10 additional grant
supported training programs have had to be discontinued. This was necessitated
by the continuing increase in funding requirements of active programs despite
an across-the-board "negotiated cut" for the third successive year, this year
at the 157o level. The plan to stabilize the number of new RCDA awards each
year at 20 is reaching fruition; if there are no further reductions in this
program area in the future, the fiscal requirements for this minimal level of
activity will be reached in Fiscal Year 1975. Fiscal Year 1971 will end with
$8.5 million approved training grant and traineeship applications unfunded and
$1.5 million approved fellowship applications unfunded. The President's Budget
for Fiscal Year 1972 will require further reductions in NINDS training activi-
ties. This will have particularly unfavorable effects on the recruitment and
training of personnel for careers in teaching and research in neurology, child
neurology, neurological surgery and otolaryngology. In addition, desperately
needed new training programs in the basic science areas of neurochemistry,
neurophysiology and neurobiology will not be activated.

B. A TEMPORARY STABILIZATION OF THE LEVEL OF RESEARCH GRANT ACTIVITY: An
increase of $5 million in research grant funds in Fiscal Year 1971 permitted
the Institute to maintain its research grant activities at approximately the
same level as in Fiscal Year 1970. The additional funds were utilized to help
offset a 77c increase in cost-of-living; to provide for a reduction to 107o of
the previous "across-the-board" budget negotiation level of 157=,; to meet a
peak in the triannual level of previously committed applications; and to
initiate feasibility studies for the establishment of acute spinal cord injury
research centers. The President's Budget for Fiscal Year 1972 reduces the
research grant activity by over a half-million dollars. This reduction will
interfere seriously with the Institute's plan to further reduce across-the-
board negotiations to the 57= level, to meet the continuing increase in research
costs, and to provide additional emphasis on selected high priority research
programs.

C. RESEARCH AREAS OF SPECIAL EMPHASIS:

(1) Stroke: The Institute's principal instrument for research grant
support of stroke research is its program of stroke clinical research centers,
eighteen of which are now active. These are centers of coordinated clinical
and basic research focused on problems such as local and regional cerebral
blood flow, cerebral hypoxia, cerebral edema, cerebral arterial thrombosis.

hemorrhage and spasm, stroke epidemiology, the pathophj'siology of the transient
ischemic episode, etc. In the area of cooperative research; The cerebral
aneurysm study has been completed with significant results; the hypertension-
stroke study is in its final phases; plans have been completed for the initia-
tion of a study to evaluate the efficacy of amino-caporic acid in the treatment
of the acute stages of intracerebral hemorrhage. As a result of the activity
of the Joint Council Subcommittee on Cerebrovascular Disease (NINDS and
NHLI) , plans have been developed for a program of coordinated research on the
problem of acute stroke care (nervous system monitoring and acute medical-
surgical intervention) through the establishment of acute stroke research units;
this activity can be initiated if the additional funds required are made avail-
able for stroke research.

(2) Trauma To The Central Nervous System: In 1968, the NINDS initiated
its program of Head Injury Clinical Research Centers by means of specialized
research center awards to plan and develop these activities. In Fiscal Years

1971 and 1972, the pilot centers will compete for future support in the cate-
gorical clinical center program. In order to provide for both a progress report
on research accomplishments to date in this area and to establish specific
scientific merit criteria for review of applications, the Institute has organ-
ized a Head Injury Research Workshop. This Workshop will be held in February

1972 and will include scientists from each of the 6 active head injury research
centers and selected scientists from other laboratories.

In the area of acute spinal cord trauma, fifteen applications for feasibility
studies have been received. These applications to develop the scientific,
medical, community and organizational feasibility of establishing up to 4
acute spinal cord injury clinical research centers in the U.S. will undergo
review during the summer of 1971 and receive final Council recommendation in
September 1971. Funds for the feasibility studies have been set aside. A
minimum of $4 million will be required to launch the 4 center activity in late
Fiscal Year 1972 or early Fiscal Year 1973.

(3) Communicative Disorders (Hearing; Speech; Language): A Task Force of
the NINDS Communicative Disorders Program Project Review Committee has completed
its report on opportunities for research in these areas. The report documents
the several research problems of high priority and opportunity which justify
increased Institute effort; included are the biological and medical effects

of noise on man; chronic and episodic vertigo; genetics and deafness; pre-school
deafness; hearing loss and chronic secretory otitis media; and traumatic neural
impairment and language. The documents supporting these specific program area
needs will serve as a basis for program planning and development in FY 1972.

(4) Parkinsonism; L-DOPA; Neurotransmitters: The availability of L-DOPA
and its acceptance as the therapy of choice for moderate to severe Parkinsonism
has redirected and accelerated the entire area of research on movement disorders,
neurotransmitters and the neural and neuromuscular synapse. L-DOPA potentiators
are now in phase 2 and phase 3 study; L-DOPA analogues, dopamine-o-mimetic
drugs and antichoreic agents are now potentially available; the delicate balance
of protagonists and antagonists of synaptic transmission is being described;

and the metabolic pathways of neurotransmitter saturation therapy are being
unraveled. The four research centers in this area supported by the Institute

2 w

are the leaders in these developments; the additional $3 million in research
projects provides for an independent but complementary research effort.

D, PROGRAM REPORTING: With the completion of the conversion of our routine
data storage and retrieval system to tape and the development of programmed
search procedures, for the first time a comprehensive search and reporting
system is now in effect. Staff scientist administrators responsible for
program- area activities now have regular reports available to them in both
the research grant and training grant programs; similar reporting will be
available in the fellowship programs in Fiscal Year 1972. Computer data
also are being used regularly for fiscal monitoring and reporting. In
Fiscal Year 1972, pilot programs will be developed and tested for the
reporting of scientific status and accomplishments.

3w

Annual Report

Administrative Report

July 1, 1970 through June 30, 1971

Extramural Programs

National Institute of Neurological Diseases and Stroke

Extramural Programs found the prohlem of diminishing staff particularly
difficult during Fiscal Year 1971- During a year when position strength was
more than 15^ below normal operating levels and the program data maintenance
and analysis organization was "being expanded to meet increasing demands
against it, the normal ability to reassign support personnel to cover absences
or heavy load demands was most difficult. The sacrifice of certain central
service positions has caused a further drain on the time and effort of exist-
ing clerical staff. Mail and messenger service and local duplication have
all become do-it-yourself tasks. The sum of this was that certain minimum
compromises with total efficiency had to be made.

The Extramural Programs participated with other Institute program areas
in two Equal Employment Opportunity conferences during FY 1971- These con-
ferences were held to identify specific problems and attitudes that foster
inequitable job situations and to develop an Institute commitment to improve
equal opportunity. An affirmative action plan has been developed and is being
implemented at this time.

The Data Analysis and Reports Unit has completed its conversion from
manual records to computer files. This new data file has begun to provide
Extramural Program's management with an information capability that enhances
management planning, analysis, and operations and improves projections of
future needs .

The full impact of the concurrent project period ruling was felt in grants
management during FY 1971- A significant amount of additional grant support
was permitted by this method of allowing the use of unexpended grant funds
during an extension of the project period.

The administration of the NINDS Extramural Programs budget was made diffi-
cult due to the late release of funds with the appropriation being signed on
January 11 and the apportionment approved by the Office of Management and
Budget on March 1, 1971. This late release of funds resulted in the delay of
the award of many new as well as competing renewal Research Grants until the
fourth quarter of the fiscal year. As in the past, reserves were withheld
from the appropriation in the amount of $2, 957 > 000 for Research Grants and
$672,000 for Graduate Training Grants, a total of $3,629,000. Of this amount
withheld from grants, $1,307,000 was allocated to NINDS direct operations for
pay raises, and the balance of $2,322,000 to other Institutes of N.I.H. and
the DHEW for pay raises. However the amounts released this year were increas-
ed over the President's Budget by $U, 211, 000 for Research Grants and $187,000
for Fellowships. There was no change in Training Grants. Of the increase
for Research Grants, $3,522,000 was for research projects and centers and
$689,000 for General Research Support Grants. Even with these increases, the
level of unfunded approvals continued to climb upward as a result of an in-

5 w

creased number of approvals, the average cost of grants increasing coupled
with a lesser amount of negotiated reduction. The amounts of unfunded approv-
als were $15,300,000 for Research Grants, $8,500,000 for Training Grants, and
$1,^50,000 for Fellowships. To assure the most efficient use of available
funds, the following steps were taken.

1. Research Grants were negotiated do^mward in an amount averaging 10^, re-
sulting in savings of approximately $U.O million which resulted in the award
of "^V^o more new and competing renewal grants than would have been possible
without negotiations.

2 . Graduate Training Grants were negotiated downward an average of 15*5^ which
made possible the award of twice as many competing grants as would have been
awarded without negotiations.

3- Additional savings were realized for Research Grants and Graduate Training
Grants by utilizing the concurrent project concept. This means that competing
renewal grants were reduced by the amount of funds remaining in the previous
project period and extended up to a year so that the grantee could use these
unexpended funds during the new project period.

6 w

Annual Report
July 1, 19T0, through June 30, 1971
Research Grants Branch
National Institute of Neurological Diseases and Stroke

Introduction

The "brain is by far the most intricate^ sensitive and
versatile organ in the body. As a result^ it has been the subject of
ejctensive study and research for centuries. However, it has yielded
only slowly to scientific exploration because of its complexity and
because of its relative inaccessibility due to being enclosed in the
skull and due to the blood-brain barrier which separates the brain
metabolic ally in many respects from the rest of the body. Nevertheless,
research is gradually bringing a greater understanding of how the
10 to 13 billion individual nerve cells in the brain, together with the
additional billions comprising the nervous system, work together to
make the human body an effective and coordinated living organism.

The economic burden of the neurological and sensory diseases
amounts to billions of dollars each year, with immeasurable human
suffering. Although the human spirit can often adjust to the effects
of physical disability, even the greatest courage may be broken by the
devastating consequences of brain injtiry or disease which may continue
or exacerbate during the remainder of a person's life.

More than 200 disorders are known to afflict the brain,
sense organs, nervous system and neuromuscular apparatus, the most
familiar of which are stroke, head and spinal cord injury, epilepsy,
cerebral palsy, aphasia, multiple sclerosis, muscular dystrophy,
parkinsonism, brain tumors, and otosclerosis. These diseases lead to
paralysis, loss of speech, paraplegia and deafness, and are among the
major causes of death and permanent disability in the United States.

The research grant programs of the National Institute of
Weiu-ological Diseases and Stroke include research projects, research
program projects, cerebrovascular and commimicative disorder outpatient
clinical research projects, clinical research centers, and grants to
establish cerebrovascular and head injury clinical research centers.
The objectives of these programs are the identification, stimulation,
and support of important research problems related to the diagnosis,
treatment, and prevention of disorders such as those mentioned above.

For many years the question of whether hypertension played a
major role in the development of stroke has been controversial. About
one year ago the Veterans Administration reported a study indicating
that antihypertensive therapy had a marked protective affect on stroke
morbidity and mortality. This whole question was then reviewed by the
Joint Council Subcommittee on Cerebrovascular Disease of the NHLI and
NINDS and by the NAIJDS Council. Both groups recommended that further

7 w

steps be taken as expeditiously and aggressively as possible to implement
the application of antihypertensive therapy on commimity bases. Since
this problem is now beyond the clinical research stage and is in the area
of commimity application, this recommendation was referred to the Regional
Medical Programs Service which was urged to give it a high priority.

Injuries to the spinal cord are occurring with increasing
frequency. Once the spinal cord degenerates in the area of injiiry,
paralysis always develops. In March 1971^ the HANDS Coimcil recommended
approval of plans to support a few Acute Spinal Cord Injury Centers. At
the first stage of development, funds would be provided only to plan the
requirements and test the feasibility of a few centers for acute spinal
cord injury. Each Center's plan would include investigation, development
and evaluation of improved methods of emergency treatment, rapid trans-
portation, diagnostic techniques, medical- surgical repair, and the train-
ing of required professional, scientific and technical personnel. Ultimately,
it is expected that a fully developed Center would contribute important
information on the prevention of degeneration of the spinal cord and on
the restoration of spinal cord function. Close liaison with other
government and non-government agencies with responsibilities in this
area is being maintained.

The importance of improved stroke acute care is obvious since
70 to 80 percent of the mortality occurs in the first ten days. Also,
the consequences of not recognizing a progression or extension of the
infarct may be catastrophic. After extended review of this problem, the
Joint Council Subcommittee on Cerebrovascular Disease, KHLI-WIKDS, agreed
that the present information about stroke acute care is exrtremely limited
and that the individual efforts on the part of investigators, singly
and in teams, to push forward with the problem have met with only limited
success to date. In view of these considerations, the NANDS Council in
March 1971 approved the organization of a Problem Commission on Stroke
Acute Care Research. The Commission will consist of 10-12 experts in
this and closely related areas and will be closely linked to the present
Stroke Clinical Research Centers. It will be the responsibility of the
Commission to (l) develop the strategy required to explore the problem
(e.g., identify the neurological variables requiring attention and
possible human and mechanical monitoring as a basis for intervention);
(2) describe the specifications of the required organization, software
and hardware; and (3) assume the responsibility for testing and evaluating
these specifications in their own Stroke Clinical Research Units.

This was the third year in which every research grant, competing
and committed, was subject to a negotiated reduction. All grants were
negotiated downward by an amount which would hopefully still allow the
work to proceed, although productivity doubtless was reduced even more
than in the previous two years because costs have increased. As a result
of this arrangement, however, over 50^0 more f\inds were available for
competing applications. Even so, approximately half of the approved
projects could not be supported. The Institute has been advised that
grants may be reduced by only a minimal amount {%) next year. In effect

this will amount to a reduction of about $2.5 million or 25 percent in the
fluids available for competing applications. Two years ago^ the National
Eye Institute was organized and all of the activities related to the
visual system and disorders of vision were transferred out of WIKDS. This
took more than 20 percent of the grants, applications and funds. Althoiigh
there has been little increase in the f\xnds available, it has been possible
to maintain about the same number of projects by reducing each grant as
described above. That is, last year 1,267 research grants were supported
at a total cost of $U8.8 million. This year 1,256 grants were supported
at a cost of $53.6 million.

Within one year after the formation of the National Eye Institute,
the number of applications to IttRDS was just as large as it was before
the operation of the two. For example, in March 1970, the RAKDS Council
reviewed about 375 applications. In June 1971 the Council reviewed about
^50 applications, a 20 percent increase over the average for the previous
year. This continued and dramatic increase in the number of requests is
due primarily to the effectiveness of the research training programs of
this Institute in which the output of fully trained investigators has
only in recent years reached its full potential.

There were two replacements in the professional staff of the
Research Grants Branch since last year. One was due to a retirement and
the other was due to a resignation. One of the new staff members is
Executive Secretary of Program Project Committee A and the other is
responsible for research grants in the areas of pharmacology, medicinal
chemistry, and toxicology. Both have proved to be mature and competent
scientists and are becoming expert administrators. Therefore, the Branch
continued to enjoy the services of an active and experienced staff.

The numbers of grants and amounts of funds in the various
disorder categories are shown in Appendix A.

Cerebrovascular Disorders

In FY 1971 support for research in cerebrovascular disease was
at the level of $5*6 million, representing 63 research grants and I9
clinical research centers.

Studies directed toward cerebral blood flow continue to be of
prime importance in research on cerebrovascular disorders. Some
investigators direct their efforts to such facets as development and
refinement of methodology and techniques for measurement of cerebral
blood flow at a regional and focal level, others to problems of regulation,
others to correlative studies of cerebral blood flow with other parameters
which might be used as predictive studies in the course of a stroke.

In one clinical research center, the relationship of cerebral
blood flow (CBF) to pH in cerebral venous blood and in cerebral cortex
is being explored. Dogs were lightly anesthetized, paralyzed and
ventilated with oxygen. CBF was meas\u:"ed by an electromagnetic flowmeter
in a shunt between the torcular and the superior vena cava. Cerebral
venous blood pH, p02^ and pC02 were measured continuously in a flow-
through within the shunt. The same parameters were measured on the surface
of the cerebral cortex by the application of flat surface counterbalanced
electrodes on the exposed brain through a parietal craniotomy. The brain
surface was protected by a shallow pool of warmed artificial cerebrospinal
fluid. Simple seizures were produced by electroconvulsive stimuli or by
pentylenetetrazol loV. and recorded by electroencephalographic monitor.
In eight dogs so studied, CBF increased immediately with the onset of
each seizure. Both brain and cerebral venous p02 increased consistently
with seizures in well ventilated animals, but fell when seizures were
accompanied by apnea. Brain pH showed little change initially during
the acute phase of cerebral hyperemia. Thereafter brain pH showed
variable changes. It decreased by O.IO-O.I5 units in most observations,
but in several experiments a primary increase in pH was found. In the
post-ictal phase brain pH was consistently increased above baseline values
at a time when CBF was still elevated. In the post-ictal phase of
impaired brains, autoregulation increases, induced by hypertension if CBF
was accompanied by increases in brain pOg and pH, and decreases in pCOg.

The lack of consistent correlation between CBF and brain pH
during and after seizures suggests that factors other than hydrogen ion
activity play a role in regulation of cerebral vascular resistance. Such
factors, whether neurogenic or metabolic, serve the important function of
augmenting CBF to meet the brain's increased oxidative metabolism during
seizures. Further studies are continuing to define mechanisms of
functional hyperemia in brain.

Another study cites experimental evidence that microparticles
present in blood during cardiopulmonary bypass cause a reduction in
cerebral blood flow and metabolism during this procedure. The extent and
distribution of ischemic cell change in dog brains following prolonged
cardiopulmonary bypass are being investigated. Six animals have undergone

10 w

thoracotomy under anesthesia and four were maintained on complete bypass
for four ho\:irs under conditions of normal temperature^ arterial pressure,
"blood gas tensions, and pH. T\<ro control animals were maintained \inder
identical conditions, "but without bypass. Thereafter, perfusion with
saline followed by perf us ion-fixation with a formaldehyde-acetic acid-
methanol mixt-ure was continued for at least 20 minutes at perfusion
press-ures of 100-110 mg Hg. Following an additional period of immersion
fixation, 20 micron sections were prepared from paraffin-embedded blocks
at 6 mm intervals throughout the cerebral hemispheres, cerebellum and
brain stem, and were stained by several critical staining methods.

The four experimental animals studied following bypass showed
early ischemic cell changes by the criteria of Brierley and Brown for
light microscopy. These changes were non-focal and present in neiirons of
layers "VI and V of the cerebral cortex, in thalamic nuclei, and in
Itirkinje cells in the cerebellum.. Although there was no absolute regional
localization, ischemic changes were more apparent within the parietal
cortex than in other cortical areas. While occasionally small and medium
sized arterioles were occluded by aggregates of red blood cells and
amorphous PTAH positive material, there were no occlusive lesions of
larger sized arterial vessels. Small punctate parenchymal hemorrhages
were present in two specimens, and two instances of intraventricular
bleeding, apparently originating in or near the choroid plexus, were
found. Control sections were histologically normal. Continuation of
these studies is planned, including microangiographic radiography of thick
sections of brain following perfusion with colloidal barium. These studies
are to be correlated with direct measiorements of microembolic material in
blood during bypass by sonar detector methods. Also, the effectiveness
of micropore filtration in preventing such changes will be tested.

A serial circular angiotomographic device has been constructed
and tested extensively on dogs during angiography. It has been possible
to demonstrate a diffuse "brain blush" in tomographic sections 1 cm.
thick. Extensive physical tests have been conducted to establish the
relation of thickness of cut to tomographic angle and the relation of
speed of rotation to resolution. The results of animal studies were so
encoiuraging that the unit was moved to the clinical area and patient
studies have been initiated. Fifteen studies have been carried out on
patients. In two cases the angiotomogram gave diagnostic information not
obtained from conventional films. Continued patient studies will be
carried out to evaluate the unit for its clinical value.

In the same radiology group, a new monitoring system for use
dioring cerebral angiography is being evaluated. The system incorporates
an industrial transducer and alarm system, and has proved to be an added
safety factor during angiography. It indicates blood clots in the needles
or catheter, sub-intimal position of the needle tip and changes of blood
pressure or pulse rate. A unique feature of the device is that the trans-
ducer may be left connected during the injection of contrast media. The
part of the pressure tracing immediately after the injection of contrast
media is of considerable interest.

11 w

In another clinical research center^ several members of the
group are continuing studies on newly developed techniques for measuring
hemispheric blood flow and metabolism in man. They have shown a bilateral
reduction in hemispheric blood flow and in metabolism in unilateral stroke
due to diaschisis. They have found that cerebral acetoacetate and alpha-
butyrate metabolism is not increased in the infarcted hemisphere. The
group now plans to direct their attention to the phosphate-oxygen ratio
which may define whether or not uncoupling is present in the human brain
following cerebral infarction.

Another group of investigators is studying new methods for
measuring regional cerebral blood flow using the Anger Camera and a tape
storage and retrieval system following the intracarotid injection of
radioactive xenon and technecium. These methods provide greater resolution
for regional measurements than previously available, and the play back
system permits measurement of up to 100 different areas of brain with a
single injection. Evidence is being provided of definitive and diagnostic
patterns of abnormal flow and changes in blood volume and blood brain
barrier in intracranial hematoma and brain tumor as well as in patients
with occlusive cerebral vascular disease and infarction.

Another group has induced experimental subarachnoid hemorrhage
by the injection of fresh blood into the subarachnoid space. This
produced a spasm of the cerebral vessels with a marked reduction of
cerebral blood flow on the release of serotonin. The relation of this
to ruptured aneiirysm and spasm in hiunan subjects is under active investi-
gation and methods of treatment are being considered.

In another set of experiments, regional cerebral blood flow is
being measured by implanted electrodes before and after occlusion of the
middle cerebral artery and the effects of various forms of treatment on
regional blood flow are under study. These studies are being combined
with measurements of regional cerebral blood flow using the radioactive
antipyrine method originally developed by Landau, Sokoloff, et a], as
modified by Reivich and Isaacs. In the human laboratory, using the
hemispheric blood flow method and the regional xenon techniques, the
therapeutic usefulness of glycerol has been shown, which reduces brain
swelling and increases blood flow in the area of cerebral edema.

Therapeutic trials are also ongoing on a double blind basis
with two different dri;igs. One is Hexobendine, a potent cerebrovasodilator.
Investigations on man and in animals have sho-vm that it increases
cerebral blood flow. The drug has been given to 11 patients intra-
venously, and it has been observed that five improved, four showed no
change and two died of unrelated causes. Wo toxic effects have been
noted. These studies are being extended to evaluation of therapy in
cases of acute and chronic cerebrovascular disease. Additionally, drug
studies with Cyclandelate are now being initiated.

Researchers in another clinical research center have completed
a study in animals of the effect of change in blood pressure on blood
flow through cerebral cortex made acutely Ischemic by occlusion of a

12W

middle cerebral artery. The effect of CO2 inhalation on blood flow
through cortex which was either acutely or chronically ischemic was
studied in animals and revealed the loss of autoregulation in ischemic
cortex. The auto-radiographic technique for the measurement of regional
cerebral blood flow in animals was used in the study of the effects of
occlusion of a middle cerebral artery on blood flow and various regions
of the brain correlated with changes in surface vessels. It was then
possible to get quantitative estimates of flow in portions of the brain
remote to those areas being visualized by the investigator. A study was
begun of the effects of stimulation of the sympathetic nerves in the neck
on blood flow through the cerebral cortex and on the diameter of s\irface
blood vessels in animals. A pilot study has been initiated in hiMians of
the measurement of cerebral blood flow by inhalation of xenon-133 and
has so far shown that the technique is useful but needs further evaluation,
particularly in patients with cerebrovascular and other neurological
disorders. This evaluation study is continuing.

Another group of investigators at this research center is
pursuing the development of clip grafts for aneurysms in small vessel surgery
and is beginning to apply the techniques in human subjects. Among personnel
of various stroke research centers where carotid endarterectomy is
performed, there continues to be active debate concerning the various
methods for keeping morbidity and mortality at the lowest possible levels.
The ability to rapidly and safely evaluate the changing pattern of regional
cerebral blood flow prior to, during and following endarterectomy would
be of potential value in making clinical correlations concerning surgical
technique, pre-operative neurological findings, including the results of
arteriography and the post-operative neurological state of the patient.

Another research group are delineating the temporal profile of
acute progressing cerebral infarction in the carotid system and of acute
progressing infarction in the vertebral basilar system. This is a problem
of practical importance since there are some patients who develop severe
worsening of clinical evidences of focal cerebral ischemia some hours
after a cerebral infarct is thought to have been "clinically completed."
The occurrence of this delayed worsening suggests certain pathophysiologic
mechanisms which might be subject to prevention by appropriate therapy.

Research to develop a method for measirrement of regional cerebral
glucose metabolism has been pursued at another research center. To do
this, the characteristics of a tracer for the proposed technique was
thought to be one which was taken up by the brain at a rate proportional
to that of glucose and whose metabolic products remained in the tissue
under study. The use of Cl^-2-deoxyglucose was selected for study and
experiments were conducted to establish a mathematical model and to
substantiate the findings by actual experiments in dogs. Favorable results
have been obtained to date, and further development to refine the technique
is being pursued. It is anticipated that this research will make possible
(1) a means of studying regional CBF simultaneously in vivo, (2) will also
make possible a direct assessment of the role of regional metabolism in
the control of cerebral circulation, and (3) finally when validated it
will make possible the extension to humian studies, by the use of Cll-2-

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deoxyglucose which can be detected with external detectors.

The investigator has also directed his efforts to an evaluation
of rapid diagnostic methods in extracranial cerebrovascular disease. Four
rapid diagnostic tests have been examined (direct thermometry^ ophthalmo-
dynamometry, bruit and pulse assessment) to discriminate for the presence
or absence of significant extracranial cerebrovascular disease. Utilized
separately^ thermometry, ophthalmodynamometry and the examination for a bruit
will find over 75^ to 85^ of cases. Pulse assessment alone reveals only kO'fo
of significant lesions. The combination of neck examination for the presence
of bruits or pulse deficit alone will discover 93^ of the vascular lesions.
In this investigator's present series, lOO/o of the cases were found by including
either direct thermometry or ophthalmodynamometry in combination with the
aforementioned two tests.

Ophthalmodynamometry accurately located the site of the major
lesion in more than 9^'fo of the cases. The direct thermometry technique
scored below kO'fo in lesion localization. It is suggested that the thermom-
etry technique, because of its ease of performance, be utilized in combina-
tion with neck examination as the preliminary step in assessing patients
with suspected extracranial cerebrovascular disease. Ophthalmodynamometry
should then be used for more accura'te localization of the lesion prior to
angiography.

An understanding of the normal control mechanisms of the cerebral
circulation is essential to any investigation of the derangement present
in various abnormal conditions. This is especially important in the study
of cerebrovascular disease. Knowledge of the important regulating factors
in both normal and pathologic states may lead to concepts that will be of
use in preventing or correcting abnormalities of the cerebral circulatory
system. The relationship between regional cerebral metabolism and regional
cerebral blood flow is one important aspect of the control system which
has not yet been able to be directly studied. The development of a method
for measuring regional cerebral glucose metabolism in vivo should be of
significance in this regard.

Considerable interest has developed in studies of cerebral
vasospasm. One research group reports progress in determining the actual
effect on regional blood flow of angiographic experimental cerebral vaso-
spasm. Vasospasm was produced both by vessel puncture (as recommended by
this group) and by subarachnoid injection of blood (as recommended by
others). It was found that, despite apparent significant cerebral vaso-
spasm (vessel caliber reduced to l/2 of control), there was still no
statistically significant reduction in cerebral blood flow. Only when
the vasospasm became intense, that is the cerebral arteries appeared to
be 1/3 or less of the original caliber, was there reduction in cerebral
blood flow. When this critical point was reached, the cerebral blood flow
dropped dramatically. In a series of 25 monkeys in which subarachnoid
hemorrhage was produced by puncture of internal carotid artery, virtually
all showed a precipitous drop in cerebral blood flow determinations made
within 1/2 hour of puncture. Cerebral blood flow determinations rapidly
stabilized and, quite -unexplainedly, increased s.lightly above control
from the first to the fifth hour after hemorrhage. Following this,
cerebral blood flow would usually stabilize despite the presence of mild

Ikv

to moderate spasm on the arteriograms. If severe vasospasm developed^
angiography would demonstrate marked arterial constriction. CBF values
at this time were often half of the control value. This second phase of
spasm (probahly part of the biphasic response described by Brawley) would
persist throughout duration of the survival of the animals. Upon the
injection of blood into the chiasmatic cistern, inmiediate angiographic
spasm was noted, associated with a significant drop in cerebral blood flow.
The spasm would never become secondarily severe, though some degree of
spasm was seen throughout the experiment. Marked reduction in CBF and this
characteristic second stage of spasm are not seen with subarachnoid blood
injections.

It is the opinion of this investigator that both rupture of an
intracranial vessel and subarachnoid injection of blood are capable of
producing an immediate transient angiographic spasm which does reduce CHF.
Apparently, the puncture technique is more likely to produce the character-
istic second phase of spasm which is probably the clinically significant
phenomenon.

Concomitant experiments carried out by this researcher related
to the determination of stability of regional CBF determinations by the
radioisotope method in experimental animals for long durations. A series
of long d-uration regional blood flow experiments (over 36 hours) were
carried out in ten animals. It was found that: (l) RBF determinations
within the first hour following the preparation fluctuate and are invalid,
and (2) the coefficient of variation among the determinations increases
with the d\iration of the experiment, within the first 5-6 hours it being
only 3-^io, but after 20 hours it may be as high as ik^o. The practicality
of this technique for inducing cerebral vasospasm may be open to question
since the technique of this investigator produced severe reduction in
blood flow in only about I/3 of the preparations, but not at all in
preparations produced by techniques described by others.

Another phase of this investigator's studies consisted of the
application of physical forces to overcome the effects of cerebral vaso-
spasm. This researcher has been able to reliably maintain regional cerebral
blood flow above control levels in normal autoregulating brains by inter-
mittently occluding the descending aorta with an inflatable balloon passed
up throLigh the femoral artery. He has determined the appropriate intervals
of inflation and deflation which allow for adequate renal perfusion, yet
can increase CBF significantly. The assumption is that in the presence of
cerebral vasospasm, increased perfusion pressure for long intervals may
adequately nourish the brain until the spasm spontaneously subsides. The
technique of intermittent aortic occlusion was performed in 35 dogs and
25 monkeys without pathologic alterations. Intermittent aortic occlusion
in the presence of spasm has been attempted. It has been shown that it is
capable of perfusing past severely constructed vessels used in the
experiment described. In this experiment there was intense spasm of the
middle cerebral artery, which barely visualized angoigraphically, associated
with a marked reduction in CBF records. On institution of intermittent
aortic occlusion, CBF values returned to near normal levels. The results
appear encouraging even though there are many pi-oblems yet to be solved.

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These include cerebral edema which can result in certain instances when the
brain is so hyper-perfused. Additionally^ certain animals develop
alterations in blood pressure after long intervals with intermittent aortic
occlusion. The importance of these studies is heavily based on the pre-
sumption that a model of cerebral vasospasm, which behaves in a manner like
zhe human vasospasm phenomenon, is necessary before any research in this
area can be continued. The significance of this model as it may apply to
problems of cerebral blood flow in cerebrovascular disease is not certain.
Fiorther studies are needed.

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Trauma (Head Injury)

Since trauma is the number one killer of man while he is in the
priTie of life^ it is only natiiral that a major aspect of trauma^ head
injury^ should receive special attention from the KINDS. During 1971,
approximately JO research grants representing $5,228^000 supported
investigators working in medical and scientific areas directly or indirectly
related to central nervous system trauma, particiilarly head injury. More
recently, this Institute has also begun to expand its support of research
on acute trauma to the spinal cord.

In general, the care and study of the seriously injured head
trauma patient has had as its primary concern the prevention of brain
swelling and therefore a search for the mechanisms that trigger the onset
of brain edema. Recent work in one of the head injury centers demonstrated
a marked diminution in arteriolar calibre (720 micra) in the injured area
following controlled impact trauma to the intact monkey skull. One day
after trauma the microangiograms showed a return toward normal in the
calibre of the larger vessels and an increase in tortuosity of these vessels
with a continuing diminution of capillary filling. Histologically, the
most significant feature was the presence of cerebral edema which became
apparent one day after injury, the edema being present in both the cortex
and the white matter. The brain swelling was well established between the
first and third days when the caliber of the arterioles was larger than
that noted in the immediate post traumatic period. This was also the
period when the capillary filling was abnormal.

Workers in another head injury center also have been investigating
cerebral vasomotor paralysis following artificial concussion in animals
and during the decompression which usually occurs in patients following
brain tumor extirpation. In the latter case, patients who had had prolonged
retraction of the brain during removal of a deep seated tiimor or clipping
of an aneurysm, had an intracranial pressure which was extremely sensitive
to changes in respiration. These patients were maintained on respiratory
assistance immediately following surgery and, quite often, removal of
the respirator was followed within two minutes by an increase in intra-
cranial pressiire of 30-50 mm Hg. The pressure promptly fell when respiratory
assistance was reinstituted. Several continued to decompensate and
ultimately the responsiveness of intracranial pressure to respiratory
control disappeared. These investigators attributed the continued rise
in Intracranial pressure to brain swelling and the lack of response to
changes in arterial p02 and pC02 to paralysis of the vasomotor tone of the
cerebral vessels.

This concept of cerebral vasomotor paralysis was developed in
monkey experiments in which pressirre waves were first identified and
studied. A series of moderately high intracranial pressure waves that had
no effect on the animal's EEG was often followed by a "terminal pressure
wave, " at which time the intracranial press\ire rose to the level of the
blood pressure, the EEG became isoelectric, and the animal passed rapidly
into shock unless fluid was rapidly withdrawn from the pressure inducing
balloon.

17w

In another series of experiments the relationship of cerebral
blood flow (CBF) and intracranial pressure (IP) was investigated. During
the control period^ when the arterial pressure was elevated with
norepinephrine, the CBF remained essentially constant. After a series of
balloon induced intracranial pressixre waves, norepinephrine was injected
again. The same increase in blood pressure now produced a greater rise
in CEF, leading these investigators to postulate that the vascular auto-
regulatory mechanism had been damaged. As with the patients discussed
above, the response of the cerebral arterioles to hypercapnia and hyper-
ventilation also disappeared. In the terminal stage, when intracranial
and arterial pressures were equal, cortical electrical activity had
disappeared, spontaneous breathing had ceased, and rapid evacuation of the
balloon produced a drop in IP to normal. This sudden expansion of the brain
following balloon deflation was attributed to rapid filling of flaccid
cerebral arterioles (vasomotor paralysis) and it was suggested that the
increase in brain volume was due to the rapid expansion of cerebral vessels,
cerebral edema, or a combination of the two processes.

Attempts to control and reverse the trend of progressive brain
edema in the head injured patient have involved the energies of scientific
personnel in a number of head injury centers. The efficacy of using
diuretics to successfully treat brain edema following head trauma has been
a subject of major concern because the results have had a wide range of
variability. Precisely controlled studies by one head injury center have
demonstrated that the brain swelling which follows experimental brain trauma
in animals is not reversed by steroid (dexamethasone) therapy. In terms
of human disease, this would certainly mean that, in those conditions
involving cerebral edema in which there is associated significant necrosis
and hemorrhage, the edema would not be expected to respond to steroid
therapy. The possible effect of steroids on inflammation and thus on
edema has not, however, been entirely ruled out.

Long-term studies on seizure incidence in children (birth to
lif years) following injury has shown that 10 percent of the head injured
children had at least one seizure during the first 2k hours after injury.
The incidence of cases with seizures dropped to 3 percent by the end of the
first year and approximately 10 percent of the children without seizures
(at the time of injury) developed EEG abnormalities, including spike foci
within 2-k years after injury. In addition, the head-injured child had
an increased incidence of negative behavioral traits, compared with their
pre-injury incidence. Among the major symptoms were hyperkinesis,
aggression, irritability and poor control of affect So

The effects of brain damage on performance and physiological
responses have been studied by another group of scientific investigators
who found that brain-injured patients have slower reaction times, diminished
alpha blocking, and disturbed orienting responses to auditory stimuli. In
addition, cold pressor stimulation to the brain-injured patient results in
depressed plethysmographic rebounds, suggesting differently tuned vascular
responsivity.

18

Another area of major concern^ and therefore major research
effort^ is regeneration of nerve tissue following central nervous system
trauma. In general most of the research models have employed the spinal
cord for study but one group has concerned itself with investigating
regeneration in the pyramidal tract. In man^ damage of the pyramidal tract
produces a total hemiplegia^ but these patients generally recover during
the following eight months with excellent control of the involved face,
arm and leg without developing the "pyramidal syndrome, " except for the sign
of Babinski. Parallel studies in monkeys revealed that the recovery of
motor function was not as complete as that recorded in man. These differing
results suggest that in man, as opposed to the monkey, there are at least
three motor systems; the first of which is the one mediated by the cortico-
spinal tract at the pyramidal level; the second being a corticospinal
pontine system; and the third, a system whose cortical origins and paths
are unknown.

An investigation concerning the origin and activity of cells
invading the damaged animal nervous tissue (spinal cord) has revealed that
they are mononuclear leukocytes. Large numbers of these cells enter the
white matter of the cord at all levels following root transection. Some
of these cells underwent cell division after entering the nervous tissue,
as evidence by the labeled mitotic figures. In addition, increased DM
synthesis was recorded in cells adjacent to the nerve cell bodies, both
in the cord and in the dorsal root ganglia.

Even more exciting are results of studies carried out in
another laboratory on regeneration following spinal cord damage. Axons
at the proximal end of the animal spinal cord following transection were
found to produce growth cones and characteristic new bouton connections on
axons and dendrites. This is the first positive data that mammalian
central nervous system neurones have the capacity to regenerate.
Additional work on regeneration in damaged animal spinal cord by another
group of investigators has demonstrated that one week following cord damage
the area of insult becomes filled with Gitter cells. At two weeks, axons
begin to appear beneath the pia, following the s\irface of capillaries, and
at seven weeks the area of destruction is filled with a heavy growth of
myelinated axons. Electronmicroscopy has shown that the cells giving origin
to the myelin are both Schwann cells and glial cells. The premise is that
the axons regenerating and encased by a peripheral type of myelin grow in
from the dorsal spinal nerve roots and blood vessels of the st-umps, while
those possessing a central type of myelin grow out from the spinal cord
stumps beyond the zone of damage.

That cultured embryonic spinal ganglion cells can form functional

synapses has been an area of endeavor in which considerable effori. has been

devoted by one group of investigators. They have shown that "typical

synapses" are formed, even though they lack other innervation territories.

The newly formed dendrites have functional meaning as recipients of input

rather than fortuitous efforts as abortive gro^rth. Intracellular records

of these ganglion cell cultures have been obtained, providing evidence

of synaptic interaction. Nerve growth factor (NGF) was employed in the

tissue culture medium and future studies are planned to answer the question

of how crucial this material is and if so, is it necessary for the formation

of functional synapses.

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OTHER NEUROLOGICAL DISORDERS
Sclerosing Disorders - General

Studies relating to sclerosing disorders in general continue to focus
;jrimarily on detailed investigations of lipids and myelin. The lipid studies
pertain to central nervous system lipids , lipid metabolism in normal and
pathological states, the relationship between brain lipids and electrolytes,
and a biochemical genetic study of bacterial lipids, phospholipids are the
subject of study with regard to their physical state, biological activity in
general and nerve-muscle function in particular, together with their metabolism
in membrane synthesis. The biochemistry of glycolipids, fatty acids, long
chain bases, inositol and phosphoinositides, along with the mechanism of ion
transport in nerve membrane and studies on subcellular fractions of developing
brain, continues in the service of a better understanding of general sclerosing
disorders. Myelin studies continue to emphasize structure, function, metabolism,
pathology and comparative studies, together with its formation and relation
to the neuron, its abnormal composition in plasma membranes and the inter-
actions of phosphoinositides in myelin itself.

Multiple Sclerosis

Multiple sclerosis is a disorder of the nervous system characterized by the
presence of tiny sclerotic areas or plaques of degenerating or destroyed
myelin in the white matter of the brain and spinal cord. Its symptoms are
referable to disruption of nerve impulses in the affected fiber tracts. Its
etiology remains to be determined and treatment is only symptomatic.

A current investigation is aimed at the etiology of multiple sclerosis and
the mechanism involved in its development. The work is directed at the
identification of infectious agents, measles virus in particular, that may be
responsible for the signs and symptoms of demyelinating diseases appearing
years after the initial illness. It is postulated that the measles virus in
some form may persist and provide active Immunity throughout life for most
people but on rare occasions may produce demyelination and symptoms commonly
associated with central nervous system disease, behaving generally in the
manner of the "slow" viruses. Evidence is now available which indicates that
the measles virus may be responsible for the pathologic changes known to be
present in various forms of measles encephalitis, a world-wide disease witn a
geographic distribution similar to that of multiple sclerosis, since its
incidence tends to be low in tropical areas and high in the more temperate
zones .

The discovery of inclusion bodies in the cytoplasm and nuclei of cells and the
formation of giant cells in areas of demyelination strongly suggest that the
measles virus is related to encephalitic manifestations. The hallmark. of the
virus has been found in individuals suffering from degenerative brain disease
years after childhood measles. Further, several patients with a classic form
of multiple sclerosis have shown inclusion bodies and giant cells similar to
those seen in acute and chronic measles encephalitis. Continuing attempts
will be made to recover active measles virus by co- cultivation with Hela and
human embryonic kidney cells. • ■

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Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis is a neurological disorder of unknown etiology
with an incidence of 8,000 to 10,000 patients in the United States. It is
characterized initially "by muscle weakness and wasting which is brought
about by degeneration of motor cells in the brain and spinal cord. Treatment

is symptomatic only.

A new study is in progress to develop an experimental model for amyotrophic
lateral sclerosis by immunomanipulation of mice infected with mouse encephalo-
myelitis virus which causes mouse poliomyelitis.

This study arose from suggestive evidence that certain progressive neurological
diseases of later life may be caused by a reactivated virus which seemed to
have been inactivated effectively many years earlier. Particularly intriguing
are some cases of amyotrophic lateral sclerosis which have appeared many years
subsequent to recovery from antecedent poliomyelitis. A not unreasonable
conjecture with regard to mechanism might be that the poliovirus has been
reactivated. Similar cases can be made for paralysis agitans, multiple
sclerosis and possibly Jakob- Creutzfeldt disease and parkinsonism.

The main objective of the program will be to develop a model system for the
reactivation of a central nervous system virus which can be applied to human
amyotrophic lateral sclerosis. The approach will be to infect animals with
mouse poliovirus so they contract the disease but ultimately recover. Follow-
ing recovery, the immunologic competence of the animals will be compromised
by various means with the expectation that latent viruses will manifest them-
selves under these circumstances.

Experimental Allergic Encephalitis

Experimental allergic encephalitis is an artificial, abnormally induced,
auto- immune disease in which the immune defenses of the organism are
manipulated and directed against its .own tissues. It has been used as a model
system for the study of immunological factors implicated in central nervous
system disease. Demyelinating diseases bearing resemblance to miiltiple
sclerosis may be induced by this means.

Serial measurement of cerebrospinal fluid beta- glucuronidase has been found
to be a sensitive method for detecting experimental allergic encephalitis and
for monitoring the course of the disease in the rabbit. Elevated levels have
been found only with clinical or histological evidence of the disease. Control
studies indicate that elevated cerebrospinal fluid enzyme does not resilLt from
induced serum increases of beta- glucuronidase levels. The suspicion is that
the increased cerebrospinal fluid enzyme levels in active experimental allergic
encephalitis reflect release of enzyme from lymphoid and other inflammatory
cells contributing to the encephalitic lesion. There are implications here
for human neurological disease states. Continuing studies indicate that cats
can develop experimental allergic encephalitis providing an elaborate
sensitization procedure is used, but so far, the disease has been induced in
a high proportion of animals only after placing them on a thiamine deficient
diet.

21 w

It was found that experimental allergic encephalitis could be inhibited in
rats by cyclophosphamide when given in daily doses beginning as early as the
first day of paralysis or as late as the fifth day of advanced paralytic
signs. It caused reversal of clinical signs and disappearance of lesions in
two thirds of the animals. Fully recovered animals showed a prompt relapse
when cyclophosphamide was discontinued. Continued remission could be obtained
with continued treatment.

Continuing investigation will determine the relative roles of different cell
types, subcellular factors and circulating antibodies in the development of
this auto- immune disease. Mechanisms underlying the inhibitory and therapeutic
effects of cyclophosphamide will be further investigated as will the apparent
ability of thiamine deficiency to enhance the susceptibility of cats to the
disease.

Experiments are being devised to discover the phylogenetic capacity to
develop auto immune disease and autoantibody along with che role of the thymus
and other central lymphoid tissue in the interest of preventing and treating
experimental auto- immune disorders.

Parkinsonism

Parkinson's disease is a progressive neurological disorder of unknown cause
affecting certain brain areas involved in the control and regulation of
movement. Its onset may be incidious and its progression may be almost
imperceptible ;, but is ultimately characterized by tremor, rigidity and bent
posture. Standard treatment in the past consisted of physical therapy, a
variety of driigs, and surgery. A highly effective form of replacement therapy
making use of large oral doses of L-Dopa or thalamic surgery are now the
treatments of choice.

A third center for research in parkinsonism has recently come into existence
and will be concerned with a correlated approach involving biochemical,
ultrastructural and neurophysiological methods in the same systems to elucidate
the role of catecholamines in the basal ganglia. The individual projects will
be concerned with anatomical studies of biogenic amines in the primate brain,
neuromelanin, neurophysiology of the substantia nigracaudate nucleus inter-
actions, blood-brain barrier to L-Dopa, amine metabolism, and motor potentials
in parkinsonism.

The anatomical studies of aminergic systems in primate brain that may be
related to the effects of L-Dopa administration and nigro- striatal pathology
will deal with catecholamine bodies in the substantia nigra and regions rich
in catecholamine terminals. They will also include mapping of nigro striatal
dopaminergic pathways in the primate. Experiments are planned which will
examine in detail the modulatary effect of the substantia nigra on the
cortico-striato- thalamic activity of the brain using both anatomical and
physiological techniques. Studies are also designed to elucidate the mechanism
of entry of Dopa or its metabolites into the central nervous system across the
blood-brain barrier and possibly across the choroid plexus. There are
suggestions that a central mechanism, not located in the motor cortex, develops
during learning of skilled movements which monitors and controls subsequent

22 w

vol\intary execution of specific motor acts. Selective inhibition of afferent
stimuli is essential to this mechansim. Interference with this system can
lead to slow behavior not unlike that seen in Parkinson's disease. It is
proposed to examine these hypotheses in animals and in Parkinson's disease
patients before and after L-Dopa therapy.

Work is in progress now to determine the effects of Dopa decarboxylase
inhibitors alone and in combination with monoamine oxidase inhibitors on the
amount of systemically administered L-Dopa that gets into the brain across
the blood-brain barrier and to explore dimethyl sulfoxide as a transport
carrier of Dopa molecules.

Finally, there is a program for the long-term follow-up of patients with
parkinsonism receiving L-Dopa and for investigations of L-Dcpa in selected
disorders other than Parkinson's disease. These studies are to be supplemented
with peripheral metabolic inhibitors and centrally acting monoamine oxidase
inhibitors when synthesized. Evaluation of dopamine-like substances, substances
which change the sensitivity of the central nervous system to L-Dopa, and the
role of serotonin as related to the involuntary movements induced by L-Dopa
will be included in these studies.

Neuromuscular Disorders

The most prominent neuromuscular diseases are myasthenia gravis (MJ) and the
muscular dystrophies (MD) which have prevalence rates of h and 6 per 100,000
respectively. While MJ has been recognized as a clinical entity since the
seventeenth century, the dystrophies constitute a variety of clinical
conditions. New dystrophic variants in both animals and humans are described
yearly and their classification constitutes a formidable neurological problem.
Myasthenia gravis has been related to impaired transmission through the
myoneural junction while the dystrophies are variously correlated with
deficiencies in muscle, nerve, intermediary metabolism, or the myoneural
junction.

MG is characterized by progressive impairment of neuromuscular transmission
with continuous exercise. Research in recent years has demonstrated that a
diminished quantity of acetylcholine is liberated from nerve terminals,
preventing adequate depolarization of the muscle membrane. Current observations
suggest that a decreased amount of the neurohumor is synthesized at the nerve
terminals, as judged by the lower activity of choline acetyltransterase in
muscle biopsies from M5 patients. It is not known whether the diminution of
enzyme activity results from the presence of an endogenous inhibitor or from
more primary structural or metabolic factors.

Treatment, for the most part, consists of the administration of anticholinesterase
drugs which act to increase the effective concentration of acetylcholine at
the endplate. However, in a significant number of patients, marked improvement
may be obtained by the chronic administration of ACTH or prednisone. The
basis for the efficacy of these latter drugs is not clear. Finally, the
surgical removal of a thymoma in a patient with M} often leads to marked
improvement. Curiously, the incidence of thymomas in MJ is approximately 15'/o
or about five thousand times more frequent than in control patients.

23 w

Recent studies continue to confirm the possibility that MG is an autoimmune
disease. The serum of any patients contain antibodies against their own
muscle, thymus, thyroid or other tissue. No antibodies have been identified
as yet as the myoneural jionction, and the presence of antimuscle antibodies
doeo not necessarily correlate with the course of the patients illness.
However, it has been reported that the serum of MG patients inhibits the
synthesis of acetylcholine by frog or rat brain.

Research efforts in the area of muscular dystrophy follow well-established
approaches. Substances which may alter the dystrophic condition are being
tested in humans, in animal models, and in tissue culture. Recently, it
has been reported that safflower oil reverses the hereditary muscular
distrophy of chickens. The testing of components of the oil as well as
structurally related analogues is currently in progress. Methods for the
detection of carriers of X-linked Duchenne muscular dystrophy continue to
improve. Approximately lOPJo of known female carriers have elevated levels of
serum creatine phosphatase. For the remaining 30fo who have normal serum
values, light and electron microscopic evaluation of biopsy material may prove
helpful. Myopathic changes in the muscle of carriers may be extensive,
minimal or absent.

The properties of dystrophic muscle continue to be elucidated. In tissue
culture, muscle cells from dystrophic chickens differ in rate of fiber growth,
rate of development of enzyme activity and strength of spontaneous contrations.
It has been shown that muscle fibers of dystrophic mice have significantly
lower resting potentials. The decrease for fast fibers is approximately
twice as large as that for slow fibers. Finally, it may be assumed that
present research on the molecular events in contraction and on the chemistry
of cell and organelle membranes will soon include dystrophic muscle.

Bioengineering Studies of Muscle Dynamics

During the past decade there was a surge in activity initiated by the joint
efforts of engineering, physical, biological, and medical scientists to
develop suitable methods for the prevention, treatment, and management of
disease.

The KINDS has played a catalytic role in this activity by providing research
grant support during the developing years of biomedical engineering, a field
which deals with the interaction between the engineering sciences in biology
and medicine. The report of the research activities of such a multidisciplinary'
team is presented below.

This group of investigators includes engineers with considerable experience in
experimental mechanics and in developing mathematical models; biophysicists
knowledgeable in systems analysis; biomedical scientists well qualified in the
field of nerve and muscle physiology; and a surgeon whose major research
interest is muscular- skeletal physiology and orthopedic surgery.

The long-term goal of their research program is to determine what differences
exist between the mechanical and control behavior of the limbs of normal
subjects and those afflicted with various known neuromuscular disorders. The
investigators' working hypothesis is that the neuromuscular skeletal system

24w

can be represented by a mechanical plus control' system model of muscle dynamics.
In disease- or disability afflicted subjects part of this system, depending
on the disease, will be functioning abnormally or not at all.

The model has undergone many modifications as experimental data have been
collected to check its various details. Since the system under analysis is
complete, the mathematical determination of the model constants is not a
simple process. A digital computer program has therefore been written to
process the experimental data and systematically determine the model parameters
from them. The program modifies parameter values in order to minimize the
error with which the model can predict the experimental data submitted to it.
The mathematical scheme used in this program, called an identification scheme,
has been treated extensively in control theory. The model is mathematically
non-linear, however, so that the development of the scheme has not been a
simple straight- forward procedure. The principal part of the successful
reduction system the investigators are now using is a non-linear simplex
method.

On the practical level, it appears that quantitative measures of neuromuscular
function in human neuromuscular disorders are almost non-existant. For example,
in connection with spasticity, it was pointed out a year ago that an objective,
accurately reproducible and easily applied test for muscle spasticity has yet
to be devised.

This group has developed control systems and mechanical models in the
relatively neglected field of muscle dynamics. The first stage of the study
was concerned with correcting, improving, and expanding conventional analyses
with particular regard to the theoretical effects of heat production on the
parameters of the model. Movement of the forearm around the elbow was studied
for this purpose. Movements of the forearm against various types of loading
in response to sensory signals were monitored both by measuring displacement
parameters and by electromyography. The study was extended to other muscle
systems of the body.

From the dynamic models of the system, the investigators hope to be able to
localize the malfunctions, and use the quantitative information obtained as a
basis for improved diagnosis and therapy.

The approach to this problem has two major components. First, the quantitative
approaches of engineering--especially in the areas of human dynamic structural
analysis, solid mechanics, and control theory--can now be applied to complex
non-linear biological systems such as human muscle. Second, such an engineering
analysis makes it possible to derive quantitative parameters.

To achieve this goal, the problem was attacked in four basic steps, three of
which are now well in hand. In the analytical and modelling phases of the
study, progress has occurred in three directions; analysis, model construction,
and parameter evaluation. In the analytical work, the investigators combined
current physiological understanding of muscle constitution and behavior with
continuum mechanics. They have thus established a useable quantitative model
for forearm movements including limb dynamics, muscle mechanics, and neural
control. It provides numerical measures of both neuromuscular parameters and

25 w

muscle tensions. A series of simple non- invasive tests provides a normal
data base.

Three types of studies designed to evaluate the parameters in the model have
been performed on human subjects. The first, a series of static tests,
demonstrates on the basis of the principles of structural mechanics that
averaged electromyogram amplitude is directly related to muscle contractile
force. This series of tests also provides numerical values for a number of
parameters in the model and for the forces in the individual muscles. The
second study, consisting of a set of constant angiilar velocity tests,
independently evaluates these same parameters as well as a damping coefficient.
The results are in agreement with those of the first study, and in addition
show that damping is small. The third study, a series of "quick- release"
tests, serves as an evaluation of the control parameters in the model. The
results indicate that direct positional control is much more important for
present experimental conditions than velocity control.

A model that includes central nervous system characteristics, local thermal
effects, wave propagation effects, and the dynamics of the skeletal frame
would be of considerable value to those physiologists, biophysicists, and
engineers who are working in the field of control theory and system performance
in other biological systems. This effort to provide a systematic account of
how muscle systems function in situ shoiild contribute basic information of
ultimate value to many medical specialties.

The investigators plan to continue this line of research to refine both the
structure and numerical constants in their model for the system so that it
represents the physiological condition more accurately.

At this time a complete testing and evaluation facility is already functioning.
Human subjects are being studies on a regular basis. The results obtained to
date indicate that numerical values for parameters, separately identifying
muscle properties and nervous system control properties, have been calculated
from measurements during a very simple (for the subject) dynamical test. This
means that changes in individual parameters in the neuromusclar systems of human
subjects can be monitored quantitatively. Such a quantitative technique is
much needed, not only for differential diagnosis, but also for monitoring
therapy.

In addition, studies of patients with a well characterized disability, e.g.,

muscular dystrophy, are planned to determine the clinical usefulness of the

model, thus leading to improved quantitative tools for diagnosis and clinical
studies .

Convulsive Disorders

Patients who suffer from convulsive disorders often conceal this fact because
it can adversely affect their social and economic status and their insurability.
It is therefore difficult to estimate numbers of persons affected. Their
number is estimated between 2 and k million. Conservatively, loss of earning
capacity caused by this disease, costs the Nation more than a billion dollars
a year. Much of this' would be unnecessary if tlae general public, by better

26w

understanding of the disorders ^ would make available to victims the social,
educational, and employment opportunities now denied them largely because of
ignorance and fear. Gnerally, persons with epilepsy have average or bex.ter-
than- average intelligence and good earning potential. A majority of them can
live normal lives because their convulsive disorders can be controlled with
anticonvulsant drugs.

Epilepsy is a disease characterized by one or more of the following symptoms:
(l) paroxysmally recurring impairment or loss of consciousness; (2) involuntary
excess or cessation of muscle movements; (3) psychic or sensory disturbances;
(k) perturbation of the autonomic nervous system. On the basis of origin,
duration of seizures , clinical and electroencephalographic evidences, epilepsy
is classified into four subdivisions.

In epilepsy known as "Grand Mai," in more modern terminology, "major motor
seizures," the attack is most violent and convulsions last longer. The
afflicted person may bite his tongue and invariably loses control of his
faculties. If the area or areas of the brain can be located from which these
abnormal electrical discharges emanate, the affliction is termed "Focal" or
Jacksonian epilepsy. In "Petit Mai," a disorder of the young, the seizures
are of short duration, and attacks occur more frequently. Seizures are
characterized by myoclonic jerks and they generally start in the extremities
and/or in one corner of the mouth. The affected part trembles violently. The
trembling movement moves upwards. It either ends up in a minor conviolsion or
the individual loses consciousness in the same way he does in grand mal.
"Psychomotor" epilepsy is caused by discharging neurons which exert their
influence upon the mental processes as well as upon the muscle movements. A
patient exhibits adversive or torsion movements, dreadful fear, flinging of
arms aimlessly, smacking of lips, and other incoherent physical and mental
behaviors. The last type is called "Autonomic" epilepsy. A person suffering
from it experiences a sharp, acute, sudden pain in the stomach. Hypertension,
perspiration and other visceral disorders are the common symptoms.

Epilepsy may be caused by several factors, such as brain damage, presence
of a scar caused by a wound or an injury, drugs, congenital malformation,
nutritional deficiences, metabolic abnormalities, fever (in infants), infectious
diseases (encephalitis, meningitis), brain tumors and abscesses. In a recent
study of the long term effects of administering a diet rich in median chain
triglyceride (MCT) on seizure control in children indicated that a diet
deriving 60 percent of its calories from MCT, when administered to a group of
children with a variety of seizures refractory to conventional modes of therapy,
was effective in controlling seizures, particularly myoclonic and akinetic types.
A less sustained control was obtained in some older children with psychomotor,
focal motor and grand mal convulsions. These studies provided very little
information regarding the mechanism of the effect of the diet on seizure
control. They only indicated that the diet produces hyperketonemia but no
metabolic acidosis.

The role that heredity plays is as yet undetermined. If one twin develops
epilepsy, the other one is likely to develop it also. However, patients with
this disease do not necessarily have epileptogenic offspring. Comparative
intelligence evaluations and electroencephalogrfim studies show that patients

27 w

with familial idiopathic epilepsy have lower intelligence quotients than their
non-epileptic relatives. It is estimated that a significant portion of the
U. S. population is epileptic with seizures that are symptomatic of a primary
disorder or of unknown idiopathic etiology. Evidence has been developed
indicating that at least a portion of the idiopathic epilepsy observed in man
has a genetic basis, although the specific mode of inheritance is not well
understood. Experimentally, using Beagle dogs with spontaneous, recurrent
convulsive seizures similar to those observed in man, evidence is being provided
indicating a strong genetic basis for conviolsive episodes. Based upon the
comparative studies of spike- and- wave EEG train in groups of patients suffer-
ing from febrile convulsions, focal EEG anomaly, or focal epilepsy, and in a
sample of normal people, it is suggested that several of the epilepsies might
liave a common genetic factor in their etiology. A number of investigators
have reported genetic involvement in other forms of epilepsy. These include
photogenic epilepsy, idiopathic epilepsy, benign familial neonatal convulsions
and partial epilepsy.

Although the exact mode of action of mechanisms underlying epilepsy is largely
unknown, the experimental evidence based on electroencephalographic studies
indicates that the neurons in the affected area build up a supply of electrical
energy through their metabolic action. By repetitive activity of charge and
discharge, the cells become overactive and start firing in synchrony. The
firing pattern may spread to other areas of the brain resulting in an epileptic
seizure. When the neurons start firing again in dysharmony, the seizure is
over. Brain waves so monitored give no indication of the individual's
intelligence, thoughts, or his mental health. However, they provide strong
clues as to whether or not a person has epilepsy. An EEG recorded during a
seizure is likely to show unusually high bursts of energy release. The
pattern indicates the type of seizure an individual has suffered.

One of the most versatile electrical instruments used in the detection of
electrical discharges from the nerve cells is an electroencephalograph (EEG)
which picks up electrical discharges from the brain cells, amplifies them and
records the impulses in the form of graphs. With its help, various areas of
the brain have been scanned and, using microelectrodes, electrical energy
discharge patterns in groups of cells or a single cell, both in vivo and
in vitro, can be recorded.

In order to understand the seizure mechanisms, epilepsy has been induced
experimentally in laboratory animals. The methods of induction can be divided
into four main groups. Methods based on electrically- induced seizures include
generalized stimulation of the intact animal to produce either threshold
(clonic) seizures or maximal (flexor-extensor) seizures, and local stimulation
of selected areas of the brain or spinal cord. Methods based on the
administration of chemicals induce seizures similar to the ones indicated for '
electrical stimiolation. Although chemicals, such as strychnine, picrotoxin,
methionine sulfoximine, and thiosemicarbazide, have limited value in inducing
seizures for evaluation of anti-convilsant drugs, they nevertheless provide
valuable tools for the study of seizure induction. The most important
chemicals used for producing seizures are convulsant drugs and irritant agents, i
For example, irritant agents, such as penicillin, applied to the cerebral
cortex or injected into various brain structures, result in an acute irritable

28w

focus, impulses from which may rapidly spread to other areas of the brain and
produce generalized seizures. Included in this section are the methods for
producing chronic epileptogenic animals by inducing various forms of brain
injury. Evidence linking acetylcholine to synaptic transmission in the
central nervous system and especially to cortical arousal systems, plus the
evidence that seizure activity can at times result from endogenously produced
acetylcholine, has led several investigators to focus their attention on this
agent with a hope to elucidate more general principles of epileptogenesis.
The injection of alumina cream in the brain of monkeys appears to produce a
reliable model which resembles human epilepsy. Investigations with iron
hydroxide, magnesium hydroxide, zinc oxide, chromium oxide and mixed earth
oxides showed that all these oxides or hydroxides are without epileptogenic
effect when applied to the motor cortex of the monkey. On the other hand,
nickel and antimony proved most epileptogenic only when implanted as pellets
into the motor cortex. Mild effects were observed with bismuth, zirconium,
tin, titanium, molybdenum and tungsten. Twenty- three more metallic powders
have been tested on the sensorimotor cortex of monkeys. Some have shown very
severe effects, while others remained ineffective. A third technique used in
inducing experimental seizures is based on sensory stimiolation, such as
elicitation of audiogenic seizures in susceptible mice and rats and photic
seizures in rabbits and baboons. Interest has recently been directed to
baboons which show a high incidence of spontaneous photic seizures comparable
to those observed in photic- sensitive epileptic patients. Lastly, seizures
may be induced by metabolic deviations, including water and electrolyte
alterations, CO2 withdrawal, hyperthermia, endocrine ablation, or hormone
administration. The aim of these studies is to elucidate the neural mechanism
involved in the transition of a localized lesion in the brain to an epilepto-
genic zone. This type of experimental approach has contributed new methods
of inducing chronic epilepsy in animals which provides an important new tool
for the study of this disease. By applying EEG techniques to these experi-
mentally induced epileptic animals, the efficacy of new drugs is being tested,
epileptic loci (foci) have been determined, and new and improved surgical
techniques for therapeutic application are being developed.

In a recent classification of the epilepsies, seizures elicited in the newborn
have been described as showing partial discharges, susceptible to change from
time to time, in both morphology and topography, from area to area and some-
times from one side to the other. In terms of epilepsy the maturing nervous
system acquires with age, an augmented capacity for generalized seizures, an
increase in the frequency, duration and spread of after discharges, and an
overall reduction of seizure thresholds. The developing nervous system has
thus been shown to be in an advantageous position in terms of protection from
convulsive disorders. Using developing brain as an experimental model,
greater insight into the pathophysiological mechanisms involved in the production
of seizures is currently being investigated.

One of the experimental leads to the study of the ontogenesis of seizure
mechanisms is based on the potassium shift across the nerve membrane. A
large increase in the radioactive potassium surface efflux prior to, and
during the early stages of seizures elicited by metrazol and electrical
stimulation of both the hippocampus and neocortex of cats and rats, was
observed. Using this as supportive evidence for the potassium accumulation

29 ^

hypothesis, a model for the regenerative, all-or-none aspects of the initiation
of seizure activity was proposed. Added strength was gained by a recent
observation that Dilantin ( diphenylhydantoin) , one of the most widely used
antiseiziire drugs, despite its great clinical utility, has thus far not been
shown to have any specific effect on the nervous system other than that of
antagonizing seizures. For this reason it has been of little value in under-
standing the pathophysiology of epilepsy. However, in later experiments
Dilantin was found to stimulate the rate of potassium uptake in an isolated
lobster leg nerve preparation. This effect was completely abolished by 2, k,
di- nit ro phenol. These observations, together with the finding that Dilantin
was without effect in changing the potassium efflux rate constant, suggested
that Dilantin may be stimulating the active uptake of potassium. The idea
that Dilantin exerts its antiseizure effect by stimulating the so called
Na-K pump is supportive of the hypothesis that accumulation of potassium
contributes to the seizure induction. By stimulating the active uptake of
extracellular potassium, Dilantin stabilizes the nervous system against
excessive changes in potassium which, according to this hypothesis, is an
effect that would be expected to diminish the likelihood for seizure activity.

Several physio- chemical differences between normal and epileptogenic brain
tissues have been found. For example, tissue from the area giving rise to
abnormal electric discharge did not bind as much acetylcholine as did the
normal tissue. This metabolite has been shown to occur in high quantities in
epileptic regions of the brain, which suggests that, when this substance
accumulates, seizures result. Furthermore, the widely accepted idea that
acetylcholine functions primarily as an excitatory transmitter in the central
nervous system is supported by the fact that intracarotid, intraventricular or
intracisternal injections, or the local application of this chemical to various
exposed areas of the brain provokes grand mal seizures. This observation is
further supported by the fact that inhibition of acetylcholinesterase led to
the same effects on seizure threshold as did the exogenously applied
acetylcholine. On the other hand, norepinephrine decreases seizure suscepti-
bility and dopamine and serotonin appear to have anticonvulsant activity.
Gamma- aminobutyrate (GABA) is of special interest in research on epilepsy
because of abundant evidence that it may function as an Inhibitory transmitter.
Applied topically to the exposed cerebral cortex or injected into the carotid
artery or the brain ventricles, it elevates thresholds for both chemically
and electrically induced seizures. In this connection, a deficiency of vitamin
B5 (pyridoxine) has induced seizures by producing a deficiency of GABA. Also,
a number of known convulsant toxins appear to interfere with the use of GABA
in the body. Disturbance of electrolyte balance may be related to the common
convulsion during fever in infancy. Information about the cause and correction
of such disturbances may be applied to reduce the severity of recurrent
epileptic attacks and to reduce the possibility of permanent brain injury
which may be caused by such episodes.

Clinical solutions to the problem of epilepsy are concerned primarily with
medical and surgical approaches. Before surgery is performed, it is necessary
to ascertain that the seizures originate wholly or in part from an area of
the temporal lobes that can be safely removed without causing serious neuro-
logical damage, the seizures occur frequently and are incapacitating, and
presently available drugs are ineffective in controlling the seizures. Also,

30w

the surgery must be accomplished so as to avoid creating a secondary scar

which may in turn cause recurrence of the seizures. Recently, it has been

found that barbituate and thiopental used in surgery can pinpoint areas of

the diseased brain tissue responsible for epileptic seizures. Locating this

tissue precisely permits it to be removed surgically.

Several investigators have confirmed the observation that sexual disturbances
in man occur frequently in temporal lobe epilepsy and less so in other varieties
of the disorder. The exception to this is the relatively rare instance in
which ant iconviils ants used in average therapeutic doses impair libido. It is
widely held that anticonvulsants are often responsible for impotence. This
may happen if the therapy is excessive. The majority of the patients with
temporal lobe epilepsy and with hyposexuality regained their normal sexuality
after operation. Attempts have been made to locate the precise region of the
temporal lobe which may be disordered in the impotent patients. Several
investigations have presented clinical evidence implicating the rhinencephalic
structures and their connections in sexual disorders, and have produced
convincing operative evidence that anterior temporal lobectomy may have a
beneficial effect on sexual functions.

Hypothermia (90 - 92 F) as a tool in therapy of patients with acute cerebral
lesions, such as cerebral contusions, cranial hemorrhage, tumors, and cardiac
arrest has been tried. Patients that were in status epilepticus, or had
intermittent seizures which could not be controlled by medication were
completely relieved of attacks or at least were benefited by cooling. Local
cooling of the epileptic brain resulted in the suppression of spiking activity
which also facilitated its response to anticonvulsant drugs. From this study
it was concluded that hypothermia is another treatment for problems of status
epilepticus where drugs are not effective or are contraindicated. Wo
complications were observed that could be attributed to the hypothermia.

The treatment of epilepsy has depended to a major extent on drugs. One of the
earliest medications was a sedative called bromide. This was followed by
another sedative, phenobarbital, which worked better, but caused drowsiness
in some cases. About twenty years ago diphenlhydantoin (Dilantin) was
introduced in the treatment of epilepsy. It has been widely used and has
proved to be a valuable anticonvulsant drug. However, it was soon recognized
that ataxia sometimes occurred as a complication of therapy with this drug.
The ataxia may develop rapidly over a period of a few days, or insidiously
over weeks or even months. One unexplained feature about this ataxia is the
fact that occasionally a patient will rapidly develop ataxia in spite of
having taken the same dose of Dilantin for several years. It has been thought
that ataxia is a benign symptom only requiring a reduction in dosage or
occasionally withdrawing of the drug. It was, however, later conclusively
shown that cats subjected to Dilantin medication had developed severe loss of
Purkinje cells in the cerebellum and cystic gliosis of the cerebellar white
matter. A number of cases of permanent damage to the cerebellum, apparently
due to this drug, have also been reported.

Further investigation on epileptic patients who had suffered Dilantin
intoxication indicated a high level of CSF protein levels during the period
of intoxication. All the patients improved when the drug was withdrawn, but

31^

abnormal signs occasionally persisted for several months. Inhibition of the
NA, K, Mg, ATPase enzyme system by Dilantin was observed which may be an
important factor in causing neuronal damage. It is suggested that Dilantin
intoxication may not be such a benign complication as was previously thought,
but this still does not alter the fact that Dilantin is probably the most
effective single drug presently available in the management of epilepsy. The
need of potent drugs of low toxicity is still paramoimt, especially in
psychomotor epilepsy. The use of Dilantin, phenobarbital, primidone, and
mephenytoin (Mesantoin) has left a relatively large group of inadequately
responding patients. Recently, considerable interest has been centered on
the use of benzodiazepines in the treatment of epilepsy. Diazepam (Valium)
I.V. has been proved to be effective in the interruption of epileptic seizures,
particularly Status Epilepticus. Similarly, chlordiazepoxide (Librium) and
especially nitrozepam (Mogadon) have been shown to be useful in prophylactic
treatment.

Oxazepam (Serax) which is a metabolite of diazepam is easily absorbed from the
intestines and is excreted quantitatively, or nearly so, as the glucuronide.
Oxazepam has been widely used as an ataraxic because of the large safety
ratio and the infrequent occurence of side effects (drowsiness, ataxia, skin
rash, headache, etc.). Clinical trials made it clear that Serax is a potent
drug in the treatment of psychomotor epilepsy and is of low toxicity compared
to Dilantin and other anticonvulsant agents. The effect is seen not only in
the reduction of seiziire frequency, but also in the KEGs. The fact that his
compound does not interact with Dilantin metabolism, facilitates its use in
combination with Dilantin.

In order to establish relationships between biological activity and molecular
structure, studies involving X-ray crystal structures are being undertaken on
anticonvulsant drugs used in the treatment of grand mal epilepsy. Recent
structural determinations of the clinically important anticonvulsants dmgs,
Dilantin and Valium, have shown that although these dn;igs are chemically
unrelated, conformationally the two drugs have many similar features. It thus
appears that their efficacies against grand mal epilepsy are primarily due to
stereochemical features. Similarly the drugs, procyclidine and triexyphenidyl,
newly important anticonvulsants, are being investigated by X-ray diffraction
methods and their molecular structure is being compared in detail with Dilantin
and Valium. Such biochemical and biophysical tools have provided new avenues
for the synthesis of more efficacious and less toxic anticonvulsants.

Infectious Diseases

Infectious diseases of the nervous system include many types of illness caused
by, or communicated by parasites, such as bacteria, protozoa, fungi or viruses.
Studies of infectious diseases are primarily concerned with epidemiology and
etiology of causative agents, their mode of transmission, host relationships,
and possibly ways to control their propagation.

Methods 'most commonly employed for recognizing the presence of viral agents in
cell cultures include; observations for cytopathic effects, hemaglutination,
hemadsorption, and interference, fluorescence and electron microscopies.
Immuno- fluorescent techniques are being used in diagnosing and studying brain

32v,'

inflammation due to viruses. Other studies are concerned with experimental
encephalitis, the epidemiology of Eastern equine encephalitis, the effects of
parasites on the nervous system, testing vaccines for protection against
arboviruses, and the possible role of viruses in acute neruological syndromes
in children.

Criteria of the responses of the experimental animal to viral infection are
established by careful monitoring of the physiologic behavior of the CNS
following inoculation with tissue extracts and whole cells derived from brains
of patients with chronic encephalopathies. The early events of the experimental
disease in animals have been detected by electroencephalography because the
EEG record becomes abnormal before any other signs of diseases appear. These
techniques are being perfected for clinical application. However, at present
it permits the selection of diseased animals for study during initial phases
of the pathogenic process. Before drawing any definitive conclusions about the
nature of the disease produced, especially about its relationship to the
original human disease, it is well to remember that a CNS disease produced in
animals may not be identical with the diseases of the patient from whom the
tissue was obtained. A dramatic example of species difference in host response
is the effect of simian virus B. In man it induces a disease that is severe,
and usually fatal, whereas for monkeys this virus is innocuous.

A considerable amount of work is being done on (l) after effects of infections
during pregnancy, where they may result in the offspring's brain damage and
mental retardation; (2) analysis of viral polioencephalomyelitis in animals;
(3) experimental measles encephalomyelitis; (k) mode and spread of a variety
of neurotropic agents; and (5) so-called "slow virus" diseases of the nervous
system. Several years ago a viral polioencephalomyelitis was identified in
pigs. The virus has now been isolated from several different organs and has
been shown to be an enterovirus quite unrelated to many other known viruses.
All isolates but one produced polioencephalomyelitis in germ free pigs
indistinguishable from naturally occurring infections.

Recently an agent causing paralysis of the CNS in rats has been isolated and
is called hemorrhagic encephalitis of rats (HER). It produces acutely lethal
encephalomyelitis when injected into suckling rats, including severe hemorrhagic
lesions of the brain and spinal cord.

Subacute sclerosing panencephalitis (SSPE) is a degenerative neurologic
disease of children and young adults. It is characterized by progressive
mental and motor deterioration, myoclonic jerks, and coma. The patients
become severely emaciated and die from intercurrent infections. The diagnosis
established during the incipient stages often shows a personality disorder or
mental retardation. At that time, the EEG shows slowing and dysrhythmia.
However, high amplitude, low frequency synchronous waves do not develop until
the patient exhibits myoclonic jerking. Spinal fluid proteins and cell counts
remain normal or increase slightly during the entire course of the disease.
Transmission of encephalomyelitis from humans to animals and further from
animal to animals, producing symiptoms typical of SSPE in the animal, has
provided an important new lead in isolating and understanding the causative
agent in SSPE. During the last few years, evidence of a relationship between
SSPE and measles virus has been established. With the help of electron

33^

microscopy, tubular structures have been seen in the brain with SSPE which
resembled the nucleocapsid.es of measles virus. Further investigation showed
a high or rising titer of measles antibody in serum, measles antibody in
cerebrospinal fluid, and measles viral antigen in the brain of SSPE patients.
On the basis of cytopathology, filtration, and serology, measles virus has been
isolated from patients with SSPE. These patients had no history of clinical
measles, but had received live measles virus vaccine; some had rubeola once
in their life time. Several characteristics common to both measles virus and
SSPE have been foiuid. They include antigenic properties, host- range,
cytopathogenic effects, temperature sensitivity, thermal inactivation and
interferon production. Although basic similarities have been established,
still unanswered are the questions of how and why the virus persists during
the long period after the patient has recovered from measles and before he
develops SSPE. Is the virus different in some way from regular rubeola? Will
measles vaccine protect against SSPE? Is the isolated paramyxovirus a neuro-
adapted strain of measles virus or a distinct but antigenically related virus?
Is a second "helper" or "co-virus" necessary to produce disease? If two
etiologic agents exist, how do they interact? How does virus reach the brain
and spread within the CNS? How is it maintained for years if indeed it is
measles virus? Are the unusually high serum measles antibody levels noted in
most SSPE patients in response to viral antigen in brain and how is the virus
protected from antibody? Does interferon play a role in limiting the disease?
Is it possible to reactivate a latent or defective virus in the CWS?

Measles encephalitis is a disease characterized pathologically by demyelination.
Evidence that the measles virus is directly responsible for the brain damaging
effects has been inconclusive. This consisted primarily of the demonstration
of cytoplasmic and nuclear inclusions, and small giant cells in brain material
from fatal cases in various stages of the disease. Presently, research is
directed towards the etiology of miiltiple sclerosis (MS) and the mechanism
involved in its development. It seems likely that the measles virus is
responsible for an acute demyelinating disease, known as "measles meningoence-
phalitis." It is postulated that MS may be a "slow" infection of the nervous
system by measles virus, i.e., that post-measles encephalitis, SSPE and MS
are different temporal manifestations of measles. It has been reported that
MS patients have significantly higher serum measles antibody titers than
patients with other neurological diseases or normal individuals. These are
the only observations that provide a possible link between measles and MS at
the present time.

The concept of slow viruses was originated by Sigurdsson in the year 195^ who
recognized in Icelandic sheep a special category of diseases with prolonged
incubation period, slow in clinical course, and restricted to a single organ
and species. Rlda (or scrapie), a chronic neiirological disease, and maedi, a
chronic pneumonia, were included. Their relevance to human diseases stems from
the similarity of sheep scrapie to human kuru. Kuru and Creutzfeldt- Jakob
Syndrome are two chronic human neurologic diseases. Kuru is a fatal neurologic
illness restricted to the Fore people of New Guinea. It has been transmitted
to chimpanzees. The transmissible agents involved have not been characterized
and studies are limited because a susceptible small laboratory animal or tissue
culture system has not been found.

31^ w

Electron microscopy has revealed particles similar to viruses of the papova
group in the brain from patients with progressive multifocal leucoencephalopathy.
No evidence has been presented on the transmissibility of this disease. Visna
virus is found in sheep and is also capable of rapid growth in tissue culture,
but pathogenesis in vivo is slow. Aleutian disease of mink is also considered
a slow virus .

Of all the slow viruses implicated in acute, subacute and chronic neuropathies,
scrapie is the only virus studied extensively because of the relative speed
and economy of using mice. Reliable criteria of its induction have been
established. Mice in advanced scrapie, when suspended by the tail, character-
istically clasp their hind legs together, whereas normal mice splay theirs
outwards. During uhe developmental stages, mice exhibit a sequence of
responses reflecting progressive central nervous system damage before the
clasping reaction is manifested. These studies have also generated many of
the seeming paradoxes of scrapie virus, such as its size relative to its
molecular weight and apparent absence of any nucleic acid. Hypotheses
explaining the special features of the scrapie agent include persisting tolerated
infection; defective virus; special auto- immune response; and a replicating
polypeptide, polysaccharide or membrane defect. Directly or indirectly these
concepts have provided rational bases for research. For example, if scrapie
is a defective virus, Sendai virus may be acting as a helper in the accelerated
course observed. If scrapie represents a membrane defect, it may be visible
by scanning electron microscopy. Scrapie virus has been successfully grown
in brain cell cultures of sheep, mouse, and mink. All workers have observed
differences in vigor and appearance between infected and normal brain cells
in vitro. Biochemical differences associated with scrapie have also been
reported. Increases in enzyme activity, such as glucosaminidase, offer the
possibility of quantitative assay using florescent- tagged enzyme substrates
and detecting the split produced with ultraviolet microscopy. Mice inoculated
with scrapie virus showed increased DNA metabolism. Inhibitors of DNA
metabolism ( idoxuridine and hydroxyurea) did not alter the course of disease.
Interferon has also been studied in scrapie. Extrinsic interferon, injected
peripherally, had no effect. Other studies showed that scrapie neither
induced interferon nor prevented its induction by viruses. Studies with
poliomyelitis and a crude predecessor of statolon, an interferon inducer,
showed that peripheral treatment was active against peripherally inoculated
polio virus, but not against polio virus inoculated intracerebrally unless
treatment was also given intracerebrally. Experiments currently in progress
indicate that scrapie can be altered in vivo. Test materials have been
selected for their relation to interferon, immunity, DNA metabolism, and
antiviral or other therapeutic activity. Wo effect on survival was noted with
adenine arabinoside, started the date before scrapie inoculation and continued
throughout the course of disease. Tentative findings indicate that short-term
amantadine treatment late in the disease did not reverse its neurologic effect.
The results of cyclophoshamide treatment varied depending on the time of
initiation. Recently, scrapie virus has been found in sheep brain inoculated
with SSPE and IVIS infected brain tissue. Also, scrapie-like illness in mice
inoculated with MS brain has been observed. Acceptance of the importance of
these reports awaits clear evidence that an agent responsible for the disease
in sheep and mice had its origin in the MS tissue and not in the experimental
animals or their surroundings.

35"

Current studies are directed towards the development of new methods of evaluating j
scrapie in vivo and in vitro, increased understanding of the disease and
etiologic~aient associated with scrapie, its related neurologic disorders,
and the means of altering them.

Myxoviruses are medium sized, ether sensitive RNA viruses, which in man have
the common effect of causing respiratory disease. Although they are not
regarded as "neurotrophic" viruses in man, mumps is the most common virus
causing central nervous system infection in rodents. In these studies (

occasional neurons are infected by the unadapted strain, and repeated passage
has yielded a strain which infects parenchymal cells of the brain, causing
acute encephalitis. However, mumps virus in hamsters has been shown to cause
a clinically inapparent infection limited largely to ependymal cells and neurons,
resulting in acute encephalitis.

Hydrocephalus may be caused by infectious agents contracted prior to birth.
Suckling hamsters infected by mumps virus developed a narrowing of the
aqueduct of Sylvius with subsequent hydrocephalus. The narrowing of this
canal, responsible for draining the ventricles of the brain, is the most common
cause of hydrocephalus in man.

The study was conducted with experimental models to determine the histopatho-
logical changes in the brain, aqueductal narrowing, and hydrocephalus induction.
The signs of clinical disease developed only after the resolution of the acute
infection (about fourteen days), during which the lining cells of the aqueduct
had been almost destroyed. Of the suckling hamsters inoculated with mumps
virus, ninety-five percent developed clinical hydrocephalus. These and other
histological changes were shown to be specifically related to the mumps virus
infection.

Although studies of experimental infections indicate that viruses may reach
the nervous system by various routes, studies of naturally occurring infections
indicate that the major pathway of virus spread to the nervous system is by the
hematogenous route. The various humoral and cellular defenses which act as
deterrents to limit the occurrence, intensity and duration of viremia, as well
as subsequent spread to the nervous system, constitute a physiologic blood-
brain barrier which must be overcome if infection of the nervous system is to
occur. It has been shown that herpes simplex virus gains access to the central
nervous system by the hematogenous route. Since blood contains both phagocytic
and immuno- competent cells, the extent and duration of hematogenous dissemination
of virus depends upon the outcome of interactions between host defenses and
virus. The characteristics of herpes simplex virus are: (l) it is a DNA virus
whose replication is susceptible cells is generally associated with formation
of easily demonstrable intranuclear inclusions; (2) it is associated with viremiaj
and central nervous system disease in both man. and animals; (3) it can cause
acute and subclinical infections; (k) it may persist in the tissue in occult
form for prolonged periods, giving rise to exacerbations of disease following
a variety of stimuli and; (5) it has been associated with chronic and recurrent
encephalitis in the rabbit, suggesting that it may also be responsible for
such illness in man. From these studies it has been ascertained that human
white cells, specifically lymphocytes will support the replication of herpes
simplex virus j^ vitro. This was the first DNA virus to be propagated in

36W

human leukocytes. Viral replication failed to -occur in the absence of
phytohemagglutinin, suggesting that lymphocytes must be in a particular growth
phase to be susceptible to infection with this agent.

Woi-K with the Sindbis encephalitis virus in mice demonstrated a precipitous
development of resistence during the second week of life, due apparently to
a limitation in the spread of infection. Wo non-specific viral inhibitors
could be detected. Studies on mumps vims encephalitis in hamsters showed
that disease and death resulted from the infection of neurons which remained
morphologically normal. It is suggested that this material may be useful in
investigating the "slow" viruses which seem to act in a similar way.

In Schilder's encephaloclastic sclerosis, virus-like particles were identified
by electron microscopy which are distinctly different from those observed in
the case with herpes simplex encephalomyelitis , but are similar to those that
occur in subacute sclerosing leukoencephalitis. The nature of these virus-like
particles is being investigated.

In 1958 there appeared the first report that free-living ameba could produce
fatal meningoencephalitis in animals. More recently a death of a boy was
reported who died five days after initiating the symptoms now being recognized
as typical of amebic nemingoencephalitis (AME). It is believed that the
invading ameba, Maegleria gruberi, enters the mouth and nose, possibly during
swimming. The victim soon suffers flu-like symptoms followed by rapid
deterioration and death. Unlike most of the parasites that live with man,
Naegleria, as a free-living organism, requires adequate oxygen. This may
explain why it has a great affinity for the oxygen- rich capillaries of lung
and brain tissue. The disease appears to be fatal and is without response to
anti- amebic drug therapy. Virus-like particles measuring about 100 mm in
diameter have been observed in some strains of these invading pathogens and
their morphological characteristics have been recorded. Preliminary experi-
ments indicate that mice are killed by introducing axenic EG ameba either
intracranially or interanasally. Other studies involving isolation, purifica-
tion, characterization of the active agent are currently underway.

In 19^3 and 19^4 a virus was recovered from mosquitoes in the San Joaquin
Valley of California. Until I965 virtually nothing was known about its
disease- causing potential in humans when the recovery of a representative
strain of this virus was reported from the brain of a child with a fatal
illness. It was apparent from serologic studies that many patients examined
were infected with California encephalitis virus coincident with rather severe
illnesses. These patients were studie very carefully to exclude other known —
causes of viral CMS diseases. All patients had encephalitic signs and symptoms
different from those due to enteroviruses. In contrast to enteroviral illnesses,
none of the patients in this study had a biphasic illness, none were associated
with upper respiratory of gastroenteric signs or symptoms, and no illnesses
occurred in siblings or other family contacts of the patients. Moreover,
seizures, coma, and disorientation were more frequent in the patients in this
study in comparison with children with CMS infection due to enteroviruses.
Behavioral correlaries of patients suffering with this virus showed difficulty
in receiving consistently meaningful, basic visual and auditory perceptual
information. They had little or no difficulty in the higher language functions.

37w

The personality of these children seemed to fit the "organic hyperkinetic
syndrome." The result is a predictable behavior pattern seemingly associated
with specific learning disorder.

Tumor C1300 has been in serial transplantation since 19^0 at the Jackson
Laboratory, Bar Harbor, Maine. This tumor arose spontaneously from the
region of the spinal cord of a albino mouse and was diagnosed tentatively as
neuroblastoma . Until recently no definite proof existed regarding its
neuronal characteristics. One of the characteristic enzymes of nerve cells,
tyrosine hydroxylase, is present in tumor C1300, confirming the diagnosis of
neuroblastoma. Recehtly it has been demonstrated that cell-free extract (CFE)
of neuroblastoma tumor induces a similar neoplasm in the dermis, mesentery,
spinal cord and sympathetic chain after subcutaneous injection. A total of
70 percent of CFE- injected mice developed neuroblastoma tumors. Like tissue-
transplanted tumors, CFE- induced tumors possess a similar amount of tyrosine
hydroxylase and, in vitro, differentiate into neuron-like structures. Electron
microscopic studies show that virus-like particles are present in CFE- and tissue-
induced neuroblastoma. The viruses are doughnut- shaped and appear to bud from
endoplasmic reticulum. Whether these viruses are causative agents in producing
neuroblastoma by CFE remains to be ascertained. The following studies using
partially- purified neuroblastoma viruses are in progress: (l) viral etiology
of neuroblastoma; (2) induction of neuroblastoma using different routes of
injection; (3) induction of neuroblastoma in different species; (k) deomonstra-
tion of the type of virus, DWA vs. RNA; (5) transformation study ±n vitro using
different mammalian cell-lines; (6) selection of a cell-line which sustains
viral multiplication without transformation; (7) exploration of the presence
of viruses in human neuroblastoma; and (8) the induction of tumor using cell-
free extract of glioma, medulloblastoma, astrocytoma and neurofibromatosis.
The animals and human tumors will be used for this purpose.

38 w

RIMAMENTAL RESEARCH M THE NEUROLOGICAL SCIENCES

Introduction

The great importance of fundamental studies on neurological, sensory, and
communicative components is frequently overlooked because they may have little
or no immediate clinical application. However, the history of science shovs
clearly that improvements in the treatment of disorders are largely dependent
on sound basic research.

It is generally accepted that the membrane around a nerve fiber plays an
essential role in the transmission of the nerve impulse along the fiber.
However, relatively little is known about the structure and mechanism of action
of the nerve membrane or of living membranes in general. There is no doubt
that research on membrane structure will contribute important information about
how the nerve impulse is transmitted along the nerve fiber . There is a
substance known as the nerve growth factor, the exact composition and source
of which is still uncertain, which has a remarkable stimulatory effect on the
growth of nerve fibers. The ultimate importance of the factor is still to be
determined, but it surely will have important functions in nerve regeneration
and it may well turn out to be useful in healing lesions and restoring function
in the brain and spinal cord.

There are several anti-convulsant drugs that are relatively effective in
controlling seizures in a good proportion of epileptic patients. Some of these
drugs are quite unrelated chemically and there is no explanation why they are
effective and why closely similar compounds are entirely ineffective. It has
been suggested that the efficacy of a drug may depend upon a highly specific
molecular structirre rather than on the chemical composition of the molecule.
Therefore, studies on molecular structure are now highly Important and involve
such sophisticated techniques as x-ray crystallography, electron paramagnetic
resonance spectroscopy, and nuclear magnetic resonance spectroscopy. On the
basis of such highly specialized information on the atomic interrelationships
and the electron activity in the effective molecules, it will be possible to
find or synthesize other molecules that are more effective in the treatment of
disease. It may also lead to an understanding of how the present drugs work,
which often is completely unknown now.

One of the great \xnknowns in neurophysiology has been the exact mechanism
by which the nerve Impulse is transmitted across the synapse from one nerve
fiber to the next or from a nerve to a muscle or other end organ. In 190^4- it
was suggested that some nerves accomplished this by the liberation of epinephrine.
In 1914 another investigator si;iggested that acetylcholine was the synaptic
transmitter in some cases. These postulates were finally proven in I92I by the
classical experiment in which two frog hearts were connected by a small glass
tube. When the beat of one heart was slowed by stimulating the vagus nerve,
the beat of the other heart also slowed a few seconds later, demonstrating
conclusively that some chemical was produced by the nerve in the first heart
which was carried in the perfusion fluid and produced a similar effect in the
completely separate second heart. It is now known that a number of compounds
(acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamlne, gamma-amlnobutyric
acid, glutamic acid, glycine) may act as synaptic transmitters. However, the
mechanism by which the transmitter may be produced and act very rapidly

39 w

(many times per second) and how the receptor nerve receives the stimulations
is still largely unknown. As mentioned at the beginning of this Report, there
are 10 to 13 billion nerve cells in the brain alone. Each then coimects with
from two to several other nerve cells. Therefore, the number of synapses is
almosc infinite and research on synaptic transmission is of the utmost
importance. Also, this is a good example of why the application of basic
research must be a time consuming process. The first evidence of the chemical
transmission across synapses was obtained more than 65 years ago. However,
the development of the area, even to its present imresolved state, had to
await the discovery of highly sophisticated techniques such as electron
microscopy and methods of chemical analysis sensitive enough to detect literally
only a few molecules of a specific compound.

The Nerve Membrane

One team of investigators has used spin labels (paramagnetic free radical
probes) to detect inside -out side conformational transitions of specific spin-
labeled lipids and proteins in biological membranes. A vai'iety of spin labels
have been synthesized and they are designed so they may be directed toward
a special region of the membrane or may serve as analogues of natural membrane
components. It was found that in membranes, particularly nerve and erythrocyte,
the motion of a series of spin-labeled fatty acid labels leads to a quantitative
estimate of the amounts of gauche and trans conformations about carbon-carbon
bonds of the fatty acid chains of phospholipids. Analysis of the magnetic
resonance spectra showed that the preferred orientation of the amphiphilic
labels I and II in nerve fibers was that in which the".long molecular axis is
perpendicular to the membrane surface. The use of spin-labels in nerves has
led to the development of a new theory of anesthesia. The magnetic resonance
spectra of lipid labels have revealed the fluidization of membranes by local
anesthetics and the tightening of the membrane in the presence of cholesterol
and antibiotics. ATPase of the cerebral cortex cell membranes is presumably
part of the active sodium potassium pump of the membranes. The fact that
biogenic amines such as serotonin and norepinephrine affect enzyme activity
and that the ATPase is foimd primarily in the synaptic region suggests that,
in addition to its function in maintaining sodium and potassium gradients,
it may be involved in nerve impulse initiation.

Another project is working toward providing a mathematical physical-
chemical theory of the activity of the axonal membrane and thereby to correlate
observed membrane phenomena and predict new phenomena. Further objectives are
to provide a theory of nerve activation by transmitter substances, to assist
in understanding the action of phannacological agents, and possibly to explain
cyclical nerve cell activity such as is detected by the EEG. The project is
designed to show that the excitability of membranes results from classical
physical -chemical forces, and does not depend upon any unusual specialized
structures, such as specific ion species channels, or special field- sensitive
gates. Some years ago it was shown elsewhere that the activity cycle consisted
first of all of a large increase in the inward Na+ current followed by an
increase in outward K+ flow. However, no attempt was made to provide a
theoretical foundation for the empirical eqimtion obtained since this was felt
to be beyond the available physical knowledge at this time.

40w

The problem is two-fold; the calculation of the steady state ion dis-
tribution, and the transient. The first requires the solution of the total
differential equation two -boundary problem, and the second, the PDE two-
value boundary problem. The total DE solution gives the steady state, from
which the activity cycle commences, and the PDE solution gives the activity
cycle. Both problems were much more complex than the investigators anticipated.
Much of the effort during the first two years was required to simply develop
the necessary numerical methods.

In this "model" of an excitable membrane, it has been shown that the
important factors are the height of the potential barriers for the ions to
traverse the two interfaces, the effect of the local electric field on these
barriers, and the blocking effect of absorbed CA++. A high negative charge
concentration near the outer surface of the membrane was found to be of
importance in giving the local field necessary at the interface. An interest-
ing and suggestive finding was the existence of oscillatory currents following
a disturbance. Their magnitude depends on factors yet to be investigated.
However, it appears that some clue to the source of EEG rhythms may be found.

Nerve Growth Factor

More than two decades ago it was observed that a crude extract of mouse
submaxillary gland coiold produce an explosive growth when applied to cultured
ganglion cells. Several years later a protein was isolated and purified which
possessed the characteristics of nerve growth factor (NGP). It was deteimiined
that the growth promoting factor was present in lower concentrations in a
number of other tissues, and that the extract of mouse salivary gland contained
a number of proteins other than NGP. Most notable among the latter is a
protein that specifically stimulates the growth of epithelial cells. Still
other growth factors may be identified in the future. The chemical and
biological inter -relationships among the various substahces isolated from the
mouse submaxillary gland are both complex and uncertain since chemical con-
stitution and biological activity are closely dependent on the isolation
procedures employed. However, it is now generally agreed that the simplest
NGP protein extractable from the gland is relatively small with a molecular
weight of 25,000 and is very basic. A larger species with NGP activity is a
combination of this basic protein with two other types of subunits, one of
which is an esteropeptidase. Whether or not other preparations with NGP activity
are actually distinct from these two, has yet to be determined. In any case
biological activity as reflected by neurite growth in tissue culture can be ob-
tained with most preparations at a concentration of 10"T grams /mililiter.

The mechanism of NGP activity is being pursued intensively in a number of
laboratories utilizing modern techniques of biochemistry and cell physiology.
We may soon know whether the NGP protein acts on the nerve membrane or moves
through it, how cellular synthesis of nucleic acid and protein is initiated by
NGP and how NGP inactivates the mechanisms which control cell size, shape and
volume. As may be expected a number or laboratories are investigating the
effects of NGP on regeneration in the nervous system.

Structural Studies of Drugs

By methods of single crystal structure determination, using X-ray and

i^iw

neutron diffraction^ one project is studying the structural details of the
■way in which barbiturate molecules associate with each other and with molecular
species of biological systems^ such as water, peptides and lipids. The use
of barbit\rrates in neurological disorders is well known. It has been assumed
that for effective action a barbiturate, initially in an aqueous medium,
must be capable of associating with the lipids of a biological membrane to
modify the normal metabolic cellular processes such as membrane transport. A
related possibility is that the barbiturate may directly inhibit the complex
enzymatic processes of oxidative phosphorylation. It is now believed that the
multi-enzyme system in electron transport is effectively blocked by barbiturate
in the area of a flavoprotein.

The detailed crystal structures of some 20 barbiturate derivatives have
been determined and tested in chemical and/or biological systems. It was
found that the barbiturate ring is planar, apart from slight distortions
depending on the crystal environment. Exceptions are found in the "half chair"
conformation of 5^ 5-<3-isubstituted barbiturates in which at least one
substituent is hydroxyl. With one exception (alloxan), all potentially hydrogen
bonding hydrogen atoms were found to be hydrogen bonded.

There was a strong preference for barbiturate imine groups to hydrogen-
bond with carbonyl groups of other barbiturate rings rather than with C(5) sub-
stituent groups or water molecules. Almost all barbitiirate structures thus
exhibited a one- or two-dimensional framework of NH. ..0-C hydrogen bonded
barbiturate rings which appears to be the dominant crystal structure determin-
ing activity. Although the nature of the C(5) substituents and the possible
presence of water of crystallization determine the manner of ring hydrogen
bonding to form this framework, their own hydrogen bonding requirements appeared
to be of secondary importance. The latter may frequently be of the "bent" or
"bifurcated" type, indicating relatively weak hydrogen bonding interactions.

The overall aim of another project is to determine the conformations of
acetylcholine (ACh) when it becomes attached to its nicotinic and its muscarinic
receptors. The approach was to synthesize analogues of ACh in which a degree
of rigidity was introduced into the amino alcohol portion of the ester.
Cyclopropane and cyclobutane rings were used to impart the desired rigidity in
the ACh analogues and congeners. Free rotation was thus prevented, and the
number of possible conformers was greatly reduced. The overall shape of the
molecules and pertinent ranges of interatomic distances could then be estimated.
This work is based on the premise that the conformation and moleciolar shape of
ACh are critical factors in its actions at the two types of cholinergic
receptors.

Some 50 analogues of ACh have been synthesized, particularly those in
which the amino alcohol portion is a part of a cyclopropane or a cyclobutajie
ring system. Each of the enantiomers of trans-2-acetoxycyclopropyltrimethylam-
monium iodide has been prepared. The absolute configurations have been
determined and have been correlated with biological data. A new approach to
the sjmthesis of c is -2 -acetoxycyclopropyltr imethylammoniuin has been developed
which Hiay permit higher yields of the compound.

In the studies on 'the cyclobutane series, cyclobutane carboxamides have

42w

been converted to cyclobutylamines with lead tetraacetate. 2-Substituted
cyclobutylmethyi ketones have been prepared from 2-substituted cyclobutane-
carbonyl chlorides, and these ketones have been shown to undergo a facile
Baeyer-Villiger reaction to give high yields of cyclobutyl acetates.

Despite its relative chemical simplicity and its importance as a neuro-
transmitter, the mode of action of ACh is still unknown. A logical hypothesis
is that the flexible ACh molecule assumes different molecular shapes and
different interatomic distances at the nicotinic and the muscarine reptors,
conditions which are imposed on the ACh molecule by the chemical and steric
nature of the receptor area. With the synthesis and testing of ACh analogues,
this project is trying to clarify the mode of stereo-chemical attachment of
ACh at its nicotinic and muscorinic receptor sites and in this way truly explain
the mechanism of action of ACh.

The Synapse and Neixrotransmitters

The identification of synaptic transmitter compounds and the mechanisms
by which lobster nerve cells regulate their accumiilation has been studied
extensively in one laboratory. Early work showed that gamma-aminobutyric acid
(gam) was the inhibitory transmitter at the neuromuscular junction, that it
was the most active inhibitory substance present in nerve extracts, and was the
only physiologically active compound selectively localized in inhibitory
neurons. Glutamate was the only excitatory substance found.

It has now been shown that GABA is actually released when an inhibitory
neiiron is stimulated and the amount released is proportional to the number of
stimuli applied. Like the release of chemical transmitters at other synapeses,
it depended on the presence of calcium in the bathing fluid. Recently, it has
been possible to localize the site of GABA uptake in lobster nerve-muscle
preparations. Certain aldehyde fixatives will covalently bond amino acids to
tissue components. With this method, using h3 labeled GABA, the site of uptake
was shown to be the connective tissue cells and Schwann cells that surround
the nerve -muscle preparation.

Using single excitatory and inhibitory axons, enzymic studies showed that
the pathway of GABA metabolism in the lobster was identicaJ. to that in the
mammalian nervous system. The substrates, glutamate and alpha -ketoglutarate,
were present in the same concentrations in excitatory and inhibitory axons,
and transaminase activity was similar in both kinds of cells. Only decarboxylase
(and GAEA) was different, with about 100 times as much activity in the inhibitory
axon. It was found that high concentrations of GABA could inhibit the decar-
bo:cylase. At the normal concentrations of GABA and glutamate in inhibitory
axons, the synthesis of GABA approximately balanced its destruction. This led
to a new mechanism to explain the selective accumulation of GABA in inhibitory
axons, with the decarboxylase being the key to accumulation and the GABA
inhibition of the decarboxylase the key to the final level to which GABA
accumulated.

In recent years, glutamate has been thought to be the excitatory trans-
mitter compound. After a prolonged series of experiments, it was possible to
demonstrate a small (lO^) release of glutamate after excitatory nerve

l^y

stimulation. The lack of adequate methods made 'it impractical to try to
obtain the necessary controls of calcium dependence on release or the pro-
portionality between frequency of stimulation and amount of glutamate released.
Quantitative comparisons of glutamate in excitatory axon extracts and the amount
of excitatory activity in glutamate units in the extracts showed that there is
about 2 to 3 times more activity than glutamate. After much work, it was
shown that most of the excess activity could be accounted for as asparate.
Asparate alone is relatively ineffective^ but it can add to the excitatory
effect of glutamate^ causing an apparent potentiation of the glutamate res-
ponse.

Studies on the post-tetanic potentiation (PTP) and post-tetanic repetition
(PTR) are particularly important because these responses disclose the activity
of motor nerve terminals. By this mechanism, another laboratory has studied
the effects of many compounds on the soleus motor nerve terminals (MNTS) in vivo.
Using increasing doses of d-tubocurarine (dTC), it was fo\md that PTR and
PTP were progressively suppressed and finally obliterated. It is impressive
that PTR and PTP were abolished by a curare (dTC) dose that had only a minimal
effect on neuromuscular transmission. This showed that the MIITS were a highly
sensitive locus of dTC action, but that this in itself was not the source of
the neuromuscular blocking action of curare. Nevertheless, this effect suggests
that the pre-jimctional structure may still be important in the action of de-
polarizing neuromuscular blocking drugs.

Because of its importance in neuromuscular studies, Mg was investigated.
It was found that Mg also depressed the PTP and PTR, This demonstrates that
the action of llg is on the MNTS. The pre- junctional effect of Mg, like the
dTC action, preceded the effect on transmission. Also, it was found that
transmission block and depression of directly stimulated muscle coincided.
Thus, Mg depressed both muscle and MNTS, whereas dTC acted selectively on MNTS.

The local anesthetics, procaine, lidocaine, tetracaine, and debucaine had
been shown by other investigators to suppress PTP and PTR. Studies on this
project showed that the respective potencies of these drx;igs to depress MNTS
correlate exactly with the order of their condution blocking action on
peripheral nerve. This result is important because analysis of procaine action
on neuromuscular transmission by in vitro quantal methods by other workers had
indicated that the major action is curariform on the post-junctional membrane.
No doubt these drugs, like Mg, will affect excitable tissues non-selectively,
but at the neuromuscular junction the first effect, as dose is increased, -^ is
the paralysis of MTS.

Diphenylhydantoin (DPH), a drug used in epilepsy, was known to suppress
PTP of the monosynaptic reflex pathway. Therefore, its effect was studied on
the hyperpolarization of MNTS that follows high frequency stim\£Lation. Anti-
conviasant doses were found to suppress MNTS PTR and consequently the muscle
PTP. Apparently, DPH opposes the hyperpolarization of MNTS just as it does in
the primary afferent endings in the 2N pathway. The DPH action on post-tetanic
hyperpolarization indicates that the drug selectively suppresses after-potential
production in the terminals.

All of this work has been done in vivo and, therefore, the results are

kk

unquestionably relevant to clinical neuropharmacology. AlsO;, the actions
of the various drugs classically associated with modification of neuromuscular
function have been seen in a new light and new understanding of ^their action
has been developed.

With the use of electron microscopy and semi-quantitative microscopic
spectrofluorometry, a team of investigators has been studying the function
of neurohumors such as serotonin (5-HT)^ nor-epinephrine (NE) and acetylcholine
(ACh). It was possible to demonstrate a relationship between the dense-core,
or granular, nerve ending vesicles and a storage form of NE in the rat vas
deferens. Vas deferens depleted of endogenous WE were exposed to WE or to
one of its precursors, DOPA or dopamine, in the presence of an inhibitor of the
conversion of precursors to WE. Only WE specifically restored the proportion
of granular vesicles to nearly normal levels. Thus, the granular vesicles were
identified as the intra-vesicular compartment of WE storage. In the presence of
an inhibitor of monoamine oxidase iproniazid, exogenous WE accumulated in the
extravesicular compartment, but did not enter the granular vesicles. Under
these circumstances, the contractions of the vas deferens usually produced by
stimulation of the nerve were markedly inhibited, presumably due to excessive
NE in the extravesicular compartment. It is clear from these studies that the
distribution of WE can be identified in its storage sites at the subcellular
level and that the subcellular localization of the amine influences physiolog-
ical and pharmacological effects.

This group has also studied neurohumors in the rat brain. They showed
that gamma-hydroxybuturic acid lactone (GBL) induced anesthesia, but had no
influence on 5-HT and gamma -aminobytyric acid (GABA) levels in the brain, and
iTke all central nervous depressants, produced an increase in brain ACh. In
regard to effects on brain catecholamines, neither gamma-hydroxybutyric acid
(GHB) nor GBL altered brain NE levels, but they did selectively increase
brain dopamine (M) levels, occasior-ally as much as 100^. In addition
gamma -methylpara tyro sine, which blocks the synthesis of both NE and DA, caused
about 100^ increase in the sleep time of animals given GHB or GBL. This
indicates that DA may be necessary for the arousal of an animal from a sleeping
state. The increase in DA during GHB-induced sleep may be due to a reduced
utilization of DA or to a compensatory activation of synthesis as a resiolt of
suppression of dopamine receptors by the GHB. Thus, a study of the ultramicro-
scopic localization of neurotransmitters may make important contributions to
knowledge of sleep mechanisms, a problem of utmost importance in itself.

1^.5w

COMMUNICATIVE SCIENCES

In communication, language is the carrier of the vast amount of what
we call culture. Knowledge of the past, techniques of food getting, science,
and social rituals are all carried in language. The social psychologist tells
us that language is important first as it relates to communication and secondly
as it functions in the socialization of the individual, particularly in the
development of his personality. Moreover, it carries for the person the social
definitions of situations, the world of discoiirse and the whole range of cul-
ture content which impinges upon and shapes the individual. The National
Institute of Neurological Diseases and Stroke is pleased to report many
important advances in knowledge in the Communicative Sciences this past year.

Sensory Processes

A physiological and psychophysical investigation in three sense modalities:
audition, touch and vision, complemented by analytical procedures involving
mathematics, electrical network modeling and digital computers, has extended
the analogy between auditory and tactile psychophysical characteristics to
cover several aspects in the frequency, time and space domain at threshold,
as well as at suprathreshold levels. The generalized functional relationship
between the firing rate produced by sensory reception and stimulus intensity,
which was discovered in the previous year and which seems to hold indepen-
dent of sense modality and animal species, was further documented with the
help of neurophysiological experiments on the Limulus lateral eye and the
auditory organ of Southern Army Worm Moths. A theorem resulting from the
mathematical formation of the relationship was confirmed by studying summation
between sinusoid and a band limited random noise. In audition measurements
of central masking characteristics have continued, and the psychophysiological
theory of central masking developed in the previous year was further validated.
In electrophysiological investigations of the auditory receptors of the
Southern Army Worm Moth, it was possible to infer the principal site of mechan-
ical stimulation. In tactile studies, it could be demonstrated that the slope
of the subjective intensity functions is negatively correlated with the thresh-
hold intensity and the density of innervation. Sensory studies continued to
focus on the Limulus lateral eye. The neurophysiological recordings from
single receptor units have brought out a nonlinear interaction between
excitatory and inhibitory processes and have made it necessary to introduce a
nonlinear correction factor into the Hartline-Eatliff equations.

Another investigator, attempting to determine the extent to which
evidence for the Stevens power law shows itself in the recorded potentials
from sensory cells, determined that in numerous sense modalities the neuro-
electric potentials can be shown to grow as a power function of stimulus
intensity. The power function exponents for two modalities, vibration and
hearing, have exhibited an interesting dependence on frequency.

Auditory Masking

In attempting to identify differences between sinusoid and noise masking.

k6^

an investigator developed a new hypothesis which accounts for some of the
differences. The observer;, faced with the problem of trying to detect a
weak sinusoid in some masking stimixlus, searches the frequency spectrum and
listens primarily at the region in frequency where the signal - to -
masker ratio is most favorable. In detecting a sinusoid in a white noise
spectrum^ there is no problem. The maximum signal - to - noise ratio occurs
at the signal frequency^ and asserting that the observer listens primarily
at that frequency^, is simply the statement of the critical band hypothesis..
Detecting a gated sinusoid in a continuous sinusoidal masker is a different
problem. The mere fact that a signal is turned on and off implies presence
of energy in regions of the spectrum other than the signal frequency. The
amount of this splattered energy depends on the exact shaping employed in
the process of gating the signal. The ratio of signal - to - masker energy
depends both on how the signal is gated and how much the masker is attenu-
ated by the observer's auditory filter. The critical item is not the rise
time of the gating filter, but the off -band attenuation rate of the auditory
filter.

Vestibular Mechanisms

One group of researchers developed an analog equivalent electrical
circuit of the cochlea that follows the principles of the electromechanical
hypothesis for the function of the organ of Corti. The steady-state
parameters of this heuristic electrical model were calculated from data
about the impedance and potential differences that exist across the walls
of the cochlear partition. The behavior of the model is such that it replicates
many observations obtained during experiments on the cochleas of guinea pigs.
The model affords a qualitative as well as quantitative approach to the study
of the direction, magnitude, and source of electric currents flowing through the
inner ear dirring acoustic stimixlation. A series of experimental investigations
on optokinetic nystagmus revealed that the oculomotor responses to optokinetic
stimulation of rabbits, cats and man were qualitatively similar when studied
by electronystagmographic methods. There were, however, some noticeable
quantitative differences. The frequency of nystagmus increased as the velocity
of the stimulus was made greater. For comparable velocities, the frequency of
the nystagmus in the rabbit is smaller than that of the cat and of the "stare"
nystagmus in man, but is larger than that of the "look" nystagmus in man. The
greater effectiveness of the optokinetic stimulus when presented monocularly
and moving in the usual field from lateral to medial direction depends on the
existence of a large number of ganglion cells in the retina that are pre-
ferentially stimulated by visual targets moving in that direction.

Another group of investigators described the discharge characteristics
of peripheral vestibular neurons innervating each of the semicircular
canals in the squirrel monkey. The average resting discharge was about 90
spikes/second. All neurons responded to angular accelerations in one direction
with an increase in the discharge and to oppositely directed accelerations
with a decrease. The response was always consistent with the morphological
polarization of the hair cells. Many units did not adapt and their response to
constant accelerations resembled the response predicted by the torsion-pendiilum
model. This and two other studies on the physiology of peripheral neurons
innervating the otolithic organs of the squirrel ;3Jonkey produced findings that

reinforced to a certain extent the current theory on the mechanics of the
semicircular canals (overdamped torsion-pendixlum model) and also provided a
basis supporting various observations in m.an and animals^ which deviated or
are not predicted from theoretical considerations. Further, these studies
may help to interpret the response of semicircular canals when exposed to
rotatory or caloric stimiolation as practiced in the clinic.

Cochlear Mechanisms

The cochlear potentials (coclilear microphonics, summating potential and
endocochlear potential) as recorded inside the cochlear duct showed much
less variability than when these potentials were recorded by one investigator
in the perilymphatic spaces. The magnitude of the cochlear microphonics
recorded in the same place in the cochlear duct showed a range of less than
+ 3 d^. around the mean. At the same time the magnitude of the cochlear
microphonic for different species varied as a function of frequency in a
manner that is different in different animals. Cats showed the greatest
resolution in their tuning curve. The summating potential behaved similarly
among different animals of the same species, but also showed quantitative
differences among species.

Another investigator working on cochlear hair cell metabolism and
cochlear potentials found that scala tympani resting pressirre in the
anesthetized guinea pig was usually slightly higher than atmospheric pressirre,
the greatest value measured being 10 mm Hg. with an average of 6 mm Hg. The
scala typmani pressure rose markedly during breathing of low oxygen concen-
trations and the scala vestibuli pressure changed in the -same direction as
scala tympani pressure diiring anoxemia. Finally, the scala tympani pressiire
during anoxemia followed a rise of cerebral spinal fluid pressure. It was
also noted that intravenously administered dinitrophenol caused an increase of
cochlear endolymphatic dc potential, in some cases 15 to 20 percent.

Inner Ear

A group involved in microscopic studies of the inner ear reported
pathological changes produced by age, disease, drugs, and noise. They found
that interest tends to focus more and more on tissues, such as the stria
vascularis and spiral ligament, that provide by their metabolic activity the
special homeostatic controls that the organ of corti requires for its func-
tioning, and the blood vessels supplying those tissues. The degenerative changes
that occur in Corti 's organ itself tend to follow a more or less stereotyped
pattern, regardless of their cause, and the sequence of changes, especially as
they affect the hair cells, is now well established. In guinea pigs treated
with Kanamycin they found there is an increase in the niimber of avascular channels
of the spiral ligament above Reissner's membrane, indicating a loss of \

capillaries. Kanamycin causes not only loss of capillaries but changes in the
cells of the stria, especially the intermediate or chromophobe cells. Exposure
to white noise for 6 to 30 hours at 120 dB. produces capillary vasoconstriction
in the spiral ligament and in the vas spiral system, as well as vacuolization
of Reissner's membrane. The hypothesis is that capillary vasoconstriction
is the primary result of noise exposure associated with the temporary elevation
of auditory threshold. Similar vascular changes are caused by quinine and

k&v

salicylates. If long continued, the ischemia causes hair cell loss and per-
manent threshold shifts.

Another group engaged in the chronological development and accumulation
of acid mucopolysaccharide and various enzymes in the endochrondrol model
Gi rodent temporal bones spent this year working out techniques which will
allow them to assess more critically acid mucopolysaccharides, phosphatases,
phosphorylase and non-specific esterase in the otic capsule. The specific
activity of these enzymes are also being investigated with controlled chemical
procedures including colorimetric determination of the various enzyme systems.

Hearing Disorders

Investigators concerned with determining the characteristics of very
low frequency auditory sensitivity observed that the rate at which auditory
sensitivity (as determined by microphonic potentials) decreases with the
decrease of stimulus frequency below approximately 100 Hz is apparently species
dependent. A combined anatomical and electrophysiological study was completed on
four species: cat, guinea pig, chinchilla, and kangaroo rat. It was demonstrated
that the sensitivity change below 100 Hz is 12 dB. /octave for cat and chinchilla,
while it is only 6 dB. /octave for guinea pig and kangaroo rat. Concomitant low
frequency cochlear microphonic phase differences were also seen. They have
shown that it is the size of the heliotrema that is primarily responsible for
the differences. The area of this opening is approximately ten times greater
in the cat and chinchilla than in the other two species.

Comparative Hearing

A group of investigators working on the problem of sound conduction in the
ear foimd that some problems could be solved by studying certain species of
lizards. Previously, they found that some chameleons, which lack an external
ear opening and lack the tympanic membrane, hear rather well. Some possess a
substitute tympanic membrane in the form of a thin plate of bone (a modified
part of the pteryoid bone). Recently, two additional species have been in-
vestigated; one ( Chamaeleo dilepis) that has this substitute mechanism, and
another ( Chamaeleo ellioti) that lacks it. The hearing was found to be
significantly better in C. dilepis. A second form of substitute for the
tympanic membrane was encountered in Cophosourus texona. It consists of an
air sac within the middle ear cavity with which the columella is in contact.
This ear performs remarkable well in sound reception. A problem of central
interest is the manner of stim\ilation of the hair cells, and the relative
effectiveness of different structures and modes of operation. In a study of the
basilar papilla of the crocodilian, Caniman crocodilus, researchers found
that the papilla differs from that known in other reptiles and in vertebrates
in general. The general morphology of the papilla bears some resemblances
to that in birds, but the structure of the short hair cells and supporting
cells appears to be unique among vertebrates in which the fine structure of the
ear is known. The Chamaeleontid shows papillar structure suggestive of a
cytologically unspecialized condition, somewhat reminiscent of that in tiortles.

49W

Echolocation

Investigators are pursuing the design of echolocation in hats and the
processing of acoustic information hy the bat brain by analyses of the
"sonar" output of several genera of hats - Hyposideros, Mo r mo ops,
Chilonycteris, and others; some during goal directed flight and electro-
physiological recording from the cochlea, auditory nerve, and inferior
colliculus. Bats using pulses of long duration, Chilonycteris parnellii and
Rhinolophic, sire now kno-^m to use pulse duration, in effect, to separate two
channels of information arriving via echo. The constant frequency portion
is processed for some 25 to 40 msec, and presumably yields velocity information
via Doppler shift while FM sweeps are actively held off until later to be
processed separately, probably for position determination. The loltimate goal
will be to understand how signals of given pitch, loudness, and timing can
be processed by the brain into a form that reveals the position, velocity and
the nature of objects in the surroundings.

Another group working in tissue transmission found that a topography of
sound transmission velocities exists in the porpoise forehead that causes
a focussing of sound within the fatty melon, either upon transmission or
reception. Sounds transmitted into the fatty melon tend to be focussed very
strongly into the right nasal plug lip. In general, velocities are slower
than in either fresh or sea water except near the plug. In the sperm whale, the
entire spermaceti organ is filled with waxy oil that transmits much faster than
sea water and which is probably the horaologue of the area adjacent to the
right nasal plug.

Noise Induced Deafness

Interaural alternated speech with and without intervening noise a.nd
interrupted speech was presented to subjects with normal and impaired hearing.
The results were found by a group of researchers to indicate little effect on
speech discrimination among normal listeners for interaural alternated speech
until intervening noise is introduced. Although there were marked individual
differences in the presence of intervening noise, the difficiilty in discrim-
inating speech increases as the rate of alternation increases. For subjects
with impaired hearing, the trend indicates that they perform all tasks more
poorly than normals even though there may be little improvement of speech
discrimination for continuous speech. Normal and hard of hearing subjects
make equal loudness balance judgements with various bands of noise at the
same and different center frequencies. The normal subjects sum loudness over
a much narrower band width than do subjects with cochlear involvement. In a
study on perception of complex auditory stim-uli by the deaf it was found that
Goarticulation of the consonants and vowels of speech produces transitions in the
frequency location of the vowel formants. The results suggest that sensorineural
damage does not necessarily impair a persons ability to use brief, small trans-
itions in formant frequency as cues for discrimination. It does seem to reduce
the ability to find cues when they occur in a speech-like environment.

Speech

A group of researchers has been investigatirg the efficiency of function

50 w

of the reinnervated canine larynx and on in vitro and in vivo tests of their
antilymphocyte serum. In addition, they have pursued other means of improving
the chances of success after transplantation of the canine larynx by removing
the offending cell, i.e., the small lymphocyte, and of accelerating rein-
nervation. They axe making a serious attempt to tissue-type their dogs before
transplantation of the larynx. Heretofore, no effort vas made to match animals
in regard to histocompatibility of antigens. They are using antilymphocyte
serum (ALS) as the immunosuppressive agent of choice in their laryngeal
transplantation. The problem of voice function of a transplanted larynx is
closely related to that of voice function of a reinnervated larynx.

Another group found that in humans, when background talk reaches a level
where it is just mildly disruptive to intelligibility for the norm.al hearer,
it can be a serious masker for the individual with sensorineural hearing loss.
They showed that competing speech causes a pathological increment in masking,
and suggest that the traditional measurements of hearing; loss, such as thresh-
hold shift and discrimination loss, as defined by reduced intelligibility
in quiet, should be supplemented with specification of the increase in the
masking efficiency of competing speech and other background sounds. In a
test of 2U subjects, measuring nonaurally discrimination of monosyllables
against competing sentences, findings showed that a third dimension of handi-
cap is imposed by sensorineural pathology. Such pathology not only changes
threshold and impairs intelligibility in quiet, but also disturbes the ability
to resist masking in complex environments containing background noise,
particularly speech.

Olfactory Communication

A team investigated the olfactory connections of the limbic system and hy-
pothalamus in the rodent. The tertiary olfactory contribution to a myelinated
bundle in the lateral hypothalamus previously identified in the rat has now been
found in both hamster and mouse. In all three species, lesions which Include
the olfactory tubercle cause degeneration of fibers which travel in the far
lateral region of the medial forebrain bundle to the posterior hypothalamus
border of the midbrain. In the rat, many regions of the olfactory cortex project
to the lateral preoptic area, mediodorsal nucleus of the thalmus, and lateral
hypothalamus. Other secondary olfactory zones (medial and cortical amygdaloid
nuclei) do not show this pattern of projection. Lesions of the medial nucleus
cause degeneration of fibers reaching the bed nucleus of the stria terminalis
through its post commissural component. Other work included electrophysiological
evaluation on hormone effects with urethane-anesthetized male rats. Comparing
recorded results from nonnal and castrated male rats to study the effects of
testicular androgens on the activity of neurons in the preoptic area, a
hypothalamic region which receives a strong olfactory input, it was found that
some units in the preoptic region reliably change their ongoing discharge rate
when the cortical EEG shows sudden transitions between activation and synchrony
characteristic of the urethanized rat preparation. Many more neurons in the
normal male than in the castrate show this correlation with the EEG. Preoptic
units which increase their discharge rate during EEG activation tend to be
those which show excitatory responses to odors and tend to change activity be-
fore the EEG change in transition from activationary to synchrony but not vice
versa.

51"

Sonic Booms

Sonic booms may damage the apical turn of the cochlea causing weakening
which in time leads to hearing loss according to another team of investigators.
Guinea pigs were exposed to 1,000 bursts of 130 dB. lasting 2., h, 5, or 125
msec, and were then retested for the Preyer reflex. Histological examinations
revealed damage to hair cells of the apical turn of all test animals.

Otitis Media

Incidence of deafness, otitis media, and perforation of the eardrum in
Guam populations is the highest reported anywhere. In school children hearing
loss was found to be four times that in most Worth American cities. Selective
Service examinations of young men in Guam showed similar incidence rates. Per-
foration of the eardrum accounted for 10^ of Guam young men being disqualified
for military service as compared with .Olfo of the Selective Service population
of the U.S.

Rubella

Another research team reported that many pre-school children may have had
congenital rubella and are likely to remain undiagnosed until a specific com-
plaint develops, such as a language or hearing defect. The findings indicate
that such conditions are recognized too often only after severe academic
retardation and emotional suffering have occurred. Therefore, they suggest a two
-part screening program as a model for identifying children with unrecognized
communication problems so that treatment or special training can begin as early as
possible. This team cooperated with Montgomery County Hesilth Department in a
program in which I36 children were located by radio, TV, newspaper notices and
letters to the locai medical society. Children with possible communication
disorders were given a clinical examination which included evaluations of growth,
speech, language, hearing, vision, and auditory memory. Blood specimens were
obtained from 11 i+ children and rubella hemogglutination inhibition (H. I. )
antibody titrations were performed on the sera.

Aphasia

In an out-patient clinical research facility for aphasic involvements
of children, studies on visual sequencing performance demonstrated that
aphasic children are generally inferior to normal children on various types
of sequencing tasks. A modality study by the same group of investigators has
produced preliminary data which indicates that aphasic children make more errors
in judging non-equivalent forms than equivalent form.s, and they also had longer
latencies for an intramodal haptic condition than for an intramodal vis\ial i
condition. Another continuing study in this out-patient facility is a series '
of directionality investigations progressing from gross visual forms to
discrimination of letters such as p and b, b and d. Findings show that aphasic
children who failed directional discrimination on the initial pre-test, were
able to complete this task after they had completed the program. Studies are
continuing to determine which of the tasks in the progression of discrimination
difficulty are essential for improved performance.

52W

Another group of investigators has been working in a multidisciplinary
effort on aphasia and related problems of "brain function. They have reported
that diagnosis and lesion site have a predictable relationship to pattern
of work comprehension and production, with respect to semantic class, word
frequency, and picturability. Scaling of syntactic tasks reflecting levels of
agrammatism is currently in progress by this group. In this area of non-
linguistic ne-uropsychological research, these investigators have presented
evidence that the left parietal lobe is found to be more critical than the
ritjht in cross-modal transfer, but less critical for short term visual memory.
Neirroanatomical studies have suggested an anatomical basis for cerebral
dominance based on the larger average size on the left than on the right planum
temporal in man. This same team of investigators has demonstrated that aphasic
patients not only vary in their ability to comprehend what they hear, but do so
according to at least four distinct patterns of auditory comprehension. They
showed that five diagnostic classes of aphasic patients differed in these
factors: (l) breadth of vocabulary; (2) auditory sequential pointing -span;
(3) comprehension of directional prepositions; (k) recognition of correct
grammatical usage of prepositions.

53w

AFFEEDIX A
RESEARCH GRANTS AWARDED IN FY 19T1 BY DISORDER CATEGORY
(Dollars in Tliousands)

DISORDER CATEGORY N0_; AMOUNT jo of $

TOTAL ALL DISORDERS I256 $53,6U5 100.0

1. NEUROLOGICAL DISORDERS

A. Neurological Disorders of 172 $ 6,200 11.6
Early Life

B. Neurological Disorders of Aging 59 2,810 5.2

C. Cerebrovascular Disorders 80 5^350 10.0

D. Epilepsy and Related Paroxysmal 52 2,500 h.G
Disorders

E. Sclerosing Disorders 63 2, 36O 4.U

F. Muscular and Neuromuscular Disorders 1^^ ^,690 8.7

G. Infectious Diseases 10 25O O.5

H. Trauma and Injury 68 3,120 5.1

J. Tumors of Nervous System 26 590 1.1

M. Neuroendocrine Studies 86 3;,050 5.7

W. Neural Aspects of Learning and ^3 1^690 3.2
Behavior

F. Nervous System Studies - li<-0 5,0U0 9.h
Normal Function

TOTAL - NEUROLOGICAL DISORDERS 9il3 $ 37,650 70.2

59

2,810

80

5,350

52

2,500

63

2,360

ihk

i+,690

10

250

68

3.120

26

590

86

3,050

^3

1,690

li^O

5,0U0

9^3

$ 37,650

52j.w

APPEEDIX A
RESEARCH GRANTS AWARDED IR FY 1971 BY DISORDER CATEGORY (Contd.)
(Dollars in Thousands)
DISORDERS CATEGORY NO. ^AMOUNT jo of $

2. SENSORY AND PERCEPTUAL DISORDERS

A, Disorders of Hearing and
Equilibrium

B. Disorders of Speech and Other
Higher CWS Functions

li+5
36

C. Disorders of Other Senses IO8

TOTAL - SENSORY & PERCEPTUAL DISORDERS 289

$ 6,605

1,890

3.750
12, 2U5

12.3
3.5

7.0

22.8

3. MULTI-CATEGORICAL

20 $ 3, 660

6.8

h, COKFERENCES

90

0.2

55w

ANNUAL REPORT
July 1, 1970 through June 30, 1971

Extramural Programs

Training Grants and Awards Branch

National Institute of Neurological Diseases and Stroke

The primary aim of the Training Grants and Awards Branch is the specialized
training of skilled professional and scientific personnel for careers in the
research and teaching aspects of the prevention, diagnosis, and treatment
of neurological and communicative impairments. To accomplish the Institute's
objective, two general types of awards are made, training grants and fellow-
ships. Included in the Training Grants category are (1) awards to superior
training institutions; (2) awards to institutions which wish to develop
programs or strengthen existing weak programs; (3) Special Traineeships ; and
(4) Teacher-Investigator Special Traineeships. Included in the Fellowships
category are (1) Postdoctoral Fellowships; (2) Research Career Development
Awards; and (3) Research Career Awards. The methods of applying for support
and the review of applications vary among the types of programs. Institutions
apply for Training Grants and Developmental Training Grants; institutions
also apply for Research Career Development Awards, except they apply for
these awards on behalf of a specific candidate. (New Research Career Awards
are no longer being made.) Special Traineeships, Teacher-Investigator
Special Traineeships, and Postdoctoral Fellowships, are awards made directly
to individuals.

To support the NINDS training activities in FY' 71, the House-Senate Conference
Committee recommended an allowance of $17,754 million which became the
appropriation. However, only $17,082 million were allocated to the Institute
which provided $14.3 million for Training Grants and Special Traineeships
and $2,782 million for RCDA' s and Postdoctoral Fellowships. The training
grant funds enabled the Institute to support 219 training programs and
201 special trainees; the fellowship funds were used to continue 12 Research
Career Awards and to award 76 Research Career Development Awards and 91
postdoctoral fellowships.

In FY' 71, the Institute made training grant awards to 55 institutions that
submitted renewal applications and to 9 institutions that submitted new
applications. In view of the large number (27) of renewal applications that
were not funded the previous year and the fact that no new awards were made,
it was anticipated that a number of institutions would submit amended
applications. In this regard, of the 55 renewal applications that were
funded, 11 were amended applications and of the 9 new applications that
were funded, two were amended applications. Thus, it was advantageous to 13
program directors who had previously approved programs to submit amended
applications. Four other amended renewal applications were recommended for
approval but again were not funded because the merit ratings they received
were too low.

Following the March meeting of the Council, when recommendations were made
concerning the funding of new and renewal applications, ten programs which

57w

had been supported for a number of years had merit ratings too low to fund.
These have been either already terminated or have been awarded phase-out
support. Eight of these programs were in neurology and two were in communica-
tive disorders. In FY' 68, the latest year for which information is available,
these ten programs provided full support to 36 trainees and partial support
to 20 trainees. In addition, 16 individuals were supported for a two or
three month period in order to give them an exposure to neurology or the
communicative disorders. The following table identifies the institutions
which were recommended for approval in Fy'71 but not funded and it indicates
the number of trainees in FY' 68 who received full, partial, or short-term
stipend support from grant funds:

Grant Number Institution

TOl NS 5049 Northwestern University
TOl NS 5099 University of Miami
TOl NS 5109 State University of New

York, Downs tate
TOl NS 5166 Wayne State
TOl NS 5182 Mayo Foundation
TOl NS 5201 University of Miami
TOl NS 5262 University of Illinois 5
TOl NS 5409 University of North

Carolina
TOl NS 5573 Mt. Sinai School of

Medicine 1

TOl NS 5574 Purdue University 6

Trainees Stipended

Full

Partial

Short-term

4

1

2

-

14

-

_

_

2

6

-

2

13

4

8

1

-

-

36 20 16

RESEARCH CAREER DEVELOPMENT AWARDS

Having made no new or renewal Research Career Development Awards in FY' 70,
FY' 71 was a banner year for this program. Of 36 applications recommended
for approval, 22 were funded. With only one exception, these 22 applications
were all new; nine were amended applications submitted in behalf of individuals
for whom support was sought the previous year.

In evaluating applications for renewal support, grantee institutions were
asked to provide a special justification for needing additional support
for the candidate. In addition, they were asked to (1) indicate their
permanent plans for the candidates; (2) state specific accomplishments of
the awardees during the initial award period; and (3) signify the need for
additional periods of training and supervised experiences. For the most
part, the institutions did not make strong cases for needing additional
support and the general philosophy of both the training committees and the
Council was to not recommend continued support for individuals who had
already received five years of RCDA support unless special justification
was provided. These candidates were considered independent investigators
and as such they had achieved the objective for which the award was initially
made .

58 w

New guidelines adopted this year, governing the Research Career Development
Award program, provide that any application terminating after June 30, 1972,
will not be eligible for renewal support. Thus, the RCDA becomes a single
five-year award with no possiblity of being renewed. Another change concerns
the evaluation of the applications. The initial review will be made by the
appropriate DRG Study Section. The National Advisory Council has requested
that all applications also be reviewed by an NINDS training review committee.

TEACHER-INVESTIGATOR SPECIAL TRAINEESHIP PROGRAM

FY' 71 was the second year of the Teacher-Investigator Special Traineeship
program. This program aims to recruit and prepare future teacher-investigators
of the highest caliber for academic careers in disciplines or areas of the
neurological, neurosensory, or communicative disorders. The award is the
most competitive and prestigious training and development award made by the
Institute to an individual.

During FY' 71, 19 teacher-investigator applications were submitted. Fifteen
of the applicants were interested in the neurological sciences and four were
interested in communicative disorders. Eleven applicants were invited to
Bethesda on February 27 to be interviewed by an NINDS Special Ad Hoc
Interview Committee.

The following individuals were selected to receive Teacher-Investigator Awards:

AWARDEE

SPONSOR AND
INSTITUTION

DISCIPLINE

Irving K. Arenberg, M.D.

Richard Torack, M.D.
Professor, Pathology and
Anatomy
Department of Pathology
Washington University
School of Medicine
St. Louis, Missouri

Oto-

neuropathology

Ira B. Black, M.D.

Fred Plxjm, M.D. Neurobiology

Professor and Chairman

Department of Neurology

Cornell University Medical College

New York, New York

Mary A. Guggenheim, M.D.

C. Henry Kempe, M.D. Neurology-

Professor and Chairman Virology

Department of Pediatrics

and
James Austin, M.D.
Professor and Chairman
Department of Neurology
University of Colorado Medical Center
Denver, Colorado

59 w

Mark E. Molliver, M.D.

Donald J. Woodward, Ph.D.

David Bodian, M.D.
Professor of Anatomy

and
Guy M. McKhann, M.D.
Professor of Neurology
Johns Hopkins University
School of Medicine
Baltimore, Maryland

Paul Horowicz, Ph.D.
Chairman, Department of

Physiology
University of Rochester
School of Medicine
Rochester, New York

Child Neurology

Neuroanatomy

Developmental
Neurobiology

D. C. UNIVERSITY CONSORTIUM: ACADEMIC NEUROSURGERY

During FY' 71, the staff held meetings with neurosurgery representatives of
the D. C, universities and other neurosurgeons in an attempt to stimulate
the development of a Ph.D. postdoctoral program in neurosurgery as a
multi-university Consortium undertaking. The organizational unit for the
program would be the D. C. Consortium and the objective of the program would
be to develop a program in the Washington area utilizing several of the
local facilities to train neurosurgeons for careers in research and academic
medicine. The meetings were attended by the following individuals:

Dr. Jesse B. Barber, Jr.
Howard University

Dr. Calvin Early

National Naval Medical Center

Dr. Ludwig Kempe

Walter Reed Army Medical Center

Dr. John Luessenhop
Georgetown University

Dr. Hugo V. Rizzoli

George Washington University

Dr» John M. Van Buren
National Institute of Neurological
Diseases and Stroke

The advantage of utilizing the Consortium is that the administrative unit
already exists and any facility in the Washington area could be utilized
to provide for a training program which would be unique to the specific
needs of the trainees.

60 1

It is anticipated that a formal application requesting support for this new
program will be submitted prior to October 1, 1971

FRONTIERS IN RESEARCH IN TEACHING
IN NEUROSCIENCE: A MINORITY TRAINING PROGRAM

In FY' 71, the Institute made an award to the Marine Biological Laboratory,
Woods Hole, to advance training in the neurosciences primarily for minority
group individuals at the postdoctoral level who are seeking opportunities
for further experience in research and who desire to strengthen their
capacities as teachers of future neuro-scientists. The program is designed
to enable trainees to carry out a personalized program in one or more areas
of neurobiology.

An award to the MBL provided support for approximately 6 trainees. Although
these individuals would be selected on a competitive basis, it was agreed
that for this pilot effort the program would be experimental in design and
it would be generally publicized with specific publicity aimed at institutions
that are composed mainly of minority ethnic groups.

Should the project be successful during the pilot period, the Marine
Biological Laboratory would submit an application for a similar grant
for long term support.

STATUS OF OTOLARYNGOLOGY TRAINING PROGRAMS IN THE UNITED STATES - 1969

During FY' 71 a report was prepared by the Committee on Education, Society
of University Otolaryngologists, on the status of otolaryngology training
programs in the United States in 1969. The data for this report were
collected by Dr. Dean Lierle and the study was supported by an NINDS grant.

The survey included 103 programs which were approved by the American Medical
Association and the Review Committee for Otolaryngology. Sixty-nine programs
were in university teaching centers; of these, 36 were independent departments
while 33 were sections of general surgery. Thirty-four were in government
or private institutions.

The report calls attention to the critical manpower situation. In 1969,
there were 253 first-year residency positions whereas there was a need
for a minimum of 500 such positions. There should be one certified
otolaryngologist for every 40,000 people in the United States and at
least 500 residency positions should be available to develop adequate
health care for the public, and provide the teacher-investigators required
for academic positions.

The formation of new medical schools (eight in 1969) added a few residency
positions, but not nearly enough to keep pace with increased needs. The
manpower situation cannot improve unless there is an expansion of many of
the residency programs. In addition to the lack of professional staff there
is also a lack of technicians in otolaryngology. The report points out that
paramedical personnel, properly trained, could be of value in lessening the
workload of faculty and residents. There appeared to be a fairly good supply
of audiologists and speech pathologists.

6iw

In the 103 institutions surveyed, a minimum of 150 more full-time teachers
are needed to improve the quality of teaching and properly supervise the
clinical and research areas. It is practically impossible for one full-
time staff member to attempt to administer, teach, and conduct research,
even in the smallest department of otolaryngology.

The report concludes b)' making the following recommendations to alleviate
the manpower situation and improve the quality of otolaryngology training
in the United States:

1. Increase the number of beds, the budgets and the faculty, in
otolaryngology, particulary in smaller departments, thus providing more
residency positions and improving the quality of teaching.

2. Utilize many available, competent young private practitioners in
areas adjacent to medical school and hospitals. These young men could
contribute greatly as part-time teachers and should be well compensated
by the institutions.

3. Train more paramedical personnel in otolaryngology to help
relieve the work load of residents and staff.

4. Make all departments and divisions of otolarjmgology independent,
particularly for budgets, in-patient beds, staff, access to the dean's
office, and membership on medical school committees.

5. Require clinical clerkships for undergraduate medical students
for a minimum period of two weeks; also, time should be provided for
electives in otolaryngology.

6. Consider postgraduate courses designed particularly for family
physicians, pediatricians, and other disciplines.

7. Provide more continuation courses for otolaryngologists in
private practice.

NEUROLOGY: A MEDICAL DISCIPLINE TAKES STOCK

In 1962 the Institute awarded a contract to Columbia University to evaluate
the status of training for research and service in the neurological sciences
and to formulate recommendations for strengthening and improving university
and non-university activities related to such training. The study was
carried out by Dr. Aura Severinghaus and during the period of the study
the following three publications by Dr. Severinghaus appeared:

Distribution of Graduates of Medical
Schools in the United States and Canada
According to Specialties, 1900-1964.
J. Med. E., 40:721-736, 1965.

A Medical Discipline Takes Stock. Arch.
. Neurol., 17:461-470, 1967

62w

Neurology and Neurological Sciences
Research and Training Study. Arch,
Neurol., 17:471-483, 1967.

Dr. Severinghaus' s study has been completed and the Institute has received
his final manuscript which is being put into final form for consideration
for possible publication.

APPENDICES

Following are four appendices which show how support for the Institute's
training programs is divided among the various training areas .

Appendix A is the anticipated number of grants and the training
grant funds awarded for FY' 71 according to the Institute's areas of
responsibility. This is an increase of 1 award over FY' 70.

Appendix B is the anticipated number of Special Traineeship Awards
and the amount for FY'71 divided among the Institute's areas of responsibility.
This is an increase of 40 awards over FY' 70.

Appendix C is the distribution of nine Teacher-Investigator Special
Traineeships among the Institute's areas of responsibility. This is an
increase of four awards over FY' 70, the initial year of this training
activity.

Appendix D is the anticipated number and the amount awarded for
RCA's, RCDA's, and Postdoctoral Fellowships in FY'71. This is an increase
of five RCDA's and 19 Postdoctoral Fellowships over FY' 70. The number of
RCA's (12) remains unchanged from FY' 70.

63''

APPENDIX A

Distribution, by Scientific Fields,
of Training Grants Awarded in FY 1971

Field

Number

Audio logy

6

Cerebrovascular

2

Child Neurology

15

Communicative Disorders

7

Neuroanatomy

5

Neurobiology

1

Neurochemis try

3

Neurological Sciences

6

Neurology

59

Neuropathology

14

Neuropharmaco logy

3

Neurophys io logy

13

Neur or ad io logy

10

Neurosurgery

24

Neurovirology

1

Otolaryngology

44

Sensory Physiology

3

Speech Pathology

3

$

Amount

368,700

68,600

613,900

533 , 100

151,200

20,800

102,000

225,400

4,197,800

513,900

166 , 100

731,700

250,300

917,400

47,500

2,680,400

128,300

162,900

TOTAL

219

$11,880,000

April 29, 1971

Gk

APPENDIX B

Distribution, by Scientific Fields,
of Special Traineeships Awarded in FY 1971

Field

Number

Audiology

5

Basic Neurosciences

3

Biochemistry

8

Biophysics

1

Cerebrovascular

10

Child Neurology

46

Communicative Disorders

1

Immunology

3

Neuroanatomy

3

Neurobiology

6

Neurochemis try

6

Neuroendocrinology

5

Neurology

9

Neuropathology

16

Neuropharmaco logy

6

Neurophy s io logy

29

Neuroradiology

17

Neurosurgery

5

Neurovirology

4

Oto laryngo logy

4

Sensory Physiology

3

Speech Pathology

2

Amount

53,500

48,400

81,200

16,700

106,900

500,600

16 , 100

35,600

31,300

74,700

65,100

49,000

114,900

171,700

63,200

293,700

198,900

41,600

44,900

47,500

37,200

28 , 100

TOTAL

192

$2,120,800

April 29, 1971

65 1

APPENDIX C

Distribution, by Scientific Fields,
of Teacher-Investigator Awards Granted in FY 1971

Field

Nvunber

Amount

Neuroanatomy
Neurobiology
Neurology
Neurophys io logy
Neurosurgery
Neurovirology
Sensory Physiology
TOTAL

19,500
15,500
35,000
35,000
22,200
21,500
15,500
$164,200

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