niaid intramural contributions, 1887-1987 - Office of NIH History ...

National Institute of Allergy and Infectious DiseasesIntramural Chronology, 1887-19871st Period : Hygienic Laboratory, 1887-19301887In August a Laboratory ofHygiene was established at theMarine Hospital on StatenIsland, New York by Dr .Joseph J . Kinyoun for thepurpose of bringing the thennewscience of bacteriology tothe study of epidemic diseases .The Marine Hospital Servicewas responsible for diagnosinginfectious diseases amongpassengers on incoming shipsto prevent the entry of diseaseinto the United States . Thebacteriological laboratory aidedclinical diagnosis . Dr . Kinyounserved as Director until 1899 .1891The Hygienic Laboratory, as itcame to be called, was movedto the Butler Building inWashington, D.C .1899Dr . Milton J . Rosenau was appointedas Director of theLaboratory and served until1909 .1901Congress appropriated $35,000to build a new building for theHygienic Laboratory and,somewhat after the fact,authorized it to investigate"infectious and contagiousdiseases and matters pertainingto the public health ."1902A reorganization act changedthe name of the Service from"Marine Hospital Service" to"Public Health and MarineHospital Service." Four newdivisions were established inthe Hygienic Laboratory : 1)Pathology and Bacteriology, 2)Zoology, 3) Pharmacology,and 4) Chemistry .1902In response to a request fromthe state of Montana,cooperative research on RockyMountain spotted fever wasbegun .1904The Laboratory occupied anew building on a five-acretract at 25th and E Streets,N.W ., Washington, D.C .1902President Theodore Rooseveltsigned the Biologics ControlAct . The function of testing aswell as regulating all vaccinesand other biologic productswas delegated to the Divisionof Pathology and Bacteriologyof the Hygienic Laboratory .1905Congress belatedly approvedpublication of the HygienicLaboratory Bulletin .Erro«, rnm e :w: 1~t,, d ~_~d, .:1900Dr . Rosenau began publicationof the Hygienic LaboratoryBulletin .1909Dr . John F . Anderson was appointedDirector of theLaboratory and served until1915 .1 . .e. .,J, i:'T rz\~i ;il~iliU nl the Butillu> I'csu?..Cr3

2nd Period : National Institute of Health, 1930-19481912The name of the Service wasshortened to "Public HealthService." The research programof the HygienicLaboratory was broadened toinclude noncontagious diseasesand water pollution .1930The Ransdell Act transformedthe Hygienic Laboratory intothe National Institute ofHealth (NIH) .1937Dr . Lewis R. Thompson wasappointed Director of NIHand served until 1942 .1942Dr . R . Eugene Dyer was appointedDirector of NIH andserved through 1948 .1915Dr . George W. McCoy wasappointed fourth Director ofthe Laboratory and served until1937, making his tenure thelongest of all Directors todate .1921The Rocky Mountain SpottedFever Laboratory wasestablished in an abandonedschool building in Hamilton,Montana, as a Public HealthService field station .1931Congress purchased the RockyMountain Spotted FeverLaboratory in Hamilton,Montana .1937National Institute of Healthwas reorganized into eight divisions. The Divisions thatevolved into NIAID or itspredecessor NMI were : InfectiousDiseases, Zoology, andBiologics Control . The RockyMountain Laboratory becamea part of the Division of InfectiousDiseases .DBC1938-41The National Institute ofHealth moved to its Bethesda,Maryland campus .1939The Public Health Service wastransferred from the TreasuryDepartment to the FederalSecurity Agency, thepredecessor of Department ofHealth and Human Services(DHHS) .:'Ah" !da n'kw .' -~j4

3rd Period :National MicrobiologicalInstitute, 1948-19554th Period :National Institute of Allergy and Infectious Diseases1955-present1948The National MicrobiologicalInstitute (NMI) was establishedNovember 1 . Constituent partswere the Laboratory of Infectious-Diseases, Laboratory ofTropical Diseases, Laboratoryof Biologics Control, and theRocky Mountain Laboratory .Dr . Victor H. Haas was appointedDirector .1953The NIH Clinical Centeropened, and the NMILaboratory of Clinical Investigationwas established .1955In June the Laboratory ofBiologics Control in NMI wasassigned to a newly establishedDivision of Biologics Standardsat NIH. Dr . Roderick Murraywas appointed director .1955On December 29, the NationalInstitute of Allergy and InfectiousDiseases (NIAID) wasestablished, replacing theNational MicrobiologicalInstitute (NMI) . Dr . Victor H .Haas continued to serve asDirector until 1957 .1956Because of expansion in theInstitute, the new position ofAssociate Director in charge ofresearch was created, and Dr .Dorland J . Davis was appointedto the post, which heheld until 1964 . From 1962-1964the position was known asDirector of Intramural Research .1957Dr . Justin M . Andrews wasappointed Director of NIAID .He served until 1964 .1957An intramural Laboratory ofImmunology was established tomeet the growing need forresearch on the mechanisms ofallergy and immunology .1957The Middle America ResearchUnit was established in theCanal Zone jointly by NIAIDand the Walter Reed ArmyInstitute of Research .1959The Institute was reorganizedto realign the larger laboratorieswith current researchendeavors . The Laboratory ofTropical Diseases was abolishedand replaced with four newlaboratories : the Laboratory ofParasite Chemotherapy, theLaboratory of ParasiticDiseases, the Laboratory ofTropical Virology, and theLaboratory of Germfree AnimalResearch . From two of thelarger segments of the Laboratoryof Infectious Diseases, theLaboratory of Cell Biologyand the Laboratory of Biologyof Viruses were created . In1986 the Laboratory of Biologyof Viruses was disestablishedwhen Dr . Norman P . Salzmanretired .1961The Laboratory of CellBiology was incorporated intothe Laboratory of Biology ofViruses .1964Dr . Dorland J . Davis wasappointed Director of NIAID .He served until 1975 .M1958The Laboratory of BacterialDiseases was established andcontinued until Dr . Norman B .McCullough retired in 1968 .5

1965Dr . John R. Seal wasappointed Director for IntramuralResearch . The title ofthis position was changed toScientific Director in 1969 . Hecontinued to serve until 1975,when he became Deputy Directorof the Institute, a post heheld until 1981 .and the Laboratory of InfectiousDiseases . A name changeoccurred when the Laboratoryof Germfree Animal Researchbecame the Laboratory ofMicrobial Immunity. TheLaboratory of TropicalVirology was disbanded in1967 . The Laboratory of ParasiticDiseases and theLaboratory of ParasiteChemotherapy were integratedinto one Laboratory ofParasitic Diseases in 1969 .1975Dr . Richard M . Krause wasappointed Director of NIAID .He served until 1984 .1978The first maximum containmentfacility (P4), forrecombinant DNA researchwas opened at Fort Detrick, inFrederick, Maryland .1967-69During these years, internalreorganization occurred . In1967, the Laboratory ofMicrobiology was established .In 1968, the Laboratory ofInfectious Diseases was splitinto two laboratories : theLaboratory of Viral Diseases1972The Division of BiologicsStandards was transferredfrom NIH to the Bureau ofBiologics in the Food andDrug Administration, but theBureau's facilities were stillmaintained on the NIHcampus .1972NIAID's portion of the MiddleAmerica Research Unit programwas transferred toGorgas Memorial Institute inthe Republic of Panama .1977The Laboratory of Immunogeneticswas established .1977Dr . Kenneth W. Sell was appointedScientific Director . Heserved until 1985 .1979The Rocky MountainLaboratory was reorganizedand renamed the Rocky MountainLaboratories . The newlaboratories created were :Laboratory of Persistent ViralDisease, Laboratory of MicrobialStructure and Function,and the Epidemiology Branch .1980The Laboratory of Immunoregulationwas established .1973The Laboratory of StreptococcalDiseases was establishedand continued until Dr . RogerM. Cole's retirement in 1981 .6

1981The Laboratory of MolecularMicrobiology was established .1984Dr . Anthony S. Fauci wasappointed Director of NIAID .1985The Laboratory of Pathobiologyat Rocky MountainLaboratories succeeded theEpidemiological Branch .1985Dr . John I . Gallin wasappointed Director, IntramuralResearch Program .1987The Laboratory of Cellularand Molecular Immunologywas established .1987The Biological ResourcesBranch was established to meetthe increasing need forsophisticated technology byIRP scientists .1987AIDS-IRP Vaccine Developmentand Treatment Centerestablished to meet the needfor a coordinated AIDS effortby IRP scientists .1985The Laboratory of Immunopathologywas established .

National Institutes of HealthScientists With EponymsLaboratory and Dates of AppointmentsJohn F . Anderson, M.D .Dermacentor andersoni, a wood tick, vector of Rocky Mountain spotted fever inPHS, 1898-1915 the west .Director, Hygienic Laboratory1909-1915E . John Bell, Ph.D . Rickettsia belli, a rickettsia that appears to be nonpathogenic to humans .RML 1948-1972Elmer G . Berry, Ph.D . Tropicorbis berryi, a potential intermediate snail host of Schistosoma mansoni .LPD 1945-1963Sara E . Branham, M.D ., Ph.D . Branhamnella catarhalis, a bacterium found in the throat .LBC 1928-1958James Brennan, Ph.D . Brennania sp ., a genus of deer flies . Chrysops clavicornis brennani, a biting fly .RML 1944-1974Willy Burgdorfer, Ph.D . Borrelia burgdorferi, the cause of Lyme disease .RML 1952-1986Herald R . Cox, Sc.D . Coxiella burneti, the cause of Q fever .RML 1936-1942G . Robert Coatney, Ph.D ., Sc.D . Plasmodium coatneyi, a species of primate malaria . Phyllodistomum coatneyi, aLPC 1942-1966 helminth .R.A . Cooley, Sc.D . Haemaphysalis cooleyi, a tick .RML 1928-1946Chester W . Emmons, Ph.D . Emmonsiella capsulatum, sexual stage of a fungus causing histoplasmosis . Em-LID 1936-1967 monsia parva, a fungus of rodents . Emmonsia crescens, a fungus of rodents .Don E . Eyles, Sc.D . Plasmodium eylesi, a species of primate malaria .LPC 1943-1963Edward Francis, M.D . Francisella tularense, the cause of tularemia .Hygienic Laboratory 1919-1936Leon Jacobs, Ph.D . Simulium jacobsi, a tropical fly .LPD 1937-1979Geoffrey M . Jeffery, Sc.D . Plasmodium jefferyi, a species of primate malaria .LPC 1944-1969William L . Jellison, Ph .D . Jellisonia sp ., a genus of fleas . Besnoitia jellisoni, a protozoan . ChrysozonaRML 1929-1962 jellisoni, a biting fly . Tabanus stonei jellisoni, a horsefly .8

Glen M . Kohls, Sc.D .RML 1931-1969Kohlsia sp ., a genus of fleas . Croypsylloides kohlsi, a flea . Clubiona kohlsi, aspider . Strebla kohlsi, a biting fly . Ploceophilus kohlsi, a cockroach . Kohlsiellasp ., a subgenus of ticks . Ixodes kohlsi, a tick . Haemophysalis kohlsi, a tick .Boophilus kohlsi, a tick . Neoschongastia kohlsi, a mite . Trombicula kohlsi, amite . Helenicula kohlsi, a mite .Ralph R . Parker, Ph .D .Director, RML 1921-1949Or-Rickettsia parkeri, a rickettsia that appears to be nonpathogenic for humans .nithodorus parkeri, a tick . Meringis parkeri, a flea . Borrelia parkeri, aspirochaete .Cornelius B . Philip, Ph .D .RML 1930-1970Director, 1962-64Martin D . Young, Sc.D .LPC 1937-1962Amblyomma philipi, a tick . Ixodes philipi, a tick . Culex philipi, a mosquito .Philipotabanus sp ., a genus of flies . Anerythrops philipi, a fly . Bolbodimyiaphilipi, a fly . Cydistomyia philipi, a fly . Haematopota philipi, a fly . Dissimasphilipi, a fly . Silvius philipi, a fly . Leucotabanus cornelianus, a fly .Plasmodium youngi, a species of primate malaria .Abbreviation KeyPHS Public Health ServiceLBC Laboratory of Biologics ControlLPD Laboratory of Parasitic DiseasesRML Rocky Mountain LaboratoryLID Laboratory of Infectious DiseasesLPC Laboratory of Parasite ChemotherapyPrepared by Dorland J. Davis, M.D.Director, NIAID 1964-19759

A Note on the Reprinted PapersThe authors of the bibliographic essays chose one classic paperthat helped to launch the area of research each was addressing .Because two of the reprinted papers were excessively long, sectionscontaining supporting data were omitted . Other papers arereprinted in their entirety .The authors of the reprinted papers are pictured here .Samuel T. Armstrong Joseph J. Kinyoun Charles W. StilesJohn F. Anderson Wade H. Frost13

ARMSTRONG AND ZINYOUN .repeated currents had been used through large uninsulatedne?dles . The result was a circular raw mass, projectingabove the surface, which refused to heal . Myxo-sarcomasact badly under electrolysis . Around the point of entranceof the needles slight ulceration at first appears, and subsegnentlyunsightly fungoid growths. The treatment in thesecases undoubtedly tends to hasten rather than to retardtheir progress . Sarcomas do not ulcerate so readily, butthey can not be treated with the same freedom as scirrhus .Fatty tumors can be treated by electro-puncture and byverv strong currents, not onlv without bad effects, but withthe prospect of very materially lessening their size in manycases, and occasionally causing them to entirely disappear .Fatty tumors are, of course, very bad conductors-muchworse than the hard fibroids which electrolyze so slowlyyetthey do disappear .This fact leads me, in conclusion, to refer to the twomethods of action through which we obtain results fromelectrolysis . The first and most apparent is the absolutedestruction of tissue which takes place at the time of treatment. It is unnecessary for the to enter into the physicaldetails preceding and accompanying the destructive actionof the galvanic current. It is suflicient to say that a molecularseparation takes place, more or less marked, accordingto the density or softness and fluidity of the tissues .Some suppuration . may follow, and thus, by an actual lossof substance apparent to the sight, the tumor decreases insize. If, however, these were the only active forces in theelectrolytic process, the method would lose much of itseffectiveness . If this be not so, how can we account forthe many well-attested cases where morbid growths haveentirely disappeared under simple external applications?Herein is the difference between the electrolysis of organicand iuor ;anie substances . In the electrolysis of inorganicsubstances the effects cease as soon as the current ceases,the substances remaining in the condition that the currentleft them . The electrolysis of organic substances, on thecontrary, starts a process that continues long after the currentceases to flow- . Besides this subsequent effect, thisretrograde metamorphosis, as it has been termed, the currentpenetrates the tissues, and induces various importantchanges beyond and beneath the eschar, and these combinedagencies do far more in many cases to diminish thesize of morbid growths and prevent further developmentthan an actual destruction of a limited area .OBSERVATIONS ON TIME C11OLERA BACILLUSAS A MEANS OF POSITIVE DIAGNOSIS .By ti . T . ARMSTRONG, M.. D ., .(-~D J . J . KINYOUN, M . D.,C\ZTED STATES MARINE HOSPITAL SERVICE .Os September 24th it was reported in the public pressthat the steam hip Alesia had arrived at the port of NewYore on the previous day, haying had 609 immigrant passeorrerson board, of whom ei ,llt had (lied of cholera en voyage,and several suffering from that disease had been admittedto the Swiuburne Island quarantine hospital on arrival. According to the captain's report, the first death hadCHOLERA BACILLUS.[N . Y . Men. JouR .,occurred September 12th, the rest of the deaths occurringand cases developing within eleven days. Subsequentlynew cases, some fatal, occurred .At the request of the writers, the health officer, Dr.William 11 . Smith, accorded the privilege of visiting thehospital, in order to examine the patients and secure materialfor bacteriological investigation . The patients werefirst seen b_v the writers on October 3d, when cultivation,were, made from excreta obtained at that visit. Plate cultivationswere made on the 5th, and characteristic coloniesof comma bacilli were found on the 6th, conforming in everyparticular to the description given by Iioch . On October9th it was reported in the public press that Dr. Shakespeare,of Philadelphia, had, from inoculations made by him fromexcreta obtained from these patients, also found the commabacillus .On October 13th the steamship Britannia, belonging tothe same company as the Alesia and coming from the samepoints in Italv, arrived at this port with 106 passengers onboard . En voyage there were three deaths of emigrants ;the diagnosis, though pointing to intestinal trouble, HealthOfficer Smith considered obscure, anti held the vessel inquarantine .On October 17th Dr . Smith requested the writers tovisit the quarantine hospital with him, in order , to takesome specimens from a suspicious case for cultivation ."The visit was made on the morning of the 18th ; thecase was that of a boy five years old, in collapse at the timeof the visit . Cultivation tubes were inoculated from washimYsfrom the bowels, as there were no ftrcal discbartyesavailable . A visit was again mude in the afternoon in orderto be present at the necropsy, the child having died at two.. .'clock . No pathological lesions were presented ; the smallintestine contained liquid faeces, with which cultivationtubes were inoculated . The necropsy was made five hoursafter death .Plate cultivations were made from the tubes as soon aspossible, and on the evening of the 19th Dr . Smith wasinformed that the comma bacillus was present in the specitnen,but a positive diagnosis would not be given until theplate cultivations had fully developed . At Dr . Smith's request,two inoculated cultivation tubes were given to himthese, it wa.s subsequently learned, were submitted to Dr .11 . M. Signs, Director of the Carnellie Laboratory, and Dr .T. Mitchell Prudden, Director of the Alumni Laboratory ofthe College of Physicians and 5ur;eons, for examination .On the morning of October 20th, typical colonies of commabacilli had developed on our plate cultivations . On October22d, at a conference with Dr. Smith, Dr. Biggs, andDr . Prudden, it was learned that their cultivations had affordedthe same results . Consequently the existence ofcholera on the steamship Britannia was demonstrated bybacteria investigations .On October~2lth the hospital was again visited on aecountof the death of a man aged fifty years. He was takenill, with diarrhoea and vomiting, on the niglit of the 22d:and died on the morning of the 24th . The necropsy wasmade eleven hours post morteut ; no pathological lesion :,were presented, excepting a slight congestion of the intes-

Nov . 12, 18h7 .11 ')IJ : TC'BFRCULAK PHTIIISIS AND THE P1VE1L1fATIC CABINET.P47tines, and the contents of the latter were fluid . Inoculationsof cultivation tubes were made, and transferred to thethird and fourth dilutions ; plate cultivations were madefrom these, and thirty hours later the characteristic coloniesof the comma bacillus were abundant .On the 35th one of the writers was placed by Dr.Smith in charge of the patients at the quarantine hospital .A woman, aged about ~wenty-two years, who had been removedfrom the Britannia on the evening of the 24th, wasin the bospital, suffering from an acute attack of cholera .A nursing child, about one year and a half old, occupiedthe same bed with her . Her case was so characteristic thatno cultivations were made from her excreta. 1oder treatmentshe recovered, and the child has not been ill up todate .On the 27th a boy, six years old, died suddenly on theBritannia . His mother stated that he had slight fever andvomiting for three days previous to his death . The necropsywas made twenty-four hours post mortem . \opathological feature presented, excepting an asearis protrudingfrom the anus, with a mass of ascarides in thecmcum, the small intestines containing white, glairy mucus .It was regretted that no cultivation tubes were at hand,and consequently inoculations were not made . But thecase presented the same appearance as those reportedabove.On the 28th a child, five months old, died in its mother'sarms while being transferred from the vessel . Site statedthat the child bad been ill for several day=, but had no diarrhoea.The necropsy was made twenty hours post mortem .\o pathological features were apparent . The small intestinecontained liquid faces ; the colon, apparently healthy faecalmatter. Inoculations of cultivation tubes were made, and,after being delayed twenty-four hours, specimens were takentherefrom and examined, the cholera bacillus being foundin great numbers ; in fact, so plenteously, and unmixedwith other organisms, that it was not considered necessaryto mike a plate cultivation .o cases nor sudden deaths have occurred since thatdate, and here we rest the case .The deductions which may be derived from these observationsare as follows1 . That in deaths among enti-rants coating from acholera-infected district, a necropsy is at)solutely essential,and cultivation tubes should be inoculated with the contentsof the intestine, for the purpose of determining tire cause ofdeath .2 . That successful inoculations may be made at leasttwenty-four hours after death .3 . . As the symptoms, in the cases examined, were byno means always well defined, the exatninati .~ns were confirntatorcevidence of the value of bacteria cultivation as ameans of positive diagnosis .-\-ember 6, 1887 .The Academy of Medicine and the Board of Health .-Followinghe reading of Dr . Br_vant's paper, an account of which we give in thisissue, Dr. Ellsworth Eliot moved the appointment of a conferencec om :nittee of five, including the president of the Academy, to co -o perate with the board of health . The motion was carried .REPORT OF A CASE OFTUBERCULAR PHTIIISIS TREATED «'ITHTHE PNEUMATIC CABINET .By WILLIAM B. WOOD, M. D,THIS paper does not enter the field in which the pneumatic-cabinettreatment has yielded the most valuable results-whichis in the control and cure of the earlier stagesof pulmonary affections-but is simply a record of what itis possible to do with the grayer forms of established pulmonarydiseases, to give relief and comfort, and to at leastprolong life ; not merely to extend an existence of chronicinvalidism, but frequently to prolong life with such improvementof health as enables a patient to resume theregular daily occupations .On the ~'+d of April, 1887, a gentleman, about forty yearsof age, presented himself at my office for examination . Hehad just returned from a winter in Florida, where he bad experienced steady, and for two months rapid, decline. His caseproved to be a typical one of tubercular pbthisis. Every oneof five specimens of sputum examined contained bacilli in veryunusual numbers . There were broncbo-vesicular respiration inthe right lung from the third interspace to the apex ; crepitantrules in the clayicular region ; moist rules and softening at theapex . There were creaking friction-sounds ; the percussionnotes were those of thickened and adherent pleura;, with someconsolidation ; there were also infra- and supra-clavicular retraction. In the left lung; erepitaot rales were heard from theclavicle to the apex . The rttional symptoms were such asalways accompany the foregoing physical signs : restless nights,rucking cough, alternate hectic and clammy stages, the septicexpression and complexion, shortness of breath, lack of appetite,loss of digestive and assimilative power, with daily expectorationof from eight to ten ounces, and a progressive loss ofvitality, weight . and lung.At this time the patient was unable to accomplish normalrespiration with sufficient vigor to meet the natural demands ofwaste-repair and up-building .By normal respiratian is meant, of course, the act offilling. the hums in inspiration with pure air and deoxygenatedblood, and in expiration that of emptying them of deoxygenatedair and oxyorenated blood, thus supplying redblood to medulla, mesentery, and mucous membranes . AsI knew of but one process-the pneumatic differentiation-which could accomplish this, treatment in the cabinetwas recommended a .s a means of temporary ameliorationcertainly, and possibly - of permanent benefit .The degree of success to be obtained with the pneumaticcabinet depends lamely upon the individualizationof the various cases and the fine adaptation of its functionsto the age, sex, particular disease, and ~ , etteral physical conditionof each patient. The personal equation of the operator is also a most important factor . The results to beobtained by the cabinet depend upon the operator's skill .It is a powerful instrument, but so ntatheiuatically - exactflint it may be perfectly controlled, while its several distinctand separate functions makti it an instrument of greatscope and power of combination ; but . those who expectedthe cabinet, per se, woul,,l work miracles, have been disap-15

A Century of Research in Bacteriologyand Mycology: Contributions ofNIAID Intramural Scientists, 1887 to 1987Roger M .ColeI was somewhat bemused, but not surprised, to notethat the program for the NIH Centennial Symposia containsno mention of bacteriology . Even in the infectiousdiseases section, the titles emphasize viruses and hostdefenses-a reflection of the times and current trends .The contributions of bacteriological science to genetics,molecular biology, and biochemistry are obscured in theburgeoning applications of these fields to problems closerto the biology of man, and unsolved non-viral infectiousdiseases are rare . It was not always so . One hundredyears ago, initiating the span of research that we nowcelebrate, bacteriological diseases and their agents providedthe impetus for establishment of the first researchlaboratory of the Public Health Service (then the MarineHospital Service) and, eventually, for the evolution thatresulted in today's National Institutes of Health .One of the latter's current components, the NationalInstitute of Allergy and Infectious Diseases (NIAID), isthe direct descendant of Joseph Kinyoun's originalLaboratory of Hygiene . Within it, through changes ofname and emphasis, many findings made by its intramuralscientists have been influential in the field ofbacteriology and mycology and often, as a consequence,in medicine and public health . Certainly, not all outstandingdiscoveries were achieved by these workers-butmany were, and it is some of these that we highlighthere as part of the NIH Centennial celebration . (Only appropriate,considering the bacteriological origins of thewhole thing!) However, since hundreds of studies havebeen carried out and published over these years, theselection of the relatively few that can be featured as examplesis a difficult task . To facilitate this, I have used,as a guide, the designation of landmark-defined as "anevent or development that marks a turning point orstage ."That seems reasonable enough, but the problem nowbecomes one of determining what constitutes a turningpoint . It is assumed that most findings that surviveeditorial reviews and thus find publication can bepresumed (perhaps charitably, in some instances) to offersomething new or different and so improve the body ofscience in one degree or another . But which of thesehave the consequences that let us recognize (sometimesretrospectively) that a turning point was reached or astage passed? The judgment of this, despite any otherknowledgeable inputs, must ultimately rest with thebeholder . Therefore, to be quite clear at the outset, Ioutline the sorts of "turning points" that guided mychoices for the accompanying bibliography .RogerM. ColeFirst and most obvious is the discovery andcharacterization of a new disease of humans . A secondturning point is the finding of its etiology, or theetiology of a disease previously known only clinically orepidemiologically _ without knowledge of the agent, thedevelopment of vaccines or specific therapy cannot evenbe attempted . The successful provisions of thesemeasures are also turning points . Also important areepidemiological findings that define disease distribution,mode of transmission, vectors, reservoirs or alternatehosts, and the like-sometimes suggesting an etiology, ifnot already known . But all of these, in respect tobacterial etiologies, are mostly things of the past .(Or arethey? Recall Legionnaire's Disease!) In any event, it isapparent that the character of bacteriological andmycological research has changed as emphasis hasgradually shifted from disease discovery toward a greaterconcentration on basic studies of the microorganismsthemselves . In addition to creating a broader literature inphysiology and taxonomy, these expanding studies gaverise to today's predominant and growing fields ofgenetics and molecular biology . They also offer newpotential "turning points," in instances when new16

laboratory techniques or basic discoveries have given newdirections to scientific thought, stimulated fruitful newavenues of research, or resulted in improved methods forsuch purposes as diagnosis, propagation of themicroorganism, and vaccine production . Obviously, thereis some overlap among these various categories . Thereare also problems in proper attribution (i .e ., who foundwhat first?), as well as uncertainties in citing a singlepublication when the impact of the ultimate discovery isreally clarified by several . However, it is not my purposehere to settle controversies nor to investigate minutiaebut rather to supply some idea of the scope and influenceof 100 years of intramural studies in bacteriologyand mycology .The easiest to deal with are new diseases and diseaseetiologies . Although bacteriology was a relatively newscience in 1887, such old and distinguishable diseases ascholera, diphtheria, tetanus, typhoid, gonorrhea,cerebrospinal meningitis, scarlet fever, lobar pneumonia,and tuberculosis were already known to be associatedwith distinct and microscopically visible microorganisms,each of which had already been named by Europeanscientists (though many designations were subsequentlyrevised-often many times) . The tasks of Kinyoun'ssmall Laboratory of Hygiene were oriented towarddiagnosis, prophylaxis, and control . For example, thefirst diagnosis of cholera in the United States was madethere in 1887 . 1 Later (1908), MCCoy2 made the firstdemonstration of bubonic plague in California groundsquirrels associated with a human case and thus initiatedthe recognition of rodents as a reservoir and source ofdisseminated infection . The laboratory had facilities forthe diagnosis of plague (the organism had been isolatedin Europe by 1894) and of diphtheria, and for productionof vaccine for the former and antitoxin for the latter. However, as the staff and their successors practicedthe methods of bacteriology as well as their clinical andepidemiological expertise, it was perhaps inevitable thatthey should find new diseases, new bacteria, and newhosts . The first and best known disease, nowvoluminously documented in all its aspects, was RockyMountain spotted fever (RMSF), delineated by Andersonin 1903 . 3 Although its association with ticks was apparent,it was not until 1906 that transmission by infectedticks (to guinea pigs) was shown by King4 andthen by Ricketts . These studies introduced a long periodof progress in what we now know to be diseases causedby rickettsiae-a field in which the contributions of PHSresearchers became well known . For example, Andersonand Goldberger, in 1909 5 , demonstrated that MexicanTyphus Fever was not RMSF (as had been previouslysuspected), but predicted its transmission by the bodylouse 6 as was demonstrated a few months later byRicketts and Wilder, and showed the cross-immunityproduced in monkeys by its agent and that of Brill'sdisease .? After the latter was shown to be a recrudescentform of the European or epidemic typhus, other workersused convalescent Brill's serum to show that the Mexicandisease was also epidemic typhus . The typhus picture wasfurther clarified by epidemiologic work of Maxcy in 1926 : 8despite his erroneous equation of Brill's Disease withDr. Cole in LID laboratory, 1953.endemic typhus, he clearly showed that the latter was adistinct disease of the southeast U .S ., with a rodentreservoir . Subsequently, the studies of Dyer, Badger, andothers 9-11 demonstrated its transmission by the rat flea .The etiologic agent was later distinguished from that ofepidemic typhus, aided by differential scrotal reactions ofguinea pigs found in 1917 by Neill, 12 and was definedand named Rickettsia typhi (as distinguished from R .prowazekii of epidemic typhus) by Philip in 1943 . 13The story of typhus, of whatever form, is a long onewith a plethora of references, many of which made smallbut important contributions to ultimate understanding ;but few of which constituted landmark studies inthemselves . A sharper picture is presented by Q Fever .This began in the U .S . with the discovery by Davis andCox, in 1938 14 of a new filter-passing agent from ticks,and the finding by Dyer 15 (from personal experience!)that the highly infectious agent caused a human diseaselike that just described as Q (Query) Fever by Derrick inQueensland, Australia . Derrick called the agent Rickettsiaburneti, Cox labeled it Rickettsia diaporica, 16 and iteventually became the only member of a new genus, asCoxiella burneti. 17A classic example of elucidation of a new disease isthat of rickettsialpox in New York . In a series of fivepapers in 1946 and 1947, 18-22 Huebner and colleaguesdescribed the disease, the agent, its reservoir in mice, andits accidental transmission to man by a rodent mite .Rarely are such landmark findings presented so clearlyand compactly . It later became apparent that the diseaseis not a major nor frequent one, that it occurssporadically in other parts of the world, and that theagent (Rickettsia akari) can be isolated from mice andmites in other regions of the U .S .A . as well as in Soviet17

.Union and Korea .Another disease historically associated with the workof PHS scientists is tularemia . Elucidation of its etiologybegan in 1911-12, when McCoy and Chapin investigateda plague-like disease of ground squirreIs 23,24 and describedBacterium tularense . It was not until two years laterthat Wherry and Lamb 25 showed that the bacterium(later named Francisella tularensis) could also causedisease in man . Francis extended the observations . 26,27Many of his other studies clarified the nature of theagent, its distribution in animals, the participation ofvarious arthropods such as ticks and deer flies as vectors,dermal and pulmonary routes of infection in man, andother aspects of tularemia .Most of the foregoing examples concern new diseasesfor which the etiology was usually soon found . In otherinstances, PHS scientists uncovered the etiology ofdiseases that had been known for sometime but whosecause remained obscure . The oldest of these, pellagra,was long considered to be of obscure, infectious origin,but was shown by Goldberger in 1914 28 to result from anutritional deficiency, thus, supplying a reverse exampleof the usual bacteria-oriented turning points consideredhere . On the other hand, psittacosis was believed to be aviral disease until Lillie, in the course of pathologic examinationof infected humans and animals, foundmicroscopically-visible rickettsia-like inclusions in 1930 . 29He named the organism Rickettsia psittaci, but it waslater placed in the genus Chlamydia . Similarly, a viruswas thought to be the cause of the predominant form ofprimary atypical pneumonia until 1962, when the "EatonAgent" was first grown on an artificial medium andrecognized as a mycoplasma by Chanock, Hayflick, andBarile . 3° It was designated as a new species and namedMycoplasma pneumoniae the next year . 31 It was the firstrecognized and remains the most prominent mycoplasmalcause of human disease . More recently (1974), a syndromeof skin lesions (erythema chronicum migrans)followed by chronic inflammatory arthritis wasdesignated Lyme disease . Despite its clear associationwith ixodid ticks and numerous investigations, itsetiology remained a mystery . The first evidence of itscause as a spirochete (since named Borrelia burgdorferiby others) resulted from the work of Burgdorfer and colleaguesat the Rocky Mountain Laboratories, andelsewhere in 1982 . 32 This study furnishes the most recentexample of the discovery of a bacterial etiology for arecognized human disease, though it may not be the last .Once the etiology of a disease was established, theusual next step considered was production of active immunityby a vaccine . This was especially true in the daysbefore the advent of specific chemotherapies, and RockyMountain spotted fever is a prime example . Spencer andParker, reasoning by analogy from results with typhusby European workers, produced a phenolized vaccinefrom ground infected ticks 33 The vaccine was widely usedand reasonably effective in Montana for 15 years . 34However, production was laborious and somewhat uncertain. The need for a better and more reliable source ofantigen in quantity was clear . The key was provided bythe cultivation of the organism in chick embryos byBengtson and Dyer in 1935, 35 followed by Cox's applicationof the method in yolk-sacs, 36 which permitted thegrowth of the rickettsiae of typhus and Q fever as well . 37This led to mass production of rickettsial vaccines : indeed,during the years of World War II, the RockyMountain Laboratory became, in large part, a vaccinefactory. The methods were licensed to commercial producers,but problems with efficacy of the RMSF vaccineled to cessation of production in 1978 . Although theavailability of effective antibiotics now decreases the needfor a vaccine, problems with adequate and earlydiagnosis of RMSF continue to stimulate an interest inprophylaxis . Most recently, MacDonald and colleagues atRML 38 applied the tools of molecular genetics to expressionand production in E. coli of a surface antigen ofRickettsia rickettsii, which they demonstrated to be protectivein mice-thus offering a new potential for productionof antigen and for immunization .Among epidemiologic studies, that of Maxcy 8 whichdistinguished endemic from epidemic typhus and otherrickettsial diseases, has been mentioned . Another fieldstudy that was influential was that of psittacosis in 1930by Armstrong, 39 which resulted in governmental regulationof the importation of psttacine birds . Also outstandingare the studies of institutional outbreaks of Q feverat two different times and in two different sites at theNIH . These, carried out by different workers, definednot only epidemiologic features of the disease, but alsonoted clinical variations, confirmed the etiology, and emphasizedthe high infectivity and capacity for airbornetransmission of this agent . 4o -43 New potentials fortransmission of the disease were revealed by the studiesof Huebner, Bell, and associates in southern California,44,45 emphasizing the hazards to human health of cattleand their milk infected with the Q fever organism . Inanother field, the findings of Emmons first pointed outthe reservoirs of histoplasmosis in soi1 46 and bats, 47 ofcoccidiomycosis in soi1, 48 and of cryptococcosis in soi1 49and pigeon droppings 5°-thus supplying crucial informationon sources of infection by these pathogenic fungi .Basic laboratory findings, unless clearly related to suchdisease-oriented apphications as vaccine or toxin production,are more difficult to assess as turning points . Notinfrequently, their influence requires the passage of timefor recognition and evaluation . An example is the firstdemonstration, by Somerson et al., in 1967, 51 thatMycoplasma pneumoniae colonies adhere firmly to aglass surface during growth, thus supplying a concentratedmass of organisms that can be easily washed andlater detached to supply antigen or organisms for a vaccine. Though originally employed to provide a clean(non-anti-complementary) antigen for complement fixationtests, the method has been tested with manymycoplasmas, and it became recognized that adherence isa characteristic that helps to differentiate some speciesfrom others . Complement fixation became supplemented(and later largely replaced) as a serologic test formycoplasmas by simpler and more specific tests devisedby Purcell, Taylor-Robinson, Wong, and Chanock in1966 . These metabolic inhibition tests, easily read and18

now almost universally used, differ according to themetabolism of the mycoplasma-being adapted to acidproducingspecies, 52 non-acid-formers," and ureaseproducers . 54Basic studies with no relation to disease often havelong-term reverberations, or the potential therefore . Oneexample that comes easily to mind (and I include thiswith some diffidence) is work done in my laboratory between1962 and 1966 . This was the first to show a differencein modes of cell wall replication between grampositive and gram negative bacteria . By using a new approachof marking the walls of living bacteria withfluorescein-labeled antibodies, we showed that the wallsof streptococci replicate in a discrete fashions and thoseof gram negative bacteria like Salmonella typhosa andEscherichia coli are replicated in a diffuse mode . 56,57Such differences were unknown and their demonstrationstimulated a rebirth of research in this rather esotericfield that led to widespread application of electronmicroscopic and biochemical techniques . Results withcocci were rapidly confirmed . Despite many variationsand ups and downs in the intervening 25 years, the mostrecent published results appear to confirm also the principleof diffuse wall replication in gram negative rodshapedbacteria .Scientific thought was also changed by new intramuraldiscoveries in mycology . It was long accepted that theagents of the systemic mycoses, histoplasmosis and cryptococcosis,were fungi imperfecti . This concept wasdestroyed when Kwon-Chung demonstrated the sexualstage of Histoplasma capsulatum in 197258 . 59 and that ofCryptococcus neoformans in 1975, 6° necessitating theredesignation of these important pathogens in new generaEmmonsiella and Filobasidiella respectively . A study ofthe mating types of the former6 l showed that both (+)and (-) types were equally distributed among soil isolatesbut that the (-) type was 7 times more common inclinical isolates . The significance of this finding remainsto be more fully explored, but its elucidation and explorationof the promising theoretical basis for geneticstudy have been hampered by loss of active matingcapacities in isolates maintained in laboratory passage .Basic research often produces unforeseen ramifications .One such example resulted from a need to identify anewly discovered microorganism found associated with adisease of citrus trees . Though previously cultured andsuspected of being a mycoplasma, it was not characterizeduntil collaborative work in 1973 by European andNIAID workers defined it as a new genus and specieswith a helical shape and rotary motility, 62,63 attributespreviously unknown among mycoplasmas . The organism,named Spiroplasma citri, became the prototype of a newgroup of mycoplasmas, previously totally unsuspectedbut now found to be widespread in nature . In retrospect,it became apparent that some of these spiroplasmas,which had never been cultured or were considered to bespirochete, had been previously observed in a disease ofcorn, in fruit flies with a sex-ratio imbalance in the progeny,and in ticks . The definition of the first spiroplasmainitiated a remarkable expansion of plant and insectpathology, and some dozen species and serovar groupsare now known from ticks, bee diseases, beetles, andmany other insects, and from plants, plant diseases andtheir insect vectors-with no end in sight . The fundamentaland continuing contributions of Tully and collaboratorsto the understanding of these and othermycoplasmas are numerous and exemplary .The foregoing examples were selected as representativeof the several categories of "turning points" previouslyoutlined . There are many other significant contributionsby intramural researchers that do not fit precisely intopredetermined slots . One of these is Armstrong's findingin 1925 64 that 25 676 of commercial bunion pads were contaminatedwith spores of Clostridium tetani, resulting incases of human tetanus (about 80°76 fatal) when suchpads were used, as was then the practice, as dressingsfollowing the scarification method of vaccination forsmallpox . His recommendations resulted in the discontinuanceof such dressings and, later, in general adoptionof the multiple pressure method of vaccination as promulgatedby Leake . 65Then, too, there are works which, taken in the aggregate,provided significant and essential knowledge ofthe subject area, often to the extent that it becamelastingly associated with the name of the researcher .Many of these findings became best appreciatedcumulatively and in retrospect . Because of this and ofthe volume of contributions over many years, I can includehere only a few sample references that indicate, butby no means encompass, the field and directions of theresearcher's work . One cannot, for example, overlookthe pioneering contributions of Evans that, from 1918on, led to differentiation of the Brucella species causingMalta Fever in man and Bang's Disease in cattle,established their occurrence in many mammalian speciesas well as in human disease, and progressively clarifiedthe serology and classification of these organisms . 66-68She also contributed extensively to the early understandingof streptococci and their bacteriophages, having initiatedthe collection and study of these bacteria in theHygienic Laboratory . In similar fashion, the name ofBranham is closely associated with early studies of thegenus Nesseria, in which she illuminated the taxonomy,described a new species in meningitis, explored propertiesof immune sera, and, especially, distinguished the threebasic serotypes of the meningococcus .69,7o A third andwidely recognized longtime association has been that ofPittman with the genus Haemophilus (and Bordetella)and with various aspects of pertussis-a field to whichshe continued to contribute after relocation from NIH tothe FDA's Bureau of Biologics and even after officialretirement .' 1,71Other lasting associations of a researcher's name witha particular organism or disease are known . One needonly recall (and I won't list them here) those intramuralworkers, already mentioned, who made continuing contributions,in more than the specific instances mentioned,to the elucidation of plague, tularemia, typhus, spottedfever, Q fever, rickettsialpox, psittacosis, mycoplasmaldiseases, and the systemic mycoses . There is no space tolist here, much less to even casually annotate, theirnumerous and collective works . Also missing, regrettably,19

must be reference to the hundreds of productive contributionsmade by other intramural scientists who, in theaggregate and over the years, studied most organismsand aspects of bacteriology and mycology . But the scenehas shifted and times have changed . Althoughmycological investigations continue here, nobacteriological laboratory now exists in NIAID atBethesda . Such research, including investigations of pertussis,gonococci, spirochetes, rickettsiae, and relatedmicroorganisms, continues at the Rocky MountainLaboratories in Hamilton, Montana . Studies of bacterialgenetics and of mycoplasmas are located at the FortDetrick facility in Frederick, Maryland, as outlying componentsof the Bethesda-based Laboratory of MolecularMicrobiology . The latter name, unintendedly, but wryly,reflects recent trends that have engendered a currentacademic controversy over the potential loss of identityof microbiology as a separate discipline and stimulatedfears (as expressed by one observer) of its "beingsmothered by molecular biology ." (See Letters, pp .355-56 ; and Microbiology By Any Other Name, pp .373-74 in ASM News, Vol . 53, No . 7, July 1987) .Whether this change has really taken place, or will doso, in NIAID and other research institutions as well as inacademic situations is a matter for debate and continuedobservation of future trends . Clearly, it is unlikely thatthe halcyon days of "pure" bacteriology will recur .However, current and future emphasis in no way lessensthe importance of the past 100 years of NIAID intramuralcontributions . May the next 100 years be asproductive!RogerM . Cole, Ph.D., M.D.In 1949 Dr . Cole joined the Public Health Service andlaunched a 32 year career with NMI/NIAID . He wasborn in Canton, Maine in 1917 . After graduating fromMassachusetts State College in 1939 with a B .S ., hereceived a Ph.D . from Harvard University in 1943, and aM.D . degree from Boston University School of Medicinein 1947 . From 1943-1946, moreover, he served in theUnited States Army . Following his internship atMassachusetts Memorial Hospital in Boston, Dr . Colecame to NMI and was assigned to the epidemiology unit .In 1951 he became chief of the respiratory bacteriologyunit within LID and was later made assistant chief . Hewas also head of the Bacterial Structure and FunctionSection, and in 1967 Chief of the Laboratory ofMicrobiology . In 1973 he was named Chief of the newlyestablished Laboratory of Streptococcal Diseases . FromSeptember 1975 until December 1976 he served as ActingScientific Director of MAID . Dr . Cole retired from thePublic Health Service in November 1981 . His publicationsnumber over 100 and his research has coveredaspects of sarcoidosis, herpangina, streptococci, bacterialcell wall replication, microbial ultrastructures, bacterialphages, streptococcal genetics, bacterial L-forms, andmycoplasmas . In retirement Dr . Cole has continued topursue a life-long interest in nature photography .

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Journal of Bacteriology115 : 367-386 .64 . Armstrong, C . 1925 . Tetanus in the United States Following theUse of Bunion Pads as a Vaccination Dressing . Public Health Reports40 : 1351-57 .65 . Leake, J . P . 1927 . Questions and Answers on Smallpox Vaccination.Public Health Reports 42 : 221-38 .66 . Evans, A . C . 1918 . Further Studies on Bacterium abortus andRelated Bacteria . II . A Comparison of Bacterium abortus withBacterium brochisepticus and with the Organism which Causes MaltaFever . Journal of Infectious Diseases 22 : 580-93 .67 . Evans, A . C . 1923 . The Nomenclature of the melitensis-abortusGroup of Bacterial Organisms . The Serological Classification ofBrucella melitensis from Human, Bovine, Caprine, and Equine Sources .Public Health Reports 38 : 1943-63 .68 . Evans, A . C . 1924 . Malta Fever : Cattle Suggested as a PossibleSource of Infection Following a Serological Study of Humans . PublicHealth Reports 39 : 501-18 .69. Branham, S . E . 1930. A New Meningococcus-Like Organism(Neisseria flavescens n . sp .) from Epidemic Meningitis . Public HealthReports 45 : 845-49 .70. Branham, S . E . 1953 . Serological Relationships Among Meningococci. Bacteriological Reviews 17 : 175-88 .71 . Pittman, M . 1953 . A Classification of the Hemolytic Bacteria of theGenus Haemophilus : Haemophilus haemolyticus Bergey et al. andHaemophilus parahemolyticus nov . sp . Journal of Bacteriology 65 :750-51 .72 . Pittman, M . 1979 . Pertussis Toxin : The Cause of the Harmful Effectsand Prolonged Immunity of Whooping Cough : A Hypothesis .Reviews of Infectious Diseases 1 : 401-12 .22

BROOKLYN MEDICAL JOURNALIlI .I Ilc\ :(1_c 111 i ;tcl l1l'"1(l ,tl( I".( r, ,~f 111(' \\ r ,t I \1K .1111(' f: l~r ill tilt- I1r , 11ttt'ti tt 1,f ill \ ,t ;l , t i " ,II1,I1, Illltllll(' 11('111 , rrll :l t' . 1,1_0111_ 11-111 Ill( \\ 111111,!I! ;t11 II\ ill, \\111111, . I Lirlll,t - , ill, ~t' \\ 111111- f rilltll('rt f 1_c . ;t f 111 ,111 t'' tilt' I1 :(n[,it(' . 111 a'l,li-II1lillt~ 1~1f :ttt :ml : f,lr Ir,uU('ri :l . \twt, 1111_ itltt ,tilutl111-\\ ;ill llllci'' 1l< :11111 it, 111~1' , till " 111_1 ;11_,' 1,tv;tl , 11111 1111 , ill 1111 ,!! .I1 , ~ : II1Cr :/\', .lr :rni,I, .\, \\c I)nwcctl 'tall frl1111 \ ir`i11i ;1 t .1 1 11r :,1 :1 \\c Intict' t1l ;II:Lilt 111I ;( III(r1 ;,"~ . 1111 :it I II II I1 : ;(\ 1,1' .li\1111,1illt , l t\\,1 ~t'IIt'r;ll rl :tc, ill IGIrIit'til :lr, 1' :11'11 cl :( 1n ill ta l,ir :tl 111 :I ct rt :[ill 1,11I11 1 f ",il . I lilt' :II It 11Ii .ti, , ltt( t a 1111 .1 ~,! ill fcc ti, ~II 111 III'1lari ;ll 11 It I It r, , ;11111tlti, i, il~ttll~l t',11t'ii ;lll\ 11 Illr Ilu .1 ( '1111 ln1\1,111,; .~ it1 .\111 ;111\, ill tilt' ,1 lttll\\,',t('rll ll :[It f( a'1,r~i :[ : tilt' , ~tllcr :ua'llli :C i, 11111' t :h1 itllr,ti11 ;11itli('rt ;,111 , .f ll(,1(li\\1,rlll-, ln1 , 11!~ill~ t,1 Ill[ .y(mir "l "1_t( ir(rri I 1111Cr-ic,Irrtr, :11111 tilt, i, pr11t'tllill('llll\ 1_tIf 1111 ,('t'tl, f, ir itl,t ;utcc,:It \\ ;(\rr,~, ., 111 tllc ~Ittllr :l,1tr11 Ir,lrt 1 1j t ; r (,r~ia,\\Il('I~t " 11101"( ;11_11 ;Illl,tlt .1, O :I~W 11f 11V 1 : \\ nil 111,( :[,c t , I I,ItC c :l,c ,I f IIi ;d :lria ; lit it \\c 1_\ :11-11 a ill's -irict \\lil t :( ,atl,l\ t( Ill )il :lIltl ;c I III rc illlllcr\- it )tt,,1111"111, :1, ;it \\ill :ic11\1110, (,IlYcc ( (IlIlI\, Irct\\cctl.\11) :ul\ alltl \\ a\cI.,l , \\r IilitI 1,11111c ;l,(- pr( \ :I1('llt . -f(Ir Tilt ,tlli, rc it,lt :tl Ir('(ltlt'llc\ (If 1110(li,ca,c, ill (I11c,ti(111 i, ;It prc,cllt ill part ()Ill\,11 ;01111\ Iti r( i,nl, ,1f cla\ t4g) (11_ ,tlll,llil tllc'11 :ltllral llr('t'lilll lrl :lcc, (If " .ln(,/Ihcl( .c \\il l illc'1_(':(,(' ill 1!tltlll1cr :[till lu r,i,t l(ol cr tllall illrt',~t,lll, . In'tlr(', \\111 1 :[II il1itial itlfccti'111 14 Illalariatllc 111,( :1,(' \\il l ,llr(' :I,l . "I llc I"CI ;0itIll ,1f nltcil1 :tria,i,t,1 tlu' , :11111 i, Il,(t (luitc ~,(, clc ;ir, lilt I \\il l cll-(I( - a\"(,r 11, t'\l)lalll this Iu,lllt, ;It Ica,t 111 part, lalt'1in tll ;~ c\cilin~ .1~..,.V ( 1 :(111 1,\ tilt f(lll :(lt :!1111 art- '1i,c1mr~-c11ill ~r1 :[I 111th!!!! r, is tin' ,t1 ,1, .i tllu II :1ti('llt< . :1f : :w \\111\1 ! ~i\t , ti~ :Ill c\1_( 11111 11t['t1111(1 1If 1,111\ - t' 111 :1~iti i,, 11 :11111 lv, a 11111'11 " t' , 11111' ( S :tilllllall,il14 Ill(' i(', 1', \,,t 1'\('r\ Illl\,iciatl, 1111\\\\111_ . , \\ Il l;1 I!licrl o11u-' : ],lit I 11 :1\c' fl~ttl111 :t ,tll titut(' tr,t\\1111_11 (_ ;Ill IW ill :[ , l(' h .% :hl\ I)t'r-11 :[1111 ill \\llicl l1110 ( rr1,r i> IIF,111 :Il11\ 11111 1 1 \ ('r =,l 111_ ,jt) I,cr icllt .ill t11c11ittill ail(1 ,c\crc illf('ctil'II, 111 lx'rll ;t11 : S(lilt (,f I ) 1_ ;I,\, lf ,licit ill icctit Its, Ill\ ,t (l, arcrc11(li,ll llrtl\\tl, :ttl11 if :1 1 , placol -)It ;Ililt'\\ 1,f \\Ilit c 1111111111 .- II ;IIlc'r flit - =c> Iti II,I IIIillnt(' ::(1111 tllctl r('tll \C',l, ;( 11i,tillrt rc1l, 1111,1111-1i1,c " [ ;tillis tl,'tircll . III this Ic,t, it i, a " ,Ilttlcll tllat \\c Ila\clu'f''rc It, a case (If attclllia lull ,ttlicritl faultItilc :s ./t( ; ('/(,~1nr1'nl (/irl rl . M), )ill :_1 It, ntr, :( fl cr t 11c(-g i, (li,cllar .L~ol ill tllc "11111,, :1 rllallllitii 1'111 k'111-hl-P111c\t .lt .l>, ; Illi, ,llcd, it " ,1 :111 after alll~ut Is,It(11tr, ; Ilict1 aln,ut ; Cla\ : later it lrtltlcr .L~tlc', aM't',)II(I ,hl',I(Illl , ;It It I Iwolllll', :[ , - cal lc, I "t'ilc\,lc(1lar\a" ; it ha, 1111\\ rc ;lrllctl tllc In fk (- t ill",ta ;;c mill Ill t It ill'Icr talc, f, I, d Ittitil it will, a1_c(' I,) a 11 , 1,1, alto \\llit'll It ,lt(Vl> It, skin ;l Illirlland a f(nlrtll tilllc and 1x'\,1111\ : :1(11111 .ktl,I/i(m ( ) j ,\tart/ 10 1_lrr("intrri(rsis .-- I\'ctttrllil1

52BROOKLYN MEDICAL JOURNAL February, 1903now to the relation of uncinariasis to the sand, Iwould suggest that when the embryo or the larvaleaves the feces and enters the ground, he stands amuch greater chance of gaining access to surfacewells in a sandy' soil than in a clay soil ; again,when lie sinks just below the surface he is morelikely to be brought to the top again by personswalking or by children playing on the sand thanon the clay, hence, chances for )land-and-mouthinfection would increase ; further, in a sandy soil,the worm would obtain more oxygen than in clay,and air isthese Nvorms .quite necessary to the development ofWhile dealin,- with the infecting stage, we inaymention briefly the methods of infection and ofpreventionInfection .-Infection may undoubtedly takeplace by swallowing the worm, either in wateror in contaminated food, as when a person withdirty hands eats a piece of bread ; infection bybiting the finger flails or by sticking the fingers,etc., when these are soiled with infested sandalso calls for credence . I_ooss has suggested thatinfection may take place through the slain, andBentley suggests that " ;;round-itch" is a resultof cutaneous infection with the larvae . Looss hasnoly dem,rnstratecl the possibility of cutaneousinfection, but I would mention the fact that"round-itch" appears to exist on clay as wellas on saridv soil, while uncinariasis is primarilyan infection of sand areas ; further, anemia cannot be said to lie a constant symptom after infectionwith ground-itch . I fence it is possible thatour Southern "groun(I-itch" is oilly in part identicalwith the "ground-itch" studied by Bentley .Prez ,ention .-As for prevention, it is clear thatthis should consist of two methods inparticular(t) Proper disposal of feces, since infection ishere present in its most concentrated form ; (2)treatment of all, even light cases of uncinariasis,to decrease the chances of disseminatinfr the infection; and to these two other points may beadded, namely (3) drink pure water, and (4)keel) hands and nails clean .Symptoms of urcciiulriusis .-I'urning now tosymptorlns, it may Ile stated that there is only onewhich is absolutely constant, namely, the presenceof hookworms in the sinail intestine . If theseworms are in the egg-laying stage, as will befound about five to six weeks after infection, ovawill be found in the feces in prol>ortiotl to thenumber of fertilizedthe small intestine .female parasites present inIn light infections, we mayhave to examine 5 or io preparations carefullyfor half an hour or more, before finding a singleegg ; in heavy infections, we may find 20 to rooeggs in the first slide .If onlv two or three parasites are present, theother syinptolns will be so light as to escape attention,in fact, it may be stated that till. patient is.practically well . As the infection increases, ht ~wever,symptoms become more proncmnced, untilwe reach the extreme stage represented by the socalled"dirt-eater ." We have now a train of symptomsmore common on a sandy than on a claysoil ; more common in rural districts than in citiesand towns ; more severe anion- women and children than anu)ng melt over 26 years old ; apparentlymore severe, Or at least more noticeablein blonds than in brunettes : more severeanion(, whites than among negrocs ; like trichinosis,several cases usually occur in the salve family,and, as a rule, also among neighboring families; above the frost-line the symptoms increasefrom spring to fall, and decrease from fall t(~s1) r ; I 1g .Anemia is one of the most common syinptimis,and is usually erroneously attributes] to malariaand pox,r food . Its degree varies, of course, 'ICcordingto the stage and the degree of infection .The skin varies from a waxy white to a tallow ora parchment-like yellow to tan . In some cases,the visible mncoiis incinbranes still retain theirreddish tinge, in others they become a marblewhite . The cardiac symptoms are of course prominent,and many of the patients speak of theirtrouble as "heart disease" ; the cervical pulsations,or "neck jerkin,-" in the sand-hill vernacular,may be seen at a distance of G to 12 feet ;therinse ranges from 8o to 132, without any Correspondinglyhigh tempcratierc ; the latter may besubnormal or normal, although loo to 1o2° h . isnot rare .\Vith the anemia we )nay have an irregularlyappearing edema, known among the patients 'is"bloat," "swelling," or "puffiness" ; the face '111(1ankles are the parts most frequently affected .Theterns "bloat" is also applied to the swollen al)(h)-men, which is very common ; this is listiall\' referredto as "pot-belly" or "buttermilk belly,"and often develops to such an extent :that nearlyall the men, women, and children of a givenneighborhood tool: as if they were about Umonths' pregnant .With the anemia, there is usually also a progressiTeeniaciotiort, so that the severest cases appearlike living pregnlant skeletons . "File musclesare soft and flabby, and naturally the patients areweak and unable to work . Partially connectedwith the muscular weakness, I may also mention

Febniary, 1()cl,iI3ROOkL}'NMEDICAL JOURNAL53the (11,1(1c WtV i)) brc" atl)ir),~ . One of the moststriking svnlptolns seen in infections of longstanding is the stunted gro ;(1h, both physical andr11cntal . 'e1 boy or girl of 14 to 18 may appearbetoonly 10 to 14 years (.f age, and I have seenmen and women 2o to 22 years old who werenot better developed than a child of 12 . Thesepatients, shall in stature, have little or - no hair(,)it their body ; the penis may be as small as theend joint of the little finger ; in a boy of 17, thetesticles have net always descended ; in a girl of16) to 22 the breast may be absolutely undeveloped,the vulva not larger than that of a chilaof 11 or 12, and the patient has perhaps neverulenstruatcd or does not menstruate over two orthree times a year, then chiefly in the wintermonths .Iwas unable to confirm the view that sterilityis very conln on in uncinariasis . but I did findthat miscarriage was exceedingly common amonginfected families . Whether these miscarriageswere actually, due to uncinariasis or to some.other cause, must be left an open question . Insome cases there was a clear history of venerealdisease ; in others, it occurred after severe workin the field ; in others, the women thought theyhad malaria and had taken large doses of quinine .These women age very fast . I have seen a womanof 26, with three children, who looked to be5o and was totally broken down ; and I haveseen a woman of 40, witll 1o children, who lookedto be 70 : one woman of about 48, with a historyof 18 children and 2 abortions, was a physicalwreck .Parents and teachers complain that the infectedchildren are backward in . their studies, and thechildren complain that to stud_\-headaches.brings on severeTile appetite and bowels are irregular .Theremay be diarrhea or constipation . The aprctitcmay be light to ravenous, and it very frequentlydevelops in some special abnormal direction : onepatient will be especially fond of strong coffee,without sugar or milk : another chews coffeemost of the tinge ; another cats salt or sticks lemons,or lemons an(] salt ; another is noted formiles around on account of his appetite for pickles; another cats wood ; another chews rags ;.,()111e cat plaster : others eat sand ; still others eatclay : and f have found a record of a negro whoate live Mire! This abnormal appetite is, I belicve,a result of the intestinal irritation and theanemia, and corresponds exactly to what we findin dogs, cattle, shed], goats, seals, and CICI)Iru1tsinfested with intestinal worms .Several recently published articles have referredto peculiar markings on the tongue incasesof uncinariasis, and this symptom has been recordedin early writings on dirt eating. 1t certainlywas more or less common in tile patient(I saw, but in a few cases I suspected that it waspossibly due more or less to tobacco or snuff.()lie very constant symptom which I found -,vasa peculiar cadaveric or fish-litre stare to the eve .If a patient is directed to stare intently at theobserver, the pupil is seen to be dilated or to dilateand the eye assumes a blank, stupid, cadavericstare, quite similar to the stare noted inpersons in extreme alcoholic intoxication or to thestare of a fish's eye . . In only two cases where Inoted this symptom, did I fail to find the eggswhen the stools were examined ; those two cases,12 and 14 years old, respectively, were sons of aconfirmed drunkard, and one of theta showed infectionwith whipwornls (Trichuris trichiura) .This peculiar symptom was not notice(], however,in any of tile severe cases of malaria which cattleunder my observation .Lethalit-if of uncinariasis.-The exact lethalityof :American in,cinariasis must be left: nndet--rminedfor the present . Sev ,2re infections ar,indoubtecil_yserious rind. often fatal, but frc;m thefact that sncil a I'll-,-c proportion of children ;ireinfected in a given locality, and some of theseseriously, Iobtained the general impression that1111Cnlarlasls per se is not quite so fatal as onetj" (n11d naturally expect . But let a patient who isserious]\- infected with this disease contract typhoid,tuberculosis, pneumonia, or severe malaria,and the second illness will be of comparativelyshort duration . While my observations have notvet extended over a long enough period to speakwith certainty upon the subject, I and not disincline((to the belief that Nvc shall eventually allunite in the view that many of tile. deaths attributedto uncinariasis in man are in reality dueto a second disease which the patient was notable to withstand because of the pr~existirlg hookwnr111infection . Further, it is evident that withthe disappearance of uncinariasis from the sanddistricts, the proportion of deaths from tubercu-1(osis, typhoid, pneumonia, malaria, childbirth, etc .,must necessarily decrease .Frc(tucrrc ~- of micinariasis.-I have alreadystated that - locally infected cases of uncinariasisin cities, towns, an(] in clay districts, are not fre(luent . 111 Cities and t(omns the conditions are unfavorableto the devcl()1n11ent of local foci of infe~(-tioll,iurallsc the strut-ts and walks are paved,~ar(ls jr, s)(l(lc(l, -111(1 s(~~-crage is present . :\s

5-t.we approach the mttskirts of tile city and entertile cdnlntr\ . paved streets andl walks decreased,r disappear ; yards :ire not sodded ; and the se\vera-csystems + are supplanted Ilk lr,x privies .Thus, cdmditidlns become more favorable four tnlcinariasisand we find that this increases in sandyre~itrtls as we leave the city andl in llrd,ln,rtual t,,the attentirnl given to tile care of the con "trll,-tion and cleaning of tile outhouses. Cas,- d,fttncinariasis fdxlnd in cities and towns hard,IN, come in from the country, hence, wiles estilllatingthe frequency of tills disease \ve must practicallyeliminate the city population from the possibilityof local infection and recall that an anemiawithin the city limits, as well as an anemia in ruralclay or stone districts, is much more likely to beclue to malaria than it is to tnicinariasis . I y this,I do no, d,f course, mean to advance the view thatmalaria is preeminently a disease of cities, butsimply to call attentidm t the fact that cities presentconditions \\ -Inc h are more favorable to thedevelopment of malaria than of ttncinariasis .1-loldin- these facts in mind, I should estimatethat in the strictly sane? districts, uncinariasis istile Most Co111111o11 disease Of tile White I)OI)LIIati0nof the South . In the negro it is less severe, and,perhaps less common, than tuberculosis, syphilis,or gonorrhea . Taking tile South as a Whole, Ishould say at present that uncinariasis should beplaced in the same general category as malaria,tuberculosis, syphilis, and gonorrhea .1.CollolrllC importance.-From an economicstandpoint, uncinariasis Must rival and probablyexceed malaria in importance . Its presence preventsthe proper education elf the children, preventsthe proper development of the youth, anddecreases tile labor-producing power of tile adult,hence, of the productiveness of the farm . It increasestile number id orphans, and the numberof people who nlnst be supported by charity . Itincreases expenses for drnr,>s and medical attendance.:\s illustrations Of these points, I have seenfatllilics Of 12, all of wlunll were affcctcd withuncinariasis ; in one falllily Of 22 Inelllbers, therehad been t t deaths . incind?ing 2 miscarried fettt,d*s; dal dale farlll of (9) h:mds, 20 \were cxalrtilledl"I'd all fdnttld 111fcetcol : the elands Of thatfarm were nd,t pr,,ducinti dryer ;dl to So) l,er colt .d,f ll,,nlml labor, and in ~oil(- family I s ;1\y d>I1 al1-,,illl-r farm, the lairs did not exceed io) per cent .14 I1d,nmd \yrk . In three orphtul asylums, If,,llnd ah,,ut H t,, t i leer cent of lll{I childircll itlicctcdltyitll uncinariasis .( - nd I'll in tilt- CdrthdH u1il1S .-I Visited threePROOKIA A' .111 :01CAL JOURNAL February . I d r , 3cl,ttcnl Mills, \vhicll I \\.IS assured l,rc>cnte , l averagetypical conditions for shell intititlltidms . allddale Mill which I was assured was far alr,ye theaverage . "faking the first three as basis for nl\remarks, it may be said that the amount )f anCHIMand the number of small children amonL" tilehands were very striking . Several children werepointed out as the typical "mill children," ofwhom we have heard so much . It took but aglance td, recognize that they presented tile tylrcOf stunted growth so common in ttncinariasis ; indluirydevelopedthe fact that they had come fromtile rural sand districts, and physical examinationgave a clear diagnosis of hrY>k\yd,rm disease .Visiting a number of mill families in companywith the contract physician, it was nr,t difficult totprove that uncinariasis was more or less common,but that with continued residence in the city, tileinfected cases improved, unless tile\- were too fargone to admit of recovery . The effects of formerinfection were clearly discernible in two- ) still menwho had been in the city, they claimed . 13 years .It is not my intention to .account for all theanemia and other conditions of the mills by attributingthem to uncinariasis, fo>r other factors,such as malaria, the frightful prevalence of tobaccochewing and snuff dipping anlollo even tileyoungest hands, the constant breathing in of tineparticles o>f cotton, etc . . come into consideration,lout I would submit that ttncinariasis represents anew factor in the still ject olf the child lalwr d,f thecotton Illills . While it is not a cd :>Inpletc cd,llltilmof tile prd,lllcins involved, it wortlcl be well for listo recall that dwse children from the sand areasare frt,ln one t'l five years older than tile\- al,-pear ; at: tile mill, they are earning more 11umec .have lighter work, are living in nu,rr hygieniclunlses, are in better health and better spirits,leave, Ilnlre regular medical attendance and betterchances for recovery than these same peoplehad on tile same farms . I did, nd,t nleall ti, picttlrt"thelll :Is giants of strength dlr their houses aspalaces . Ins td, prd,lu . rly judge them we r1ttlst coInllarethe edlnditums :It tilt' Mills with tile col1ditid,llsdltl the farms, and the Mill harldls \\Itl l tilefarm hands .Ccuerul d " fio d, ts on Mo . inlduhilurlls (Is n ClUss.-\Ve ticsare all familiar with the generalatlribntcd to tile white popillathm illcertain parts of tilt' Sdnlth, nalllcly, that class sotfredlllelltl . rcfcrrcdl tot 11Mler tile Iu,t yet - \ - rllrtoricalexl,ressidm d,f "tile pd,d,r \yllite trasll .- 'I'dlfully allllrecialc jest what tht-e people are andhow they lire, we Must sec thel11 ill t1wir 1umics .N- low it 11215 hd'ell Ill\. exl,d'I"lt .11cc tlIal them' people

t clnnarr, 1, It I . ;prc-scllt ;1 1114 Irk , t\pica] picture in tllc wild rc-i1nls111 ;111 ill t'1 :1\ 11)calllles . ' I ' llr ll()()rk . r \\1111t'~ ill lilt .cla\ rcgII I Is alld ill the cities :it . ( , 1lcalillicr alld11t(1re ;ICli\c Ihall those ill saild districts . I .et :1falIIil\ 1111)\'e fl-1)III salld t(1 il :l\, atld it lllll)1'(I\'CS ;I('t it . lmi\'e fn ml (-I ;l\ it I sa11(Lmid I1 I - tills 11()\\'11,'1'llis iilva III :II a cI ;I\ soil is IIc ;IIIIIic-r tllall ;1saml\ soil is ('()I11111(Ili kll()\\lcdgc :1I11()ng maul, (Ifthese lw(I1Ilc . Wit 11 tit(' diso~\er\ If the pr(\alencc()f mlcillariasis ill tit( . sand 1(ICalitics, \\cha\e all cxplatlatii Ill (1f tltese facts \\ hick . tll()Itgllwhichit so lutlds ( " \trelllc :(11(1 scilsati(nlal . Is 1It( .demands scri4itts ;itILIlti(ill . 1 1crs(mall \, after acareful sttul\ (if right weeks ill the 1( .)calitics inIt in . and ;Iftcr seeing Iltilllcn)Ils cases (If ttncillariasis,1 see 11(:1 lit )SO)le escape fl-Iarl ill(' Collclttsi(Inthat this disease Offers 11 .1; a patlx)logicbasis for the facts mentioned, and that this is one()f the most important, if II t the most important .factor in the inferior mental and physical develolpnientof these people . Fradicate tincinariasis andthese poorer Whites Will, in one or two generati(Ins. be a different people : or let tit(- conditionsremain as tile\- arc . an(] any whites who go) tothe sand farms and live as these people d(1, willdeteriorate to the level of the so-called "dirt eaters"and "poor white trash ."No.,' to chars,-c the rrcsrnt conditions.-To alterpresent conditions, the first thing not to do isto try to teach the people to boil their water andkeep the hands of their children clean . The propositionof boiling or filtering drinking water isabsurd to the average inind of the lalx)rinclasses; and the average mother of three to tenchildren on a "one-horse farm" has somethingelse to do besides washing the hands of her childrenevery tithe they eat . Any campaign Ill thismatter which \\e adopt nnlst he practical, notacademic . By pursuing an academic course, \\eshall lose all our influence with the average 1111-educated roan,The first point is t(1 disseminate witlespreadanulng the country physicians an account of thecause, symptoms, cure, and prevention ()f uncinariasis. This should not he obscured 1)\ technicalwords which not half of the physicialts ofill(- world understand .The next point is tot c(lucate tit(, rural populationto a more general and a better construction,in(] care of privies, which should he located insuch a place that the drinking water call not beinfected . This can pest be done 1)\ ill(- familyphysician, \%-]tol has the confidence of the people .Efforts by strangers will meet with suspicion, butthe fanlily physician can present tot the faIltll\Ill( . 11lill ot11ieclcd "Ith ill( . \\ ;Itel, c''l1U1111111ation;lid ill( . fillalwial si(Ic of tllc (lox>ti(nl ill ill(- 1(Issof ill0)1lle, Cxpcllsc for drugs, etc . ()lie practical1Ie111ollstralit'll ill a Ilcigh1P)I'llo''d \\Il l (11) Illore111 ;111 ;Ill \' ;11111)'lllt ()f agltatl1)Il . 11 :11111 " Ill halld\\it h these IlWasllrc . (If IIre\('IltII)I1, fine (If the tiledittlllsevere cases in tit( , famil\ shield be treated;IS a practical object lcss(nl . .After the rec(wery()f tills patient, ill(- faIIIIIY will be 1)111\ - tow) anxl,,)ItsI, I f()ll(i\\' IIlit directi , rits, and with ; tit .impr(Ivementill tit( , general condition of the familC, Illc cleanlinesswill more ()r less take care (If itself . especiallyif the children go) top school . 1 doubt, how-C\cr . Mletlicr the average farm hand will evercome t() ill(- point ()f boiling or filtering the drinkingwater .7'rcotrm'nt.-The method of treatment is verysimple . lint contains one prominent pitfall . name-1y, the impression that a single (lose of medicinecan be administered and the patient dischargedas cured. As a rule, large (loses of thvntol orlarge (loses of male fern are given . If the forliter,the physician should absolutely prohibit theuse of alcohol during the da\- of treatment . Onemethod is as followsR A. \I . 2 grains ( ;1 grains) pot'dcr :d thymolin capsules .to A. M . Repeat .12 \a)11 . Dose of salts or of castor oil .Repeat treatment once a week until stoolsshow it() evidence of eggs .If stale fern is used, comparatively high (andsometimes fatal) (loses are usually given, namely,from io .o 12 1 _fuidranis) to ,,o.o (~ fluidrants) .UntInari(lsis ill _\'orthern Practice.-With thepossible exceptions of mining districts, probablymost of the cases of hcx)kwonn disease observedby Northern practitioners will be imported frontIll nrope, the Philippines, the Southern States,Cuba, Porto Rico, Central and South America .If from I':urope, Asia, Africa, or the Philippines,.l,~ih~'lol,ctl)ulol (1uodcnalc will be usually found .I f from the American continent, ('ncinariu ulllcri-CUR(1 is More likel\ t(1 he found . To the \orthrrllJiltvsician, who has the patient isolated frontIllc s(nlrce of infection, it is, of course, of imporlanceto) determine llow lot, the parasites \\il lpersist in tit( , intestine . Itearilw upon this pointI can say that I nave one cast', which is free fromcriticism, where 1 ' / ICill(11'1U (I)M'1'1,C011(1 has lived (1Years and 7 In(alths : t\\() cases, likewise free fromcriticism, \\here the infection has persisted ()Years : finally, ()lit- case . not free from criticism,Where the infection slur existed I(1 \cars .It is needless for nle to insist upon tit(- practi-

;ni"N()()R1.VA. ,tll :/ll( :Il. JOE RIVAL Fcl)rttary, 1y)3c ;ll applicati,)Il 11f this point . Here ill I~n)okl~1l\1)it IIllt\ Il ;t\c a case of ()1)scurc allclllia ; ill sucha ca>c it is necessary to go Nick hit- tit Vicars i11the Ili,tor_\ I,) sec \\Itcthc r tilt patient has ,luringthat tillIC " \isitctl all\ sallcl areas ill tilt' ~MIIIICI_11State's ()r clsc\\ll,~rc ill \\arntc r cliillatus .INFANT DIARRHEAL MORTALITY IN BROOKLYN .ITS CAUSE AND PREVENTABILITY .fly - I'M Is C. A(& .N, N1 .1) .121-0,1 at the \t"tinw of Ow hit" t nnwy NOW- :,I A-Whm,I)c,.on .to,, t, . 1q,-' .111e till,' ,)f tilt .; paper pr,,pcrl~ - cltll)rac,., t\\~~y ;tc,tit , Its1 . IIIC cattle and 11rc\ctlt ;tl)ilitc ,1f ill, ; highsttlluncr ill f ;ult in, )rtalit\ ill all lame chit s .11 . The call " ( : and III I-\ I lllahilit\ 1,f tllc cxc,')f I~nu,kl\It's ,ulllnicr ill i :1111 in, Ilalit\ \c1- that.\Iallll :ttlan .'Ill,' Pa1x r \\ il l I),' lllailll\ do-\i 'led I, tilt ,on,l Jilt>tioll, htn 111'"t , if tilt' ~lc , lncti ,, ll all'ityt'ai,nl, will ;I1 )111 \- c,lu :Illc t . . ,filler l ;ir~c chic, .I ile ,t ;tti,li,~, art' ,It'ri\e,l it , ntl tlur,,' ,~nirccs ;f . I Il, l - . ~ . ( t.lI

Significant Contributions from the Division ofZoology and Its Successor Laboratories at the NIH,1902-1970 .Leon Jacobs.The history of medical parasitology at the HygienicLaboratory starts with Charles Wardell Stiles . As a studentat the University of Leipzig and other institutions,Stiles acquired from a number of distinguished Germaninvestigators, in addition to enormous amounts of information,the attitude of the "Geheimrat." He returned tothe United States and took a position in the Bureau ofAnimal Industry of the U .S . Department of Agriculture(USDA) . A prolific worker, Stiles published a considerableamount of information on parasites oflivestock . His attention was drawn to hookworm byBailey K . Ashford, a former student working in PuertoRico, and by Thomas A . Claytor, a Texas physician whodescribed the first clinical case of human hookwormdisease in the United States . Stiles identified the parasite,naming the new species of hookworm Uncinariaamericana, now known as Necator americanus, which isan important parasite of human beings .' This informationcame to the attention of Milton J . Rosenau, Directorof the Hygienic Laboratory, and Surgeon GeneralWalter W . Wyman of the U .S . Public Health andMarine Hospital Service, precursor of the U .S . PublicHealth Service (PHS) . Rosenau and Wyman recruitedStiles to initiate programs in medical zoology within theHygienic Laboratory . Stiles must also be credited withinteresting John D . Rockefeller, through the efforts ofWalter Hines Page, in the problem of hookworm in theSouth, an interest which led eventually to the establishmentof the Rockefeller Foundation . The originalRockefeller campaign against hookworm was also an impetusfor the development of strong parasitology programsat the Johns Hopkins School of Hygiene andPublic Health and elsewhere .Stiles's interests in parasites were broad . He publishedextensively on helminths and also recorded the first caseof giardiasis in the United States . However, towards theend of his career he spent most of his energy recordingthe literature of parasitology in the Index Catalog ofMedical and Veterinary Zoology, which is still extant .After Stiles's retirement, George W McCoy, director ofthe then singular National Institute of Health, againturned to the USDA Bureau of Animal Industry to finda new leader for the program in medical zoology . In1936 Maurice C . Hall, a veterinarian with a Ph.D . inzoology, was appointed as Chief of the Division ofZoology . He brought with him from the USDA a groupof investigators who had already made significant contributionsin veterinary parasitology, including WillardH . Wright, Eloise B . Cram, Myrna F . Jones, and JohnLeon JacobsBozicevich . Hall outlined a program for this group, basedon his perception of parasitic diseases which continuedto surmount our sanitary barriers . These weretrichinosis, oxyuriasis, and amebiasis . After Hall's suddendeath in 1938, Wright was named to direct the Divisionof Zoology . Wright was also both a veterinarianand Ph .D . in zoology . With imagination and considerableability he continued the earlier programs andadded new ones . The Division of Zoology incorporatedmalaria programs conducted elsewhere and eventuallyevolved with the incorporation of arbovirology, into theLaboratory of Tropical Diseases . From 1938 until hisretirement in 1958 the Laboratory of Tropical Diseaseswas headed by Wright .It is well to pause here and make note of the fact thatin the early days the Hygienic Laboratory and the NationalInstitute of Health were oriented not only toresearch but also to the prevention and control ofdisease . Thus, some of the early studies on theprevalence of trichinosis in the United States, first doneby Stiles and then continued and extended systematicallyby Hall and Wright, outlined the importance of diseaseas a public health problem and defined the characteristics29

of the nematode parasite and the methods for its control. Indeed, it was not until after World War II that adelineation of functions, between research and control,began to develop in the Public Health Service . Thisdivergence resulted in the creation of the CommunicableDisease Center, now the Centers for Disease Control(CDC) . The CDC has acquired its own highly respectedreputation for excellence, but areas of cooperation betweenthe two institutions remain . It is important, afterthese parenthetical remarks, to make clear that some ofthe early studies done at NIH were motivated by itslarger mission as the principal PHS research and controlinstitution . This explains some of the more practicalpapers that will be cited henceforth .It is also important to note that specific research goalswere established and pursued by some of our earlyleaders . Whether an individual research project was basicor applied mattered little, so long as it fitted into theoverall objectives . This was true, indeed, of mostbiomedical research at that time . The purpose of researchwas to accrue more and more information that wouldlead to the conquest of disease . The individual paper wasa contribution to that end . Each paper representedanother building block in the necessary structure of thescience . In the words of the distinguished RockefellerFoundation malariologist, Lewis W . Hackett, who servedfor some years as editor of the American Journal ofTropical Medicine and Hygiene, "We build like coralpolyps ." The exposition which follows is based on thisidea : that a number of contributions have to be cited toreveal the development of major landmarks .Maurice C . Hall and his colleagues instituted a seriesof studies of trichinosis after Hall came to NIH from theUSDA Bureau of Animal Industry . These studies firstdelineated the problem of the extent of infection anddisease due to Trichinella spiralis,2,3 and some studieswere done on the control of infection . 4 . 5 Also, Bozicevichand his colleagues did some of the early work on the immunodiagnosisof parasitic infections 6 and introduced thebentonite flocculation test for trichinosis which was widelyused for many years .? In the course of some of thestudies of immune reactions in trichinosis, Bozicevichand Detre made one of the earliest demonstrations of theoccurrence of circulating antigens in recently infectedanimals .$ This was a precursor to later work on the occurrenceof antigens and antigen-antibody complexes in avariety of infections and disease states .During World War 11, Wright directed some of the effortsof the Division of Zoology to appraising the risksof importation of vector-borne parasitic diseases to theUnited States in returning troops . Studies were conductedon potential mosquito vectors of filariid nematodes, 9 andsimilar work was done on North American snails aspossible vectors of schistosomiasis . 10,11,11 The work onfilariid vectors was considered especially important,because a focus of endemic filariasis had previously existedin Charleston, South Carolina, having probably arrivedoriginally from Africa . It was not to be consideredunlikely that other strains of the same filariid from otherparts of the world, or different filariids, could findDr . Jacobs, second row, far right, with Division of Zoology in 1938.suitable mosquito vectors here . The most significantwork on schistosomiasis vectors was the later demonstrationby Newton of the role of genetics in observed straindifferences in susceptibility and other host-parasite interactionsof snail vectors . 13,14 This genetics work waslater expanded by Richards to show characteristics of differentstrains of snail, and also genetic differences in theinfectivity of schistosomes to strains of snails . 15Other work done in the Division of Zoology andLaboratory of Tropical Diseases involved the efficacy ofsewage- and water-treatment in the control of parasiticinfections . Basic work on the effects of a number ofagents and treatments on parasite eggs and cysts wascontributed . 16,11,11 Some of the early data were importantin military operations during World War II forbivouacking in the field .Enterobiasis was another early focus identified by Hallfor the Division of Zoology . Its pathogenesis as apediatric infection had not been established, and thisdepended on a reliable diagnostic technique, because theeggs of the worm are not passed in feces . The techniquedeveloped at the NIH relies on the demonstration of eggson the skin of the perineal region, where they aredeposited by the female . Developed by Hall, it is knownas the "NIH swab" and is still the accepted technique . 19Other work done on the pinworm involved the incidenceand epidemiology of the infection, including such factorsas the viability of the pinworm egg, and on treatment. 20,21,22 In connection with attempts at control earlywork on the effects of radiation on living organisms wasdone in the Division of Zoology with AlexanderHollaender who was in physical biology . This involvedtests of monochromatic ultraviolet radiation on the pinwormeggs . 23,2430

Additional important work was done after World War11, when radioisotopes first became available . FrederickJ . Brady collaborated with Dean Cowie of the CarnegieInstitute of Terrestrial Magnetism and others on some ofthe first uses of radioisotopes in pharmacology . 25 Thisinvolved synthesizing drugs with radioactive elements toidentify the sites of activity of such drugs in animals infectedwith filariid parasites .A considerable problem in studying amebiasis andamebic dysentery before 1961 was the inability of investigatorsto grow pure cultures of the amoeba, Entamoebahistolytica, or any of its close relatives . Cultureswere always contaminated with bacteria and could not bemaintained without bacteria . Starting about 1940, a seriesof studies at NIH resulted in the development of culturesof the amoeba with single species of bacteria, by thetechnique of micro-isolating cysts of the parasite,washing them repetitively in sterile water, and inoculatingthem into cultures with selected bacteria . 26 Later, whenantibiotics became available, it was possible to eliminatebacteria from cultures and to maintain them brieflywithout bacteria ' 27 or to substitute other bacteria, orprotozoa 28 instead of bacteria . Finally, cultures of theamoeba without any other organisms were obtained andmaintained indefinitely . Thus, a whole series of studieseventually culminated in a great landmark . 29 Similartechniques led to the cultivation of other species ofparasitic protozoa in axenic cultures, 3 ° ,31 which subsequentlyallowed studies on the biochemistry of theorganisms . 32,33Physiology of parasites was the principal effort of onedistinguished scientist recruited by Wright . Displaced bythe Nazis before World War 11, Theodor von Brandmade major contributions in describing the aerobicfermentation of blood trypanosomes and the anaerobicmetabolism of parasites . It is difficult to single out individualpapers as his most significant contributions,because his work and that of contemporaries in the samefield is voluminous and very detailed . Many of his trulyremarkable contributions, however, are summarized intwo books . 34,35In malaria research, the volume of work done bymembers of the staff of the Laboratory of ParasiticDiseases and its antecedent organizational componentswas likewise large and important . A group of investigatorsin South Carolina, exploiting the then-currentclinical use of induced malaria to control syphiliticdementias, studied the characteristics of strains ofmalaria obtained from many different parts of the worldto elicit information on the relapse patterns ofPlasmodium vivax strains . 36 They provided informationon other species of malaria37 of considerable importanceregarding the occurrence of dormant forms of themalaria parasite, and this information contributed to theeventual description, in 1948, by Short and Garnham, ofexoerythrocytic stages of P. vivax . Citations of some ofthese early studies are found in the symposium alreadycited ,36 on human malaria published by the AmericanAssociation for the Advancement of Science in 1941 . Agreat deal of the work on malaria during the years ofWorld War 11 was a coordinated effort on drugdevelopment,headed by James A . Shannon, who was tobecome, years later, the Director of NIH . A record ofthe findings on almost 4000 substances studied in achicken malaria system at NIH from 1941 until 1952, existsin a Public Health Service Publication, No . 193,which appeared concurrently with Public Health Reports,v . 68, no . 1, in January 1953 . 38Early work on malaria involved careful and accurateidentification of the stages of the parasite in its naturalhosts in the field and in experimental hosts in thelaboratory . In 1942, in recognition of the need for identificationof the parasites' stages, the NIH sponsored thepublication of a manual describing them . This manualwas prepared by Aimee Wilcox, with the assistance ofthe NIH resident artist, Inez Demonet . Its color plates ofthe morphology of the malaria parasites have been thestandard for years and have been reproduced many timesfor different texts . 39 Contributions to the study of candidateantimalarial agents in man were also made byNIH researchers . One example is a short paper describingthe method by which a great deal of basic informationon drugs and on relapse patterns of the malariaswas obtained in the period from World War Il throughthe 1970's . 40In addition to drug-screening techniques and clear cutclinical studies, field work on malaria was also carriedout by members of the Laboratory . In the course ofsuch work in Malaya, one of the staff discovered thetransmission of a simian malaria parasite to man, byvectors that were colonized in the laboratory . The principalinvestigator, Don Eyles, suffered himself from theinfection, due to Plasmodium cynomolgi bastianelli . 41Another facet of malaria pharmacology involved theresistance of the parasites to drugs . The first rigorouslydocumented study of resistance of Plasmodiumfalciparum to the drug chloroquine was provided by NIHinvestigators, after the drug had been in use for fifteenyears . 42Other progress in malaria research involved collaborationof malaria specialists with individuals interested inthe immunology of parasitic infections . One major contributionwas the demonstration that the technique ofimmunofluorescence could be applied to the diagnosis ofmalaria infections . 43,44 The later development of effortstowards malaria vaccines derives from the original observationson antigenicity of the parasites . In addition,research on malaria therapy contributed to the treatmentof at least one other disease . The drug, pyrimethamine(Daraprim) had been shown in malaria studies to haveconsiderable usefulness . One of the malaria investigators,Joseph Greenberg, suggested to Eyles that it be triedagainst Toxoplasma gondii . Eyles was then undertakingchemotherapeutic trials against T. gondii in a Memphis,Tennessee laboratory field station of the Laboratory ofTropical Diseases . Eyles and Coleman45 not onlydemonstrated the efficacy of pyrimethamine against Toxoplasmabut very neatly showed that the drug wassynergistic with sulfa drugs . This paper was the first tooutline a treatment for toxoplasmosis and the drugs stillremain the most accepted treatment .In the field of helminthic infections, some additional31

mention should be made of the filarial parasites . Workon testing the potential of domestic mosquitoes to serveas vectors of Wuchereria has already been described . Inother work, LPD investigators studying Dirofilaria infections in dogs as models of human disease found that, inaddition to D . immitis, another dog filariid occurs in theU . S ., and that this parasite is less pathogenic to thedog . 46,47 This finding has considerable significance inveterinary medicine, because the heartworm (D. immitis)causes a great deal of morbidity and mortality while theother filariid poses no severe clinical problems . In addition,this discovery demonstrates how disease studies inanimal models contribute materially to our knowledge ofanimal diseases (a point to make to anti -vivisectionists,even though it is hardly likely that they will listen) .Research on onchocerciasis in Guatemala was performedby LPD scientists working in collaboration with thePan American Health Organization (formerly the PanAmerican Sanitary Bureau) . The most significant resultof this work was a monograph on the blackfly vectors ofthe filariid nematode, Onchocerca volvulus . In additionto the description of species, this work also providedvital information on the breeding places of the flies, ontheir flight ranges (which are very long), and on theirvector potential . 48 Other studies on filariasis, pursued byLPD scientists in the South Pacific, resulted in significantcontributions to our understanding of the bionomicsof the vectors of human aperiodic filariasis . Another importantdiscovery, however, derived from the presence ofone of our scientists in that locale . Leon Rosen identifieda clinical disease there called eosinophilic meningitis andwas able later to recover a nematode worm from thebrain of a fatal case of the disease . It was identified as alarval form of a worm ordinarily found as an adult inthe rat . Rosen and colleagues also worked out theepidemiology of the human disease, which involves invertebrateanimals capable of serving as intermediatehosts of the nematode .49Additional work on helminths involved thepathogenesis of human schistosomes and some of theiravian relatives . In early work, Olivier was able to showthat the phenomenon of schistosome dermatitis was dueto sensitization to larvae of avian schistosomes that wereattempting to invade human skin . This was the first wellcontrolledstudy of the phenomenon, demonstrating thatlesions increase with repeated exposure .5° Similarly,Cheever found, in the first careful quantitative study ofhuman schistosomiasis, that lesions increased in size aswell as number with increase in worm burden . 51 Weinbach,working on the use of the pentachlorophenols asmolluscicides for the control of schistosomiasis,demonstrated that the mechanism of action of thesecompounds is the uncoupling of oxidative phosphorylationin mitochondria . He and a colleague laterdemonstrated that rates of oxidative phosphorylation inmitochondria varied with the age of the animal fromwhich the enzymes were derived . 52,53In 1956 Weinstein and Jones contributed landmarkresearch on helminths when they cultivated larvae of anematode parasite to the adult stage in vitro . 54 This providedthe basis for cultivation of a number of otherhelminths later and for significant contributions to thephysiology of these organisms . 55Jacobs and colleagues contributed information on thebiology of the intracellular parasite, Toxoplasma gondii,on the resistance of the cysts of this parasite, 56 and onthe occurrence of the cysts in the flesh of meatanimals57-all of considerable significance in theepidemiology of the infection . They developed thehemagglutination test for toxoplasmosis as a substitutefor the difficult and dangerous dye test which was thenthe only accepted diagnostic procedure . 58 They alsodemonstrated the occurrence of T. gondii in the eye ofadult human beings, and through the interpretation oflow serological titers, were able to demonstrate thesignificance of toxoplasmosis as a cause of oculardisease . 59 .6° Later, Sheffield and Melton contributed tothe elucidation of the life cycle and morphology of Toxoplasma,providing convincing evidence of the coccidiannature of the parasite and of the existence of an intestinalstage in the cat .ConclusionThe aim of this essay has been to present an overview ofthe evolution of studies in medical zoology throughoutthe early years of the Hygienic Laboratory and NIH .Although other authors might have chosen differentpapers, the selections here were made according to thelarger context of developments in basic or appliedresearch or in the epidemiology of disease . These papersare, indeed, the individual coral skeletons to which LewisHackett referred--together they comprise a body ofknowledge . In some instances, the citations are givenprincipally to indicate the varied directions in which thework of the zoology laboratories went . (Members of theLaboratory of Parasitic Diseases could undoubtedly questionthe choice of citations, but the goal was merely toindicate the type of research .) In other instances, as hasbeen pointed out, the citations represent highly significantadvances of which we are all proud . It may be ofinterest to note that when the editor of the Journal ofParasitology solicited from the Institute for Scientific Information(ISI) a listing of the ten papers in the Journalof Parasitology that were most cited during the period of1961 through 1982, seven of the ten papers identifiedwere from the NIH . All of these were published beforeor in 1970 (Journal of Parasitology 69(4) : 774) . Itshould, of course, be noted that this essay has focusedsolely on contributions from the laboratories at NIH andof necessity has neglected other sources . As this reviewends with 1970, the many new and technicallysophisticated methods used today in the study ofparasites and the diseases they cause, are reflected in theannotated bibliographies prepared by the currentlaboratory chiefs, which indicate that medicalparasitology continues to expand in scope and depth .32

Leon Jacobs, Ph.D.Dr . Leon Jacobs worked for 42 years at NIH both asa bench scientist and as an administrator . Born inBrooklyn, New York in 1915, he graduated fromBrooklyn College in 1935, and two years later, joined theDivision of Zoology . After serving in the Army from1943-46, principally as a Malaria Control Officer in theSouth Atlantic Theater of Operations, he obtained aPh .D . in 1947 from George Washington University .From 1946 to 1966 he held various positions inNIH/NMI/NIAID . In 1959 he became Chief of theLaboratory of Parasitic Diseases and in 1964-65, he wasnamed Acting Scientific Director of NIAID . Dr . Jacobsleft NIH in 1967 to serve as Deputy Assistant Secretaryfor Science, DHEW . Returning to NIH in 1969 he wasAssistant Director for Collaborative Research . From 1972to 1978 he was Associate Director for CollaborativeResearch . In July 1978 he was appointed Director of theFogarty International Center . Dr . Jacobs retired fromFederal service in 1979 . He has published over 100papers, and edited several journals including the Journalof Parasitology . As Scientist Emeritus at NIH, he has anongoing research project on toxoplasmosis at LPD andretains an active interest in studying amebiasis and otherparasitic diseases .References1 . Stiles, Charles W . 1903 . Hookworm Disease (uncinariasis), A NewlyRecognized Factor in American Anemias . Brooklyn Medical Journal 17 :51-6 .2 . Hall, M . C . and Collins, B . J . 1937 . Studies on Trichinosis . I . TheIncidence of Trichinosis as Indicated by Post-mortem Examination of300 Diaphragms Public Health Reports 52 : 468-90 .3 . Wright, W . H . ; Kerr, K . B . ; and Jacobs, Leon . 1943 . Summary ofthe Findings of Trichinella spiralis in a Random Sampling and OtherSamplings of the Population of the United States . Public HealthReports 58 : 1293-1313 .4 . Wright, W . H . 1944 . The Relation of Municipal Garbage Disposal tothe Trichinosis Problem . Public Works Engineers Yearbook 103-17 .5 . Wright, W . H . 1944 . Control of Trichinosis by Refrigeration ofPork . Journal of the American Medical Association 155 : 1394-95 .6 . Bozicevich, John . 1938 . The Diagnosis of Trichinosis by ImmunologicalMethods . Revista de Medicina Tropical y Parasitologia 4 :(Bacterio y Clinica y Labortorio, Habana) 4 : 155-57 .7 . Bozicevich, John ; Tobie, John E . ; Thomas, Elizabeth H . ; Hoyer,Helen M . ; and Ward, Stanley B . 1951 . A Rapid Flocculation Test forthe Diagnosis of Trichinosis . Public Health Reports 66 : 806-14 .8 . Bozicevich, John, and Detre, Laszlo . 1940 . The Antigenic Phase inTrichinosis . Public Health Reports 55 : 683-92 .9 . Newton, W . L . ; Wright, W . H . ; and Pratt, Ivan . 1945 . Experimentsto Determine Potential Mosquito Vectors of Wuchereria bancrofti in theContinental United States . American Journal of Tropical Medicine andHygiene 25 : 253-61 .10 . Files, Virginia S ., and Cram, Eloise B . 1949 . A Study on the ComparativeSusceptibility of Snail Vectors to Strains of Schistosoma mansoni. Journal of Parasitology 35 : 555-60 .11 . Cram, E . B . ; Jones, M . F . ; and Wright, W . H . 1944 . UnsuccessfulAttempts to Infect Eleven Species and Subspecies of Domestic Snailsfor Schistosoma mansoni. Proceedings of the Helminthic Society ofWashington 11 : 64-66 .12 . Cram, E. B . ; Jones, M . F . ; and Wright, W . H . 1945 . A PotentialIntermediate Host of Schistosoma mansoni. Science 101 : 302 .13 . Newton, Walter L . 1953 . The Inheritance of Susceptibility to Infectionwith Schistosoma mansoni in Australorbis glabratus . ExperimentalParasitology 2 : 242-57 .14 . Newton, W . L . 1944 . The Establishment of a Strain of Australorbisglabratus which Combines Albinism and High Susceptibility to Infectionwith Schistosoma mansoni. Journal of Parasitology 41 : 525-28 .15 . Richards, Charles S . 1975 . Genetic Studies of Pathologic Conditionsand Susceptibility to Infection in Biomphalaria glabrata . Annals of theNew York Academy of Sciences 266 : 394-410 .16 . Wright, W . H . ; Cram, E . B . ; and Nolan, M . O . 1942 . PreliminaryObservations on the Effect of Sewage Treatment Processes on the Ovaand Cysts of Intestinal Parasites . Sewage Works Journal 14 : 1274-80 .17 . Newton, W . L . and Jones, M . F . 1949 . The Effect of Ozone onCysts of Entamoeba histolytica. American Journal of Tropical Medicine29 : 669-81 .18 . Newton, W . L . 1952 . Water Treatment Measure in Control ofAmebiasis . American Journal of Tropical Medicine 38 : 88-89 .19 . Hall, M . C . 1937 . Studies on Oxyuriasis 1 . Types of Anal Swabsand Scrapers, with a Description of an Improved Type of Swab .American Journal of Tropical Medicine 17 : 445-53 .20 . Jacobs, Leon, and Jones, M . F . 1939 . The Chemistry of the Membranesof the Pinworm Egg . Proceedings of the Helm inthological Societyof Washington 6 : 57-60 .21 . Wright, W . H . and Brady, F . J . 1938 . The Treatment of Oxyuriasis. Revista de Medicina y Parasitologia (Bacterio y Clinica yLabortorio, Habana) 4 : 151-53 .33

22 . Wright, W . H . and Brady, F . J . 1940 . Studies on oxyuriasis XXII .The Efficacy of Gentian Violet in the Treatment of Pinworm Infection .Journal of the American Medical Association 114 : 861-66 .23 . Hollaender, Alexandra ; Jones, Myrna F . ; and Jacobs, Leon 1940 .The Effects of Monochromatic Ultraviolet Radiation on Eggs of theNematode, Enterobius vermicularis . I . Quantitative response . Journal ofParasitology 26 : 421-32 ..24 . Jones, Myrna F . ; Hollaender, Alexandra; and Jacobs, Leon . 1940 .The Effects of Monochromatic Ultraviolet Radiation on Eggs of theNematode, Enterobius vermicularis II. Journal of Parasitology 26 :435-45 .25 . Lawton, A . H . ; Ness, A . T . ; Brady, F . J . ; and Cowie, D . R . 1945 .Distribution of Radioactive Arsenic Following Intraperitoneal Injectionof Sodium Arsenite Into Cotton Rats Infected with L . carinii . Science102 : 120-22.26 . Chinn, Ben D . ; Jacobs, Leon ; Reardon, Lucy V . ; and Rees, C . W .1942 . The Influence of the Bacterial Flora on the Cultivation of Endamoebahistolytica . American Journal of Tropical Medicine 22 :137-46 .27 . Jacobs, Leon . 1947 . The Elimination of Viable Bacteria fromCultures of Endamoeba histolytica and the Subsequent Maintenance ofSuch Cultures . American Journal of Hygiene 46 : 172-76 .28 . Phillips, B . P . 1950. The Cultivation of E. histolytica with T. cruzi.Science 111 : 8-9 .29 . Diamond, Louis S . 1961 . Axenic Cultivation of Entamoebahistolytica . Science 134 : 336-37 .30 . Diamond, Louis S . 1957 . The Establishment of VariousTrichomonads of Animals and Man in Axenic Cultures . Journal ofParasitology 43 : 488-490 .31 . Diamond, Louis S . 1968 . Techniques of Axenic Cultivation of Entamoebahistolytica Schaudinn 1902 and E . histolytica-like Amoebae .Journal of Parasitology 54 : 1047-56 .32 . Weinbach, E . C . and Diamond, L . S . 1971 . Entamoeba HistolyticaI . Aerobic Metabolism . Experimental Parasitology 35 : 232-43 .33 . Weinbach, E . C . ; Harlow, D . R . ; Takeuchi, T . ; Diamond, L . S . ;Claggett, C . E . ; and Kon, H . 1977 . Aerobic Metabolism of Entamoebahistolytica : Facts and Fallacies . Proceedings of the International Conferenceon Amebiasis, Mexico City, Oct . 27-29, 1975, 190-203 .34 . von Brand, Theodor . 1952 . Chemical Physiology of EndoparasiticAnimals . Academic Press, New York, 339 pp .35 . von Brand, Theodor . 1966 . Biochemistry of Parasites . AcademicPress, New York, 429 pp .36 . Coatney, G . Robert and Young, M . D . 1941 . The Taxonomy of theHuman Malaria Parasites with Notes on the Principal AmericanStrains . AAAS Publication No . 15 Washington, DC .37 . Young, M . D . and Coatney, G . Robert 1941 . The Morphology,Life Cycle and Physiology of Plasmodium malariae (Grassi and Feletti,1890) . AAAS Publication No . 15 Washington, DC .38 . Coatney, G . Robert ; Cooper, W . C . ; Eddy, N . B . ; and Greenberg,Joseph . 1953 . Survey of Antimalarial Agents . Chemotherapy ofPlasmodium gallinaceum Infections ; Toxicity, Correlation of Structureand Action . Public Health Service Publication No . 193 v . 68, no . 1 .39 . Wilcox, Aimee . 1942 . Manual for the Microscopial Diagnosis ofMalaria in Man . NIH Bulletin No. 180 U .S . Government Printing Office,Washington, D.C .40 . Coatney, G . Robert; Cooper, W .C . ; and Ruhe, D .S . 1948 . Studieson Human Malaria VI . The Organization of a Program for TestingPotential Antimalarial Drugs in Prisoner Volunteers . American JournalofHygiene 47 : 113-19 .41 . Eyles, Don E . ; Coatney, G . Robert ; and Getz, M .E . 1960 . VivaxtypeMalaria Parasites of Macques Transmitted to Man . Science 132 :1812-13 .42 . Young, M . D . and Moore, D . V . 1961 . Chloroquine Resistance inPlasmodium falciparum . American Journal of Tropical Medicine andHygiene 10 : 317-20 .43 . Tobie, John E . ; Coatney, G . Robert ; and Evans, Charles . B . 1961 .Fluorescent Antibody Staining of Human Malaria Parasites . ExperimentalParasitology 11 : 128-32 .44 . Tobie, John E . 1964 . Detection of Malaria Antibodies-Immunodiagnosis . American Journal of Tropical Medicine and Hygiene13 No . 1, Part 2 : 195-203 .45 . Eyles, Don E ., and Coleman, N . 1953 . Synergistic Effect ofSuefadiazine and Diaprim Against Experimental Toxoplasmosis in theMouse . Antibodies and Chemotheraphy 3 : 483-90 .46 . Newton, W . L . and Wright, W . H . 1956 . The Occurrence of a DogFilariid other than Dirofilaria immitis in the United States . Journal ofParasitology 42 : 246-58 .47 . Newton, W . L . and Wright, W . H . 1957 . A Re-evaluation of theCanine Filariasis Problem in the United States . Veterinary Medicine 52 :75-78 .48 . Dalmat, H . T. 1955 . The Blackflies (Diptera, Simuliidae) ofGuatemala and their Role as Vectors of Onchocerciasis . SmithsonianMiscellaneous Collection v . 125, no . 1, Washington, DC, 425 pp .49 . Rosen, Leon ; Chappel, R ; Laquer, G . L . ; Wallace, G . D . ; andWeinstein, P . 1962 . Eosinophilic Meningitis Caused by a MetstrongylidLung-worm of Rats . Journal of the American Medical Association 179 :620-24 .50 . Olivier, Louis . 1949 . Schistosome dermititis, a SensitizationPhenomenon . American Journal of Hygiene 49 : 290-302 .51 . Cheever, Allen W . 1968 . A Quantitative Post-mortem Study ofSchistosomiasis in Man . American Journal of Tropical Medicine andHygiene 17 : 38-64 .52 . Weinbach, Eugene C . 1957 . Biochemical Basis for the Toxicity ofPentachlorophenol . Proceedings of the National Academy Sciences,USA 43 : 393-97 .53 . Weinbach, Eugene C . and Garbus Joel . 1969 . Mechanism of Actionof Reagents that Uncouple Oxidative Phosphorylation . Nature 221 :1016-18 .54 . Weinstein, P . P . and Jones, M . F . 1956 . The In Vitro Cultivationof Nippostrongylus muris to the Adult Stage . Journal of Parasitology42 : 215-36 .55 . Weinstein, P . P . 1960 . Excretory Mechanisms and Excretory Productsof Nematodes : An Appraisal . In Stauber, L . A ., Ed ., Host Influenceon Parasite Physiology, pp . 65-92 .56 . Jacobs, Leon ; Remington, Jack S . ; and Melton, Marjorie L . 1960 .The Resistance of the Encysted Form of Toxoplasma gondii. Journal ofParasitology 46 : 11-21 .57 . Jacobs, Leon ; Remington, Jack S . ; and Melton, Marjorie L . 1960 .A Survey of Meat Samples from Swine, Cattle, and Sheep for thePresence of Encysted Toxoplasma . Journal of Parasitology 46 : 23-28 .58 . Jacobs, L . and Luna, M . N . 1957 . A Hemagglutination Test forToxoplasmosis . Journal of Parasitology 43 : 308-14 .59 . Jacobs, L . ; Cook, M . K . ; and Wilder, H . C . 1954 . Serologic Dataon Adults with Histopathologically Diagnosed Chorioretinitis . Transactionsof the American Academy of Ophthalmology and Otolaryngology58 : 193-200 .60 . Jacobs, L . ; Fair, J . R . ; and Bickerton, J . H . 1954 . Adult OcularToxoplasmosis . American Medical Association Archives ofOphthalmology 52 : 63-71 .61 . Sheffield, H . G . and Melton, M . L . 1970 . Toxoplasma gondii : TheOocyst, Sporozoite, and Infection of Cultured Cells . Science 167 :892-93 .34

The JournalOFTHEAmerican Medical AssociationA MEDICAL JOURNAL CONTAININGTHE OFFICIAL RECORD OF THE PROCEEDINGS OF THE ASSOCIATION, AND THE PAPERS READ ATTHE ANNUAL SESSION, INTHE SEVERAL SECTIONS, TOGETHER WITH THEMEDICAL LITERATURE OF THE PERIODEDITED FOR THE ASSOCIATION UNDER THE DIRECTION OF THE BOARD OF TRUSTEES BYGEORGE H. SIMMONS, M.D .VOLUME LVIJANUARY-JUNE, 1911AMERICAN MEDICAL ASSOCIATION, CHICAGO, 1911

y -ot.UME 1.\'lN, - VBE[t 9ABORTIVE POLIO111'E1'L171S-AIVDERSOY-FROST G ; ;3used by nose and throat men for their reflected light .The detection of albumin is most satisfactorily clone inconnection with the heat and acid method . after heatinoand adding acid to the specimen the test-tube containingthe fluid is held directly in front of the bull'seyelantern and the specimen vieyced from a point atright angles to the rays of light . In specimens in whichI and others have failed to `detect any cloudiness in allother lights, the cloudiness has stood out phlinly whensubmitted to the above procedure . The urine shouldalways be filtered, especially for the contact test . anybull's-eve lantern \gill suffice . The great ady-antage ofthis method lies in the fact that it can be clone at anytime-night or dad- .ABORTIVE CASES OF POLIOMYELITISAK EXPERIMENTAL DEMONSTRATION OF SPECIFIC III\IC\EBODIES IN THEIR BLOOD-SERC'\IJOHN F . ANDER',0N, M.D .Director Hygienic Laboratory, 1' . S . Public Health and Tlarine-Hospital ServiceAND\FADE A. FROST, M.D .Passed Assistant Surgeon, 1' . S Public Health and Marine-HospitalServiceWASHINGTON, 1) . C .It has been pretty generally recngnir_ed, since the publicationof Wicknwn's studies on epidemic acute anteriorpoliomyelitis in Sweden in 191) .1-0G .t that cases of thisdisease may run their course without resultant paralysis .The evidence on which the occurrence of Filch so-called"abortive" cases is predicated is, briefly, the following :Especially in epidemics, cases of acute anterior polionlyelitisare encountered showing all gradations in thedegree and extent of paralysis . In the same group maybe found cases resulting in extensive and lasting paralysis; cases with permanent paralysis of slight extent ;cases in which the patients have transient paralvsis,recovering completely within a few weeks or even a fewdays ; other cases in which there is no definite paralysis,but merely muscular weakness, of short duration ; stillothers in which the only motor disturbance is ataxia,tremor or a transient ocular disturbance such as diplopiaor nystagnlus . Finally, within the salve group are seencases of illness exhibiting only the symptoms of a generalinfection, usually accompanied by symptoms indicativeof meningeal, spinal or encephalitic irritation, butwithout definite motor disturbances .The diagnosis of acute anterior poliomyelitis in thesecases is suggested usually by their close association withtypical cases of the disease rattler than by the distinctiyenessof their symptoms . The symptoms are . howe\-er,sufficiently similar to the pre-paralytic symptomsobserved in paralytic cases, and in many instances suficientlydifferent from the sy- nlptorns observed in themore common infectious diseases to justify a provisionaldiagnosis of poliomyelitis . The similarity of theseabortive cases to eases of franc: poliomyelitis in the earlystage, the signs of involvement of the nervous system,their close and constant association with frank paralvtiecases, and the gradation of synlptonls above mentionedhad convinced many observers of the etiologic identit\'From the Hygienic Laboratory1 . Wickman, Ivar : Beitr:ige zur Kenutniss dot' I1~ " ine-)fedinschenICrankheit (Poliomyelitis acuta and verwandte Erkrankungenl .Berlin . 1907, S . Karger .of abortive and paralytic cases of poliomyelitis evenbefore in light had been throicn on the subject by thestudy of experimental poliomyelitis .It has recently been noted by several observers thatmoukevs inoculated with file virus of poliomyelitis occasionallypass through an indefinite illness without resultantparalysis, clinically similar to abortive attacks asobserved in man .It has been shown by- Leyaditi and Landsteiner .1Romer and Joseph' and Flexner and Ley\is' that theserum of monkeys which have recovered from polionly- cl-.itis . Mixed in suitable proportions with an emldsion ofthe virus and allowed to remain in contact for a suflicientlength of time, renders the virus inactive, so that \\- he nin - jcctcd into fresh monkeys it fails to produce thediscitsc .\(-tier and Levaditi .' and Flexner and Lewis,' havedemonstrated the same property in the serum of humanlivings vvho have recovered from acute poliomyelitisThis property has been found b}- all the above observersto be absent from the serum of normal persons andmonkeys, and has therefore been considered a specific."property . the demonstration of which in the serino justifiesthe inference that the subject has passed through theinfection of acute anterior poliomyelitis .\otter and Leyaditi,a availing themselves of this test,have also demonstrated this germicidal propert'- againstthe virus of poliomyelitis in the serum of a suspectedabortive case, thereby giving additional confirmation ofthe etiologic identity of such cases with frank poliomyelitis.The clinical history of the abortive case tested byfictter and Leyaditi is as follows (translation from theFrench)"('ASV: 3 .-Henriette G ., sister of Emile (see below), was witha nurse in L'Aveyron, returning in good health Dec 15, 1909 .Three tteeks after her return the child fell off . lost appetite,becnine cross . showed less disposition to run about . cotupfainedof pain behind the knees when the legs were touched, and oftht arms when they were pulled . These symptoms, whichattracted the attention of Dr . Ergbischoff, caused no uneasinessto the rather heedless parents, who refused to show us thechild on February 7 . The mother thought the child was sufferin', front mat du pays . Whatever the trouble, it all disappearedin the course of three weeks . The child regained her normalappearance and showed every appearance of health the latterpart of March ."A brother, Ensile, G years old ryas taken 111 either afew days before or a fey\ clays after this child (tile datesgiven are not clear as to the sequence of cases) with , Itypical attack of acute anterior poliomyelitis, resultingin lasting paralysis of a leg and an arm .The experiments which ive here report are similar tothe above experiment of -Netter and Levaditi . undertakenOn a more extensive scale, designed to throw somelight. on the diagnosis and epidelniologic significance ofa class of cases encountered by one of us,' in a field Ftildyof n( ute anterior poliomyelitis at Mason City, Iowa . andother points in that state during the summer of 1910 .' . Le,aditi . (' . . and I .sndsteiner, K . : Compt .-rend . Soc . d e biol .,10111 Ixviii . 311 a . INlmer. 1' . . and Joseph, K . : Uiinchen mod . Wchnschr, 1916,3 . I'lest)er . S ., and Le"- is, Paul A . : Experimental 1'oliotnveliIisIn \loukeys, 'rtIF. JotA . M . A ., \lay 1S. 191(1, p . 17\ONutter . A-, and Lovaditi . C . : Comp .-rend . Soc . d e biol ., 1'.110,Ixvili . GI 7 .6, \otter . A ., and Luvaditi, C . : Compt .-rend . Soc . bb d . . 7976,Ixviii, S ..5 .- . feast, \y . tl . : 'flu- Field Investigation of Epidemic Polio -mci'liti, 1 \\lis t the Health Officer ('an Iio Toward Solving a,National Problem) . 1'116 . Il(mith Pop- Nov . 18 . 1!ilo, 1) 1f;( ;3 ;1N-ussion of the Epidoniic of Anterior Poliomyelitis at a >h"etingof the Iowa State Board of Health, Iowa Med . jour ., Nov, 15 . 1910,I) . 1136 .

C64 ABORTIVE POLIO_1fYE,'TLITIS-ANDERSOA'-FROSTJ0L'a . A . Df . AMeacti 4, 1911In Mason City, Iowa, a town of about 14,000 inhaltitants,there occurred from April to September, 1910,forty cases of undoubted acute anterior polioniYelitis,resulting in typical flaccid atrophic paralysis . At thesame tinge there occurred a considerable number of casesof illness with symptoms suggestive of the early sylriptolnsof acute poliomyelitis, but not resulting in paralysis,and often of such mild character as not to comeunder the observation of any physician .REPORTS OF CASESBlood-specimens were obtained in the latter part ofNovember from a number of these cases, whose clinicalhistories are given in abstract helow . For the historiesin several of these cases we are indebted to various ph\sic'ians'of1Nfason City . Other patients were not seen byall 'N, physician during their illness and the only historiesavailable were such as could be given several weeks laterbY the patients and their families.CASE 1.-Control-Reported by Dr . C . 'if . Sxcale . Mn-niCity . C . T ., male . aged 48, lahoier, ryas taken sick .tune 24The spasticity of the paralyzed limbs, exaggeration ofreflexes and absence of atrophy in this case indicated alesion in the upper motor segment, either in the motorcortex of the brain or in the pyramidal tracts of thecord . The case was included in our series in order t , tascertain the diagnosis, since it represents a rare clinicaltype of poliomyelitis, the diagnosis of which has allyaybeen uncertain, and whose occurrence leas been a matterof some dispute .CASE 3 .-Reported be Dr. C . E . Dakin . Maso n City . 11 . C . .a iyoman aged 23 . nurse . was taken sick suddenly June 24, aftera long walk in the ]lot situ, with violent occipital Ileadaclic . %ertigoand stiffness of the neck, causing some retraction of thebead . She had chilly sensations . vomited . was extremely 1)rU ,-trated . and felt "crazy ." Temperature was normal, puts( , fill .The next day site had pains in the legs and back . with teudernessalong the spine . and was very restless . There iyns n,ifurther vomiting : flit, patient was constipated . The third dayshe complained of cramps and a sensation of numbness in theleft leg . and found . on attempting to get out of bed, that thisleg was weak . These symptoms gradually improved . s o that ina week the patient was able to be up viRll no sign of paralysis .Vie spine, however . remained tender for several days and sen-TABI,r: 1 .-I\OCITLATIO\S IS SERIFS 1T~pnkcpResult 2N0. Serum from Description of Case Ap,In lifonl:evs1G (`ass 1 (control) . . . . Frank poliomyelitisAdult . Has remained well .17 Case 2 . . . . . . . . . . . . Suspected poliomyelitis with Adult . Fins rcmninod well .spastic paralysis .is Case 3 . . . . . . suspected poliomyelitis withoutparalysis (ahortiv, " ) .Adult . Hits remained well .19 Clse 4 . . . Suspected polionivolitis vvitlioutparalysis (abortivoi .Adult . flax remained well .20 Case . . . . . . . . . . Suspected poliomyelitis xvithoutparalysis (alutrtin " 1 .Adult . Has remained well .21 Case fi Suspected poliotnpelitis withoutparalysis labnrtiv, ) .Adult . ll :is remained well .2-, Car 7 Suslncted Iioliom-liti, iv'ithoutparalysis (ahortive) .I c, Paralysis all extremities on sixth day .211 Case N ; . . . . . . . . . . Suspected poliomyelitis withoutparalvsis (abortive) .Child . Paralysis all extremities . trunk and neck .__ ('ass 9 . . . . . . . . . . suspected poliomyelitis withoutpilralysis (abortive) .Adult . Ilas remained icell .2 :t Ca- 111 . . . . . . . . - . . Susp (. led poliomyelitis withoutparalysis (abortive) .Adult . 11ns remained24 Ca', 11 ; . . .- . .- . . Suspected poliomyelitis with-Paralysis all extremities on eighth d»p .27 (")title] .11t . . . . . . . Not Dial_ . . . . . . . . . . . . . . . .27A Control JII . . . . . . . . Normal . . . . . . . . . . . . . . . . . . . . .Adult .Adult .I'araly,fs of hind legs t,nlh day . ell extremities twelfthday : chloroformed .Paralvsis of all extremities tenth dax' .Each enonkev inoculated intracorebrallc Nov. 30, 1910 . with 0 .5 c .c. of :i mixture of ;, per cent, emulsion of fresh spinal cord(of Monkey 15) o. ., cx . : -runt to lie t-o " d . C.c . : fresl, n,n'nial serum. 11.1 Mixtures allowed to tand one hour at 37 C. andtwenty - hour, at 15 C . before injection . :~: It leis to salon " serum in Series - .vvitli a chill, fever . pains in the legs and hack . stiffness of neck .some delirium . On the third day there was paralysis of I,otllthighs and legs and both upper arms . and partial paralysis )fthe forearms . When last seen . Nov . 24 . 1910. th e lower lirubaand upper amts were still paralyzed, flaccid and atrophic . lintshowing some improvement . Tllis is a typical case of acuteanterior poliomyelitis . included in our series as .i control .CASE 2-Reported by Dr . R . F . R eston . Alacon ('ity . Mrs .W . . 22, vyaitie, \vas taken sick the latter part of June . 1!)111,with fever and indefinite general 'Nuptonl, . _After severaldays site beennie paralyzed in both lower linihs . Site \%a,admitted to a hospital about one week after onset . -he w,l,said to have load . a t that tinie . a flaccid motor paralysis of bothloxc(" r extremities ichieh . IIOweyer . becxtne sl)iistiC within e fexydays . When the patient ica .s seen the latter part of Inlc .1)10 . both legs and thighs were quit(, sl)a,tic . \o activemotion wits po,silde except of tlw toes and slight flexion (If theleft knee . Pa .ice motion veto, limited to parli :ol fle-ioti of thethighs and slight flexion of left knee-liltnost none (of tile li p lltknee . The patellar reflexes greatly exaggerated on both ,ode, :ankle-clones on right side : sensation for toitcb and pain xca,normal . Examination xcas othervii,e negative . The paticut"general health \\a, good .Nov . 20 . 1910 . Hit, left leg could tie waned . lint rather axx - k-wardly . Tlie riglit leg showed little . if any, improvement .Patellar reflexes were still exaggerated . more so oil tile rightside . There was no ankle -clones and no atrophy' .sitive to jarring . A week later she had a slight recurrence ofsimilar symptoms . with a pharyngitis .There was no elevation of the temperature at any time inthis case . which was seen daily by the attending physiciatt-Tlle patient knew of no direct association vvitli case, of poliomieliti,,within a month prior to her illness . She was, however. a t the time of ]let illness living at the )louse of a plix- sicianvvho had recently attended patients A\-ho were sufferingfront this discilse .('As ; 4,-Dr . C . E . Dakin, of Alason ('ity, gives the followingncconnt of his on - n illness . the nature of which was liardlvsuspected at the time . lie ivas taken sick about July l . 1910,the onset being gradual . Ile lost appetite, xcas constipated .lolcl a coated tongue and foul breath . Ile suffered from a pl " r-sistent occipital licadaclle . not relieved by treatment, and frontsevere pains in the upper dorsal and lumbar region . The neckxc ;is stiff, so that it vvas painful to bend the head forwardalthough it could lie rotated w- itllont pain . No retraction ofthe ]lead ix- as noted . but it xxas found that the mo,t conifortaldeposture" was lying on the batik xvith the head extended over ;opillow placed under the shoulders . lie felt extremely nervou, .irritable and inclined to worry over trifles . There ivas no ( " b" -vation of temperature . Tliese symptoms, xi- hich reached theirheight about July 7, gradually subsided iiithin the next iyeel: .(kept the general nervousness and irritabilitc . which per_sited a good deal longer . During this time Dr . Dakin xiasnot conliued to bell, and continued logo practice .

COLT" N E LV INLxtaea 9ABORTIVEA child of his family- had an attack of acute poliomyelitiswith slight peroneal paralysis in May . Also about June 23Dr . Dakin attended a child suffering from what was clearlyan abortive attack of acute poliomyelitis .CASE: 5.=The patient, Mr . S ., aged 30, merchant, in DlasonCity, was not seen be a physician . The history- was obtainedsome two months later from the patient and his family . Hewas taken sick rather suddenly about June 19, with headache,pain in neck, back and legs . The temperature was not determined. The patient felt ill for a week or more, but was notconfined to bets ; thought lie had the influenza . For more thana week after recovery one leg remained weak and "rheumatic ."No previous association with any case of poliomyelitis wasknown . About three weeks later a 3-year-old son (the onlychild trf the family) developed a typical case of poliomyelitis,resulting in paraly,i ; of the left thigh and leg .CASE 6 .-Reported by Dr . C . E . Dakin . :tlason City . who firstsaw the patient October 25- E . D . . 45, farmer, residing about 7miles nortliwest of \lason City, about Oct . 1t3, 1910,began to feel fleeting, neuritic pains in various parts of hisbody. There was no special pain . tenderness or stiffness of neckor back . A chronic indigestion became much aggravated, causinggastric distress, especially after eating . The patient lostappetite, and was constipated ; the tongue was coated andbreath foul . He was unusually nervous, irritable and sleep-POL103IYETLITI"-ANDEPSON-FROST.TABLE 2.-INOCULATIONS IN SERIESMonkeyNo Serum from Description of Case Age30 Control JFA I . . . . . Normal . . . . . . . . . . . . . . . . . . . .31 Control ('111, . . . . . . Normal . . . . . . . . . . . . . . . . . . . .32 Control WH1"' . . . . . . Normal . . . . . r . . . . ., . . . . . . . . .33 Control w' t , . . . . . . Normal . . . . . . . . . . . . . . . . . . .34 Control It I . . . . . . . Normal . . . . . . . . . . . . . . . . . . . .35 Case 11 Q . . . . . . . . . Suspected abortive case . . . . .3G ('a

666 ABORTIVE POLIOJIFETI,ITIS-ANDERSON-FRO,~TF ., had a severe occipital headache, pain andspine,tendernessand someof theinflammation of the tonsils . Thethe temperaturenc\twasdaynormal and tit headachetendernessand pa ,of tlie; ,dspine had disappe,ired .Two other children in the same housebetween Juoe 29'taken -ickand July_l ': : " ,, o srmilar ' s}mptom ; . thinrecoveredt, tvithia a- :f~ :days, while the other developed , pa~al'si, of the arms .Cases 3 to 11 inclusive are representative of a class ofcases which were reported to be quite common in nltdaround Mason Cit .- during the epidemic of poliomyelitis,While there is a considerable variety in their synlptoinatclogythere is, in most cases, a fair]), distinct syndrome,yiz ., headache, more commonly occipital ; pain and tendernessalong the spine ; neuritic pains and It yperesthhsiaof the limbs ; a marked degree of restlessness or irritability; prostration out of proportion to the severity of tileother symptoms ; some gastro-intestinal derangement(anorexia, nausea, constipation or diarrhea) ; occasionallyother symptoms of nervous derangement, as vertigo,photophobia, delirium, muscular paresis, disorderedreflexes . The temperature was definitely determined tobe normal in Cases 3, 4 and 10, and found elevated(102 .5° F .) in Case 11 . In the rest of the cases noexact information is available, as the patients were notunder the observation of a ph`-sician during the acutestage . Dr . Dakin,e of Dlason City, who has reportc(Ielsewhere 36 cases of poliomyelitis, including Caseand 10 of tile above series, describes . a s very characteristic,a staring, terror-stricken facial expression, whichhe noted also in Cases 3, 6 and 10 of our series .Blood-specimen; were obtained from all these cases 1)yvenepuncture during the latter part of November . Theserum was withdrawn after coagulation and kept inclosely stoppered sterile bottles . Repeated tests showedall specimens of serum to be sterile when used .The virus used was originally obtained from theRockefeller Institute for Dledical Research throueli thecourtesy of the director . Dr . Simon Flexner . It leadbeen propagated through a long series of monkeys at theRockefeller Institute and found very uniformly virtdent .Al'e have found it virulent for alltilefresh monkeyswhich we have inoculated other than those mentioned inthe following series .SERIES l.- ;donkey 15, inoculated with file above virus .developed complete paralysis of the hind legs on the s "venlhday ; it was chloroformed and necropsy performed the samedap . A 5 per cent . emulsion of the fresh spinal cord was preparedin no-mal salt solution, and filtered through paper toremove gross particles . One-half cubic centimeter of thisemulsion was added to an equal amount (0 .5 c .c .) of each ofthe serums to be tested .To each mixture was added 0.1 c .c . of fresh normal humanserum (J . R .) . As controls, two mixtures were made withnormal human serum (J . R .) . The mixtures of serum andvirus were kept 1 hour at 37 C., then placed over night (about20 hours) at 15 C . A series of monkeys was inoculated inthe brain, each monkey receiving 0.5 c .c . of one of the mixturesof virus and serum, Nov . 30, 1910 .The two control monkeys receiving the mixtures of normalserum and virus developed typical paralysis on the eighth andtenth days respectively .The monkeys receiving the mixtures of virus with the serumof Patients 7, 8 and 11 also developed equally typical paralysison the seventh, fifth and seventh days respective] '\ . All tileothers have remained well up to tile present time (Fell . 3,1911) .It will be noted that the three specimens of serumwhich failed to affect the virus were from young persons(Patient 7, aged 16 ; Patient 8, aged 10 ; Patient 11,8 . Dakin, C. E. : Iowa Med . Jour., Nov. 15 . 1910, p. 221) .J,t,t A . M . :\ .3lntteo 4, 1;111o2cd 7) . Tlie remainder of the serums were from adults .Tiiis suggested two possiljilitics :1 . The sertnn of normal adults may have considerablegermicidal properties for the Virus .2 . The serum of children, even when diet have lead anattack of polionlvelitis, may not develop germicidal antilxltlies; or inay develop them in smaller amounts tli ;inthe serum of adults .Sraii:s 2.-To test these hvpotbeses, we inoculated anotlu" rseries of morikev= with exactl} the same teclmic, ex c(" Irt dint,instead of a ~i per cent . emulsion of spinal cord we u,e1 aJ per ccut . emulsion (fresh cord of 'llonkev 29, inoculatedDecember 13 ; paralysis of hind legs December 23 ; chloroformedand cord removed December 2 :3) .In this series we used again the serum of Patients 7 . 8 and11, and for controls tool: tile serum of three normal adults(J . F . A . . C . 11 . L ., and W . 11 . F .) and of two normal children(R . and 1y .) . Granting an equal concentration of the virus inthe two cords used in series 1 and 2 (\lonkev 15 and Dlonkev29), each specimen of serum vs- ould now be mixed Nvitb thesame aolame of emulsion as in Series 1, but vvitli one-fifth offire amount of t-iru .e . Also . the amount used in inoculation(0 .5 c .c .) would represent only one fifth of the aruount ofvirus inoculated into the animals of series 1 .Tire results in this series were somewhat irregular . Tilemonkeys receiving the serum of two of tile normal adults (C .11 . L . and W . 1-I . F .) developed poliomyelitis on the fifth andthirteenth days re.specticelc . Tile monkey which received theserum of Patient 7 developed paralysis of all extremities ontile eighth day . None of the other monkeys . including thosewhich received the serum of Patients 8 and 11, the serum ofone normal adult (J . F . A .), and of the two normal children,have developed paralysis up to this time . Two of the monkeys .however (those receiving the serum of the two normal children),appeared about a week after inoculation to have -anindefinite illness . manifested by slight rise, of temperature . lossof appetite, nervousness . and undue excitability . Whether thesemay be considered abortive attacks of polionlvelitis is extreruelyquestionable .These results would indicate that in this series we hadapproached the limit of proportions in which norn)alhuman serum may exert a neutralizing effect on tilevirus of polioln .velitis, or else had approached theminimum effective amount of virus for inoculation .They indicate pretty- clearly that the serum of Patient 7had no unusual germicidal properties, but leave the othertwo cases (8 and 11) in doubtful status .SE1AEs 3.-Three specimens of normal serum having beenfound capable of neutralizing the virus in the proportion usedin Series 2, that is, against equal volumes of a 1 per cent .emulsion of virus, it remained to test these three specimen,again under the conditions of Series 1 . Accordingly we inoculatedanother series of monkeys, using a a per eeiit . emulsionof virus (fresh cord of Tlonkey 9, inoculated Jan . 7, 1911 ;died fourth day ; gross and bistologic lesions typical) .The serums used were the same specimens which had slmwnneutralizing properties in Series 2, viz ., from one normal adult(J . F . A .) and two normal children (R . and \C .) . The technicand proportions were exactly as in Series 1 .All three monkeys came down with typical paralysis betweenthe eighth and the twelfth days .The three series of experiments are presented in theaccompanying tables .SuM\IARY.The results of these experiments may be summarizedas followsNormal Human Servin .-The results in Series 2indicate that normal human serum inay have a germicidalaction on the virus of polionnelitis If this isso, however, the action has quantitative limits whichclearly differentiate it from the action exercised by tlwserum of persons who have had poliomyelitis as shown iii

IULI'\I F: LVIN~'usea 9Series 1 and 3 . 'N'o appreciable difference has beendemonstrated between the normal serum of adults ando1' children in regard to their action on the virus .,Scrunt of Patients Who Have Recorered-As shownby the workers above cited and by our results, the serumof persons who have recently recovered from frankattacks of poliomyelitis exhibits a germicidal action onflap virus considerable greater than that exhibited bynormal serum . Serum from a person suffering fromparalysis of spastic the showed the same properties .thus confirming the clinical evidence that acute polioiuyclitismay cause paralysis of this type.The serum of six out of nine patients (66.7 per cent .)ivho had recently recovered from suspected poliomyelitiswithout paralysis (abortive cases) showed the samegermicidal action as the serum from a frank case ofpoliomyelitis .In the serum from the other three suspected abortivecases of polionivelitis we were unable to demonstrateany germicidal property beyond that shown by normalserum . These three specimens of serum were all obtainedfrom 'young persons . These three eases clinically reserubledpolionivelitis more than did some of the adult cases ;and the symptoms were, on the ivliole, equally severe .The following possibilities suggest themselves :1 . The cases niay not have been polionivelitis .2 . They may have been poliomyelitis, but, if so, antibodieswere either formed in less amount, or disappearedmore rapidli than in the adult cases .The experimental evidence on which the specificity,constancy and quantitative relations of this reactionmust be estimated is scant . So far as it goes, however .i t justifies the inference that the reaction as demonstratedin our experiments is specific .We feel justified in concluding that the diagnosis ofacute polionivelitis has been established in six of ournine suspected abortive cases . The diagnosis in the otherthree is not cleared up by the experiments . The clinicaland epidemiologic evidence of poliomyelitis in thesecases must he weighed against the absence of a serumreactionof unknown constancy .The facts here presented have a significant bearingon the diagnosis and epidemiology of acute anteriorpoliomyelitis .DIAGNOSISThe generally accepted criterion for diagnosis ofpoliomyelitis has been the development of paralysis orat least definite weakness . It has been urged that theattempt to make a diagnosis on any clinical evidenceless than this would result in hopeless confusion ; andthis is partly true in the present state of our knowledgeof the disease . In the cases of our series, however,poliomyelitis had been suspected on clinical evidencealone and confirmed by biologic test in 66 .7 per cent ., afact which would indicate that the diagnosis of abortivecases of this class is not wholly a iuatter of guess-work .provided special attention has been directed to polioniyelitis,as was the case in -Mason Citv . \o laboratorydiagnostic methods of denionstratcd reliability and universalapplication have been evolved . The earl- examinationof cerebrospinal fluid is the most reliable laboratorymethod at present known . Lumbar puncture for diagnosisis hardly justifiable, however, unless some considerationof the safety, either of tlie patent or of thecommunity, makes an accurate diagnosis of specialimportance . Total and differential leukocyte countsoffer a promising aid to diagnosis, but much more workwill be necessary to establish the reliability of thisTHERA PEUTICS 66method . But even in the absence of any specific diagnosticmethods, much light can doubtless be thrown onthe subject by a careful and conservative study of allcases which may reasonably be suspected of being casesof poliomyelitis, paving especial attention to examinationof the nervous system and to association with casesof frank polionivelitiss .Until the clinical recognition of poliomyelitis is p1,,(-('don a more reliable basis there will remain an indeterminahlefactor in its epidemiology, a factor wliose significancecan only be surmised at present . Conclusions fromepidemiologic data will have to be correspondinglyconservative .A more detailed discussion of abortive cases, asobserved in Iowa, and of their epidemiologic relationswill be repoited in a forthcoming bulletin of theHygienic Laboratory .TherapeuticsPROPHYLAXIS OF SCARLET FEVEREvery physician, no matter how limited or specializedhe may attempt to make his practice, is likely toencounter a case of scarlet fever, and to have his opinionasked in regard to what should be done to prevent thetransmission of the disease to others . Even if he doesnot personally encounter the disease, he cannot avoidbeing asked the same question, apropos of some case hehas not seen, by an anxious mother or a father seekingfor positive information from one whom they believe tohave the knowledge of an expert . It reflects no crediton his scientific attainments, it does not increase theconfidence in him of his patients, if he cannot statepromptly, clearly, concisely and positively what shouldbe done by the family in which the disease occurs to preventits spread to other individuals, what the familywhich does not have it, but is afraid of getting it, shouldthemselves do . as well as what they have a right to expectothers to do, in order to prevent the spread of the disease.To the striking development of knowledge of sanitaryscience and preventive medicine which has taken placeduring the last three or four decades we are indebtedfor the wider recognition of the fact that scarlatina,scarlet rash and scarlet fever are synonymous terms,that they do not indicate different diseases, but one andthe same disease . Formerly, many of the laity and nota few physicians believed, or at least said, that s^arlatinaand scarlet rash were different from scarlet fever .But numerous careful observations have shown, and theprofession generally has learned, that while scarlet fevermay be, and often is, a very serious disease with hightemperature, severe sore throat, intense and widelyspread eruption . followed by copious desquamation, thefever may be slight or entirely absent, the throat maynot show more than slight congestion, the eruption . ifnot entirely absent . may be not very pronounced inappearance' not widely spread over the hodv and ofrather transient duration, while the desquamation niaybe so slight as to be hardly recognizable .Furthermore, it is now generally recognized not onlythat the very mild cases may be followed by the mostserious sequels , which are ,observed after the severeforms of the disease . and particularly by inflammationof the kidneys, but also that severe forms of scarlet40

Noteworthy Papers on Viral DiseasesDorland J .Davisin the last decade of the 19th century when manybacteria were found to cause disease in many animalsand plants, it was discovered that some biologicalmaterials were still infectious after being passed througha porcelain filter that removed all bacteria . These infectiousagents came to be known as "filterable viruses"and included those of smallpox, vaccinia, yellow fever,foot and mouth disease of cattle, and other animal andplant infections . They were also invisible by the usualstains in a light microscope . Among these infections waspoliomyelitis then commonly called infantile paralysis,which was beginning to occur in epidemic form inEurope and the United States . Physicians at the HygienicLaboratory of the U .S . Public Health Service were calledupon for assistance in understanding this disease and inthe attempts to control it . Thus poliomyelitis was thefirst viral disease investigated by medical scientists at theHygienic Laboratory .Studies by Anderson and Frost demonstrated "specificimmune bodies" in blood serum of non-paralytic cases in1911, one of the earliest attempts to understand immunityin this infection . This paper is noteworthy because itdemonstrates for the first time laboratory evidence ofpolio infection in persons with non-paralytic disease(abortive cases) .' Written by two very influential medicalscientists at the Hygienic Laboratory, it linked experimentalwork with clinical and epidemiological observations. A few years later Frost described in detail theclinical course and epidemiology of outbreaks occurringin Iowa and Cincinnati, and with Leake an outbreak inBuffalo and Batavia, NY .z Leake reported a winterepidemic in West Virginia . 3Although the infectious agent of poliomyelitis could bepropagated in monkeys, investigation was hindered bythe lack of a convenient and cheap experimental animaluntil Armstrong in 1939 1,5 adapted the Lansing strain tocotton rats and then to laboratory mice . This opened theway to quantitative measurement of virus and neutralizingantibody on a large scale . Progress was rapidthereafter as two other strains were also adapted to mice .Eventually the polio virus was cultivated in tissue culture,leading to the development of effective vaccines .Encephalitis outbreaks appeared in the early 1930s andNational Institute of Health scientists were called to investigate. During the course of the 1933 epidemic in St .Louis, Armstrong and Lillieb recovered a virus that produceda lymphocytic choriomeningitis in monkeys butwas shown not be the cause of the epidemic . This virus,termed LCM (lymphocytic choriomeningitis), was foundDortand J. Davisto cause disease primarily in rodents (house mice) and inhumans exposed to infected mice (La Maladie de Armstrong). Later Bengtston and Wooley 7 cultivated the virusin embryonated eggs . This virus is now classified withthe arenavirus family . Members of this family areresponsible for some of the most severe infections inman, such as Bolivian hemorrhagic fever .During World War 11, most NIH investigations wereoriented to war-time problems . By then better techniquesfor laboratory work had been developed such as the useof embryonated chicken eggs and improved serologicalmethods . Mumps was always an important disease inmilitary recruits . Habel 8 succeeded in cultivating the virusin embryonated eggs and devised serological tests for itspresence . Later vaccines were developed and are nowwidely and effectively used .Measles (rubeola) was also an important war-timedisease . In 1911 Anderson and Goldberger 9 at theHygienic Laboratory had transmitted the infection withgreat difficulty to monkeys by contact . Progress and avaccine, however, had to await the development ofspecific tissue culture techniques in the 1950s . Rubellawas investigated by Habel'° who succeeded in passaging41

an isolate through chick embryos and then to monkeys .In the 1960s, an effective vaccine was prepared byseveral laboratories that reduced the threat of infantmalformation due to this infection .Following World War II, interest turned to respiratorydiseases such as influenza and the common cold . Agroup of agents termed the Coxsackie viruses was intensivelyinvestigated . Heubner and his associates" in 1951showed that herpangina, an illness characterized by fever,sore throat, and anorexia that mainly affected children,was caused by several types of Group A Coxsackie virus,which could be isolated in suckling mice . They also implicatedCoxsackie B with cases of pleurodynia inTexas . 12 Later Coxsackie A-21 was found to be an importantcause of acute respiratory disease in militarypopulations which led to development of a vaccine thatreduced the burden of this infection in training camps . 13NIAID scientists continued to investigate respiratoryinfections, and Rowe and others were the first to isolatean adenovirus from patients . 14 Virus was found inadenoid tissue that was observed to degenerate in successivetissue culture passages . A large number of typeshave now been recovered from tonsils and adenoids .Several types, 4, 7, and 14 are frequently associated withrespiratory diseases 15 and type 8 with keratoconjunctivitis. Rowe, Heubner, Bell, and their associates 16clarified the role of the adenoviruses in respiratorydisease and described the epidemiology and clinicalcharacteristics of these infections .Further studies of these viruses by Heubner 17,18 andothers demonstrated virus-free adenovirus antigens inanimal tumors that led to extensive investigations on oncogenicity. Rowe 19 secured evidence for a genetic hybridbetween adenoviruses 7 and SV 40, a papovavirus ofmonkeys . This work was expanded to recombinants ofadenovirus hybrids . Heubner and associates20 found oncogeniceffects in hamsters when injected with humanadenovirus types 12 and 18 .The influenza epidemics of 1957 and 1968 stimulatedintense investigation of the antigenic characteristics of influenzatype A virus with the goal of improved vaccines .Murphy and associates2l developed temperature sensitivemutant influenza strains that may eventually lead to aneffective and safe live virus vaccine .Chanock and his group also investigated the cause ofinfantile croup and other respiratory childhood infections. They first established the etiologic role ofcytopathic myxoviruses, parainfluenza type 1 and type 3,in these conditions . 22,23 NIAID scientists24 also studiedclinical conditions caused by the rhinoviruses, the causeof the common cold and with others showed that thegroup was composed of over 100 different serologicaltypes .Hepatitis had been differentiated into infectious (A)and serum (B) soon after the extensive experience ofWorld War II . Hepatitis B, the most lethal, commandedprincipal attention particularly after the identification ofthe Australia antigen as a component of the infectiousagent . Gerin, Holland, and Purcell25 contributed importantlyto its purification from human serum and tobiochemical studies of its proteins .Dr. Davis, standing on right, in 1937 at Johns Hopkins School of PublicHealth.Hepatitis A, a frequent cause of epidemics, particularlyin tropical areas, is widely prevalent but less lethalthan B . Feinstone, Kapikian, and Purcell, 26 using immuneelectron microscopy were able to visualize HAVviral particles clumped together with immune serum inthe stools of hepatitis patients and to measure HAV antibodies. This provided an excellent means of diagnosisand a basis for epidemiological and natural historystudies of human infection .Usually following transfusions, some cases of clinicalhepatitis occurred that could not be diagnosed as eitherA or B . These infections were called hepatitis non-Anon-B and were found by Feinstone and associates to betransmissible . 27 They still form a major part of posttransfusionhepatitis as well as sporadic hepatitis .In the early 1960s investigation of arbovirus infectionsbegan, based at a-laboratory in the Panama Canal Zoneknown as the Middle American Research Unit or MARUsponsored by NIAID and the Walter Reed Army Instituteof Research . One important result was the studyof Bolivian hemorrhagic fever, a devastating disease insome rural areas of Bolivia and other South Americancountries . Johnson and his co-workers 28,29 recovered avirus from patients and from wild rodents living inhuman habitations . They called it Machupo and laterstudied similar agents from Africa . The group, includingthe LCM virus, is now known as the arenaviruses .Continued study of respiratory viruses in childrenyielded infectious agents related to animal viruses .McIntosh 3 ° ,31 and his associates studied the antigenicrelationships and pathogenicity of these coronaviruses, asthey came to be called, and found they were a significantcause of childhood respiratory illness .42

The most important cause of lower respiratory tractdisease in infants and children is the respiratory synctialvirus (RSV), first recovered in 1957 by Chanock andassociates . 12,13 RSV is now considered to cause thousandsof infant deaths in this country alone . Attempts todevelop a vaccine through intensive research thus farhave not been successful .Kapikian extended the technique of immune electronmicroscopy to other agents and detected and identifiedan agent, the Norwalk virus, associated with acute nonbacterialgastroenteritis . These observations have led togreat advances in knowledge of the common butheretofore poorly understood intestinal infections,especially in children . The Norwalk virus is now knownto be responsible for one-third of all epidemics of nonbacterialgastroenteritis . 34At the Rocky Mountain Laboratory in Hamilton,Montana, where excellent animal facilities were available,Hadlow35 studied the "slow virus" of scrapie, a sheepdisease . He compared the condition to Kuru, a fataldisease in New Guinea natives that has been carefullystudied by Gajdusek in recent years . Eklund, Kennedy,and Hadlow 36 later described the pathogenesis and viralreplication of scrapie virus in the mouse . The "slowviruses" are now recognized as a distinct form ofmicrobial pathogens that may be the cause of somechronic diseases .The role of viruses in production of tumors has beenof constant interest, beginning with studies of thepolyoma virus of mice by Eddy and Stewart . 31 Habel"discovered a virus specific transplantation antigen oncells transformed by polyoma virus . Rowe, andassociates 39,4° ,41 studied extensively animal leukemia andsarcoma viruses in tumors and the immunologicalreactions .Underlying all the tissue culture work with viruseswere the very careful studies by Eagle42 of the specificamino acid requirements of a mammalian cell in tissueculture . It made possible the rapid development of linesof various cell types for cultivation of a wide variety ofviruses . Eagle's defined media for tissue culture cells hasbeen a major contribution to the study of viral diseases .Detailed studies on the molecular structure of virusesand the processes of replication have laid a foundationfor understanding and manipulating them . Moss andSalzman43 analyzed the protein synthesis of vaccinia virusand the process of virus assembly . Sebring44 andassociates provided one of the first demonstrations of thestructure of replicating DNA molecules . Shatkin45 andhis associates gave the first demonstration of a transcriptaseactivity in an RNA virus . Later Weir and Moss 46began further investigation of molecular structure of vacciniavirus leading to important practical results in constructionof a variety of immunizing agents on amolecular basis .An ever expanding array of techniques, instrumentationand concepts has lead to the remarkable unfoldingof our knowledge of viral diseases in the last 80 to 90years and to effective methods of control of many ofthem . As demonstrated by the work cited here, scientistsof the early Hygienic Laboratory and those in the currentNIAID intramural program have played key roles inthe world-wide explosion of medical science .Dorland J. Davis, M.D., D.P. H .Born in Chicago, Illinois in 1911, Dr . Davis received aB .S . degree from the University of Illinois in 1933, andan M .D . from Johns Hopkins University in 1937 . Twoyears later, shortly before receiving a D.P .H . from JohnsHopkins University, he joined the Public Health Serviceas a researcher with NIH's Division of InfectiousDiseases . His entire career was spent atNIH/NMI/NIAID, except for duty in North Africa forthe Department of State from 1943 to 1944 as a memberof a medical team investigating endemic diseases particularlymalaria and typhus . In 1954 he was appointedChief of the Laboratory of Infectious Diseases . Hisresearch interests in the 1940s were poliomyelitis,American trypanosomiasis (Chagas' Disease), viralhepatitis, and psittacosis . In the late 1940s and early1950s, he studied acute epidemic conjunctivitis inchildren, elucidating its cause, epidemiology and treatment. Dr . Davis next turned to studies on influenza . Inthe late 1950s he travelled to Central and SouthAmerica, Alaska, and McMurdo Sound areas of Antarcticaevaluating research opportunities and disease problems. In 1956 he was appointed NIAID's first AssociateDirector in Charge of Research, a position later calledDirector of Intramural Research . From 1964 until 1975he served as Director of NIAID, retiring in 1975 after a36 year career .43

References1 . Anderson, John F ., and Frost, Wade H . 1911 . Abortive Cases ofPoliomyelitis : An Experimental Demonstration of Specific ImmuneBodies in Their Blood-Serum . Journal of the American MedicalAssociation 56 : 663-67 .2 . Frost, Wade H . 1913 . Epidemiologic Studies of Acute AnteriorPoliomyelitis . Hygenic Laboratory Bulletin No . 90 Washington, D .C . :U .S . Government Printing Office, 252 pp .3 . Leake, J . P . ; Bolten, Joseph ; and Smith, H . F . 1917 . Winter Outbreakof Poliomyelitis, Elkins, West Virginia, 1916-17 . Public HealthReports 32 : 1995-2015 .4 . Armstrong, Charles . 1939 . The Experimental Transmission ofPoliomyelitis to the Eastern Cotton Rat, Sigmodon hispidus, hispidus.Public Health Reports 54 : 1719-21 .5 . Armstrong, Charles . 1939 . Successful Transfer of Lansing Strain ofPoliomyelitis Virus From Cotton Rat to White Mouse . Public HealthReports 54 : 2302-05 .6 . Armstrong, C . and Lillie, R . D . 1934 . Experimental LymphocyticChoriomeningitis of Monkeys and Mice Produced by a Virus Encounteredin Studies of the 1933 St . Louis Encephalitis Epidemic .Public Health Reports 49 : 1019-27 .7 . Bengtson, I .A ., and Wooley, J . G . 1936 . Cultivation of the Virus ofLymphocytic Choriomeningitis in the Developing Chick Embryo . PublicHealth Reports 51 : 29-41 .8 . Habel, Karl . 1945 . Cultivation of Mumps Virus in the DevelopingChick Embryo and Its Application to Studies of Immunity to Mumpsin Man . Public Health Reports 60 : 201-12 .9 . Anderson, John F ., and Goldberger, Joseph . 1911 . ExperimentalMeasles in the Monkey : A Preliminary Note . Public Health Reports 26 :847-48 .10 . Habel, Karl . 1942 . Transmission of Rubella to Macacus MulattaMonkeys . Public Health Reports 57 : 1126-39 .11 . Huebner, Robert J . ; Cole, Roger M . ; Beeman, Edward A . ; Bell,Joseph A . ; and Peers, James H . 1951 . Herpangina . Etiological Studiesof a Specific Infectious Disease . Journal of the American MedicalAssociation 145 : 628-33 .12 . Huebner, Robert J . ; Risser, Joe A . ; Bell, Joseph, A . ; Beeman, EdwardA . ; Beigelman, Paul M . ; and Strong, James C . 1953 . EpidemicPleurodynia in Texas . A Study of 22 Cases . New England Journal ofMedicine 248 : 267-74 .13 . Bloom, H . H . ; Johnson, Karl M . ; Mufson, M . A . ; and Chanock,Robert M . 1962 . Acute Respiratory Disease Associated with CoxsackiA-21 Virus Infection . II . Incidence in Military Personnel : Observationsin a Non-Recruit Population . Journal of the American Medical Association179 : 120-25 .14 . Rowe, Wallace P . ; Huebner, Robert J . ; Gilmore, Loretta K . ; Parrott,Robert H . ; and Ward, Thomas G . 1953 . Isolation of aCytopathogenic Agent from Human Adenoids Undergoing SpontaneousDegeneration in Tissue Culture . Proceedings of the Society for ExperimentalBiology and Medicine 84 : 570-73 .15 . Bell, J . A . ; Rowe, Wallace P . ; Engler, Joseph I . ; Parrott, RobertH . ; Huebner, Robert . 1955 . Pharyngoconjunctival Fever .Epidemiological Studies of a Recently Recognized Disease Entity . Journalof the American Medical Association 157 : 1083-92 .16 . Huebner, Robert J . ; Rowe, Wallace P . ; Ward, Thomas G . ; Parrott,Robert H . ; and Bell, Joseph A . 1954 . Adenoidal-Pharyngeal-Conjunctival Agents : A Newly Recognized Group of Common Virusesof the Respiratory System . New England Journal of Medicine 251 :1077-86 .17 . Huebner, Robert J . ; Rowe, Wallace P . ; Turner, H . C . ; and Lane,W . T . 1963 . Specific Adenovirus Complement-Fixing Antigens in Virus-Free Hamster and Rat Tumors . Proceedings of the National Academyof Sciences, USA 50 : 379-89 .18 . Huebner, Robert J . ; Chanock, Robert M . ; Rubin, B . A . ; andCasey, M . J . 1964 . Induction by Adenovirus Type 7 of Tumors inHamsters Having the Antigenic Characteristics of SV40 Virus . Proceedingsof the National Academy of Sciences, USA 52 : 1333-40 .19 . Rowe, Wallace, P . and Baum, Steven G . 1964 . Evidence for aPossible Genetic Hybrid Between Adenovirus Type 7 and SV40Viruses . Proceedings of the National Academy of Sciences, USA 52 :1340-47 .20 . Huebner, Robert J . ; Rowe, Wallace P . ; and Lane, W . T . 1962 . OncogenicEffects in Hamsters of Human Adenovirus Types 12 and 18 .Proceedings National Academy of Sciences, USA 48 : 2051-58 .21 . Murphy, Brian R . ; Chalub, Elias G . ; Nussinoff, S . R . ; Kasel,Julius ; and Chanock, Robert M . 1973 . Temperature Sensitive Mutantsof Influenza Viruses : III . Further Characterization of the TS-1(E) InfluenzaA Recombinant (H3N2) Virus in Man . Journal of InfectiousDiseases 128 : 479-87 .22 . Chanock, Robert M . ; Parrott, Robert H . ; Cook, Katherine M . ; Andrews,B . E . ; Bell, J . A . ; Reichelderfer, T. ; Kapikian, Albert Z . ;Mastrota, F . M . ; and Huebner, Robert J . 1958 . Newly RecognizedMyxoviruses from Children with Respiratory Disease . New EnglandJournal of Medicine 258 : 207-13 .23 . Chanock, Robert M . 1956 . Association of a New Type ofCytopathogenic Myxovirus with Infantile Croup . Journal of ExperimentalMedicine 104 : 555-76 .24 . Cate, T . R . ; Couch, R. B . ; Johnson, Karl M . 1964 . Studies withRhinovirus in Volunteers : Production of Illness, Effect of Naturally AcquiredAntibody and Demonstration of a Protective Effect NotAssociated with Serum Antibody . Journal of Clinical Investigation 43 :56-67 .25 . Gerin, J . L . ; Holland, P . V . ; and Purcell, Robert H . 1971 .Australia Antigen : Large Scale Purification from Human Serum andBiochemical Studies of Its Proteins . Journal of Virology 7 : 569-76 .26 . Feinstone, S . M . ; Kapikian, Albert Z . ; and Purcell, Robert H .1973 . Hepatitis A : Detection by Immune Electron Microscopy of aVirus-Like Antigen Associated with Acute Illness . Science 182 : 1026-28 .27 . Feinstone, S . M . ; Kapikian, Albert Z . ; Purcell, Robert H . ; Alter,Harvey J . ; and Holland, P . V . 1975 . Transfusion-Associated HepatitisNot Due to Viral Hepatitis Type A or B . New England Journal ofMedicine 292 : 767-70 .28 . Johnson, Karl M . ; Niebenga, N . H . ; Mackenzie, R . B . ; Kuns, M .L . ; Tauraso, N . M . ; Shelokov, A . ; Webb, Patricia A . ; Justine, S . G . ;and Beye, H . K . 1965 . Virus Isolations from Human Cases of HemorrhagicFever in Bolivia . Proceedings of the Society for ExperimentalBiology and Medicine 118 : 113-18 .29. Johnson, Karl M . ; Kuns, M . L. ; Mackenzie, R . B . ; Webb, PatriciaA . ; and Yunker, Conrad E . 1966 . Isolation of Machupo Virus fromWild Rodent Calomys callosus. American Journal of Tropical Medicineand Hygiene 15 : 103-6 .30 . McIntosh, K . ; Dees, J . H . ; Becker, W . B . ; Kapikian, Albert Z . ;and Chanock, Robert M . 1967 . Recovery in Tracheal Organ Cultures ofNovel Viruses from Patients with Respiratory Disease . Proceedings ofthe National Academy of Sciences, USA 57 : 933-40 .31 . McIntosh, K . ; Kapikian, Albert Z . ; Hardison, K. A . ; Hartley, JanetW . ; and Chanock, Robert M . 1969 . Antigenic Relationships Among theCoronaviruses of Man and Between Human and Animal CoronaViruses . Journal of Immunology 102 : 1109-18 .32 . Chanock, Robert M . ; Roizman, Bernard ; and Myers, Ruth . 1957 .Recovery from Infants with Respiratory Illness of a Virus Related toChimpanzee Coryza Agent (CCA) . I . Isolation Properties andCharacterization . American Journal of Hygiene 66 : 281-90 .33 . Chanock, Robert M ., and Fineberg, Laurence . 1957 . Recovery fromInfants with Respiratiory Illness of a Virus Related to Chimpanzee CoryzaAgent (CCA) . II . Epidemiological Aspects in Infants and YoungChildren . American Journal of Hygiene 66 : 291-300 .44

34 . Kapikian, Albert Z . ; Wyatt, R . G . ; Dolin, Raphael ; Thornhill, T .S . ; Kalica, Anthony R . ; and Chanock, Robert M . 1972 . Visualizationby Immune Electron Microscopy of a 2 .7 nm Particle Associated withAcute Infectious Nonbacterial Gastroenteritis . Journal of Virology 10 :1075-81 .35 . Hadlow, William J . 1959 . Scrapie and Kuru . Lancet 2 : 289-90 .36 . Eklund, C . M . ; Kennedy, R . C . ; and Hadlow, William J . 1967 .Pathogenesis of Scrapie Virus Infection in the Mouse . Journal of InfectiousDiseases 117 : 15-22 .37 . Eddy, Bernice, and Stewart, Sara E . 1959 . Characteristics of the SEPolyoma Virus and a Virus that Induces Malignant Turmors in Mice,Hamsters, and Rats and Benign Growths in Rabbits . American Journalof Public Health 49: 1488-92 .38 . Habel, Karl . 1961 . Resistance of Polyoma Virus Immune Animalsto Transplated Polyoma Tumors (26453) . Proceedings of the Society forExperimental Biology and Medicine 106 : 722-25 .39. Huebner, Robert J . ; Armstrong, D . ; Okuyan, M . ; Sarma, PadmanS . ; and Turner, H . C . 1964 . Specific Complement-Fixing Viral Antigensin Hamster and Guinea Pig Tumors Induced by the Schmidt-RuppinStrain of Avian Sarcoma . Proceedings of the National Academy ofSciences, USA 51 : 742-50 .40 . Hartley, Janet W ., and Rowe, Wallace P . 1966 . Production ofAltered Cell Foci in Tissue Culture by Defective Moloney SarcomaVirus Particles . Proceedings of the National Academy of Sciences, USA55 : 780-86 .41 . Pincus, Theodore ; Hartley, Janet W . ; and Rowe, Wallace P . 1971 .A Major Genetic Locus Affecting Resistance to Infection with MurineLeukemia Viruses . I . Tissue Culture Studies of Naturally OccurringViruses . The Journal of Experimental Medicine 133 : 1219-33 .42 . Eagle, Harry . 1955 . The Specific Amino Acid Requirements of aMammalian Cell (Strain L) in Tissue Culture . Journal of BiologicalChemistry 214 : 839-52 .43 . Moss, Bernard, and Salzman, Norman, P . 1968 . Sequential ProteinSynthesis Following Vaccinia Virus Infection . Journal of Virology 2 :1016-27 .44 . Sebring, Edward D . ; Kelly, Thomas J ., Jr . ; Thoren, M . M . ; andSalzman, Norman P . 1971 . Structure of Replicating Simian Virus 40Deoxyribonucleic Acid Molecules . Journal of Virology 8 : 478-90 .45 . Shatkin, A . J ., and Sipe, J . D . 1968 . RNA Polymerase Activity inPurified Reoviruses . Proceedings of the National Academy of Sciences,USA 61 : 1462-89 .46 . Wei, C . M ., and Moss, Bernard . 1975 . Methylated NucleotidesBlock 5' Terminus of Vaccinia Virus Messenger RNA . Proceedings ofthe National Academy of Sciences, USA 72 : 318-22 .45

TREASURY DEPARTMENT .Public Health and Marine-Hospital Service of the United States .HYGIENIC LABORATORY .--BULLETIN No . 43 .MARCH, MS .THE STANDARDIZATION OFTETANUS ANTITOXIN(AN AMERICAN UNIT ESTABLISHED UNDERAUTHORITY OF THE ACT OF JULY 1 . 1902)BYM . J . ROSENALandJOHN F . ANDERSON .WASHINGTON :GOVERNMENT PRINTING OFFICE .igo8 .46

CONTENTS .Introduction . . . . . . . . . . . . . . . . . . . . . . . . ._ . . . . . . . . . . . ._ . . . . . . ._ . . . . . . . . . . . . . . . 5American unit, and tnethOd< (If standardization . . . . . . . . . . . ----------- . . _ . . . . _ 6Definitions ._ .__ . . . . . . . .__ . . . . . ._ . ._ . . . ._ ._ . . . . . . . . . . . . .___ . . . . . ._ . . . . . . . . 6Methods . . . ._ . . . . . . . . . . . . . . . . . . . . . . . . . . . ._ . . . . ._ . . . ._ . . . . . . . ._ . ._ . . .___ 8Necessity for this standard --------- .--------- .---------- .--------------- 10Historical review . . .__ ._ . . . . . . ._ . ._ . . ._ . . . . . . . . ._ . . . . . .__ . . . . ._ . . . . ._ ._ . . ._ . 12The tetanus toxine---------------- .--------------------------------------- 24The stability of tetanus toxine ----------------- ----------------------- 16Mode of action of tetanus toxine ----------------------------------------- 32German method of standardizing tetanus antitoxin . . . . . . . _ . . . . . . _ . _ _ _ . . . . _ . . _ _ 41Italian method of standardizing tetanus antitoxin .----------------------------- .46French method of standardizing tetanus antitoxin . . . . . . _ . _ . . . . . . . . _ _ . . . . _ . _ _ _ 49Tables for diluting the toxine and antitoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4948

THE STANDARDIZATION OF TETANUS ANTITOXIN[An American unit established under authority of the act of July 1, 1902 .]I3\' ?k111 .T(~N J . ROHFNAV,Surgeon, Director Hygimlic Laboratory . I` . S . Pnblir Health and Marine-Hospital Serwc,andJOHN F . ANDERSON,Passed Assistant Surgeon, .4ssistan .t Director Hygienic Laboratory, [T . S . Public Healthand Marine-Hospital Service .INTRODUCTION ..There are. now four methods of measuring the strength of tetanusantitoxin : (1) The German method described by Behring, (2) theFrench method of Roux, (3) the Italian method after Tizzoni, and(4) the American method described in this bulletin .The European standards are admitted to be unsatisfactory and,for the most part, not accurate ; further, they are complicated anddifficult to carry out . The American method adopted officially underthe law of July 1, 1902, for this country is the result of several years,work upon this subject in the Hygienic Laboratory and it is believedwill commend itself for its simplicity, directness, and accuracy .The standard toxines and antitoxins are preserved under specialprecautions to prevent deterioration and are tested against eachother reciprocally, so that the least alteration in either may bedetected .NPhile the unit is based upon the neutralizing value of an arbitraryquantity of antitoxic serum, the antitoxin is not issued to other laboratoriesfor the purposes of test, as is the case with diphtheria . Astable precipitated tetanus toxine, the test dose of which has beencarefully determined, is given out . All the tetanus antitoxic serumsfor use in man upon the American market are now measured againstthis same standard toxine and have therefore precisely comparablestrengths .The need of uniformity in standardizing this valuable prophylacticserum is evident from the table on page 11, from which it will be seena Manuscript submitted for publication March 4, 1908 .49

sthat before the establishment of an American standard the tetanusserums upon the market varied extravagantly in the unit strengthclaimed, and were, for the most part, comparatively weak in trueantitoxic potency . Since the promulgation of this standard unit theserums on the market have decidedly greater antitoxic value . Thefeebleness of the foreign serums compared with those of Americanmanufacture is also evident from the table .The object of this bulletin is to describe in detail the methods usedin preparing and using this standard . A discussion of the theoreticalconsiderations is introduced for a better understanding of the principlesinvolved .AMERICAN UNIT, AND METHODS OF STANDARDIZATION .DEFINITIONS.-"The immunity unit for measuring the strength oftetanus antitoxin shall be ten times the least quantity of antitetanicserum necessary to save the life of a 350-gram guinea pig for ninetysixhours against the official test dose of a standard toxin furnishedby the Hygienic Laboratory of the Public Health and Marine-Hospital Service."The unit was thus officially defined October 25, 1907, in TreasuryDepartment Circular No . 61 . This circular amended the regulationspromulgated in accordance with the act approved July 1, 1902,entitled "An act to regulate the sale of viruses, serums, toxins, andanalogous products in the District of Columbia, to regulate interstatetraffic in said articles, and for other purposes ."Based largely upon the work on the. standardization of tetanusantitoxin done in the Hygienic Laboratory, a special committee. ofthe Society of American Bacteriologists, which niet in New YorkDecember 27 and 28, 1906, made the follosc-ino, report, which wasunanimously adoptedThat tetanus antitoxin be standardized by the tetanus toxin furnished by thePublic Health and Marine-Hospital Service . The unit is ten times the least amountof serum necessary to save the life of a 350-gram guinea pig for ninety-six hours againstthe official test dose of the standard toxin . The teat dose is 100 minimal lethal doesof a precipitated toxin preserved under special conditions at the Hygienic Laboratoryof the Public Health and ?Marine-Hospital Service . It was decided that the minimalimmunizing dose for a case of possible infection through a wound should be 1,500 ofsuch units . It was decided that after April 1 the new unit should be adopted by allproducers of tetanus antitoxin .J . J . Iu-,rouN, Chairman .THEOBALD SMITH .HERBERT D. PEASE .E . M . HOUGHTON .JOSEPH McFARLAND .M . J . ROSENAU .WILLIAM H. PARK, Secretary .50

The American unit for measuring the strength of tetanus antitoxinmay be defined as the neutralizing power possessed by an arbitraryquantity of antitetanic serum preserved under special conditions toprevent deterioration in the Hygienic Laboratory of the PublicHealth and Tlarine-Hospital Service . This arbitrary quantity nowcontains ten times the amount. of tetanus antitoxin necessary toneutralize somewhat: less than 100 minimal lethal closes of a standardtoxine for a 350-grain guinea pig-that is, one-tenth of a unit mixedwith 100 minimal lethal closes of the standard toxine contains justenough free poison in the mixture to kill the guinea pig in four claysafter subcutaneous injection .The standardization of tetanus antitoxin does not differ radicallyfrom the standardization of diphtheria antitoxin . The toxines andantitoxins are measured against each other reciprocally, so thatchange or deterioration of either the standard toxine or the standardantitoxin may readily be, determined . Duplicate toxines and antitoxinsmade, from time to time will act as checks against deteriorationof either the standard toxines or antitoxins .The value of an unknown serum is measured indirectly throughthe toxine, using the L+ close as the test dose . The L+ dose isthe smallest quantity of tetanus toxine that will neutralize one-tenthof an immunity unit, plus a quantity of toxine sufficient to kill theanimal in just four days .The toxine and not the antitoxin. is given out to licensed manufacturersand others interested for the purposes of standardizing theirserums . The L+ or test close of the particular toxine (A) nowdispensed contains just 100 minimal lethal closes for a 350-gramguinea pig . This particular toxine is very stable and has not changedappreciably in two years . As soon as it alters or is exhausted thenext toxine that will be issued may contain considerably more or lessthan 100 minimal lethal doses, but the test close will contain preciselythe same neutralizing power .The antitoxic serum, for the purposes of this standard, was obtainedfrom a single horse . The serum was reduced to dryness and groundto an impalpable powder . The powder so obtained is preserved inmany vacuum tubes under the influence of _ pentaphosphoric acid .These tubes are kept in absolute darkness at a constant temperatureof 5° C. Every two months, or oftener, one of these tubes is opened,dissolved in a solution of glycerin 66 parts and isotonic salt solution34 parts, and tested .While the-tetanus antitoxin preserved in dry powdered form underthe conditions above named is stable, duplicates of the antitoxinsmade from time to time so as to guard against loss or change willinsure the permanence of the standard which has now been established.51

8METHODS .In order to obtain reliable and comparable results, it is necessaryto take into account all the factors concerned-the composition ofthe poisons, their concentration, the diluting fluid, length of timethe mixtures are allowed to stand, the site of inoculation, etc . Allthe known factors have been considered in our method of standardizingtetanus antitoxin, so that it is our belief that we now haveas accurate and satisfactory a standard for this antitoxin as is thecase with diphtheria .Careful regard must be had for the following points in order toobtain uniform resultsThe diluting fuid.-Salt solution containing 0 .85 per cent chemicallypure sodium chlorid sterilized by boiling is used for dilutingboth the toxine and the antitoxin .Time and temperature.-The mixtures of toxine and antitoxin arekept one hour at room temperature in diffused light before injectioninto the guinea pig .Amount.-A total of 4 c . c . of the toxine-antitoxin mixture isinjected into the guinea pig . If the toxine-antitoxin mixture doesnot equal 4 c . c ., salt solution equal in amount to the differenceis used to wash out the syringe, so that, the total amount injectedand the pressure effects are always equal .Concentration of the toxine .-One-tenth of a gram of the driedtetanus toxine (A) is dissolved in 166 .66 c . c . salt solution . One c . c .of this dilution equals the test dose (L + dose) .Concentration of the antitoxin.-The dilution of the antitoxicserum should follow the tables on page 50 .Guinea pigs.-The guinea pigs should weigh 350 grams . Animalsweighing from 300 to 400 grams are allowable in preliminary roughtesting .Site of injection.-The injection is always given subcutaneouslyinto the tissues of the abdomen about the level of the umbilicus .°Time of death.-The number of immunity units contained in theserum is determined from the amount given the animals that areliving ninety-six hours after the inoculation of the mixtures .An example of a test.-Carefully tare a weighing bottle, then addapproximately 20 to 50 mg . of the dried poison . Again carefullyweigh . Dissolve the toxine in the weighing bottle with salt solution(0.85) in the proportion of 0 .1 gram of the dried poison to 166.66c . c. of the salt solution . This proportion is used for the reasonthat each cubic centimeter of this solution will represent 0 .000,6gm . of the original dried poison ( =100 111LD's) . This proportiona it is very important to inject the animals in the same place on account of therelation to the motor nerve endings and the distance to travel to the central nervoussystem .52

9is taken because it is very convenient, in measuring out the testdose, which represents 1 c . c . of the solution . Thus :44 . 5692 gm ., bottle + toxine .44 . 5300 gm ., bottle ..0392 gm . . toxine .0 .1 gin . : 166 .66 c . c . : : 0.0392 : x .x = 65 .33 c . c .In other words, if the quantity of toxine . placed in the weighingbottle should weigh, as in this instance, just 0.0392 gin ., carefullydeliver from an accurately graduated burette just 65 .33 c . c . saltsolution into the Weighing bottle ; and, as before stated, each cubiccentimeter of this solution Will be the 1, + or test (lose .Now dissolve the serum of unknown value in accordance with thetable of dilutions (page 50) and mix aliquot parts of the serum withthe test dose of toxine, as follows :TABLE IN- 0 . 1 .No . ofguineapig-Weight ofguineapig(grams) .Subcutaneous injection ofa mixture of--_iToxine (testdose) . Antitoxin .Time of death .. Gram .r c .1 . . . . . . . 300 0. 0000 0.001 2 day's, 4 hours .2 . . . . . . . 350 . 0(X)G . 0015 4 days, 1 hour.3 . . . . . . . 350 . 0006 . 002 Symptoms .4 . . . . . . . 360 .0006 .0025 Slight Symptoms .5 . . . . . . . Wo .0000 .003 No symptoms .According to tliis series the guinea 1)ig which received 0 .0015 c . c .of the serum died in four (lays an(] one hour . Therefore 0.0015 c . c .of the serum contains one-tenth of an immunity unit, as the unit hasbeen defined as ten times the least amount, of antitetanic serum necessaryto save the life of a 350-gram guinea pig 96 hours against theofficial test dose . This serum would, therefore., contain just 66 unitsper cubic centimeter .On account of the importance of the L+ or test dose the followingtable is given showing the results of these tests with Toxine A.TABLE No .2.-Showing the results of the test (L+) dose of tetanus toxine A from January17, 1907, to April 11, 1908 .Guinea pig No .Weightin grams . Date . Amount oftoxine A .Amount ofantitoxin .Time ofdeath.I Gram . Days . Hours .306 . .__ . . . ._ . . . . ._ ._ ._ . . . . . . . . . . . . . . . . . . 290 Jan . 17,1907 326 . 0.0006 1 unit . . . . . ._ ._ . . 4 0300 .334 Jan . 24,1907 .0006 1 ._ . ._ unit .__ . . 3 3.__ . . ._ . . . . ._ . . . . . . . . . . . . . . . . . . . 315. . . .do . . . . . . . .0006 1 unit . . . . . 3 7387___ . . . 350 Feb . 20,1907 .0006 1 unit . . . . . 4 19388 . . . . . . . . . ._ . . . . . . . . .__ . . . ._ .___ ._ . . ._ 350 I j . . . . . do------- .0006 1 unit .___ . 4 8389 . . . . . . . ._ . . . . . . . . . . . . . . . . ._ . ._ ._ . . .__ ----.do ._ .__ . . .0006 1 unit ._ ._ . 4 1653

i,210TABLE No .2.-Showing the results of the teat (L+) dose of tetanus toxine A from January17, 1907, to April 7, 1908-Continued .Guinea pig No.Weightin gramfa . Date. Amount of Amount oftoxine A . antitoxin .Time ofdeath .Gram . Days . Hours.400 .__ .__ _ . ._ . . . ._ . ._ .___ . . . . . .__ .__ . . .I 330 Feb . 25,1907 0.0006 1 unit . . . . . 4 21401 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 . . . . .do . . . . . . . 0006 I 1 unit . . . . . 5 7407 . . . . . . . . . . . . . . . . . . . . . . . ._ . . . . . . . . . . 335 ----- do . . . . . . . . 0006 I 1 unit- . _ . . 4 16408 . . . . . . . . . . . . .__ . . . . ._ .__ . . . ._ . . . ._ . . .I 315 .- . . .do . . . . . . . .0006 1 unit . . . . . 4 9415------- . . . . . . . . . . . . . . . . . . . . . ._ ._ .__ . .I 300 Mar . 5, 1907 .0006 1 unit . . . . . 3 041fi----- . . . . . . . ._ . . . . . . . . . . . . . . . . . . ._ . . .I 325 . . . . .do . . . . . . . .0006 1 unit . . . . . 3 10422 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 I . . . . .d . . . . . . . . .0006 1 unit . . . . . 3 9..' ~!..lI' ;...423 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I 325 ~ . . . . .d . . . . . . . . .0006 I 1 unit . . . . . 3 104 .30 .__ .______ . . . ._ . . . . . . . . . . . . . . . . . . . . . I 290 Mar. 22, 1W7, .0006 1 unit . . . . ., 4 11431 . .___ .___ . .__ . . ._ . . . . ._ . . . . . . . . . . . . . . I 290 --do ------- .0006 1 unit . . . . . 4 14454___ ._ .___ ._ .----------- . 300 Mar. 28,1907 . 0006 I 1 unit4 1455---- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 II 509 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 Apr . 13,1907 .0006 1 unit_ . . ._ 3 17510 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 . . . . .do .____ . . .0006 1 unit . . . . . 3 17524 ._ .__ . .__ .__ . . . . . . . . . . . . . . . . . . . 280 Map 3,1907 .0006 1 unit_ . . . . 3 18525__ . ._ .___ ._________ ._ ._ . .__ . . . . .__ . . 290 . . . . . do_ .____ . .0006 1 unit . . . . . I 3 11538 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 May 17,1907 .0006 1 unit . . . . . 3 20320 . . . . . do___ . ._- .0006 i 1 unit . . . . . 3 7556 .____ . . ._ . . . . . . . . . . . . . . . . . . . . . . . .__ . 330 May 24,1907 0006 1 unit . . . . . 2 20557 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 ~----- do___ ._ ._ .0006 1 unit . . . . . 3 10615 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 ~ July 28,1907 .0006 1 unit . . . . . 22616 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 . . . . .do . .____ . .0006 1 unit . . . . . 3 10625 . . . . . . . . . . . . . . . .--------- . . . . . . . . . . . .' 350 July 31,1907 .0006 1 unit __ ._ . 3 13626 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .350 . . . . .do . . . . . . . .0006 i 1 unit . . . . . 4 12350 Sept . 6,1907 .0006 1 unit . .--- 3 4640----- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 ._ . . .do . . . . . . . .0006 1 unit . . .__ 3 2654------ .__ . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Sept . 24,1907 .0006 1 unit . . . . . ; 3 17655--------- . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 I----- do ._ .__ .- .0006 l unit . . . . . 6 19679 . . . . . . . . . . . .__ . .___ . ._ . . ._ ._ . . . . . .__ . 325 Nov . 11, 1907 .0006 i 1 unit . __ . . 3 19680I. . . . . . . . . . . . . . . ._ . . . . . . . . . . . . . ._ . . . . 350 ~---- _do . . . . . . . .0006 1 unit . . . . . 4 0350 Dec . 7,1907 .0006 1 unit- . . . . 3 15360 do . -----. . . . . . .0006 1 unit . . . . . 3 1370-------- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 Dec . 19, 1907 .0006 1 unit . . . . . 3 23757 . . . . . . . . . . .__ . ._ . . ._ . ._ . . . ._ . . . ._ . . . .I 350 Apr . 7,1908 .0006 1 unit . . . . . 4 1758___ . . .__ .__ ._ . ._ ._ . ._ ._ . . . . . . . . . ._ . . 350 .1 . ._ -do . . . . . . . .0006 1 unit . . . . . 3 187r59 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 ~ . . . . . do . . .__ . . .0006 1 unit . . . . . 4 0NECESSITY FOR THIS STANDARD .We now know that when the symptoms of tetanus have developedthe toxine, has combined with the motor nerve cells and that theunion between the cell and the poison is too strong for the antitoxinto break up . However, if the antitoxin is given in time the toxineis neutralized and rendered harmless . Tetanus antitoxin is thereforean exceedingly valuable prophylactic and for this purpose iscoming into more general use both in human and veterinary practice .Whether used as a prophylactic or curative agent it is important.to use a potent serum . The law of July 1, 1902,a not only insures54

serums of useful potency, but also establishes uniformity amongthe producers of tetanus antitoxin in America . The necessity ofestablishing uniformity for this product in American markets is evidentfrom the following table :TABLE No .3.-Showing the differences in the methods of testing tetanus antitoxin beforethe adoption of the American unit .Name.Labeled.Contents(cubiccentimeten).Unitsclaimed accordingtospecialmethods ofstandardization,percubic centimeter.Units accordingtothe Americanstandard,per cubiccentimeter., New York department of health, Albany One. immunizing dose '. . . . . . . . . (15 434(submitted by Dr . H. D. Pease for com- (No. 2929F) .parison) .New York City department of health (sub- . . . . . . . . 700 166mitted by Dr. W. H. Park for comparison).H. F. Mulford Co . (i>ought on open mar- 1,000,000 immunizing 10 100,000 77ket) . units (No . 2121) .Farbenfabrik vorm. Meister Lucius & Tetanus Antitoxin, 333Bruning, Hoechst, a/M (submitted for 5 fach . Normal,comparative tests) . Priifungs dose =1/500.H. S. Mulford Co ., No . 9971 (submitted for 60,000,000 units------- 10 6,000,000tests) .Parke, Davis & Co . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 .000 700New York department of health, Albany . . . . . . . . . . . . . . . . . . . . .75 789(submitted by Dr . H. D. Pease for comparison).Serum Antitetanique, Pasteur Institute, Unit value not stated . 10 Not stated . 4010 c . c . fluid .Serum Antitetanique, Pasteur Institute . . Unit value not stated . 10 Not stated . 40.Tizzoni, Antitossina del Tetano, No . 2912, Unit value not stated . Not stated . b8a 3DryPasteur Institute, Serum antitetanique, Unit value not stated . 10 Not stated . 66II-7 .Institute Bacteriologique de Lyon et du Unit value not stated . 10 Not stated .Bud-Est .a Dissolved in 26 . c . c .b This equals "i units per gram of the dry serum .c Less than 50 .Tetanus antitoxin is made by the following laboratories in theUnited States :Parke, Davis & Co ., Detroit, Mich . (license No . 1) ; H . K. MulfordCompany, Philadelphia, Pa . (license No . 2) ; Lederle Antitoxina An act regulating the sale of viruses, serums, toxins, and analogous products, etc .Approved July 1, 1902 .55

12Laboratories, New York, N . Y . (license No . 17) ; New York Statedepartment of health, Albany, N . Y . ; and the department of healthof the city of New York, N . Y .HISTORICAL REVIEW .Compared wiuh the major plagues of man lockjaw- has always been arare disease . It is on account of the peculiar and characteristicspasms that it early attracted attention . In the writings of Hippocratescases of tetanus are described, diagnosed, and prognosis given .From the learned Aretaeus of Kappadoza we have a description oftetanus that holds until modern times .a Aretaeus described anopisthotonos, an emphrothotonos, and a tetanus, depending uponwhether the muscles of the back, the abdomen or the general muscularsystem were affected . To this classification pleurothotonos waslater added when the muscles of one side were especially affected .Throughout the Middle Ages our knowledge of tetanus remainedat a standstill . The symptoms of the disease so plainly indicatedthat the lesions are localized in the nervous system that, with theintroduction of studies in pathologic anatomy, the brain, spinal cord,and nerves were studied to determine the seat and nature of theillness . It was not, however, until about the end of "the sixties" inthe last century that experimental studies in wound infections beganto throw light upon the chaos of theories . A review of the historicaldevelopment of these theories is exceedingly interesting, for theymirror the prevailing thought upon the nature of disease, as itdeveloped from that of evil spirits, through the "humoral " theory,the realm of miasm and noxious effluvia, to the germ theory .It was long known that tetanus was a coinplication of wounds .Micheles, 1797, thought it was clue to irritation of the peripheralnerves from foul secretions in certain wounds (''wound insults") .Tetanus could not escape the rheumatism theory, which has beensuch an alluring catch-all for symptoms and diseases difficult ofexplanation . In cases of wound tetanus in which no plausibleexplanation seemed possible it was called rheumatic wound tetanus ."Taking cold" was assigned its usual r61e here as elsewhere . Whenno assignable cause seemed at hand the disease was given the learnedtitle "idiopathic tetanus ."About 1860 Heiberg and Rose, and Billroth and Spencer Wellsbattached tetanus to the list of zvmotic diseases and believed it due toa miasm, the spasms being caused by a poison in the blood similar tostrychnine .a A part of the historical data are taken from Von Lingelsheini's article on Tetanus inKolle & Wassermann's Handbuch der pathogenen Mikroorganisinen, vol . 2, p . 566-600 .b Billroth and Spencer Wells . Wiener coed . Presse, 1869, p . 36 .56

13E . Rosea 1870, described a particular form of tetanus followingwounds in the area of the twelfth cerebral nerve which he called"head tetanus" or "tetanus hydrophobicus ."An important milestone in the history of tetanus was passed whenStrfimpell,b 1876, declared tetanus to be an infection . The firstexperiments to prove. this view failed .Arloing and Tripier (lid not succeed in transferring the disease . todogs and rabbits by carrying over the blood and the pus from tetanuswounds .Billrothd and Schultz-5 also obtained negative results with dogs .Carle and Rattone, f 1884, -ere the first. t o obtain positive results indemonstrating the transmissibility of the disease . They injected anumber of rabbits with the washings from the neighborhood of anacne pustule from which the tetanus originated . The rabbits wereinjected either into the sciatic nerve or into the muscles of the back .After an incubation period of two to three days 11 of the 12 rabbitsshowed typiottl tetanic spasms . They also succeeded in carrying theinfection from animal to animal .By injecting earth taken from different, places into 140 mice,rabbits, go-viinea pigs, etc ., in studying the microorganisms of soil(ground tetanus), Nicolaier, s 1884, produced symptoms of tetanus in69 of them and always found a . slender rod in the pus . Therefore heconcluded that there exists a. bacillus that causes tetanus in deepwounds of mice, rabbits, and guinea pigs . In cultures Nicolaier wasunable to separate this bacillus from the other microorganisms withwhich it was associated, but. inoculation experiments with mixedcultures produced tetanus .The next year (1885) Nicolaier,h working under FhIgge in the GottingenHygienic Institute, made, noteworthy advances upon the subject. He showed that each injection into mice, guinea pigs, andrabbits caused a disease with tetanic spasms, while dogs were refractory. Nicolaier saw in the pus of the wounds this slender bacillus,.:a Nicolaier, Arthur : Zur Aetiologie des hopftetanus (Rose) . Virch. Arch ., vol . 128,1892, p. 1-19 .b Strumpell : Ueber die Uraachen der Erkrankungen des Nervensystems . Arch . f.klin . Med., vol . 35, p . 14-15 .cArloing and Tripier : Gaz fined . de Paris, 1870, 1' . 337 .Billroth Allgemeine chirurgische Pathologies and Therapic . Berlin, 1882, p -504 .r Schultz : Ueber einer humulation von Tetanusfallen im Stadtkrankenhause zuRostock . Rostock, 1876, p. 13 .f Carle and Rattone : Studio sull' enziologia del tetano (communicazione preventiva). Giorn . dell' R. Accad . di med . di Torino, Marzo, 1884 .gNicolaier, Arthur : lieber infectiosen Tetanus . Deut. med . Woch ., vol . 10, 1884,p . 842-844.h Nicolaier . Arthur : Beitraae zur Aetioloaie des Wundstarrkrampfes . Inaug . dis-57

14which he considered the cause of the infection, but again was unableto obtain it in pure culture, although he carried on a mixed cultureupon coagulated calf's blood serum .Soon afterwards Rosenbach, a 1886, found a similar bacillus with around terminal spore in a case complicating frost gangrene in manand transferred the infection to animals in the pus .In 1889, with the aid of anaerobic technic, Kitasato b for the firsttime grew the bacillus in purse culture and by successful inoculationexperiments finally proved that this bacillus was the real cause oftetanus . Kitasato succeeded in isolating the tetanus bacillus, firstdescribed by Nicolaier and later by Rosenbach, not alone by understandinganaerobic methods but by taking advantage of its resistingspore . This bacillus appears in the pus of wounds of man and animalshaving tetanus . It often forms spores in the pus, but if the pus isexamined early it may contain spore-free rods . He planted thematerial containing the bacillus upon agar slants, which were incubatedone to two days . The mixed cultures so obtained were heatedone hour at 80° C . and then transplanted to agar plates . Kitasatodesigned for this purpose a special shaped apparatus in which hydrogenwas used to replace the air .Kitasatoc further showed that the tetanus bacillus is not found inthe heart's blood of mice dead of tetanus and therefore concludedthat we are dealing with an intoxication and not with an infection .It was in the following year, 1890, that Behring and Kitasato dpublished their epoch-making work upon the tetanus toxin andtetanus antitoxin, laying the foundation of serum therapy . Onaccount of the great historical and practical, importance of this documenta transcription of its principal features is given :The immunity of rabbits and mice which have been immunized against tetanusdepends upon the power of the cell-free serum to render the toxic substances whichare produced by the tetanus bacillus harmless .Experiments show :1. The blood of tetanus immune rabbits possesses the property of destroying thetetanus poison .2. This property is destroyed by the extravascular blood and the cell-free serumobtained from it .3. This property is of such a stable nature that it is also effective in the bodies ofother animals, so that we are in a position, by means of the transfer of blood serum, toaccomplish noteworthy therapeutic results .a Rosenbach : Zur Aetiologie des Wundstarrkrampfes beim Menschen . Arch . f .klin . Chir ., Berl ., vol . 34, p. 306-317 .b Kitasato, S . : Ueber den Tetanusbacillen . Zeit . f . Hyg., vol. 7, 1889, p . 225 .c Kitasato, S . : Experimentelle Untersuchungen fiber das Tetanusgift . Zeit . f. Hyg.,vol . 10, 1891, p. 304 .d Behring, E., and Kitasato, S . : Ueber das Zustandekommen der Diphtherie-Immunitat and der Tetanus-Immunitat bei Theiren . Deut . med . Woch ., vol. 16,1890, p. 1113-1114 .58

154 . The property of destroying the tetanus poison is absent in the blood of animalswhich have not been immunized against tetanus . If the tetanus poison be given tosusceptible animals it may be demonstrated in.the blood and other body fluids afterthe death of the animal .For instance : A rabbit was immunized against tetanus to such a high degree ofimmunity that it, was able to withstand 10 c . c . of a virulent culture containing thetetanus organism, 0 .5 c . c . of which was sufficient to kill a normal rabbit . This rabbitwas not only able to withstand the infection of living tetanus bacilli, but was alsoimmune to the tetanus poison . Thus it withstood without any symptoms twentytimes the quantity of tetanus poison sufficient to kill a normal rabbit .This rabbit was bled from the carotid . and before the blood clotted 0 .2 c . c . wasgiven to a mouse and 0 .5 c . c . to another . After twenty-four hours both of these treatedmire and two control mice were inoculated with a virulent tetanus bacillus . Theamount injected was so great that the control animals showed tetanus in twenty hoursand were dead in thirty-six hours . Both treated mice, on the other hand, remainedwell .The larger quantity of blood was allowed to stand until the serum separated . Of thisserum six mice were injected with 0 .2 c . c . each into the peritoneal cavity . Twentyfourhours later they were infected and remained well, while the control mice died inless than forty-eight hours .It was further found that the serum had a therapeutic value in that the mice werefirst infected and afterwards protected by injecting the serum into the peritonealcavity .They further made experiments to demonstrate that the serum has an enormouspower of destroying the poison .Of a tetanus culture ten days old which was freed from bacilli by filtration, 0.00005c . c . was sufficient to kill a mouse in four to six days and 0.0001 c . c . was sufficientto kill a mouse with certainty in two days . However, 5 c . c . of the serum from thetetanus immune rabbit was mixed with 1 c . c . of this culture, and the serum wasallowed to act twenty-four hours upon the culture containing the tetanus poison . Ofthis mixture, four mice were given each 0 .2 c . c ., containing 0.0033 c . c . of the culture,or more than 300 times the dose otherwise fatal for mice . All four of the miceremained well . The control mice, on the other hand, died in thirty-six hours with0 .0001 c . c . of the fluid . The mice which were treated as above described, as well asthose which had received the mixture of tetanus poison with serum, so far as can betold, remained well . Later they were injected repeatedly with virulent tetanusbacilli, which they withstood without a trace of symptoms .This fact is of very particular note, because in the innumerable experiments nomouse, no rabbit, in fact no animal so far tested has shown a natural immunity againsttetanus . We therefore may draw the conclusion that the above-mentioned explanationof the conditions of immunity which may be obtained promptly and withoutdifficulty, and which may be accomplished without harm to the animals in the processof immunization, will prove of far-reaching value . It is to be understood that controlanimals were tested with the blood and serum of nonimmune rabbits . The same maybe said to be true of cattle and sheep .We permit ourselves from our results to venture the conclusion that, after finding atherapeutically potent substance useful for animals, it will also be useful in the treatmentof diphtheria and tetanus in man . We only desire to call attention to one thingin conclusion : In former times the transfusion of blood was considered to be a heroic,but in certain cases a very powerful, curative agent . In later times it was thoughtthat physiological salt solution was able to accomplish the same result . On the otherhand, our experience makes memorial the phrase "Blut ist ein ganz besonderer saft."59

Summary of Remaining Pages in Foregoing PaperPrepared by Margaret PittmanStability of Tetanus Toxine (pp 16-18)Tetanus toxine does not remain constant in solution .Success was obtained "by precipitating the filteredbouillon culture with ammonium sulphate, drying theprecipitate over sulphuric acid, grinding to impalpablepowder, and preserving it in vacuo under the influenceof pentaphosphoric acid-a very strong dehydratingsubstance-in a cold, dark place ." The dried toxineshows no loss for at least 25 months .The Stability of the Standard Tetanus Toxine (PP 18-24)The dried standard toxine (No . A) showed markedstability after exposure to sunlight for one and one-halfhours, to 37 °C for several days, to room temperature forgreater than 11 days, etc .The pioneer investigators reported destruction of thefluid toxine at 65°C and susceptibility to mineral acids,alkalis and salts ; agar culture is the most poisonous ;chickens, snakes, turtles and tritons are immune to toxin,formalin detoxifies the toxin . Anderson (Hyg . Lab . -Bull . No 39, 1907) reported the concentration of formalinexposure that rendered 100 MLD nonlethal for theguinea pig .The Tetanus Toxine (pp 24-32)An interpretative review of the published investigationson the toxine is given . Remarkable basic information hasbeen reported . Some of the pertinent points covered werethat (1) "tetanus toxine" is a complex substance-two toxins, tetanolysin and tetanospasmine ; contains both a toxophoreand a haptophore group ; induces protectiveimmunity-a free receptor in the blood combines withthe toxophore and neutralizes the toxine ; produces symptomsafter a definite period of incubation, never lessthan 8 hours, and is inversely related to the size of thedoses ; one of the most poisonous of substances ; susceptibilitywithin animal species is remarkably constant butnot between species-the MLD's for different species aregiven; harmless by mouth ; and chemical nature isunknown . Remarks are made in regard to the best mediato produce the toxine and the drying of toxine with theuse of ammonium sulphate .vestigations relating to neuronal action of the toxine itwas stated"the weight of evidence is in favor of the vithat the toxine produces its effect solely by the adsorpof the toxine by the nerves, whose axis cylinder substahas a specific affinity for the poison . They transport ilto the cord and brain through axis cylinder substanceitself ."The next three sections give the German, Italian, andFrench methods for Standardizing Tetanus Antitoxin41-49) .The first two methods are very complex and are difficultto interpret . The strength of the French antitoxirnot stated on the label . "It is stated that the antitoxindose of serum amounts to 1,000,000,000 ; that is, to prtect a mouse against the smallest fatal dose it is sufficientto use 1/1,000,000,000 of its weight of serum ."The last section gives Tables for diluting the Toxine atAntitoxin (pp . 49-55) ."They are published not only because they will savetime and the possibility of error incident to such calcultions, but because it is of some importance that the saimethods of dilution be used by different laboratories iiorder that the results may be strictly comparable ."Mode of Action of Tetanus Toxine (pp 32-41)Since ancient times it has been known that tetanus affectschiefly the nervous system . After a review of the in-60

The Regulation of Biologic Products, 1902-1972Margaret PittmanThe acorn planted a century ago, when the Laboratoryof Hygiene was established, has grown into an astoundingtree . One of the spreading branches, the Regulationof Biological Products, has been of key importance inprotecting the lives of millions of Americans . This articlecovers briefly the developments in biologic products atthe National Institutes of Health from 1903, when the1902 Biologics Control Act became effective, until thetransfer of Regulations of Biologic Products to the Foodand Drug Administration in 1972 .I . Background : The Biologics Control Act of 1902Biologics control in the United States followed pioneeringresearch in Europe that opened the way to preventionand specific cure of infectious diseases by vaccination ortreatment with antitoxins . In 1796 Jenner had introduceda vaccine against smallpox, and, by the late nineteenthcentury, Pasteur, Koch, Ehrlich, von Behring, Roux, andothers were making progress against other diseases . In1894 von Behring and Roux reported that diphtheria antitoxincould cure diphtheria . Very shortly, laboratoriesacross Europe and the United States sent researchers tolearn this technique . In 1894-95 1 New York CityLaboratories began producing the antitoxin, using theunique strain of Corynebacterium diphtheria isolated byAnna Williams ("Park 8") . Production was also startedat the Hygienic Laboratory after Kinyoun visited theKoch and Roux laboratories in 1894 . 2 The antitoxin wasbeing produced also by other laboratories . Sometimes itworked, other times not at all .There were no requirements for potency or safety . Atragedy in 1901 provided the stimulus for Congressionalpassage of the Biologics Control Act in 1902 . The city ofSt . Louis had engaged a bacteriologist to preparediphtheria antitoxin for free distribution to physicians . In1901, during a serious epidemic of diphtheria, tenchildren treated with antitoxin died of tetanus-notdiphtheria . The horse from which the antitoxin was obtainedhad contracted tetanus . In the rush to provide theneeded antitoxin, no safety test had been performed . 3This tragedy convinced Congress and the public that producingantitoxin or vaccine was not a simple matter likeweighing out a dose of a drug on a scale .Under the provisions of the 1902 Biologics ControlAct, which is reproduced in Appendix A, enforcementwas delegated to the Division of Pathology andBacteriology of the Hygienic Laboratory . Thus began thework of regulating biologicals . 4 In 1937 the work wasplaced in a newly established Division of Biologics Control .Margaret PittmanIn 1944 this division was redesignated the Laboratory ofBiologics Control, the name under which in 1948 itbecame a part of the National Microbiological Institute .In 1955 it was given separate division status within NIHand renamed the Division of Biologics Standards . InAugust 1972 the regulation of biologics control wastransferred to the Food and Drug Administrations TheChiefs or Directors of the control activities were MiltonJ . Rosenau, 1903-1909 ; John F . Anderson, 1909-1915 ;George W . McCoy, 1915-1937 ; Walter T . Harrison,1937-1940 ; Milton V . Veldee 1940-1949 ; William G .Workman, 1949-1955 ; and Roderick Murray, 1955-1972 .High tribute is due to these directors for theirsagacious handling of the development of regulationsfrom the beginning, when the state-of-the-art inmicrobiology and immunology was in its infancy . Thistradition has been continued through the complexity ofcontrols of viral vaccines and their clinical evaluation .Praise also is due to the dedicated scientists and technicianswithout whom, the work could not have beenaccomplished .61

II . General AspectsResponsibilityThe organization designated to regulate the control ofbiologics is responsible for establishing and maintainingstandards of safety, purity, and potency of all biologicalsas stated in the Biologics Control Act . The standards areset forth in prescribed regulations . "To apply our presentknowledge to the improvement of biologics now on themarket, to find better ways of producing and testingthese biologics, and to help in the development of newimmunizing agents against the infectious diseases that, sofar, have baffled science - these are the ultimate aims ofthose concerned with the control of biologics,"wroteRoderick Murray, Director of DBS in 1955 . 6Development of StandardsTwo forms of licenses are required : for establishment tosell a product and for each product sold . Licenses areissued only upon showing that the prescribed standardsfor safety, purity, and potency have been met . To ensurecompliance with Public Health Service Regulations,specially trained members of the scientific staff inspectestablishments applying for licenses . Those already licensedare inspected annually .Specific requirements for a particular product aredetermined by the nature of the product and the state ofknowledge about it . Two of the products first licensedsmallpoxvaccine and diphtheria antitoxin, provide examplesof this . Standards for smallpox vaccine initiallyemphasized bacterial content on the ivory points (by colonycount) and the use of 50°10 glycerin to preventgrowth of non-spore forming bacteria, yeasts, andmolds ;? animal test for freedom from tetanus spores ; anda limit on the dating period of no more than threemonths "if stored at 5°C ." 8 Close vigilance over thevaccine was demonstrated by the quick action takenwhen foot-and-mouth disease appeared in the calves usedby two producers of smallpox vaccine : their licenses wererevoked and their products condemned and destroyed .No human infection occurred . 2 For diphtheria antitoxin,requirements specified a sterility test for bacterial contamination,animal tests for safety (preservative, tetanusspores or other obnoxious poisons), and potency assaythat ensured a specified number of immunity units perdose . 9Early requirements were promulgated on the basis ofknowledge gained from the pioneer European investigators,often modified by research at the HygienicLaboratory . The U .S . official immunity unit ofdiphtheria antitoxin, for example, was based on Ehrlich'snormal serum unit with improvements in the precision ofthe assay . 9 The safety, purity, and potency tests weresimilar to those prescribed in the German official methodfor diphtheria antitoxins . The standardization of tetanustoxin was based on the American unit which was differentfrom the German, Italian, and French units .'° Ithas remained the standard unit .Technical methods for evaluating a product havelikewise depended upon its nature . Except for smallpoxand rabies vaccines, classical bacteriological methodswere followed until 1941 when new techniques wereDr. Pittman, with assistant, in 1930's DBC laboratory.required for typhus vaccine, which was produced withrickettsiae propagated in the embryonated hen's egg . Inthe early 1950s the virus for poliomyelitis vaccine waspropagated in animal tissue cultures . This procedurecaused revolutionary changes to insure absence of adventitiousagents that might be present in the animal tissue .For development and application of the new tests, a considerableexpansion in the number of skilled researchscientists and in space was required . It was at this timethat the Division of Biologics Standards was established .ResearchFrom the beginning, research has been vital in thedevelopment of standards . Members of the Division haveconstantly worked to improve existing biologic productsand to develop standards for new provisional products .In order that biologic standards be uniform throughoutthe world, these scientists serve on World HealthOrganization expert committees involved in the establishmentof international standards, and they cooperate withscientists worldwide . Through their research, significantcontributions have been made to medical science .III . Biologic ProductsChronology of Product LicensesThe chronology of product licensing clearly reflects thestate-of-the-art in microbiology, immunology, and theuse of biologics in the practice of medicine . In 1903 fourproduct licenses were issued-smallpox vaccine (6 U .S ., 2foreign) diphtheria antitoxin (6 U .S .), anti-streptococcusserum (1 U .S ., 1 foreign), and toxins (1 U .S .) before requireddesignation of specific toxin in the license . Between1904 and 1906 only antibacterial sera were licensed .By the end of 1907 nine bacterial species wererepresented : dysentery, gonococcus, meningococcus,plague, pneumococcus, staphylococcus, streptococcus,62

typhoid, and tubercle bacilli . A few more were addedlater . There also was thyroidectomized goat serum andhorse serum . In 1907, anti-tetanic serum,-later changedto tetanus antitoxin, (3 U .S ., 4 foreign) and a secondviral vaccine, rabies (1 U .S .), were licensed . Later, additionalmanufacturers were licensed for rabies vaccine .The year 1907 was a turning point : manufacturers changedfrom producing "anti-bacterial" serum products exclusivelyto include "anti-bacterial" vaccines . Althoughmost of the individual anti-bacterial sera were producedby only one manufacturer, many manufacturers producedthe same bacterial vaccine . For example, thirteencompanies were licensed for the "Colon-Bacillus" vaccine,and ten each for Staphylococcus albus and typhoid,and even seven for the nonpathogen Micrococcus catharrhalis. Some 30 bacterial species, representing practicallyall of those associated with bacterial diseases, had beenincluded by the 1930s when the sulfa drugs, followed bythe antibiotics, were introduced . One shortcoming ofcontrol during this period was that potency tests relativeto efficacy had not been developed .Beginning in 1917 licenses were issued for immunizingtoxin products, for additional kinds of antitoxins, andfor human bacterial and viral antisera . The toxin productsincluded diphtheria toxin mixed with antitoxin(1917), diphtheria toxoid (1926), tetanus toxoid (1933),and scarlet fever streptococcus toxin (1925) . Differentcombinations of diphtheria and tetanus toxoids with pertussisvaccine began in the 1940s, a period when the useof aluminum absorbed vaccines also became prominent .The antitoxins included botulinus (1921) perfringen andother gas gangrene species (1931-1939), scarlet feverstreptococcus (1925) and gonococcus (1937) . The humanantisera included pertussis, poliomyelitis and mumps(1939-1941) .In 1941 the first rickettsial vaccine, typhus, producedby growing the microorganism in embryonated hen's egg,was quickly followed by other rickettsial and viralvaccines-influenza (1945), mumps (1950), and yellowfever (1953), or tissue cell culture from embryos formeasles (1963), and rubella (1969) . Viral vaccines producedfrom virus grown in propagated animal tissue culturewere poliomyelitis vaccine (1955) and adenovirus (1957) .Other viral vaccines included equine encephalitis (easternand western) and Coxsackie 21 . Bacterial vaccines includedBCG (1950), anthrax (1970) and a number ofbacterial vaccines for allergen extracts .During World War II blood and blood-derivativeswere licensed, including whole blood for transfusion,plasma, albumin, and other derivatives . Since 1972, whenthe functions of DBS were transferred to FDA, 5 licenseshave been issued for four bacterial capsularpolysaccharides-the meningococcus Groups A and C,pneumocococcus-23 valent, and Hemophilus influenzaetype b . In addition, hepatitis B antibody' for screeningbloods, for transfusion and derivatives, for the presenceof hepatitis B antigen has been licensed ."Classification of LicensesBy 1972, 450 establishment licenses had been issued but=nly 235 (16 of these were foreign) were active . 12 Productlicenses numbered around 1300, covering approximately300 specific products that could be classified asallergenic extract, antisera, antitoxins, bacterial vaccines,toxins, toxoids, viral and rickettsial vaccines, blood andblood derivatives, immune serum globulins, diagnosticsubstances for skin tests, venoms, and antivenoms .IV . Selected ContributionsAllergenic ProductsAllergenic products are administered to man for thediagnosis, prevention or treatment of allergies . Sourcesof allergens are numerous, and identification of thespecific antigen has progressed slowly . In 1960, DBS initiatedthe development of standards under the directionof Harold Baer, Chief, Laboratory of Allergenic Products. General Additional Standards for all allergenicproducts with specific standards for only one product,short ragweed pollen extract, have been prescribed."Descriptions of this product, 13 of fire ant venom protein,14 and of poisonous Ancardiaceae (e .g ., poisonivy), 15 illustrate different kinds of reactive substances .The technic for performing the skin test, prepared by acommittee on standardization of which Baer is a member, 16has contributed significantly to the evaluation of results .AntiserumMeningococcus . Deficiency in type-specific antibodies wasone cause of ineffectiveness of some bacterial antisera .One was antimeningococcus serum . In the early 1930sthere was a massive epidemic of meningitis . The antiserumformerly thought to be efficacious was largely ineffective .NIH invited Sara E . Branham to investigate the cause .In her 1956 review, Dr . Branham described the two earlyclassification of the meningococcus and the final groupspecificclassification .17Encapsulation of the organismwas discovered . Type IIa was identified as Group C . UsingPetrie's observation that a precipitin halo developsaround a colony on immune serum agar plates, group(type) specificity was shown . 18 A recommended- mouseprotection test likewise showed specificity . 19 Mouseassayed potency and plate precipitation of Group I antibodiesshowed high correlation . 20 By the early 1940s,however, treatment with meningococcus antiserum wassuperceded by treatment with sulfonamides .AntitoxinThe precise methods used to develop standards fordiphtheria antitoxin by Rosenau 9 and for tetanus antitoxinby Rosenau and Anderson)° provided a model for thedevelopment of other products . Following the standardmodel for diphtheria antitoxin, for example, Ida A .Bengtson established the immunity units for botulinusantitoxins, A, B, and C . Previously the unitage rangedfrom 2 to 450 per m1 . 22 Bengtson also established thestandards for the gas gangrene antitoxins-Clostridiumhistolyticum, 23 C. perfringens, 24 a C. septicum,24b C.oedematiens, 25 and C . sordelli. 26 She also supplied thefirst international standard preparations-the respectivetoxins and antitoxins-to the League of Nations Committeein Copenhagen, Denmark . 27 Although the UnitedStates was not a member of the League, George W .63

McCoy, Director of NIH, served as an invited unofficialmember of the Committee .In addition to the development of standard units,Rosenau and Anderson investigated the phenomenon ofsensitivity to horse serum following antitoxin treatment .Their research on such reactions, which caused concernabout the safety of antitoxins, provided pioneer contributionsin the study of anaphylaxis . 21Bacterial VaccinesEfficacy of only a few of the many bacterial vaccineslicensed before the advent of antibacterial drugs was encouragingbut variable . Those containing a toxin (toxoid)or capsular substance proved to be the most effective . Abrief discussion of the cholera, pertussis, and typhoidvaccines will illustrate both successes and difficulties .Cholera Vaccine . The first pathogenic bacteriumisolated by the Hygienic Laboratory was Vibrio cholerae,successfully obtained in 1887 from an immigrant on aship in New York Harbor . In the 1960s, during theeighth pandemic of cholera, the World Health Organization(WHO) and the newly established Pakistan-SEATOCholera Research Laboratory in Dacca, East Pakistan(Bangladesh) stimulated work on a potency test forcholera . This potency assay 28 correlated with human efficacyof the vaccine, 29 although of relatively short duration. This SEATO program was organized by Joseph E .Smadel, then Associate Director of NIH (1956-60) . From1960 to his death in 1963 he was Chief of theLaboratory of Virology and Rickettsology, DBSLater it was shown that pathogenesis and immunity ofcholera is effected by a cholera toxin . Studies continueelsewhere on how to provide prolonged immunity againstcholera .Pertussis Vaccine . Pertussis vaccine regulation wasconcerned with (i) control of the bacterial content ; (ii)development of a potency assay, (iii) standardization ofpotency unitage ; (iv) relation of unitage to protectionagainst pertussis, (v) toxic substances of Bordetella pertussis,(vi) the concept that pertussis is toxin mediated,(vii) an animal model, and (viii) neurotoxicity of B .pertussis .In order to obtain uniformity in the bacterial contentof pertussis vaccines, an opacity standard was preparedwith fine pyrex glass particles . It was equivalent to 10 X109 pertussis bacteria, in aged vaccine, as determined bydirect count . In comparison with the standard, theaverage of lots per manufacturer differed from 0 .4 to 1 .6fold . 3 oFor many years attempts to develop a potency assayfor pertussis vaccine had failed . The first successfulpotency assay for pertussis vaccine was developed as aconsequence of John F . Norton's observation that thevaccine protected against intracerebral challenge of B.pertussis . The original potency requirement was based onrelative potency of the bacteria in the vaccine and in thereference (1949) . In 1953, a value of 12 units per totalimmunizing dose was prescribed . Pittman 3 l described thedevelopment of the potency assay, standardization, andthe rapid drop in mortality of children that followed . Inthe collaborative assessment of the vaccines in Britishfield trials, it was shown that 12 units (three injections)afford about 85°7o protection against home exposure, 32with a high correlation between unitage and protectionagainst home exposure . The World Health Organizationadopted the criteria that a single dose should contain nofewer than 4 units .B. pertussis induces a diversity of pathophysiologicalreactions . Although the toxic factor(s) had not been identified,a mouse toxicity test was prescribed in the first requirementsfor pertussis vaccine . Pittman and COx 33reported on analysis of the results of 9 .5 years of testing .Some years later Pittman proposed that both the harmfuleffects of pertussis and its prolonged immunity werecaused by an exotoxin . 34 Study of experimentalrespiratory infection in the mouse emphasized thedisparate pathophysiological responses, 35 and this wasfollowed by the elucidation of pertussis as a toxinmediateddisease . 36 Finally, accrued evidence suggestedthat encephalopathy is caused by neuronal necrosis effectedby a toxin-excited neurotransmitter, such asglutamate or aspartate . 37Typhoid Vaccine . Evaluation of the potency oftyphoid vaccine has been complicated by the lack of asusceptible animal . However, with the use of the socalledN ip assay it was confirmed that Vi antigen is amajor protective antigen and that the relative potency ofthe vaccines reflected efficacy . 3 s The results of thelaboratory assay of WHO field trial vaccines, which includedthe reference NIH 4, prompted the selection of aVi containing vaccine as the U .S . standard and promulgationof Additional Standards for Typhoid Vaccine .Later Wong et al . 39 by enzymatic removal of capsuleconfirmed and emphasized the importance of the Viantigen .Invasion of the blood by salmonellae is limited toSalmonella typhi and only this species has a capsule (Vipolysaccharide) . Infection and purified Vi stimulate a risein Vi antibody . It is reasonable to consider that Vi antigenalone or conjugated to .a protein may provide immunityand thereby eliminate the undesirable pyrogenicreaction after whole cell vaccination .Mycobacterial and Fungal AntigensMany questions about various aspects of tuberculosis remainunanswered . Sotiros D . Chaparas, Chief and hiscolleagues in the Laboratory of Microbacteria andCellular Immunology demonstrated the correlation of invitro cellular transformation and skin reactivity ofmycobacterial antigens .4o ,41 Chaparas, Fuller andSeligmann investigated methods for sensitization ofguinea pigs in the control testing of fungal antigens . 42Additional Standards for BCG Vaccine and Tuberculinhave been prescribed ."MycoplasmaMycoplasma are frequent contaminants of continuouscell lines and, less frequently, of primary cell cultures . 43In 1962 Michael F . Barile, Chief of the Laboratory ofMycoplasma, developed the definitive test forMycoplasma, required during the manufacture of viralvaccines." Barile et al . have identified more than 100064

mycoplasma from contaminates in cell cultures, anddetermined their source . 44 After it was shown thatMycoplasma pneumoniae was a primary cause of atypicalpneumonia and that vaccination against the disease appearedencouraging, Barile and colleagues developed ahamster potency assay for the evaluation of M.pneumoniae vaccine . 45ToxinM . F . Barile directed the investigation for a suitable toxinto be used as the U . S . Standard for the Schick Test .The guinea pig erythema potency assay, established asthe officially prescribed 'assay, is significantly morereproductive than the minimum lethal dose method . 46ToxoidThe effects of adjuvant tetanus toxoids on the durationof the protective level of antitoxin (0 .01 u/ml) againstneonatal tetanus in New Guinea, were studied by R .MacLennan, F . D . Schofield, and DBS colleagues .Results showed that oil-adjuvant toxoid (one injection)and AIP04 toxoid (two injections) protected againstneonatal tetanus . The average protective antibody levelpersisted for more than 3 yrs, but fewer than 4 years .Plain toxoid (three injections) was protective, but durationwas less than 1 year . Unacceptable side-effects wereinduced by subsequent lots of oil-adjuvant toxoid,however, and this has precluded the use of thisadjuvant . 47a .b ,c,d ,eViral VaccinesSmallpox Vaccine . The first vaccine against any infectiousdisease, smallpox vaccine, was also the first licensedvaccine in the United States (1903) . A few years ago thisonce prevalent and dreaded disease was declared byWHO to be eradicated . The Laboratory of Control Activitiescontributed to eradication by establishing standardsfor dried, potent, stable vaccine that was used tovaccinate large populations . Potency was standardized asthe number of pocks produced on the chorioallantoicmembrane of chick embryos relative to the U . S .reference vaccine . This method, proposed by Fuller andKolb ' 48 replaced the rabbit scarification method . Hornibrookand Gebbard 49 first showed that smallpox vaccinecould be dried without loss of potency .Rabies Vaccine . Rabies vaccine was first produced inthe United States by a few widely scattered laboratoriesknown as "Pasteur Institutes." Exposed persons oftentravelled several hundred miles to be treated at theselaboratories . In 1906, the Hygienic Laboratory resumedstudy of the vaccine, and by 1908 it was treating personsin the Washington, D .C . area . Shortly thereafter, theLaboratory began shipping rabbit spinal cord preservedin glycerin to state health officials who had laboratoryfacilities for treatment . This practice was discontinued in1921, after several commercial manufacturers of biologicshad developed facilities for treatment . 2 Later advancesincluded desiccation of the cord, in vacuo, over P 205 ,after which it was preserved with glycerin . Further progresswas made by achieving inactivation with UV, aeplacement for phenol . To eliminate the infrequentneurological reaction caused by the rabbit brain tissue,the virus was propagated in embryonated duck egg .Presently, the preferred method is propagation of thevirus in cell culture . In 1948 Habel and Wrightdeveloped a mouse test for potency assay . 5 ° Since 1953,the modified Seligmann test which is the NIH test hasbeen used . A comparison of the NIH test with anantibody-binding test by Fitzgerald, et al., suggests promisefor an in vitro assay test . 51Other Viral VaccinesAmong the challenging problems encountered in standardizinglive viral vaccines propagated in animal cellculture was the detection of adventitious agents in mammalianor poultry embryo tissue cells . 52 In 1962, for example,Eddy et al. identified simian virus 40 in rhesusmonkey cell cultures . 53 For live oral poliovirus vaccine, amonkey neurovirulence test was developed by Ruth L .Kirschstein and colleagues to provide assurance that seedviral strains and the vaccine were not neurovirulent . 54,55Trials of a live measles virus vaccine in Upper Voltaby Meyer et al . showed the feasibility of combining livemeasles, smallpox and yellow fever vaccines and their administrationby jet inoculation . 56 As many as 300 injectionscould be made in one hour ; approximately 730,000children were vaccinated .Three articles in the 1960s from the Laboratory ofViral Immunology described remarkable progress in protectingagainst the so-called German measles, from theattenuation of the rubella virus to the standardization ofa live rubella vaccine . Parkman et al . 57 described itsdevelopment and laboratory characterization . Myer eta1 .51 reported on production and clinical trials, andStewart et al. 59 described the development of the simple,inexpensive rubella-virus hemagglutination test that is usedeffectively for diagnosis and for determining immunitystatus . Its use in preventing of disease and congenitaldefects has been very successful .Influenza vaccine was one of the first viral vaccines inwhich the virus was propagated in hen embryos . BerniceE . Eddy developed the first potency assay . 6° The hemagglutinatingand neuraminidase antigens that determine thespecificity of the strains tend to change, hence the formulafor the vaccine is assessed yearly .Rickettsial VaccinesThe microorganisms that cause typhus, Rocky Mountainspotted fever and other rickettsial diseases are propagatedin embryonated hen's egg for vaccine production .Typhus vaccine was the first to have extensive use .Transmitted by lice, typhus caused devastating epidemicsthat ravaged southern Europe during World War I andthe years immediately following . During World War II,every serviceman in North Africa and Italy (and manyliberated civilians) were vaccinated against typhus .Among U . S . military personnel there were only 64 casesof typhus and no deaths .In 1941 Bengtson and Topping used rickettsial antigensto develop a complement fixation test that specificallyidentified antibodies of epidemic typhus fever . 6165

Blood and Blood DerivativesThe first program on blood and blood products wasdeveloped during World War II by Roderick Murray andJ . T . Tripp . They established standards and supervisedplasma production in commercial establishments fromblood collected by the American Red Cross . Some13,000,000 pints of blood were processed to plasma andalbumin .A most disturbing development was the presence of anicterogenic agent in some pools of plasma . In 1942,28,000 servicemen injected with yellow fever vaccineprepared with human serum as a stabilizing agent fell illwith jaundice, and 100 died . Murray et al . confirmedthat post-transfusion hepatitis was caused by whole bloodfrom carriers of hepatitis . 6 z,63 Currently, the risk oftransmission of serum hepatitis is practically nil . Allbloods for transfusion or for preparation of other productsare screened for presence of hepatitis B antigenwith use of "Antibody Hepatitis B Surface Antigen ."There are 34 whole blood products and 45 diagnosticsubstances, 41 of which are for blood grouping and typing. Among blood products are four human serumglobulins : Immune Globulin for general prophylactic useand Measles, Mumps and Tetanus Immune Globulins .Of the approximately 5,000 blood banks in the country,at least 250 operate under federal license and are inspectedeach year .In addition to preventing hepatitis, research in theblood program has continued to be vital . When, for examplethe military planned to stock pile large amountsof albumin in caves for emergency use, Finlayson et al.developed assays to detect the effect of long-term storageon the albumin . 64,65Aronson made fundamental observations on the proteolyticnature of the conversion of human prothrombinto thrombin . 66,67Coral Snake Venoms and AntiveninAbout 50 species of coral snakes inhabit southern NorthAmerica, Central America, and South America . Theirneurotoxic and hemolytic venoms may be fatal to man,hence antivenom is needed . Cohen, Berkeley, andSeligmann, studied the serological relationship betweensix coral snake venoms from North, Central, and SouthAmerica . 68 Micrurus frontalis venom, which elicited themost cross-reactive neutralizing antibody of the venomstested, would be the most suitable for the production ofpolyvalent coral snake antivenin . Two other antivenomsare licensed-Crotalidae polyvalent and Latrodectusmactans .V . General Standards for BiologicsSterility TestingSterility testing is essential to assure freedom frombacterial contamination in biological products . At thebeginning, the culture medium was dextrose meat infusionbroth that was used in the Smith fermentation tubeand temperature of incubation was 37°C . 69In the early 1940s, Brewer or Linden fluidthyoglycollate medium was prescribed . It promotedaerobic and anaerobic growth and neutralized the in-hibitory action of mercurial preservatives . Reports thatthe Eh indicator, methylene blue, inhibited growth of afew contaminants prompted an investigation of growthpromoting activity of each ingredient in the media . Alarge number of bacterial contaminants and twotemperatures of incubation, 37 °C and 25°C, were used .Resazurin replaced methylene blue . With one slightchange, reduction in 1-cystine from 0 .75 g to 0 .5 g/liter,the medium developed in 19467° has remained one of theofficial media for sterility testing . It was adopted by theU .S . Pharmacopeia and internationally by the WorldHealth Organization .Another important change was the specification of alower incubation temperature of 30° - 32°C or the use oftwo temperatures 37 ° C and 25°C . The reason for therevision was that some bacteria isolated from wholeblood or plasma that had caused a severe reaction, attimes fatal, would not grow at the incubationtemperature of 37 ° C but would grow at 4°C . 71Pyrogenicity TestingIn the early 1940s, the increasing use of intravenoustherapy, particularly blood, plasma and other blood products,drew attention to the role of bacterial contaminantsas the cause of fever reactions (pyrogenicity) .Probey and Pittman studied the pyrogenicity of 28bacteria isolated from plasma . 72 All were capable of inducingfever in rabbits, but reaction varied both quantitativelyand qualitatively . This study, which also showedthat growth in serum enhanced pyrogenicity and thatthe number of bacteria should not exceed 5000 per ml .before filtration to avoid pyrogenicity, contributed to thepromulgation of the rabbit pyrogen test .The Limulus test for endotoxin (pyrogen), shown byCooper et al . to be satisfactory for radiopharmaceuticalsand biologics 73 was adopted officially." Endotoxin affectscoagulation of the lysate of amebocytes of thehorseshoe crab, Limulus polyphemus .MethodsFreeze-Drying . About 1937, NIH purchased its firstequipment for freeze-drying bacterial cultures . Thismethod has been invaluable for references and standardsin the control of biologics, also for certain products .Seligmann and Farber further enhanced its usefulness byexamining the influence of residual moisture and recommendingtechniques . 74Mice . To obtain reproducible results in mouse potencyassays of vaccines, the mouse strain used is very important. Lajos Csizmas showed a correlation between theprotective response to tetanus toxoid and pertussishistaminesensitizability (HS) of the mouse strain ; Pittmanreported similar results for pertussis vaccine . 75 Themouse HSFS/N line strain was selectively bred for highHS from the N:NIH(SW) strain by Manclark et al. 76Safety (oncogenicity)1 . Biologics showed no evidence of oncogenicity . In the1960s, oncogenic screening of biologic products was initiatedunder NIH contracts to DBS . All bacterial andviral vaccines, toxins, toxoids, and venoms as well as the66

seven preservatives used in these products were testedunder subcontracts for oncogenicity in animals . Theresults were negative .2 . Mineral oil adjuvants may be hazardous. In 1966,DBS initiated a series of studies under contract toevaluate the safety of mineral oil adjuvants . At thattime, there was much interest in use of oil adjuvants inallergens and other products . Untoward reactivity oftetanus toxoid containing mineral oil had been reportedin 1965 by MacLennan et al . 47 a The 1972 final report byMurray, Cohn, and Hardegree states "these data suggestthat the use of mineral oil adjuvants in human populationsmay be hazardous and that they should not berecommended for general use in man." 77IV . SummaryThe unfolding of advancements in knowledge has madepossible the development of biologics products (and theirregulation) that are applicable to the prevention, treatment,or cure of diseases or injuries of man . Graduallythere was understanding of the specific immunizing antigensof vaccines and their relation to human efficacy,and of the groups and types of blood that make possibletreatment with blood and blood products .Benefits are exemplified by the control of infectiousdiseases by routine vaccination with toxoid and viral vaccines,also other vaccines as indicated, and life savingtreatment with blood expanders : whole blood and bloodderivative . Millions and millions of lives have beensaved .The purpose of the Public Health Service is to contributeto public health . The Regulations of BiologicalProducts has served well . 78The outstanding progress was made possible not onlyby in-house scientists but by cooperation of scientistsworldwide and of industry, all in a research context .It is not possible to recognize all of the contributors .Omission does not diminish in any way theircontributions .Appendix AExcerpts from the Public Health Service Act,As AmendedTitle III - General Powers and Duties of the PublicHealth ServicePart F - Biological ProductsRegulation of Biological Products42 U.S.C . 262Sec . 351 . (a) No person shall sell, barter, or exchange, oroffer for sale, barter, or exchange in the District of Columbia,or send, carry, or bring for sale, barter or exchangefrom any State or possession into ahy other Stateor possession or into any foreign country, or from anyforeign country into any State or possession, any virus,therapeutic serum, toxin, antitoxin, or analogous product,or arsphenamine or its derivatives (or any othertrivalent organic arsenic compound), applicable to theprevention, treatment, or cure of diseases or injuries ofman, unless (1) such virus, serum, toxin, antitoxin, orother product has been propagated or manufactured andprepared at an establishment holding an unsuspendedand unrevoked license, and prepare such virus, serum,toxin, antitoxin, or other product for sale in the Districtof Columbia, or for sending, bringing, or carrying fromplace to place aforesaid ; and (2) each package of suchvirus, serum, toxin, antitoxin, or other product is plainlymarked with the proper name of the article containedtherein, the name, address, and license number of themanufacturer, and the date beyond which the contentscannot be expected beyond reasonable doubt to yieldtheir specific results . The suspension or revocation of anylicenses shall not prevent the sale, barter, or exchange ofany virus, serum, toxin, antitoxin, or the productaforesaid which has been sold and delivered by thelicensee prior to such suspension or revocation, unlessthe owner or custodian of such virus, serum, toxin, antitoxin,or other product aforesaid has been notified bythe Secretary not to sell, barter or exchange the same .(b) No person shall falsely label or mark any packageor container of any virus, serum, toxin, antitoxin, orother product aforesaid ; nor alter any label or mark onany package or container of any virus, serum, toxin, antitoxin,or other product aforesaid so as to falsify suchlabel or mark .(c) Any officer, agent, or employee of the Departmentof Health, Education, and Welfare, authorized by theSecretary for the purpose, may during all reasonablehours enter and inspect any establishment for the propagationor manufacture and preparation of any virus,serum, toxin, antitoxin, or other product aforesaid forsale, barter, or exchange in the District of Columbia, orto be sent, carried, or brought from any State or possessioninto any other state or possession or into anyforeign country, or from any foreign country into anyState of possession .(d) Licenses for the maintenance of establishments forthe propagation or manufacture and preparation ofproducts described in subsection (1) of this section maybe issued only upon a showing that the establishmentand the products for which a license is desired meetstandards, designed to insure the continued safety, purity,and potency of such products, prescribed in regulations,and licenses for new products may be issued onlyupon a showing that they meet such standards . All suchlicenses shall be issued for the maintenance of establishmentsfor the propagation or manufacture and preparation,in any foreign country, or any such products forsale, barter, or exchange in any State or possession shallbe issued upon condition that the licensees will permitthe inspection of their establishments in accordance withsubsection (c) of this section .(e) No person shall interfere with any officer, agent,or employee of the Service in the performance of anyduty imposed upon him by this section or by regulationsmade by authority thereof .67

(f) Any person who shall violate, or aid or abet inviolating, any of the provisions of this section shall bepunished upon conviction by a fine not exceeding $500or by imprisonment not exceeding one year, or by bothsuch fine and imprisonment, in the discretion of thecourt .(g) Nothing contained in this Act shall be construed asin any way affecting modifying, repealing, or supersedingthe provisions of the Federal Food, Drug, and CosmeticAct (U .S.C ., 1940 edition, Title 21, ch .9) .Preparation of Biological Products42 U.S.C . 263Sec . 352 . (a) The Service may prepare for its own useany product described in section 351 and any productnecessary to carrying out any of the purposes of section301 .(b) The service may prepare any product described insection 351 for the use of other Federal departments oragencies, and public or private agencies and individualsengaged in work in the field of medicine when suchproduct is not available from establishments licensedunder such section .Margaret Pittman, Ph.D .Dr . Margaret Pittman was born in 1901 in PrairieGrove, Arkansas . Following her graduation from HendrixCollege with an A.B . degree in 1923, she earned anM .S . and Ph.D . in Bacteriology from the University ofChicago in 1929 . Before coming to NIH she worked atthe Rockefeller Institute from 1928 to 1934 . From 1936until 1971 she was a Microbiologist at NIH, in NMI andDBS . In 1957 she became Chief of the Laboratory ofBacterial Products-one of the first women to becomeChief of an NIH laboratory . While at NIH Dr . Pittmanhas been recognized and honored for her research contributionsin respiratory infections, meningitis and conjunctivitisand the standardization of bacterial vaccinesand toxins, with emphasis on pertussis as a toxinmediated disease after she retired .In 1971 she officially retired from Federal service, butshe has continued as a Guest Worker in the Division ofBiologic Standards and the FDA Center for Drugs andBiologics located at NIH .References1 . O'Hern, E . M . 1986 . Anna Wessels Williams . In Profiles of PioneerWomen Scientists . Washington, D . C . Acropolis Books, Ltd ., pp . 33-452 . Stimson, A . M . 1938 . A Brief History of Bacteriological Investigationsof the United States Public Health Service . Public Health ReportsSupplement No . 141, Washington, DC : U . S . Government Printing Office,p . 163 . The Coroner's Verdict in the St . Louis Tetanus Cases . 1901 . NewYork Medical Journal 74 : 977 ; Special Article : Fatal Results fromDiphtheria Antitoxin . 1901 . Journal of the American Medical Association37 : 1260-61 ; Minor Comments : Tetanus from Anti-diphtheriaSerum . Ibid, pp . 1255-564 . Legislative History of the Regulations of Biological Products . Divisionof Biologics Control, National Institutes of Health, 1968.5 . Biological Products : Part 273-Biological Products : Transfer ofRegulations . Federal Register, vol 37, No . 154 . Wednesday, August 9,19726 . Murray, R . 1968 . The Division of Biologics Standards . Public HealthService Publications No . 17447 . Rosenau, M . J . 1903 . The Antiseptic and Germicidal Properties ofGlycerin . Hygienic Laboratory Bulletin No . 16 Washington, DC : U . S .Government Printing Office, 30 pp8 . Rosenau, M . J . 1927 . Preventive Medicine and Hygiene, 5th ed .,New York : D . Appleton and Company, pp 17-189 . Rosenau, M . J . 1905 . The Immunity Unit for StandardizingDiphtheria Antitoxin (Based on Ehrlich's Normal Serum) . HygienicLaboratory Bulletin No . 21 Washington, DC : U . S . Government PrintingOffice, 92 pp10 . Rosenau, M . J ., and Anderson, J . F . 1908 . Standardization ofTetanus Antitoxin . (An American Unit Established Under the Authorityof the Act of July 1, 1902) . Hygienic Laboratory Bulletin No . 43Washington, DC : U . S . Government Printing Office 55 pp11 . Food and Drug Administration . 1987 . CFR 21 Part 600Washington, DC : U . S . Government Printing Office12 . Establishments and Products, Licensed Under Section 351 of thePublic Health Service Act . Division of Biologics Standards, National Institutesof Health, 1972 . DHEW Publication No . (NIH)72-20813 . Baer, H . ; Godfrey, H . ; and Maloney, C . J . 1970 . The Potency andAntigen E Content of Commercially Prepared Ragweed Extracts . Journalof Allergy 45 : 347-5414 . Baer, H . ; Liu, T .-Y . ; Anderson, M . C . 1979 . Protein Componentsof Fire Ant Venom (Solenopsis invecta). Toxicon 17 : 397-40515 . Baer, H . 1986 . Chemistry and Immunochemistry of PoisonousAnacardiaceae. Clinics in Dermatology, 4 : 152-259 .16 . Report of the Committee on Standardization : I . A Method ofEvaluating Skin Test Responses . 1971 . Annals of Allergy 29 : 30-34 .17 . Branham, S . E . 1956 . Milestones in the History of Meningococcus .Canadian Journal of Microbiology 2 : 175-88 .18 . Pittman, M . ; Branham, S . E . ; Sockrider, E . M . 1938 . A Comparisonof the Precipitation Reaction in Immune Serum Agar Plateswith Protection of Mice by Antimeningococcus Serum . Public HealthReports 53 : 1400-8 .19. Branham, S . E ., and Pittman, M . 1940 . A RecommendedProcedure for the Mouse Protection Test in the Evaluation of AntimeningococcusSerum . Public Health Reports 55 : 2340-2346 .20 . Pittman, M . 1943 . Mouse Protective Values of AntimeningococcusSerum in Comparison with Precipitation in Immune Serum Agar Plates .Public Health Reports 58 : 139-42 .21 . Rosenau, M . J ., and Anderson, J . F . 1908 . Further Studies UponAnaphylaxis with Special Reference in the Antibodies Concerned .Hygienic Laboratory Bulletin No . 45 Washington, DC : U . S . GovernmentPrinting Office, 65 pp .68

22 . Bengtson, I . A . 1924 . Studies on Organisms Concerned as CausativeFactors in Botulism . Hygienic Laboratory Bulletin No . 136 Washington,DC : U . S . Government Printing Office, 90 pp .23 . Bengtson, I . A ., and Stewart, S . E . 1931 . The Official UnitedStates and International Unit for Standardizing Gas Gangrene Antitoxin(histolyticus) . Public Health Reports 51 : 1263-72 .24a . Bengtson, I . A . 1934 . The Standardization of Gas Gangrene (perfringens)Antitoxin . Public Health Reports 49 : 525-29 .24b . Bengtson, 1 . A. 1934 . The Official United States and InternationalUnited for Standardizing Gas Gangrene Antitoxin (Vibrion septique) .Public Health Reports 49 : 1557-69 .25 . Bengtson I . A ., 1936 . The Official United States and InternationalUnit for Standardizing Gas Gangrene Antitoxin (oedematiens) . PublicHealth Reports 51 : 266-75 .26 . Bengtson, Ida A ., and Stewart, S. E . 1939 . Studies on Standardizationof Gas Gangrene Antitoxin (sordellit) . Public Health Reports 54 :1435-41 .27 . Biological Substances . International Standards . Reference Preparationsand Reference Reagents . 1975 . Geneva, Switzerland : World HealthOrganization .28 . Feeley, J . C ., and Pittman, M . 1965 . Laboratory Assays of CholeraVaccine Used in the Field Trials of East Pakistan . Lancet 1 : 449-50 ..29 . Mosely, W . H . ; Feeley, J . C . ; and Pittman, M . 1971 . The Interrelationshipsof Serological Responses in Humans, and the Active MouseProtection Test to Cholera Vaccine Effectiveness . In Symposium Series,Immunobiology Standards, vol . 15, Basel/New York : A G . Karger, pp .185-196 .30 . Pittman, M . 1976. History, Benefits and Limitations of Pyrex GlassParticles Opacity References . Journal of Biological Standardization 4 :115-25 .31 . Pittman, M . 1956. Pertussis and Pertussis Vaccine Control . Journalof the Washington Academy of Sciences 46 : 234-43 .32 . Pittman, M . 1958 . Variations du Pouvoir Protecteur des DifferentsVaccins Anticoquelucheux leur Rapport avec la Protection de L'etreHumain . Revue d'Immunologie 22 : 308-22 .33 . Pittman, M ., and Cox, C . B . 1965 . Pertussis Vaccine Testing forFreedom-from-Toxicity . Applied Microbiology 13 : 447-56 .34 . Pittman, M . 1979 . Pertussis Toxin : The Cause of the Harmful Effectsand Prolonged Immunity of Whooping Cough . A Hypothesis .Reviews of Infectious Diseases 1 : 401-12 .35 . Pittman, M . ; Furman, B . L . ; and Wardlaw, A . C . 1980 . Bordetellapertussis Respiratory Tract Infection in the Mouse : PathophysiologicalResponses . Journal of Infectious Diseases 142 : 56-66 .36 . Pittman, M . 1984 . The Concept of Pertussis as a Toxin-MediatedDisease . Pediatric Infectious Disease 3 : 467-85 .37 . Pittman, M . 1986 . Neurotoxicity of Bordetella Pertussis. Neurotoxicology7 : 53-68 .38 . Pittman, M ., and Bohner, H . J . 1966 . Laboratory Assays of DifferentTypes of Field Trial Typhoid Vaccines and Relationship to Efficacyin Man . Journal of Bacteriology 91 : 1713-23 .39. Wong, K . H . ; Feeley, J . C . ; and Pittman, M . 1972 . Effect of a VI-Degrading Enzyme on Potency of Typhoid Vaccine in Mice . Journal ofInfectious Diseases . 125 : 360-65 .40 . Chaparas, S . D . ; Sheagren, J . N . ; DeMeo, A . ; and Hedrick, S .1970 . Correlation of Human Reactivity with Lymphocyte TransformationInduced by Mycobacterial Antigens and Histoplasmin . AmericanReview of Respiratory Diseases 101 : 67-73 .41 . Chaparas, S . D . ; Good, R . C . ; and Janicki, B . W . 1975 .Tuberculin-Induced Lymphocyte Transformation and Skin Reactivity inMonkeys Vaccinated or Not Vaccinated with Baccile Calmette-Guerin,then Challenged with Virulent Mycobacterium tuberculosis . AmericanReview of Respiratory Disease. 112 : 43-47 .42 . Chaparas, S . D . ; Fuller, V . J . ; and Seligmann, E . B . 1972 .Laboratory Procedures for Potency Testing of Blastomycin,Histoplasmin, and Coccidioidin . Proceedings of the Society for ExperimentalBiology and Medicine 140 : 556-59 .43 . Barile, M . F. 1968 . Mycoplasma and Cell Cultures . National CancerInstitute Monograph 29 : 201-4 .44 . Barile, M . F. ; Hopps, H . E . ; Grabowski, M . W . ; Riggs, D . B . ;Delgiudice, R . A . 1973 . The Identification and Sources of MycoplasmasIsolated from Contaminated Cell Cultures . Annals of the New YorkAcademy of Sciences 225 : 251-64 .45 . Barile, M . F. ; Yoshida, H . ; Chandler, D . K . F . ; Grabowski, M .W . ; Harasawa, R . 1982 . The Hamster Immunization-Protection-Challenge-Potency Assay for Evaluation of Mycoplasma pneumoniaeVaccine . In Seminars in Infectious Disease. Bacterial Vaccines, vol . 4,ch . 28 ., R . B . Robbins, J . C . Hill, J . C . Sadoff, eds ., New York :Theme-Stratton, pp . 202-11 .46 . Barile, M . F . ; Kolb, R . W . ; and Pittman, M . 1971 . United StatesStandard Diphtheria Toxin for the Schick Test and the ErythemaPotency Assay for the Shick Test Dose . Infection and Immunity 4 :295-306 .47a . MacLennan, R . ; Schofield, F . D . ; Pittman, M . ; Hardegree, M . C . ;and Barile, M . F . 1965 . Immunization Against Neonatal Tetanus inNew Guinea . 1 . Antitoxin Response of Pregnant Women to Adjuvantand Plain Toxoids . Bulletin of the World Health Organization . 32 :683-97 .47b . Hardegree, M . C . ; Barile, M . F . ; Pittman, M . ; Schofield, F . D . ;MacLennan, R . ; and Kelly, A . 1970 . 2 . Duration of Primary Responsesto Adjuvant Tetanus Toxoids and Comparison of Booster Responses toAdjuvant and Plain Toxoids . Ibid ., 43 : 439-51 .. 47c . Barile, M . F . ; Hardegree, M . C ; and Pittman, M . 1970 . 3 . TheToxin-Neutralization Test and the Response of Guinea-Pigs to the Toxoidsas Used in the Immunization Schedules in New Guinea . Ibid ., 43 :453-59 .47d . Hardegree, M . C . ; Barile, M . F. ; Pittman, M . ; Maloney, C . J . ;Schofield, F . ; and MacLennan, R . 1970 . 4 . Comparison of Tetanus AntitoxinTitres Obtained by Haeaggultination and Toxin Neutralization inMice . 1970 . Ibid ., 43 : 461-68 .47e . Pittman, M . ; Kolb, R . W . ; Barile, M . F . ; Hardegree, M . C. ;Seligmann, E . B ., Jr . ; MacLennan, R . ; and Schofield, F . D . 1970 . 5 .Laborator y Assayed Potency of Tetanus Toxoids and Relationship toHuman Antitoxin Response . Ibid ., 43 : 469-78 .48 . Fuller, V . J ., and Kolb, R . W . 1968 . Comparison of Titrations onthe Chorioallantoic Membrane of Chick Embryos with the RabbitScarification Technique for the Potency Assay of Smallpox Vaccines .Applied Microbiology 16: 458-62 .49 . Hornibrook, J . W ., and Gebbard, W . H . 1951 . Dried SmallpoxVaccine . Public Health Reports 66 : 38-43 .50 . Habel, K ., and Wright, J . T . 1948 . Some Factors Influencing theMouse Potency Test for Rabies Vaccine . Public Health Reports 63 :44-55 .51 . Fitzgerald, E . A . ; Green, O . L . ; and Seligmann, E . B ., Jr . 1974.Rabies Vaccine Potency Testing : A Comparison Between the Antibody-Binding Test and the NIH Test . In Symposia Series in ImmunologicalStandardization, vol . 21, Basel : S . Karger, A . G ., pp . 300-07 .52 . Murray, R . 1964 . Biologicals for the Control and Therapy of VirusDisease . Bacteriological Reviews 28 : 493-96 .53 . Eddy, B . E . ; Borman, G . W . ; Grubbs,G . E . ; and Young, R . D .1962 . Identification of the Oncogenic Substance in Rhesus Monkey CellCultures as Simian Virus 40 . Virology 17 : 65-75 .54 . Kirschstein, R. L ., and Clark, G . 1966 . Reproducibility andNeurovirulence Test . In Immunological Standardization, vol 2 ., Basel :S . Karger A . G ., pp . 141-50 .55 . Kirschstein, R. L . 1984 . I . The Virus and Poliomyelitis Vaccine . InNIH: An Account of Research and Its Laboratories and Clinics. D .Stetten, Jr ., and W . T . Carrigan, eds ., New York : Academic Press, pp .380-86 .69

56 . Meyer, H. M . Jr . ; Hostettler, D. D. Jr . ; Bernheim, B . C . ; Rogers,N . G. ; Lambin, P., Chassary, A., Labusquiere ; and Smadel, J . E.1964 . Response of Volta Children to Jet Inoculation of Combined LiveMeasles, Smallpox, and Yellow Fever Vaccine. Bulletin of the WorldHealth Organization 30: 783-94 .57 . Parkman, P . D. ; Meyer, H. M., Jr . ; Kirschstein, R. L. ; Hopps, H .E. 1966 . Attenuated Rubella Virus . I . Development and LaboratoryCharacterization . New England Journal of Medicine 275 : 569-74 .58 . Meyer, H. M., Jr . ; Parkman, P . D . ; Panos, T. C. 1966 . AttenuatedRubella Virus . II . Production of Experimental Live-Virus Vaccineand Clinical Trial . New England Journal of Medicine 275 : 375-80 .59 . Stewart, G. L . ; Parkman, P . D. ; Hopps, H . E . ; et al . 1967 .Rubella-Virus Hemagglutination-Inhibition Test . New England Journalof Medicine 276 : 554-57 .60. Eddy, B . E. 1947 . A Study of Influenza Vaccine by a Serum VirusNeutralization Test and Active Immunization . Journal of Immunology57 : 195-202 .61 . Bengtson, I . A., and Topping, N. H. 1941 . The Specificity of theComplement Fixation Test in Epidemic Typhus Fever Using RickettsialAntigens . Public Health Reports 56 : 1723-27 .62. Murray R. ; Diefenbach, W. C. L. ; Ratner, F . ; et al. 1954 . 2. Confirmationof Carrier State by Transmission Experiments in Volunteers .Journal of the American Medical Association 154 : 1072-74 .63 . Murray, R. 1955 . Viral Hepatitis . Bulletin of the New YorkAcademy of Medicine 31 : 341-58 .64 . Finlayson, J . S . ; Suchinsky, R. T. ; and Dayton, A. L . 1960 . Effectsof Long-Term Storage on Human Serum Albumin . 1 . Chromatographicand Ultracentrifugal Aspects . Journal of Clinical Investigation 39 :1837-40 .65 . Finlayson, J . S . 1965 . Effects of Long-Term Storage on HumanSerum Albumin . II . Follow-Up of Chromatographically and UltracentrifugallyDetectable Changes . Journal of Clinical Investigation 44 :1561-65 .66 . Aronson, D. L . 1964 . N-Terminal Amino Acid Changes During theconversion of Human Prothrombin to Thrombin . Proceedings of the IXCongress of the International Society of Hematology, 2 : 309-14 .67 . Aronson, D. L., and Menache, D. 1966 . Chromatographic Analysisof the Activations of Human Prothrombin with Thrombokinase .Biochemistry 5 : 2635-40 .68 . Cohen, P . ; Berkeley, W. H. ; and Seligmann, E. B., Jr . 1971 . CoralSnake Venoms . In Vitro Relation of Neutralization and PrecipitatingAntibodies . The American Journal of Tropical Medicine and Hygiene20 : 646-49 .69 . Bengtson, I . A. 1918 . The Nature of Contamination of BiologicalProducts . Hygienic Laboratory Bulletin No . 112 Washington, DC : U.S . Government Printing Office, pp . 18-23 .70 . Pittman, M. 1946 . A Study of Fluid Thioglycollate Medium for theSterility Test . Journal of Bacteriology 51 : 19-32 .71 . Pittman, M. 1953 . A Study of Bacteria Implicated in TransfusionReactions and of Bacteria Isolated from Blood Products . Journal ofLaboratory and Clinical Medicines 42 : 273-88 .72 . Probey, T. F., and Pittman, M. 1945 . The Pyrogenicity of BacterialContaminants Found in Biologic Products . Journal of Bacteriology 50 :397-411 .73 . Cooper, J . F . ; Hochstein, H. D. ; and Seligmann, E . B., Jr . 1972 .The Limulus Test for Endotoxin (Pyrogen) in Radio-Pharmaceuticalsand Biologicals . Bulletin of the Parenteral Drug Association 26 : 153-62 .74 . Seligmann, E. B., and Farber, J . F . 1971 . Freeze Drying andResidual Moisture . Cryobiology 8 : 138-144 .75 . Pittman, M. 1967 . Mouse Strain Variation in Response to PertussisVaccine and Tetanus Toxoid . In Proceedings of International Symposiumon Laboratory Animals 1966 . In Symposium Series ImmunobiologyStandards, vol . 5 ., Basel: Karger A. G., pp . 161-66 .76 . Manclark, C. R . ; Hansen, C . T. ; Treadwell, P . E. ; and Pittman,M. 1975 . Selective Breeding to Establish a Standard Mouse for PertussisVaccine Bioassay . Journal of Biological Standardization 3: 353-63 .77 . Murray, R. ; Cohen, P. ; and Hardegree, M. C. 1972 . Mineral OilAdjuvants : Biological and Chemical Studies . Annals of Allergy 30:146-51 .78 . Banta, P. M. 1958 . Federal Regulations of Biologics Applicable tothe Diseases of Man. Washington, DC : Department of Health, Education,and Welfare (General Council, DHEW . An Address Before theNew York State Bar Association . New York, NY, January 29, 1958) .70

TREASURY DEPARTMENT .Public Health and \larine-Hospital Service of the United States .NVALTER Nh'YINIax, Surgeon-General .HYGIENIC LABORATORY .-BULLETIN NO . 29 .APRIL, 1906 .A STUD' OF THE CAUSE OF SUDDEN DEATHFOLLOWIV'r THI; INJECTION OFHORSE SERUM .BYM. J. ROSENALTANY)JOHN E . aNDF,FZSON .WASHINGTON :GOVERNMENT PRINTING OFFICE,I9o6 .71

TABLE OF CONTENTS .Page .Introduction____________________,_________________________________ .________ 71 . Control experiments___-------------- 91--The action of normal horse serum up - nial guinea pigs_------------ 91-'he action of antitoxic horse serum upon normal guinea pigs_ _ _ _ _ _ _ _ _ _ 10G-I . Iiorse serum is poisonous to a "treated" or "used" guinea pig_ _ _ _ _ _ _ 11The symptoms caused by the injection of horse serum into a susceptibleguineapig__________14The poisonous action of horse. serum acts upon the respiratory center_ _ 141/ 111 . The toxic action bears no relation to diphtheria_-------------------------- 16The poison is nottoxon_---------- _-------------------------------- 16_.-Guinea pigs can not he rendered susceptible with previous infections withthe B . diphtheii_18/-''Diphtheria toxine can not render guinea pigs susceptible_ 19Diphtheria antitoxin plays no role in this action_ 21IV . The toxicprinciple_25Is the poison specific?_____________________________________________ 25Are other blood serums equally toxic?_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 28There.lationofliemolysis__.______,___31The relation of precipitin to the toxic action_ 35The effect of heat upon the toxicity of horse serum_-------------------- 38L_--The toxic principle in horse serum is filterable through porcelain_-------- 38Drying does not injure the toxic principle------ 39The toxic principle can not be separated by precipitation and dialysis_ _ _ 39The effect of various chemical substances upon the toxic principle ofhorse serum._39The influence of antiseptics__ .._42The effect of old horse serum upon susceptible guinea pigs_ _ _ _ _ _ _ . _ _ _ _ _ 42The effect of X rays upon the toxic principle- 43V . The influence of time------------------------------------------------- 45(~- The time necessary to render a guinea pig susceptible_ 45-- The guinea pig remains susceptible a very long time--- 45VI . Dosage as a factor---------------------------------------------------- . 47,~ The minimal amount of horse serum necessary to render a guinea pigsusceptible47The minimal amount of horse serum necessary to poison a susceptibleguinea 48The influence of large quantities in rendering guinea pigs susceptible . 50VII . The sensitizing substance______________________________________________ 51Guinea pigs may be sensitized with precipitated and dialyzed serum_ _ _ 51Drying does not injure the sensitizing substance_ _ _ _ _ _ _ _ _ _ _ _ : _ _ _ _ _ _ _ _ _ 51Small quantities of horse serum may render guinea pigs more susceptiblethan large quantities_._____52The sensitizing substance is not free in the blood serum_ 54The effect of heat upon the sensitizing substance_ 5472

VIII . . : . . . . . . .>56IX .Other 57. .____ .______ .__ .__ . .____ .______ . .____ . 59ActiveGuineaserum 59Passive . ._ .____ 62TheofThe.guinea . 64X .._________ .____.67Guinea 67Guinea 69May 70XI . . 73XII . . . . . . 75XIII . . . . . 77XIV . 91

A STUDY OF THE CAUSE OF SUDDEN DEATH FOLLOW-ING THE INJECTION OF HORSE SERUM .By A11LTON J . ROSENAU,Passed Assistant Surgeon, Director Hygienic Laboratory, l7 . S. Public Health and Marine-Hospital Service.andJOHN F. ANDERSON,Passed Assistant Surgeon, Assistant Director Hygienic Laboratory, I7 . S . Public Health andMarine-Hospital Service .It has long been known that the blood of certain animals ispoisonous when transfused or injected into certain other species .Many instances might be cited showing that the blood serum ofone animal has poisonous properties when injected into an animalof another species . But the blood serum of the horse apparentlylacks such poisonous action . Very large quantities of the bloodserum of the horse may be injected into man, rabbits, guinea pigs,and many other animals without serious inconvenience, exceptoccasionally a slight reaction at the site of inoculation .In a certain proportion of cases the injection of horse serum intoman is followed by urticarial eruptions, joint pains, fever, swellingof the lymph nodes, edema, and albuminuria . This reaction, whichappears after an incubation period of eight to thirteen days, hasbeen termed by Pirquet and Schick the "serum disease ."In exceptional instances sudden death has followed an injection ofhorse serum in man .These studies were taken up in October, 1905, in order to throwlight upon the cause of this unfortunate accident . We have shownthat ordinarily horse serum is a comparatively bland and harmlesssubstance when injected into certain animals ; but these animals maybe rendered so susceptible that an injection of horse serum mayproduce sudden death or severe symptoms . For example, largequantities of horse serum may be injected subcutaneously or into theperitoneal cavity of a guinea pig without apparently causing theanimal the least inconvenience . However, if a guinea pig is injectedwith a small quantity, say 2 -1 6 c . c ., of horse serum and after the74

8.expiration of a certain interval is again injected with horse serumthe result will probably be fatal . The first injection of horse serumhas sensitized the animal in such a way as to render it very susceptible to a toxic principle in horse serum. It is probable that whenthe guinea pig is injected with the first, or sensitizing, quantity ofserum the strange proteid contained in the horse serum develops inthe body of the guinea pig " antibodies " which, when brought intocontact with more horse serum given at a second injection, produceeither a union or a reaction, which causes the toxic action .A certain time is necessary to elapse between the first and secondinjections of horse serum before this toxic action is able to manifestitself . This "period of incubation" is from ten to twelve days,and corresponds suggestively with the period of incubation of theserum disease which Pirquet and Schick place at eight to thirteen days .Guinea pigs may be sensitized with exceedingly small quantitiesof horse serum. In most of our work we used quantities less thanyJa c . e . and we found in one instance that c . c . of horseserum was sufficient to render a guinea pig susceptible .It also requires very small quantities of horse serum, when givenin a second injection, to produce poisonous symptoms . One-tenthc . c . injected into the peritoneal cavity is sufficient to cause the deathof a half-grown guinea pig . One-tenth c . c . of horse serum injectedsubcut,aneously is sufficient to produce. serious symptoms . The factthat this toxic action may be developed by such small quantitiesof serum and the fact that exceedingly small quantities are sufficientto produce symptoms and death upon a second injection, a prioriplaces both the sensitizing and the toxic principle in the horse serumin the "haptin group" of substances in the sense used by Ehrlich .A still further indication that, the side-chain theory in its broadestsense may be applicable is the. further fact that immunity may beproduced against the toxic action by multiple injections of theserum .While at first we thought that diphtheria antitoxin had somerelation to this action, we, are now able to state positively that ithas nothing whatever to do with the poisonous action of horse serum ;further, that diphtheria antitoxin in itself is absolutely harmless .The toxic action which we have studied is caused by a principle innormal horse serum and is entirely independent of the antitoxicproperties of the serum .75

SUMMARY AND CONCLUSIONS.Normal horse serum, when injected into the peritoneal cavity of anormal guinea pig . produces no symptoms . When injected subcutaneouslythere may result at most a slight local reaction consisting ofswelling and edema ., which gradually disappears .Antitoxic horse serum is equally harmless for normal guinea pigs .Horse serum is, however, poisonous to a guinea pig which has previouslybeen injected with horse serum . Tne "period of incubation"or time necessary to elapse between the first and second injection isabout ten days . Under these circumstances, horse serum is poisonouswhether injected subcutaneously or into the peritoneal cavity .The first injection of horse serum renders the guinea pig susceptible .The symptoms caused by the injection of horse serum into a susceptibleguinea pig are respiratory embarrassment, paralysis, and convulsions,followed by death . The symptoms come on usually withinten minutes after the injection, and when death results it usuallyoccurs within one hour, frequently in less than thirty minutes, andsometimes within a few minutes .The poisonous principle in horse serum appears to act upon therespiratory centers . The heart continues to beat long after respirationceases .The toxic action of horse serum bears no relation to diphtheria .The poison is not toxone . Guinea pigs can not be rendered susceptibleby previous infections with the B . diphtheria or by previousinjections with diphtheria toxine .It seems from our work, however, that guinea pigs first injectedwith a mixture of diphtheria toxine plus horse serum are more sensitiveto subsequent injections of horse serum than are guinea pigssensitized with a first injection of horse serum alone .DIPHTHERIA ANTITOXIN PLAYS NO PART IN THIS POISONOUS ACTIONAND IN ITSELF IS HARMLESS .As soon as we realized that the toxic principle in horse serumexerts its action in quantities so minute as to place it almost in thecategory of the ferments and, further, when we concluded fromour work that this toxic principle is doubtless one of those highlyorganized and complex proteid substances belonging to the "haptingroup" in the sense used by Ehrlich, we recognized how futile itwould be with present methods to attempt to isolate this substance .(9I)76

92Nevertheless we devoted much time and study to the relation ofthis toxic principle to various chemical, physical, and electricalinfluences . The practical importance of eliminating or neutralizingthis toxic principle in horse serum is at once evident .It is probable that when the strange proteid is introduced intothe guinea pig it causes a reaction resulting in a production of"antibodies," so that when a second injection of horse serum isgiven there is probably either a union or a reaction between theantibodies and a substance in the horse serum which produces thepoisonous effect .This poisonous principle is quantitatively specific ; that is, guineapigs treated with horse serum are rendered somewhat susceptibleto the subsequent injection of the serum of another animal . Guineapigs treated with the serum of another animal are slightly sensitiveto the toxic action of horse serum .Guinea pigs treated with the serums of various animals and subsequentlyinjected, are much more susceptible to homologous serumsthan to heterologous serums .This poisonous action has no relation to hemolysis . Our workproves that blood serum may contain an acute poison entirely independentof any hemolytic action Normal horse serum has no lyticpower upon the red corpuscles of the normal guinea pig .This poisonous action has no relation to the specific albuminousprecipitins .The poisonous principle in horse serum is not affected by a temperatureof 60° C . for 6 hours, but it is destroyed at 100° C . for 15minutes .The poisonous principle is filterable through porcelain, is notinjured by drying, and can not be separated by precipitation withammonium sulphate and subsequent dialysis .The following chemical substances do not oxidize, neutralize, orprecipitate the poisonous principle in horse serum : Butyric acid,permanganate of potash, citrate of soda, alcohol, succinic peroxideacid (alphozone), hydrogen dioxide, and ammonium sulphate . Thepresence of chloroform or trikresol (0 .4 per cent) does not interferewith this poisonous action .Serums eight years old are as toxic as those freshly separated .Exposure to X-rays does not affect the poisonous action of horseserum .It requires about 10 days after the first injection of horse serumfor a guinea pig to show susceptibility to a second injection . Aguinea pig remains susceptible a very long time, at least 160 days .As small a quantity as 1 , , 0Mc . c . of horse serum was sufficientin one instance to render a guinea pig susceptible . Quantities77

93varying from gJ-6 to ~ b c . c . almost invariably render guinea pigshighly susceptible when given in the toxine-antitoxine mixture .One-tenth c . c . of horse. serum injected into the peritoneal cavityof a susceptible guinea pig is sufficient to cause death . The samequantity inoculated subcutaneously may cause serious symptoms .There is some evidence to show that the sensitizing substance inhorse serum is the same as the poisonous substance . The sensitizingsubstance is not affected by precipitation with ammoniumsulphate and dialysis .Guinea pigs may be sensitized with horse serum that has beendried and redissolved .The sensitizing substance is not affected by a temperature of 60°C . for 6 hours .It is probable that small quantities of horse serum render a guineapig more susceptible than do large quantities . If this be true, it isdue, perhaps, to the fact that large quantities, owing to slow absorptionor prolonged reaction, partly immunize the guinea pig at thesame time that it is being sensitized .The sensitizing substance apparently is not free in the bloodserum of guinea pigs .An active immunity against this toxic principle may readily beestablished by repeated injections of horse serum, at short intervals,into a guinea pig . Although guinea pigs may be immunized activelyin this manner we have not yet succeeded in transferring this immunity in the blood serum or body juices to another guinea pig . Ittherefore appears that the immune bodies, if such exist against thetoxic action of horse serum, are not free in the blood and body juicescontrary to the case in diphtheria .Guinea pigs may be sensitized to the toxic action of horse serumby feeding them with horse serum or horse meat .The fact that guinea pigs may be rendered susceptible by the feedingof strange proteid matter opens an interesting question as towhether sensitive guinea pigs may also be poisoned by feeding withthe same serum given after a proper interval of time . If man canbe sensitized in a similar way by the eating of certain proteid substancesmay not this throw light upon those interesting and obscurecases in which the eating of fish, sea food, and other articles of diethabitually cause sudden and sometimes serious symptoms?The susceptibility to the toxic action of horse serum is transmittedhereditarily from the mother guinea pig to her young .These results upon the hereditary transmission of the susceptibilityto the poisonous action of horse serum in guinea pigs maythrow light upon the well-known hereditary tendency to tuberculosisin children born of a tuberculous parent . There. are certain analogiesbetween the action of tuberculosis and horse serum . Both78

94may produce a hypersusceptibility and also a certain degree ofimmunity . Now that we have proved that this hypersusceptibilityor anaphylactic action in the case of horse serum may be transmittedhereditarily in guinea pigs, may it not throw light upon the fact thattuberculosis "runs in families? "Demonstrations of the hereditary transmission of acquired charactersare comparatively rare in biology . While there are severalrecorded instances demonstrating that immunity to certain infectiousdiseases may be. transmitted from a mother to her young, yet, as faras we know, this is the first recorded instance in which hypersensitiveness,or anaphylaxis, has been experimentally shown to be transmittedfrom a mother to her young .Other albuminous substances, such as skimmed milk, peptone,hemoglobin, egg albumin, and vegetable proteids possess no poisonousaction upon guinea pigs sensitized with horse serum . Whetherguinea pigs are rendered susceptible to a subsequent injection withthe same albuminous matter with which they have been sensitizedwill be reported in a later paper .We believe that the substance which sensitizes the animal is identicalwith that which later poisons it . However, the substance mustfirst cause a reaction in the organism resulting in a production ofantibodies . We have found that small quantities of horse serum produce,after a definite period of incubation, a condition of anaphylaxis ;multiple or repeated injections produce immunity . We thereforepossess in horse serum a substance capable of causing both anaphylaxisand prophylaxis .It may be that man can not be sensitized in the same way that wehave shown to be the case with guinea pigs . Children have, in a numberof instances, been injected with antidiphtheric horse serum atshort and long intervals 'without, so far as we are aware, causingdeath . Certain serums, for example, the antitubercle serum ofMaragliano and the antirheuinatic serum of Menzer, are habituallyused by giving injections at intervals of days or weeks . In all suchcases of frequent and repeated injections the amount which has beeninjected and the interval between the injections must be taken intoaccount in relation to our work . Von Pirquet and Schick have shownthat a second injection of horse serum into children causes an "immediate"or an "accelerated" reaction . Both the immediate and theaccelerated reaction in children are characterized by symptoms of"the serum disease ."We might conclude that children may not be sensitized to the toxicaction of horse serum by eating horse meat, for horse meat is a favoritearticle of diet in certain European countries and there is nothing uponrecord to show that the injection of horse serum in those countries isfraught with more danger than where this diet is not used . It should,79

95however, be borne in mind that our work has shown that guinea pigsmay be sensitized with exceedingly minute quantities of a strangeproteid, and that repeated injections cause an immunity, and it doesnot seem impossible that the same action may be true of food .Man reacts to the first injection of horse serum after a period ofeight to thirteen days ; guinea pigs show no reaction as a result of thefirst injection ; both man and guinea pigs react to a second injection .The reactions in man and the guinea pig differ, however, both in severityand in kind . The relation, therefore, that our observations uponthe guinea pig inay have in their application to man must await furtherstudy .The fact that other animals beside man and the guinea pig react toa second injection of horse serum would seem to indicate that we aredealing with one and the same action .We believe that our results make . i t probable that man may be renderedsensitive to the injection of a strange proteid, as is the case withthe guinea pig and other animals, and that this explanation must beconsidered as well as the status ly?nphatacus, which has heretofore beenassigned as the cause of sudden death following the injection of horseserum .[POST SC$IPTuiu.-After the galley proof of this article had left ourhands an article by R . Otto entitled " llas Theobald Smithsche Phanomender Serum-Ueberempfindlichkeit," reprinted from Leuthold-Gedenkschrift, band 1, first came to our notice . His paper dealswith some of the problems we have studied and his results are inharmony with many of our conclusions .]O80

Immunology and MAID (1887-1970)Sheldon G . Cohen and William R . DuncanR osenau, Anderson, and the Beginnings (1899-1915)The first appearance of immunologic studies in theHygienic Laboratory reflected the state-of-the art of thetimes . At the turn of the century, identification of therole of bacterial organisms in the definition of infectionwas just beginning to emerge as a new pursuit in thefield of pathology . The unfolding of new knowledge offeredcorresponding new insights and opportunities todesign specific attacks on unmasked microbial agents ofdisease, but not without creating a new array of problemsand challenges for clinical medicine . In the evolutionof this development, the revelation of immunologicphenomena in host defense was brought into focuswithin the Public Health and Marine Hospital Service .It all began eighteen years after the Laboratory'sestablishment, during the tenure of Milton J . Rosenau,second Director of the Hygienic Laboratory . In 1899,after serving for twelve years as the HygienicLaboratory's founding Director, Joseph Kinyoun left hisposition to join the H.K . Mulford Laboratories andRosenau was named to replace him . Just eight yearsearlier, the Laboratory had been relocated from itsoriginal site in the Marine Service Hospital at StatenIsland, New York, to a new facility in Washington, D .C .Here, as subsequent events would prove, the seeds wereplanted for immunology's growth and ascendancy to itscurrent day position of preeminence among biomedicalscience endeavors housed within the National Institutesof Health-the ever-expansive, ultimate evolvement ofthat very same Hygienic Laboratory .Rosenau, after receiving an M .D . degree from theUniversity of Pennsylvania in 1889, pursued postgraduatestudies at the Hygienische Institut in Berlin, theInstitut Pasteur in Paris, and the Pathologisches Institutin Vienna . Commissioned in 1890 with the rank of AssistantSurgeon, he joined the Marine Hospital Service onreturn from training experiences at the famed medicalcenters of Europe .Shortly after Rosenau had assumed the Directorship,an expansion of the Service was authorized in order totake on responsibilities in the newly-developing area ofbiological products that involved the preparation andstandardization of vaccines and antitoxins .' This chargeresulted from a tragic series of deaths in St . Louis attributedto tetanus contamination of administereddiphtheria antitoxin . Working closely with Rosenau inthis exciting initiative was John F . Anderson, who joinedthe Hygienic Laboratory as Assistant Director in 1902 .Anderson, a medical school graduate of the University ofVirginia, also had a European background of training inbacteriology at the medical centers of Vienna and Paris .Sheldon G . CohenWilliam R. Duncan

Then, in 1898, following studies in England at theSchool of Tropical Medicine in Liverpool, he received acommission in the Marine Hospital Service .In laboratory endeavors, deaths were being noted inguinea pigs used in protocols for standardization ofdiphtheria and tetanus antitoxins raised in foreign animalspecies . Relevant and especially frightening were reportsof occasional severe reactions, and even some fatal accidents,that had occurred among patients treated withthese antisera . Out of Anderson's undertaking to determinea possible common cause and basic mechanism forthese incidents, classic studies on anaphylaxis werespawned in the Hygienic Laboratory . As a result, creditfor the NIH's first landmark publication in immunologycan be given to Rosenau and Anderson for their 1906paper on the study of the cause of sudden death followinginjections of horse serum .zThe Laboratory's follow-up series of studies on thesubject contributed measurably to the development of anunderstanding of anaphylactic phenomena . 3 It is pertinentto appreciate that these investigations were undertakenand interpretations established independently,without awareness of the dog and rabbit experiments ofRichet and Portier in France in 1902 . Additionally,Rosenau and Anderson characterized features of themechanism of sensitization and demonstrated the requirementsboth for antigen-antibody specificity and foran incubation period of several days between sensitizingand shocking doses . They also noted the phenomenon ofmaternal transmission and the potential of the oral routefor the induction of sensitization .Although anaphylactic shock traditionally is referred toas the "Theobald Smith phenomenon" as a result of Otto'sreference to Smith's unpublished observations, it isimportant to note that Rosenau and Anderson's findingslikely represent the first valid demonstration of hyperreactivityin guinea pig experimental models . In contrastto the Hygienic Laboratory workers' critical experiments,factors of toxicity were evident in the induction of reactionsin Smith's series, i .e . use of successively administeredlarge volumes of horse serum and the absenceof requisite sensitizing-challenging intervals . Smith's protocolswere followed without control groups, and theobservations lacked significance . Hence Rosenau andAnderson's contribution remains the first definitivepublication on the subject of anaphylaxis . Also of interestis their introduction of the term "allergin . "3However, it should be appreciated that this term, coinedby the Hygienic Laboratory staff, referred to allergic antibody(also named "anaphylactin") and, thus, is not tobe confused with "allergen," a current-day term used todefine a sensitizing antigen in the etiology of atopic orclassic human allergic disease .Integrated with and a by-product of interest and involvementin the investigations of Rosenau and Andersonwere the studies of William H . Schultz . After serving asan instructor in the Department of Physiology and Pharmacologyat the University of Missouri, Schultz joinedthe Public Health and Marine Hospital Service in 1908as Associate Pharmacologist at the Hygienic Laboratory .There Schultz's orientation to cardiac physiology andbiological assay of drugs was directed to the study of thepharmacodynamics of anaphylactic shock . Out of thispioneering investigation came Schultz's classic contributionand landmark publication on contraction of theisolated strip of sensitized guinea pig ileum whensuspended in a bath of physiological solution andchallenged by specific corresponding antigen . 4 Thefollowing year, the English physiologist and pharmacologistSir Henry H . Dale described a variation ofthis technique, utilizing the guinea pig uterine strip andits muscular contraction effected by a-iminazolyethylamine(histamine) . Thereafter known as the Schultz-Dalephenomenon, the application of this technique foundconsiderable use for many years in the study of localanaphylactic phenomena, identification of mechanismsand mediators of sensitization, and assay of pharmacologicanti-allergic and antihistaminic agents . After astay of five years, Schultz left the Hygienic Laboratoryin 1913 to accept the Professorship of Pharmacology andMateria Medica at West Virginia University .When Rosenau accepted Harvard Medical School's invitationto assume the Chair of Preventive Medicine andHygiene in 1909, Anderson was named the third Directorof the Hygienic Laboratory . After publishing an exhaustivereview on anaphylaxis, 3 Anderson turned hisprimary attention to other aspects of bacteriology, includingcholera, typhus, tuberculosis, and poliomyelitis .Especially important was his collaboration with JosephGoldberger, the Public Health and Marine Hospital Serviceofficer who later achieved recognition as a pioneerin nutrition and the conquest of pellagra . Their workresulted in the development of an experimental animalmodel for measles by transmission of the virus tomonkeys . Subsequently, in 1915, Anderson resigned fromthe Public Health Service to assume the directorship ofE.R . Squibb and Sons Research and BiologicalLaboratories .The In-between Years (1915-1957)During the 32 years that followed Anderson's tenure,from immunology's standpoint, the Hygienic Laboratoryand its successor,-the National Institute of Health, continuedto reflect the state-of-the-art of the times . In thisevolving period investigations on immunity were undertakenwith primary emphasis on unraveling themechanisms of host- defense . Innovative studies on structureand function of the immune system would have towait for the requisite base of information to begenerated, first with the advent of applicable,sophisticated advances in biochemistry to provide the impetusfor inquiry .For all intents and purposes, the immunology of thatperiod could be equated with its defined component ofserology . The exquisite specificity of antisera was used asan aid in the study and classification of microorganismsand cleverly employed as a probe in unraveling the intricaciesof pathogenicity . Highlighting the work of these82

years were the contributions of Rolla E . Dyera, who in1925 defined the unit for scarlet fever streptococcus antitoxin;s Kenneth F . Maxcy, who in 1928 published oninfection and immunity in the epidemiology ofdiphtheria ; 6 Ida A . Bengtson, who standardized gasgangrene antitoxin in 1934 ; 7 John Bozicevich, who in1938 developed a reagent for the in vitro diagnosis of ahelminth parasite infection ; 8 and Karl Habel in 1940,who worked with yet another class of infectious agents,the rabies virus . 9Typical of the contributions from studies based uponserologic analysis was the definition of serotypes ofvarious antigenic bacteria . As an outgrowth of theselaboratory endeavors, investigators were provided withthe means to identify microbial-derived specific antigeniccomponents . In the case of the pneumococcus, thebiochemically-isolated capsular polysaccharides providedopportunity for another landmark contribution from theNIH laboratories . For the first demonstration of "immunologicalparalysis" that subsequently evolved into thephenomenon known as immune tolerance, credit must begiven the findings of Lloyd Felton in 1942 . 1 °Upon receiving a P.H .S . commission in 1938, Feltonjoined the NIH Division of Infectious Diseases's staffafter faculty positions in preventive medicine and hygieneat Harvard and in bacteriology and pathology at JohnsHopkins . Special interest in microbial infection, especiallypneumonia, led him to undertake studies on thepneumococcus . From this line of investigation, Feltondemonstrated the role of capsular polysaccharide indecreasing pneumococcul infection and the persistance ofthis microbial material in tissue, accounting for an expressionof immunological paralysis, defined during hisNIH tenure .The demands of World War II years directed the attentionof the NIH Division of Infectious Diseases professionalstaff to special needs of the military services,i .e . development of vaccines and the problems of diseasesindigenous to tropical regions . Subsequently, with cessationof hostilities came the opportunity for recruitmentof scientific staff from among the pool of those beingdischarged from military obligations . It then becamepossible for the expanding resources of NIH' to supportthe new directions and expanding horizons of evolvingbiomedical disciplines . In this setting in 1948, areorganized NIH gave birth to the NationalMicrobiological Institute (NMI) .Although the NMI's mission in infectious diseases hadthe potential to expand beyond infection and microbialimmunity, its horizons were still limited by the absenceof a single focused unit capable of initiating investigativeendeavors in a field that was beginning to take its owndistinct shape as immunobiology and immunochemistry .Further, in 1955, a change in the NMI's name to the NationalInstitute of Allergy and Infectious Diseasesalater served as NIH Director, 1942-1950(NIAID) added still another dimension to the Institute'smission . With the new component's aegis, NIH hadcome full circle in recognizing its roots in the pioneeringendeavors of Rosenau and Anderson in hypersensitivity .The change from NMI to NIAID represented one inname, rather than in mission ; for from its very beginning,implicit in NMI's charge were all aspects of infectiousdiseases, including invading organism and hostresponse .This nominal change from NMI to NIAID was inkeeping with the pattern of identifying NIH componentInstitutes with disease categories . To the public, to concernedCongressional committees upon whose appreciationand insights NIH support depended, and to othersof the laity working for health causes, microbiology as adescriptor evoked little in the way of practical understandingor sympathy for NMI's mission . The substitute insertionof infectious diseases would circumvent the lackof appreciation of a good number of illnesses that wereinherent in the National Microbiological Institute's orientation. However desirable in designating the Institute'smission from a scientific standpoint, adding the descriptorimmunology could be expected to accomplish little, ifanything, in further clarifying the issue of the Institute'sname to the laity . Instead, in the choice of allergy forthe Institute's new name, consideration was given to thescientific acceptability of this term within a global interpretationthat would include the study of immune functionsand disorders . At the same time, it would satisfythose who believed that NIH had lacked involvementwith and concern for that large segment of the public afflictedby allergic disorders .b Henceforth, the focus ofthe Institute's public visibility would be on an array ofdiseases within the new NIAID purview, rather than on abiomedical discipline, whatever the earlier intent toascribe the very same role to the predecessor NMI .Nevertheless, it was some 18 years later that the first appearanceof the singular name allergy became identifiedformally with an Institute laboratory section, and yet anadditional six years later that a clinical section on allergywas created .It was in this setting that Dorland J . Davisd in 1956assumed the position of Associate Director of NIAIDand was given the responsibility for intramural research(a position later renamed Scientific Director) . Aftergraduation from medical school and advanced studies inpublic health at Johns Hopkins University, Davis receiveda commission as a PHS officer and came to NIH in1939 to work in the Division of Infectious Diseases .Following World War II service as a medical officer inNorth Africa, he returned to NIH and in 1954 was appointedChief of the Laboratory of Infectious Diseases .His background of experience in research on a variety ofb Haas, V .H . Director, NMI, 1943-55 ; Director, NIAID, 1955-57, personal communication tothe authors, 1987 (see note on last page of this chapter) .c the Section of Allergy and Hypersensitivity, initiated within the NIH Laboratory of Immunologyin 1962, and the Section of Clinical Allergy and Hypersensitivity, established withinthe Laboratory of Clinical Investigation in 1968, are discussed in later sections of thisaccount .d later served as Director of the National Institute of Allergy and Infectious Diseases,1964-1973 .83

parasitic and virus diseases provided him with additional insightsto appreciate immunology's potential to assume anever-expanding, interdisciplinary role in the basic biomedicaland clinical sciences . It was, therefore, evident to Davisthat the absence of focused immunologic research constituteda distinct void, and that a structured program couldassume a prominent position in the facility that served asthe nation's showcase of medical research endeavor .At this time, moreover, the field of immunology hadbegun to expand with the emergence of new knowledgegenerated by research, but little in this area of investigationhad developed within NIH . In later reminiscences,Davis notede that, during the period of the late 1940s tothe early 1950s at NIH, there were only two investigatorsdealing with immunologic areas, Lloyd Felton andFrederick G . Germuth . As previously noted, Felton'swork on pneumococcal polysaccharides had provided experimentalleads for his classic studies on immunologicalparalysis . In 1946, Germuth, in the position of Immunologistwith a Laboratory of Infectious Diseases grouphoused at the Ft . Detrick facility in Frederick, had initiateda series of studies on hypersensitivity." His interestin this area of research could be traced to earlier trainingand association in the Department of Pathology at JohnsHopkins with Arnold R . Rich, whose pioneering studieshad centered on the delineation between immunity andhypersensitivity responses . Germuth subsequently leftNIAID in 1951 for Washington University in St . Louis,the first of a series of positions in which his studies onimmune complex phenomena and experimental vascularsensitization were extended . Although there was indeed aplace for immunology, per se, there was an equal needfor experts in immunologic research to interact, associate,and collaborate with counterpart basic and clinicalscientists who were contributing to the widening spectrumof biomedicine at NIH .During one of his periodic meetings with the Chiefs ofthe NIAID component intramural laboratories, Davishad occasion to introduce this subject . Followingfavorable discussion of Davis's proposal, a committeewas charged with exploring all possibilities to secure aplace for immunology in the Institute . Between twoviable alternatives, the Committee favored establishing anew and independent laboratory of immunology, ratherthan promoting the generation of immunologic studiesscattered throughout several existing laboratories .Establishing and implementing plans for a new unit andto direct studies at the fore of immunologic research,however, would require the proven leadership and experienceof a recognized and accomplished immunologicscientist . Joseph E . Smadel, who had just moved fromthe position of Chief of the Department of Virus andRickettsial Diseases at the Walter Reed Army Institute ofResearch to assume the Associate Directorship at NIH,was especially familiar with people in the field of immunologyand in an informed position to offer thenames of several candidates . On Smadel's recommendation,Jules Freund was recruited in 1957 .Laboratory of Immunology: The Freund Years(1957-1960)Jules Freund had had a long and distinguished career asa medical scientist that began on the faculty of preventivemedicine at the University of Budapest . He thenserved as bacteriologist at the Von Ruck ResearchLaboratory and the Henry Phipps Institute of theUniversity of Pennsylvania, as a member of the facultyof Cornell University Medical College Department ofPathology, and as Assistant Director of the Bureau ofLaboratories and Chief of the Division of Applied Immunologyat the Public Health Research Institute of theCity of New York . He was well known at NIH in hisrole as a consultant to the Public Health Service, as apast president of the highly selective American Associationof Immunologists, and for his original studies onantibody formation, toxin-antitoxin reactions, andautoimmunity in aspermatogenesis . He was also creditedwith the development of the most potent agent known tothe present day for the enhancement of immuneresponsiveness .f,12These were the early years of the unraveling ofknowledge of biochemistry applicable to the new dimensionthat quantitative methods were bringing to chemicalimmunology . With an appreciation of immunochemistryand yet not neglecting beginning revelations in cellmediatedimmunity, Freund justified the confidence placedin his appointment . As remembered by Davis andformer associates, Freund proved to be the right personto assume the requisite responsibility for the developmentand direction of the Laboratory of Immunology . Therespect for his fund of knowledge was enhanced by thepersonal element of character he brought to the position .Although scientifically demanding in maintaining standardsof exactness and excellence, at the same timeFreund was ever ready to extend assistance to associatesand colleagues with gentility . Against the background offirm, thoughtful leadership by a keen, expertly informedscientist and kindly man, NIAID's Laboratory of Immunologywas formally initiated in a setting of harmony,excitement, and mutually-supportive efforts .One of the very important and evident strengths thatFreund brought to the construction of the Laboratory ofImmunology (LI) was his ability both to identify and tobring together those with expertise whom he found inplace within NIH and to guide those he had recruited inundertaking original pursuits with immunologic bent .Constituting the original LI staff were the one associatewho had accompanied Freund from New York, SanfordH . Stone, and three previously established NIH medicalscientists, John Bozicevich, Sheldon Dray, and John E .Tobie . Although these appointments, effected through aregrouping process, represented a spectrum of varyingexperiences and orientations, a common denominator ofinterest and purpose guided the shaping of theLaboratory's thrust . At the outset, a deliberate decisionfFreund's adjuvant, consisting of killed mycobacteria, an emulsifying agent, andmineral oil for the incorporation of an immunizing antigen .epersonal communication to the authors, 1987 .84

was made by Freund and those administratively concernedto focus on and emphasize laboratory research inbasic immunology . The fact that this plan for the Institute'sinnovative venture was designed also to providethe foundation for an ultimate clinical arm was evidentin the following notation that appeared in the NIAIDAnnual Report of Program Activities for 1957 :The new research program of allergy and immunologyis one year old . The immediate objectives have beento initiate research projects in problems which couldbe attacked by laboratory methods . These are nowunderway and the new Laboratory of Immunologylooks towards the beginning of clinical studies concurrentlyconducted with fundamental laboratoryinvestigations .With this mission in view, the recruitment process foradditional staff with appropriately relevant interestsbegan . Among the first new names to be added in 1958were those representing a younger group invited to embarkon the new NIAID venture : Wilton E . Vannierfrom the California Institute of Technology, Curtis A .Williams from the Rockefeller Institute for MedicalResearch, Philip Y . Paterson from the University ofVirginia, and Philip R .B . McMaster, just out of medicaltraining at the Cornell-New York Hospital MedicalCenter .Upon setting themselves up in NIAID's newLaboratory of Immunology, Freund and Stone continuedthe line of investigation they earlier had pursued in NewYork . This had consisted of studies on animal models ofadjuvant-induced immune-mediated disease, includingaspermatogenesis as the first fully authenticatedpathologic demonstration of experimental autoimmunity,experimental allergic encephalomyelitis, and thyroiditis .In 1958 the more classic concept of allergy first appearedat NIAID with Stone's investigations on experimentalanaphylactic shock, representing an extension of earlierwork initiated with Freund .I 3It was during this period that increasing attention wasbeing directed to the guinea pig as an especially suitableexperimental model for the study of delayed-type hypersensitivity. Adoptive cell transfer of this immunemediatedphenomenon by lymphoid cells from sensitizeddonors to naive recipients had recently been demonstratedin the Rockefeller Institute laboratories of Merrill W .Chase . Freund, perceiving that genetic control could influencethe outcome of such experiments, uncovered aunique opportunity in a NIH resource that for some timeapparently had been forgotten . Dating back to the workof the geneticist G.M . Rommel in 1906 at the Departmentof Agriculture, perpetuation of the two survivinglines of inbred guinea pig strains, numbers 2 and 13, hadbeen continued as a matter of routine by a NIH animaltechnician . When he learned of the existence of this nonutilizedanimal colony, Freund appreciated the great advantageinherent in homozygous states for studying lymphoidcell functions ; accordingly he seized the opportunityto develop applicable protocols . The resultant series ofFreund and Stone experiments, based on the efficacy ofcell transfer of tuberculin sensitivity and allergicencephalomyelitis, demonstrated the special value of theinbred guinea pig in the study of cell-mediated immunity .In later years, these inbred strains resurfaced and, whenbrought to the fore of immunologic research by Freundand Stone, proved especially valuable to NIH scientists inpioneering studies on genetic control of the immuneresponse .Of the original Laboratory of Immunology Staff,Sheldon Dray had first served as a P .H .S . commissionedofficer for two years in the Nutrition Section of theBureau of State Services . Next joining the PhysicalBiology Laboratory of the National Institute of Arthritisand Metabolic Diseases in 1949, Dray initially focusedstudies on the physical chemistry of membranes andmembrane transport . Newly-developing interests in noninfectiousimmunology then led him in 1955 to seektransfer within NIH to the NIAID Laboratory ofClinical Investigation . In this setting, Dray planned totake up his earlier interests in antigen-antibody reactions,and in the field of allergy to give special attention tochemical procedures for the isolation of ragweed pollenantigenic fractions . These were subject areas to which hehad been introduced in the laboratory during medicaland graduate school years . 9In another redirection of orientation, Dray's interestagain was diverted from attraction to the ragweed systemto the potential of the technique of immunodiffusionthat recently had been developed by Jacques Oudin .hFirst to gain experience at NIH in this new technologyand its utilization in the separation and study of serumproteins, Dray soon served as a NIH-wide resource forthe work of colleague scientists . By coincidence, this occurredabout the time Oudin came to NIH as a visitingforeign scientist . Mutual interests in this common scientificendeavor led to additionally rewarding interactionsand Dray was influenced to pursue yet another line ofinvestigation that Oudin had been pioneering, allotypy .Then an invitation to join Freund's Laboratory of Immunologyfacilitated his undertaking these new studies .As in Freund's case, Dray found in NIAID a uniqueanimal research resource, a large breeding colony of rabbitsthat provided the necessary stock for protocolsdesigned to study allotypy . Unfortunately, reality offeredthe first roadblock . Dray learned that the large numbersof animals required for this line of investigation werehoused in a facility intended only for production andsupply, not for research . However, with administrativeinsight, this issue was resolved . In this unique setting,Dray not only confirmed Oudin's seminal findings butalso expanded the scope of this particular area of immunogenetics. In attempting to extend these studies to the definitionof human allotypes, Dray employed protocols basedupon immunization of primates with human sera, and, asa fortuitous by-product, discovered IgG subclasses .'4gAs a graduate student with William H . Welker, Chairman of the Department ofPhysiological Chemistry at the University of Illinois College of Medicine and a pioneer in thefield of immunochemistry .hChief of the Analytic Immunochemistry Service of the Institut Pasteur, Paris, 1959-1978 .85

One important contribution to medicine that wasespecially concerned with clinical laboratory endeavororiginated in the Laboratory of Immunology through thework of John Bozicevich . His description of the technicalmethod for adsorbing DNA to bentonite particlesrepresented an innovative approach to serologic identificationby visible flocculation . This procedure, whichlent itself to the detection of anti-DNA antibodies, wasfound to have extremely useful applications as alaboratory aid in the diagnosis of systemic lupuserythematosus and rheumatoid arthritis .l s Reported inBozicevich's article-which evoked requests for 3,000reprints-the technique was widely used in hospital andclinical laboratories throughout the country . A co-authorin this publication, Donald E . Kayhoe, at the time a LCIsenior investigator, subsequently joined the NIAID CollaborativeResearch Program in 1966 as head of theTransplantation Immunology Branch (TIB) . Through thework of the TIB, NIAID pioneered the development of ahistocompatibility typing serum bank, as a unique extramuralresearch resource for the scientific community, andthe large-scale, multicenter Kidney TransplantationHistocompatibility Study .Coming from an appointment in tropical medicine atTulane University in 1943, John Tobie's first associationwith NIH in the Division of Zoology lasted but a fewmonths . Entering military service that same year, Tobiejoined the U.S . Army Sanitary Corps and was assignedto the South Pacific theater as Chief of the Departmentof Parasitology in an Army medical laboratory . Uponreturning to NIH, he worked in the Division of InfectiousDiseases' Laboratory of Tropical Diseases (laternamed the NIAID Laboratory of Parasitic Diseases) onserologic flocculation and complement fixation studies inparasitic diseases . After joining Freund's group, Tobiebecame more involved with basic immunology and in thisarea was responsible for introducing the technique offluorescein attachment to antibodies .' 6The newly-recruited group of Vannier, Williams, andPaterson were placed in a curious circumstance : thefacilities of the Laboratory of Immunology were notready to provide adequate laboratory space for all . Aninterim plan was developed . Although carried on NIAIDstaff rolls, by special arrangements they were temporarilydetailed to extramural laboratories and given concurrentvisiting or affiliated institutional appointments . Throughspecifically-effected contracts to the cooperating institutions,NIAID paid their official salaries and providedsupport for the research projects undertaken .Prior to his NIAID recruitment, Vannier had pursuedadvanced training at the California Institute ofTechnology with Dan Campbell, a pioneer in immunochemistryand in the development of the basic sciencefoundations of allergy . Under the terms of the temporaryarrangement, Vannier's LI appointment was initiatedwhile he continued to work in Campbell's laboratory onthe isolation and characterization of allergenic fractionsof house dust .Paterson had come to Freund's attention throughPaterson's earlier associations with Smadel at the WalterReed Army Institute of Research and with Lewis Thomasat Tulane University . The Thomas-Paterson studies onexperimental allergic encephalomyelitis (EAE) served as alink with Freund's identical research interest in autoimmunity. Until adequate animal quarters for Paterson'sproposed investigation could be put in place at NIH,Freund arranged for him to work with Alwin M . Pappenheimer,Chairman of the Department of Microbiologyat New York University (NYU), on delayed-typehypersensitivity . While at NYU, Paterson haddemonstrated to Freund that EAE could be inducedwithout mycobacteria in the mineral oil adjuvant(Freund's incomplete adjuvant) if xenogeneic, rather thanallogenic, spinal cord was used as the autoimmune inducingantigen ; also, Paterson was the first to adoptivelytransfer EAE .I 7In New York, Nobel Laureate Rene J . Dubos and theRockefeller Institute provided the Freund-Williams connection. Freund had known Williams and was familiar withhis studies on mycobacteria with Dubos and his earlierwork on immunoelectrophoresis . Williams's LI appointmentby interim assignment in Dubos's Rockefeller laboratoryand his pursuit of studies on immunologic properties ofmethanol extracts of tubercle bacilli constituted anotherlink with Freund's research interests .McMaster, in his neophyte scientist status, began workat NIAID on experimental models of autoimmunity tocomplement the thrust of Freund and Stone's ongoingstudies . Of the off-site group, Vannier subsequently didcome to NIH in 1960 to continue and further his workin immunochemistry . Paterson and Williams, however,terminated their NIAID appointments after two years .It was anticipated that construction of the requisitespace and facilities at NIH would be completed within ayear after Paterson's arrival at NYU in 1957, but by1959 this still had not been effected . Paterson's decisionto resign his LI position was further influenced byFreund's development of serious illness and the offer ofa faculty appointment by his former Tulane mentorThomas, who had since come to NYU and taken overthe chairmanship of the Department of Medicine . WhenFreund died in 1960, it became apparent to Williams thata new Chief of LI would not be appointed for sometime . Preferring a more clearly defined option, he resignedfrom his NIAID position in favor of remaining for arappointment at Rockefeller with Edward Tatum in theLaboratory of Biochemical Genetics . Thus, the Patersonand William episode constitutes a unique experience inthe history of NIAID immunology . Although theirpublications appeared under the official NIAID aegis, exceptfor periodic visits to prepare quarterly reports anddiscuss progress with Freund, they enter the record as activescientific staff who had never worked within thestructural confines of the Institute .As programs in basic immunology were taking shapein the Laboratory of Immunology, Freund began to looktowards possibilities for developing clinically-relevant investigations. In this connection, his attention was drawnto Howard C . Goodman, who was moving into immunologyfrom a clinically-oriented base . Goodman's initialNIH appointment in 1953 was as a research associate atthe National Heart Institute (NHI), where he86

came after serving as a research associate at the Cedarsof Lebanon Hospital Institute of Medical Research inLos Angeles . At NHI, with James A . Baxter, Chief ofthe Laboratory of Cellular Physiology and Metabolism,Goodman's work involved studies on experimentalMasugi nephritis . Additionally, his studies identifyingserum factors in patients with lupus erythematosusbrought him into collaboration with immunologistswithin other NIH component Institutes .' Especially importantwere associations with John L . Fahey of the NationalCancer Institute (NCI} and Sheldon Dray ofNIAID . Influenced by these interactions, Goodmanrecognized that his interests were turning increasingly toimmunology and appreciated the fact that further trainingwould be required if he were to progress in this area .Consequently, he arranged to spend a year (1959-1960) inthe laboratory of Pierre Grabar at the Institut Pasteur .While in Paris studying serum proteins, utilizingGrabar's pioneering technique of immunoelectrophoresis,Goodman received an invitation from Freund to join theNIAID Laboratory of Immunology on his return toNIH . He eagerly accepted this opportunity, but a shorttime later Freund developed a terminal illness and diedbefore Goodman completed his stay with Grabar . Nevertheless,upon return to NIH in 1960, Goodman followedthrough on original plans and joined the Laboratory ofImmunology whose direction had been since given toJohn Tobie as Acting Chief .In retrospect, the three years of Freund's tenure areremembered and recognized as the period when immunologygained its own identity within NIAID . In additionto bringing the LI into organizational being, Freundleft his personal mark indelibly stamped on the shapeand direction taken by immunology as it developed intoan independent Institute endeavor, beyond and apartfrom microbial infection . Insightful guidance and encouragementof initiative characterized Freund's quiet,but strong, influence on associates and investigativeundertakings . At the same time, his otherwise optimisticplans for the start-up process were tempered as Freundfaced the realities of limitations on available space andpositions . The placement of newly-joined junior staffwho attempted to initiate their work and expand theirhorizons at distant laboratory sites that were separatedfrom day-to-day contact and vital interactions withNIAID mentor and colleagues, and, ultimately, the appearanceof terminal illness reduced Freund's active roleas LI Chief to even less than his three years of record .Were it not for these circumstances, perhaps a morelengthy documentation of productivity and objective accomplishmentsin the building process, and a more successfuloutcome in the recruitment and retention of newresearch in basic immunology at the National Cancer Institute was especially concerned withimmunoglobulins, whereas studies at NIAID tended to focus progressively on areas of cellularimmunology.(After leaving the NCI in 1971 to take up the position of Chairman, Department ofMicrobiology and Immunology at the University of California, Los Angeles, Fahey servedNIAID as a consultant in several areas relevant to the Institute's mission in Allergy and Immunology,including membership on the National Advisory Allergy and Infectious Diseasesouncil.staff might have been possible . Regardless, the contributionof the Freund years is unquestioned : a newdisciplinary effort at NIAID was launched and the patternof its laboratory base set to keep pace with the advancingforefront of immunobiology andimmunochemistry .Laboratory of Immunology : Tobie and a Holding Action(1960-1962)All concerned at the leadership level had agreed that thecourse set by Freund for the Laboratory of Immunologyshould be continued . The search for a Laboratory Chiefwould concentrate on identifying and recruiting a scientistwith an extensive background of experience andcredentials in basic immunology . For the interim, Tobieagreed to take on the responsibilities of Acting Chief .For the next two years, this arrangement worked welland to the satisfaction of all involved in contributing tothe Laboratory's best interests . During the transitionperiod, the work of the LI progressed . Stone's studies onanaphylactic shock and experimental allergicencephalomyelitis were furthered, and Dray's extendedstudies on allotypy resulted in the description of allotypicsuppression and the advancement of a new immunologicconcept ."An important and innovative development during thetwo-year Tobie period was the staff initiative that effectedmeaningful, interactive efforts between theLaboratory of Immunology and the Laboratory ofClinical Investigation (LCI), then under Vernon Knightas LCI Chief . Not only were collaborations effected, butalso organizational lines were crossed and official jointappointments were made . Although maintaining hisprimary staff assignment in the Laboratory of Immunology,Goodman's concurrent association with theLaboratory of Clinical Investigation was made formalwhen a position was created especially for him . Identifiedas Head of a newly-established Section on Clinical Immunology,he played a pivotal role in establishing thissection as a new prototype and model for clinical immunologyunits in research and academic institutions .Before leaving NIAID in 1963 on detail in Geneva to theWorld Health Organization as Chief of its ImmunologyUnit, Goodman focused his studies on experimentalthyroiditis, purification of human serum gammaglobulin, and the interaction of serum factors from lupuspatients with chromosomal material .I 9 The firstdemonstration of antibody activity of certain immunoglobulinclasses and subclasses by Fahey andGoodman 20 was representative of NIH's developing opportunitiesand unique resources for collaborative efforts .It was just such endeavors, crossing organizational linesof the categorical Institutes, that measurably contributedto immunology's disciplinary expansion and increasingrelevance to clinical medicine .A new recruit joining the Laboratory of Immunologyduring this period was Arthur J .L . Strauss, who,although a neophyte investigator, already had anestablished relationship of his name to a seminal observation. Having developed a special interest in neurologicaldiseases during a postdoctoral fellowship at Columbia87

University, Strauss, with co-workers, undertook clinicalinvestigative studies on myasthenia gravis . In 1960 hewas the senior author of a publication reporting theoriginal demonstration of a muscle-binding complementfixingglobulin by immunofluorescence . Eager to pursueand extend this productive line of investigation, Straussarrived at the Laboratory of Immunology in 1961,specifically to work with Goodman . During the ensuingyears, Strauss's studies were primarily focused on myastheniagravis as an immunologic disorder, and by 1962he could report on serologic studies from 100 myasthenicpatients . 21Tobie's own work also was furthered by collaborativeefforts with the Laboratory of Clinical Investigation . In1960, Sanford Kuvin arrived at NIAID to begin anappointment as Clinical Associate in the Laboratory ofClinical Investigation . During a previous period ofgraduate school studies in medical microbiology at theUniversity of Pennsylvania, he had developed a specialinterest in immunology . However, at NIAID, he foundthat immunologic investigation relevant to his area of investigativeinterest in parasitic infection had yet to beundertaken . At that time, studies in the Laboratory ofParasitic Diseases centered on the morphology oforganisms and those in LCI on other clinical features ofparasitic diseases . By initiating a collaborative associationwith Tobie and applying the latter's technologic approaches,Kuvin launched a series of immunologicstudies on antibody responses in human malarial infection. Special credit was given to this LI- LCI group fordemonstration of the first reliable technique for thedetection and titration of human antibody responses tomalarial organisms by the indirect method of immunofluorescence. 22 In 1963, Kuvin left for the LondonSchool of Tropical Medicine and Hygiene to furtherspecial scientific and professional interests that had beennurtured at NIAID .Laboratory of Immunology: The Landy Years(1962-1967)To aid in the recruitment of a permanent Chief for theLaboratory of Immunology, a search committee wasformed . Several able external candidates were identifiedand contacted . Although some candidates expressed initialinterest in exploring possibilities, serious negotiationswere another matter . In addition to the ever-presentdeterre,,, of salary reduction that senior academic scientistshad to face when electing to enter government service,another factor contributed to the attractiveness ofexternal candidates' existing situations . These were thebright, beginning years of support for immunologicresearch . Because the outlook for continued availabilityof ample, if not generous, NIH grant awards for extramuralresearch was optimistic, potential candidateswere provided with a valid reason to decline consideration. Attention then turned internally to one of thesearch committee members, Maurice Landy, who, at thattime, was Head of a National Cancer Institute Sectionon Immunology . Ultimately, Davis offered the positionto Landy, and in 1962 a new Chief for the Laboratoryof Immunology was in place .Landy had come to NIH six years earlier from theWalter Reed Army Institute for Research, where he servedas Chief of the Department of Bacterial Immunology .As head of the Section of Immunology of the NationalCancer Institute's Laboratory of Chemical Pharmacology,his research interests were focused on mechanismsof host defense, especially on the role of microbialproducts in precocious and natural immunity .When Landy assumed directive responsibility, theLaboratory's primary thrust began to reflect his interestsin natural immunity . 23,24 However, other areas of basicimmunology that were in the process of acceleratingascendancy to the forefront of the biomedical scienceswere not neglected . Sections were created to accommodateand further the special interests of senior staff.Tobie, who had served in an acting capacity during theprevious two years assumed responsibility for a newlycreatedSection on Applied Immunology within the LI .The following year, in further pursuit of special interests,Tobie moved to the NIAID Laboratory of GermfreeAnimal Research as Chief and served in this capacity untilretirement in 1972 . Additionally, in 1969 he took onconcurrent responsibilities as NIAID Assistant ScientificDirector for Laboratory and Clinical Research .In addition to the Section on Applied Immunology forTobie's work, research on autoimmunity previously introducedby Landy's predecessor, Freund, was continuedby Stone in a Section on Allergy and Hypersensitivity .Others Sections included Immunochemistry, headed byDray ; Clinical Immunology, headed by Goodman inassociation with the LCI ; and Autoimmunity withStrauss .In the Section on Immunochemistry, Dray continuedstudies on immunoglobulin allotypes . His report on allotypesuppression stands as an especially important contributionto this subject . 25 The influence of Dray's development ofthis research area at NIAID was felt as intramural laboratorylines were crossed in collaborative efforts with LCIstaff. 26 In 1965, Dray left NIAID to head the Departmentof Microbiology at his alma mater, the Universityof Illinois College of Medicine, and was succeeded byVannier as Head of the Section on Immunochemistry .The line of investigative work that Dray had initiatedwas carried forward by one of his former trainees, RoseMage . Mage had come to NIH as a postdoctoral fellowin the Laboratory of Immunology after completing workfor a Ph .D . with Elvin Kabat at Columbia University . kIn view of her primary interest in genetics, Mage was attractedto special studies on allotypy under Dray's directionand, in 1964, she received a staff appointment asCareer Investigator . In this area of endeavor, her studiesincluded allotype suppression and the genetic and chemicalbasis of antibody diversity . Especially noteworthy wasMage's contribution to understanding the organization ofgenetic information coding for the variable and constantregions of immunoglobulin chains . 27A Professor of Microbiology, Human Genetics and Development at Columbia University Collegeof Physicians and Surgeons . Kabat's formal affiliation with NIH was later initiated as aFogarty Institute Scholar in 1974, later with a NCI appointment as Expert in 1975, and continuedsince 1981 at NIAID . In this connection, his studies have been concerned with thedevelopment of a computer base containing published amino acid and nucleotide sequences ofimmunoglobutins and other immunologically relevant molecules .88

A major Landy recruit joining Dray in the Section onImmunochemistry in 1965 was Ralph A . Reisfeld . Afterreceiving his Ph.D . in 1957, Reisfeld spent two years atNIH in the position of Biochemist in the NCI EndocrinologyBranch . Four years as a chemist in the pharmaceuticalindustry with Merck and Company followed,after which he joined Dray in the LI ImmunochemistryBranch . An analytic chemist, Reisfeld collaborated withDray on chemical characterization of rabbit immunoglobulinallotypes and their correlation withbiologic function of genetic determinants . He also workedwith Mage on chemical differences among the differentallotypes . After Dray's departure, Reisfeld undertookindependent studies on the isolation andcharacterization of transplantation antigens and contributedsome of the first biochemical information onhuman histocompatibility antigens . 28 Additionally, his LIinvestigations on structure and function of humantumor-associated antigens, centering on malignantmelanoma, provided the foundation for future years ofwork at the Scripps Clinic and Research Foundationafter leaving NIAID in 1970 .Another Landy recruit who made major contributionsto LI research endeavors in immunochemistry was JohnInman . Following work in the pharmaceutical industrywith Ortho and with Johnson and Johnson, Inman helda special NIH fellowship at John Hopkins, after whichhe joined NIAID in the position of Research Biochemist .During this early period in the LI, his work and contributionswere concerned largely with the developmentof innovative technologies, e .g . immunoabsorbants andmethods for peptide isolation, sequencing and synthesis . 29Within the Section on Autoimmunity, Strauss advancedhis inquiries on the immunologic aspects ofmyasthenia gravis, especially anti-muscle factor (AMF),with whose identification he had been credited . Out ofthis work came the first indication of the reactivity ofAMF with identical components found in thymic myoidcells and skeletal muscle . 3 ° This finding was of considerableclinical interest in view of the association ofthymomas and frequency of thymic hyperplasia in patientswith myasthenia gravis .An early problem Landy faced was in recruiting additionalstaff. He sought to expand the LI's researchhorizons by creating a "critical mass" of investigatorswith appropriate diversity and experience . The aim ofthis infusion of additional talent was to enhance productiveinteractions among staff . However, it was difficultto attract qualified, established investigators with the requisiteexpertise and sophistication . First, there werelimitations on salary and level of career positions builtinto the Federal government system that made it extremelydifficult, not only to attract, but also to retainaccomplished senior medical scientists . Second, a ceilingon allotted positions further complicated this issue .Although the LI had been provided with a more thanadequate budget, most of it was designated for supplies .In this regard, however, Landy proved to be asresourceful and inventive as he was imaginative and insightful. When competitive salaries and positions couldnot be offered, he brought in visiting foreign scientistsand guest workers supported by external funds frompharmaceutical companies and international organizations,e .g . Rotary International . In this undertaking,Landy's wide-ranging international associations wererevealed . Among the collaborations between notablevisiting scientists and LI staff were those of RuggeroCeppellini of Italy with Landy on bacterial polysaccharides,3 J Payotis Liacopoulas of France with Stone onanaphylaxis, 32 Jan B . Borjeson of Sweden with Reisfeldon pokeweed mitogen, 33 and Gideon Goldstein ofAustralia with Strauss on the thymus in myastheniagravis . 34 Also included was Shlomo Ben-Efraim, who extendedthe series of studies initiated in Israel on syntheticantigens, 35 and from Czechoslovakia, Tomas Hraba andJaroslav Rejnek . 36 Not only did a qualitative change occur,transforming the intellectual environment by providingopportunities for collaborations and interchangesin this international-like atmosphere ; also a quantitativefactor soon became apparent . By 1965, the Europeanvisiting scientists outnumbered the LI permanent staffuntil the addition of more career positions restored thebalance .The LI Clinical Immunology Section, as originally conceivedby Freund with Knight's support, with provisionfor active associations with the Laboratory of ClinicalInvestigation continued to be productive into the earlyLandy years . In addition to Goodman's own work, therewere evident spin-off benefits . Clinical Associates in theLCI who wished to gain experience in immunologicresearch had opportunities to work with Goodman onimmunosuppression, with Landy on endotoxin, withDray and Vannier in immunochemistry, and with Stoneon autoimmunity and hypersensitivity .When Goodman left the LI to take up his WHO appointmentin Geneva, Landy sought to replace him inthe position of Section Head . However, as an experimentallaboratory with a non-physician chief, the LI couldnot be provided with beds in the NIH Clinical Center .Without the promise of access to patient involvement forthe hands-on study of disease entities, Landy was unsuccessfulin recruiting attempts to fill the void created byGoodman's departure, and the original plan graduallydissipated . Elsewhere within the LCI, the study of immunologicdiseases generated increasing staff interest,and a structured program on clinical immunology andallergy eventually emerged . An account of this developmentis given in a following section .In retrospect, it was only to be expected that a changein LI leadership would be accompanied by a variety oforganizational and functional changes reflecting the newLaboratory Chief's scientific interests and goals and hispersonal and operational style . Where Freund of necessityhad to focus on initiating the process of organizationand Tobie on maintaining progress, Landy was presentedwith the opportunity to further and expand theLaboratory's development and to influence the directionof its investigative components .In the LI that Landy took over, he perceived the existenceof only a beginning . He appreciated the need toconsolidate and strengthen existing resources, introducenew initiatives and fill the voids left by the incomplete89

ealization of Freund's plans for staff development . Thecreation of LI Sections and designation of established investigatorsto continue independent pursuits of special interestsas Section Heads kept the LI on course . At thesame time, this move minimized any sense of threat andovershadowing by the new Laboratory Chief's introductionof studies on microbial-related immune phenomena as anarea of major research emphasis . Yet it would have beenquite unrealistic to expect that a change-over to newstyle, objectives, and design could have been madewithout creating unrest and a whole new set of stresses .The accelerating pace of research advances in immunologyrequired those who would move with the fieldto generate new insights and exploit newly-developed experimentalleads . In retrospect, a forward-looking,expertly-informed Laboratory Chief with evident convictionshardly could be less demanding of the potentialgiven to his charge than offered by the opportunities andchallenges of the times . Quiescence had passed with theFreund years ; Landy and staff shared in the ferment ofintellectual forces, expressions, and differences that characterizedthe Laboratory of Immunology in transition .As Landy approached his fifth year in the position ofLI Chief, a shifting of scientific and career interests wasin the making . He had worked to move the LI from thefading era of descriptive immune phenomenology to amore demonstrably analytic and theoretical discipline .Further, the ever-expanding field of immunology wasrapidly providing new knowledge on the biology,chemistry, and genetics of the system of cells responsiblefor immune function . Landy's perceptions and insightsled him to appreciate that within this changingbiomedical science lay opportunities for intellectual excitementand reward beyond the actual conduct oflaboratory-based original investigation . To him, NIAIDappeared to offer just the right setting for judiciousutilization of unique resources and for involvements toinfluence and enhance the development of the field .With John R . Seal,( who had since replaced Davis inthe position of NIAID Scientific Director, Landy beganto explore possibilities . Both Landy and Seal agreed thatto meet the challenges of immunology's emerging advances,NIAID needed an informed and accomplishedscientist to direct the Institute's extramural research programin this field . Accordingly, in 1967, Landy undertookthe task that required a career change and movedinto the position of Chief of the Allergy and ImmunologyBranch of the NIAID Extramural Program . In thiscapacity, until his retirement from NIH in 1972, Landyutilized this sophisticated base and his wealth of associationsto excellent advantage . Especially noteworthy werehis unique contributions and assistance to biomedicalscience through his role in designing the extramural networkon Program Projects on Lymphocyte Biology andIWhen Dorland Davis was named NIAID Director in 1964, John Seal (Capt . M .C ., U .S .N .),then Director of the Naval Medical Research Institute in Bethesda, was recruited for the positionof Deputy Director for Intramural Research (title later changed to Scientific Director in1969) . Seal later served as Acting Director of NIAID in 1975, following Davis's retirement,and as Deputy Director (1975-81) during the subsequent tenure of Richard M . Krause .Brook Lodge conference series . The latter highlightednew, advancing research areas of the day, i .e . mediatorsof cellular immunity, immunologic intervention, immunologicaltolerance, immune surveillance, and geneticcontrol of the immune response .Laboratory of Immunology : The Benacerraf Years(1968-70)With Landy's move to take on new responsibilities andchallenges of the NIAID Extramural Allergy and ImmunologyBranch, the position of Laboratory of ImmunologyChief was once again open . John R . Seal, asIntramural Scientific Director, had definite convictions ashe simultaneously viewed both the rapidly-changing fieldof immunology and the considerable intellectual resourcesof the NIAID . Within the LI he perceived the potentialto move to the forefront of advancing research in thefield it represented to the community of basic biomedicalscientists . For the LI in transition, the search was on forstrong, imaginative leadership combined with scientificexpertise . But first, the direction most advantageous forthe LI to take needed to be charted .After surveying the rapidly-changing field of immunobiology,in the Search Committee's judgmentcellular immunology and immunogenetics were pinpointedas the particular areas offering the greatestpotential and opportunities . To meet this objective, BarujBenacerraf of New York University was identified andthe recruitment process was initiated .In his presentation to Benacerraf, Seal emphasizedNIH's character as an institution dedicated solely toresearch and research training, the availability ofresources that at the time seemed almost limitless, andthe benefits associated with scientific interactions in alarge, multidisciplinary, intramural program . An additionalattraction was the availability of the NIH largecolony of inbred guinea pig strains that earlier hadcaught Freund's attention and that was important forBenacerraf's pioneering studies on immune responsegenes .As in the case of the LI's founding Director, Freund,Benacerraf came to NIAID already established as a majorfigure in the research world of immunology . For theprevious twelve years he had been a member of thenationally-recognized group of immunologists assembledat NYU by Lewis Thomas . During that expansive, excitingperiod, Benacerraf had established a strongresearch program on genetic control of the immuneresponse and cellular interactions involved in the inductionof antibody production . These would be the areashe would continue to pursue during his forthcomingtenure at NIAID .With the thrust of LI research turning in the directionof cellular immunology and immunogenetics, a realignmentin Institute programs ensued . One change of interestwas the relocation of investigations on autoimmunity,one of the earliest studies in LI's history . Stonein 1969 transferred the setting for his studies to a newlycreatedSection on Immunology in the Laboratory ofMicrobiology . In 1968, following newly-developing interests,Strauss pursued training in psychiatry and joined90

the staff of a PHS neuropsychiatric facility, St .Elizabeth's Hospital in Washington, D .C .During the early Benacerraf period, research studiesfocused on several areas of basic immunology, primarilythe genetics and chemistry of immunoglobulins, geneticcontrol of immune responses, and characterization offundamental mechanisms of cellular immunology . Continuingin LI were the investigations of Mage, Reisfeld,and Inman . There was Mage's demonstration of separateloci that encode for the variable and constant regions ofrabbit IgG heavy chains .z7 Reisfeld, with expertise inanalytic immunochemistry and in collaboration withMage, pursued studies on the characterization of rabbitimmunoglobulin chains at immunochemical andbiochemical levels and the definition of chemical differencesbetween allotypes . 36 Other areas of Reisfeld'swork, concerned with the isolation and characterizationof transplantation antigens, contributed some of theearliest information on the biochemistry of humanhistocompatibility antigens .z$In Rose Lieberman's ongoing studies, initiated in theLCI, Benacerraf identified an area relevant to LI intereststo be encouraged and furthered . Accordingly,Lieberman was recruited to the LI ; with her transfer, thestudies of Mage and Lieberman provided the LI with astrong program on the genetics and biochemistry of immunoglobulins. In Lieberman's collaborative series withPotter of NCI, important contributions were made instudies on murine immunoglobulin heavy chains .m ,3'In line with the aims of the LI reorganization,Benacerraf recruited two of his former NYU trainees,who were in the process of developing independentcareers as investigators, William E . Paul and Ira Green .For Paul, this constituted a reassociation with NIH, sincehe had served as a clinical associate in the NCI EndocrinologyBranch in 1962-64 . Green's credentials inbasic immunology were supplemented by a clinicallyrelevantresearch background in hematology .Of Paul's research interests in cellular immunology, hefocused on cellular interactions in the immune responseand to antigen-binding receptors of leukocytes . WithBenacerraf, Paul described the role of carrier-specificcells in anti-hapten responses . 3 sWhile at NYU, Green had initiated studies on geneticcontrol of the immune response . Continuing this line ofinvestigation with Benacerraf in the LI, he demonstratedthe limiting influences of the major histocompatibilitycomplex on immune response (Ir) genes in guinea pigmodels . 39 In another rewarding collaboration, Green andFrank of LCI opened up a new area for extended studyin their demonstration of the C4 deficient guinea pigstrain . 4oBenacerraf's tenure as LI Chief covered only a twoyearspan . During this period, he continued to extend thelines of investigation initiated earlier at NYU . Especiallyinventive was the series of studies based upon his earlierseminal observation on Ir genes that probed the geneticm described in a following section on the Laboratory of Clinical Investigationcontrol of the immune response to simple antigens .With Green and other LI associates, he documented theclose linkage of Ir genes and the guinea pig majorhistocompatibility complex (MHC) . 39 This line of investigation,extended into later post-NIH years, earnedBenacerraf the Nobel Award for Physiology orMedicine in 1981 . Additionally, during his LI years, heinitiated another pioneering, experimental series on thecellular collaboration required for induction of immuneresponses to hapten-carrier complexes . 41Having fulfilled his commitment to reorganize andfurther the development : of a vibrant and highly productiveLaboratory of Immunology, Benacerraf left NIH in1970 to accept the Chair in Pathology at HarvardMedical School . He was succeeded as LI Chief by Paul .In retrospect, Benacerraf's major contributions toNIAID involved the reorganization of the LI ; realignmentand focusing of staff interests ; and introduction ofstudies on one of the most important problems of immunologyof the day, cell-cell-interactions . These accomplishmentsset the stage for continued sophisticatedresearch in immunobiology and immunogenetics atcellular and molecular levels . His influence was responsiblefor creating an intellectual environment which attractedmany high quality research fellows to the LI,many of whom advanced to staff positions and in turnenhanced the impact of Benacerraf's relatively shortstay at NIAID .Laboratory of Germfree Animal Research (1959-1969)Laboratory of Microbial Immunity (1969-72)In 1959 the Laboratory of Germfree Animal Research(LGAR) was established as one of four NIAID laboratories. At the start and during the early years of itshistory, its investigations were devoted to the biologicaspects of experimental animals born and maintained inmicrobial free environments . In particular, the LGARinitially directed its concern to technical problemsassociated with keeping germ free animals alive . However,it was only to be expected that the potential ofthis facility for advancing immunologic research wouldsoon be appreciated . Within the settings offered by theLGAR were unique opportunities to study the structureand function of immune systems that had escaped antigenicchallenges of microbial agents and their productsat varying periods from birth through senescence .Within three years after its creation, an immunopathologistwas added to the LGAR staff . In 1962, EdwinM . Lerner moved from his position in the NationalInstitute of Arthritis and Metabolic Diseases to take upthis NIAID appointment . In collaborations that crossedlaboratory organizational lines, Lerner, with McMasterand Stone of the LI, conducted investigative studies onthyroiditis and experimental allergic encephalomyelitis . 4zThe pace of immunologic investigations within LGAR,introduced by Lerner in 1962, soon picked up momentum. The following year, Richard M . Asofsky, aftercompleting training in pathology and immunology atNew York University, joined the Laboratory staff tobegin his early studies on immunoglobulin synthesis .91

One year later, in 1964, after completing his obligationto serve as interim Acting Chief of the Laboratory ofImmunology, Tobie moved to the LGAR to become itsnew Laboratory Chief. Also accompanying Tobie ontransfer from the LI to LGAR appointment wasMcMaster . With this change in leadership and staff additions,immunology became the center of the Laboratory'sresearch endeavors . During Tobie's tenure as LGARChief, he continued to pursue studies on the biology ofthe human immune response to malarial infection .McMaster's series of studies on EAE similarly wereextended . 43Further realignments in staff associations and thecharacter of investigations occurred . In 1965, Lernertransferred . to the LI to pursue his work in immunopathology. In 1968, Philip J . Baker, after completinga staff fellowship in the LI, joined the LGAR tofurther his investigational interests in microbial immunity,and Harvey M . Cantor came to NIH to work as astaff fellow with Asofsky .Baker's studies centered on the induction of antibodyresponses and tolerance to microbial-derived polysaccharides. These lead to a seminal observation on theregulation of immune responses . In an analysis of T-independent responses to type III pneumococcal polysaccharidein mice, Baker independently demonstrated anew regulatory cell type, the suppressor T cell . Also, forthe first time, the role of T cells in putative T-independent responses was documented . 44Asofsky and Cantor, in graft vs . host reaction (GvH)studies, were the first to describe T-T cell interactionsdemonstrating the synergistic actions within mixtures ofyoung and old lymphoid cells in the induction of GVH . 4 sThis seminal finding set the stage for the identificationof T cell populations involved in the augmentation andsuppression of immune responses .Because of the continuing change of focus in LGARresearch activities, it was appropriately renamed theLaboratory of Microbial Immunity (LMI) in 1969 . Thefollowing year, when Tobie was given additional NIAIDwideduties as Assistant Scientific Director of Laboratoryand Clinical Research, Asofsky was named AssistantChief of the LMI . Two years later, upon Tobie's retirementin 1972, Asofsky replaced him as LaboratoryChief .Laboratory of Clinical Investigation (1952-1970)The Laboratory of Clinical Investigation (LCI) dates itsestablishment to 1952, coincident with the opening of theNIH Clinical Center . Since, at that time, the founding ofthe Laboratory of Immunology by Freund was still sixyears away, two of NIAID's meritorious early entries intothe arena of immunobiology and immunochemistrywere launched and nurtured in the LCI . Later transferinto the more appropriate setting of the LI for basicstudies became possible . An account of the LCI genesisof Sheldon Dray's studies on immunoelectrophoresis andrabbit immunoglobulin allotypy is contained in apreceding section on history of the Ll . In a similar manner,LCI also offered the requisite base and resource forRose Lieberman to initiate her work in a comparablearea of research endeavor .Lieberman's opportunity to work at NIH in the positionof Microbiologist was of necessity delayed, pendingthe opening of the Clinical Center . During the three-yearwaiting period, she spent two years as a Medical Bacteriologistat the Dayton, Ohio, Veterans AdministrationHospital, and a third as a Research Bacteriologist at theFt . Detrick, Frederick facility of the U .S . Army BiologicalCorps . Joining the original LCI staff in 1952, Liebermanspent the first year on detail to the Clinical Center settingup a clinical microbiology laboratory .Whereas Dray's pioneering studies on allotypy utilizedrabbit immunoglobulin, Lieberman's contributions onallotypes had their origin in her work with mice . In initialprotocols employing Freund's complete adjuvant inintraperitoneal routes of immunization to raise antistaphylococcusantibodies, she was able to produceascites 46 where the attempts by others were unsuccessfulbecause of strain differences in the mouse models . Applicationof this system in various mouse strains withDray's collaboration led to the identification of mouseallotypes . 47 In addition to the original plan to utilizeascites containing antibody for the allotype studies, anassociated finding contributed to a whole new line of investigation. Recognition by Michael Potter of NCI thatthe BALB/c strain concurrently was developing plasmacell tumors led to fruitful collaborations among LCI, LI,and NCI investigators . As a result of the excitementgenerated by this new finding, Lieberman, Dray and Potterlaunched a long series of studies on allotypes,idiotypes, and the use of the plasmacytomas as a sourceof pure H chains . 48 With Benacerraf's arrival at NIH andhis expressed special interest in this line of investigation,Lieberman transferred her position of Research Microbiologistto the LI where these studies were expanded .Having served an important and valuable function ininitiating research studies in immunobiology, immunochemistryand immunogenetics before the advent ofthe Laboratory of Immunology, the LCI awaited the appropriatestaff and time to dedicate resources to clinicallyrelevant immunologic investigations . Its first approachestablishinga Clinical Immunology Section in 1960through Goodman's joint LI-LCI appointments and inproviding opportunities for collaborative projects byKuvin, Tobie, and other LCI-LI staff-were described inan earlier section of this account . Although the time wasright for LCI to develop its own independentimmunologically-relevant program, the requisite leadershipawaited the advent of appropriate circumstances forthe initiative to be taken by Sheldon M . Wolff .Wolff's arrival at NIAID in 1960 as a clinical associatewas linked by circumstance to the appointment of hismentor, Vernon Knight, at a senior level within NIAID .Association with Knight dated back to Wolff's trainingin internal medicine during the preceding few years atVanderbilt University, where Knight had served on thefaculty and hospital staff. In 1959, upon being namedNIAID's second LCI Chief and NIAID Clinical Directorto fill the position formally held by Norman McCullough,92

Knight offered Wolff the opportunity to join him at NIHand to extend his interests to infectious diseases . Wolff'splans and career goals in endocrinology were then putaside .Knight directed his energies and expertise tostrengthening and expanding the LCI's program in infectiousdiseases . The initiation of clinical studies in immunologydepended on developing intra-laboratoryassociations between LCI and Ll . Wolff's first exposureto this new area of endeavor was fostered by theestablishment of the joint LI-LCI Section, effectedthrough the Knight-Tobie agreement, and a later opportunityto pursue cooperative studies when Landy replacedTobie as LI Chief . With Goodman, Wolff conducted investigationson antimetabolite agents in immune systemrelated diseases and with Landy, developed the beginningsof an extensive series of studies on endotoxin andfever . 49 These proved to be expanding areas that wouldengage his major research interests and expertise duringsubsequent years . However, with Goodman's taking up anew position with WHO and departure for Geneva in1963 and the resultant disappearance of the joint Sectionon Clinical Immunology, these early active and vibrantLI-LCI associations in immunologic research began tofade . However, clinical immunology would reappear asan expanding LCI research endeavor when Wolff waspresented with the opportunity a few years later .Advancing within LCI through appointments as SeniorInvestigator and Head of the Clinical Immunology Section,Wolff was named Acting LCI Chief and ActingNIAID Clinical Director six years after his arrival, whenKnight departed to take up the position of Chairman ofthe Department of Microbiology and Immunology atBaylor College of Medicine . Two years later, Wolff'spositions were converted to permanent appointments,which he held until 1977, when he left to assume theDepartment of Medicine chairmanship at TuftsUniversity .The period of Wolff's tenure witnessed an escalationin the quality of research in immunology, achievingcreditable recognition parallel to counterpart NIAIDlaboratories of basic immunology, microbiology, and infectiousdiseases and related developing activities at NCIand other Institutes . The mechanism of dual appointmentswith other NIAID intramural laboratories wasutilized to further opportunities for interchanges of ideasand techniques in immunology and to encourage collaborativeefforts . When Wolff took over as LCI Chiefand NIAID Clinical Director, excellent virology andmycology services remained from previous tenures .However, there was not much more in the way of otherstrong efforts . Staff departures for academic opportunitieshad depleted personnel in all but two senior levelpositions for supervision of clinical investigations relatedto the 52 Clinical Center beds allotted to NIAID . Wolffthen made the decision to interface immunology with infectiousdiseases through a new LCI program to examineadult host defense mechanisms . Accordingly, a highlyselective recruitment process went forward for appropriatelyqualified staff associates and for fellows withpotential for development as independent investigators .This initiative proved to be both successful and rewarding.During the period through 1970 covered by this account,important original work at the forefront ofresearch in clinical immunology was carried out by Wolffand clinical associates who advanced to LCI staff positions,e .g . Harry R . Kimball, John N . Sheagren, andRichard K . Root . 5 ° Their highly productive studies includedstudies on febrile tolerance, bacterial endotoxins,phagocytic functions, neutropenia, Chediak-Hygashi syndromein man and animal models, experimental immunefever, and human leukocytic pyrogen .Also during the period covered by this account through1970, senior staff associates who had been recruited byWolff to undertake investigations in clinically-relevantareas of immunology initiated their series of independentstudies . This was only the beginning of Michael M .Frank's long association with LCI and his early researchcontributions to complement biology . 51 This proved to bean area of special research interest in which he wouldgain distinguished recognition . Frank, who joined NIAIDin 1968 as Senior Investigator after leaving a positionwith the National Institute of Child Health and HumanDevelopment, subsequently succeeded Wolff as LCIChief and NIAID Clinical Director in 1977 . It was alsothe beginning for Charles H . Kirkpatrick, recruited in1968 from the University of Kansas to develop a Sectionof Clinical Allergy and Hypersensitivity, and his series ofstudies on cell-mediated immunity and chronicmucocutaneous candidiasis . 52It is pertinent to note that, in the immediate years thatfollowed, a number of LCI clinical associates who trainedat NIAID with Wolff engaged in highly productiveand rewarding endeavors in clinical immunology . Theirimportant work placed LCI at the fore of studies onWegener's granulomatosis and mid-line granuloma, thehypereosinophilic syndrome, Chediak-Hygashi syndrome,necrotizing vasculitis, granulomatous liver disease, cyclicneutropenia, neutrophil biology and phagocyticmechanisms, corticosteroids and cytotoxic immunosuppressiveagents in the treatment of collagen vasculardiseases, and immunoregulatory mechanisms . Among thisgroup and remaining at NIAID was Anthony S . Fauci,who advanced to the position of NIAID Deputy ClinicalDirector in 1977, was named to head a newly-established,independent Laboratory of Immunoregulation in 1980,and succeeded Richard M . Krause as NIAID Director in1984 . Also, John I . Gallin, Head of the LCI Section onBacterial Diseases, succeeded Kenneth Sell as Director ofthe NIAID Intramural Program in 1985 .93

AllergyA period of 59 years elapsed from the time of Rosenauand Anderson's classic studies on anaphylaxis' before thedesignation allergy was incorporated in the name of anNIH component Institute in 1955 . During this interval,the only recognition of allergic mechanisms as a subjectfor study was the work of Germuth on hypersensitivityin the Division of Infectious Diseases in the late 1940searly1950s . 11 Three years after the mission given toNIAID, studies on anaphylaxis appeared in experimentalprotocols of the newly-created LI with the resumption ofFreund's and Stone's studies initiated earlier at thePublic Health Research Institute of New York . It wasstill an additional four years before the name of allergybecame associated with LI endeavor and a Section onAllergy and Hypersensitivity headed by Stone wascreated by Landy after replacing Freund as LaboratoryChief.Meanwhile, two years earlier, the first indication ofNIH's intent to serve the interests of human allergicdisease appeared apart from NIAID in a new programwithin the NIH Division of Biologics Standards (DBS) .In 1960, Harold Baer was recruited from the Departmentof Microbiology at Tulane University to take on theresponsibilities of Chief in the newly-created Section onAllergenic Products in the DBS . Baer's early interestsand studies in cell-mediated immunity and delayed-typehypersensitivity reactions to mycobacterial fractions,BCG and tuberculin, led him to consider the pressingnature of problems in human contact dermatitis . Justfive years earlier, in 1955, the DBS forerunner, BiologicsControl Division, was detached from NIAID and expandedto Division status within NIH . Had this developmentbeen otherwise, Baer's investigations on delayedcontact sensitivity to various chemical sensitizersespeciallyconcerned with the chemistry of catechols inpoison ivy dermatitis-and his leadership in organizingan international effort for the standardization ofallergenic extracts would have been effected under anNIAID aegis .Baer's expertise, nevertheless, was made available toNIAID through interactions in a special project undertakenin 1959 . At that time a collaborative group headedby the noted immunochemist, Dan H . Campbell of theCalifornia Institute of Technology, attacked the problemof characterization of allergens . As mentioned earlier,Campbell had been associated with NIAID intramuralstaff investigations when Vannier was detailed to work inhis laboratory while awaiting the availability of intramuralspace after recruitment to the LI . 53 To initiatethe work of Campbell's three subcommittees onchemistry, animal testing, and clinical trials, respectively,ragweed was chosen as the prototype for the fractionation,isolation and identification of allergenic fractions ofetiologic importance in seasonal hay fever and asthma .Baer, in this cooperative DBS-NIAID effort, assumed theresponsibility for quality control and supply of ragweedpollen derived-starting materials .In any event, 15 years would go by after NIAID wasestablished before the identity of a program on clinicalallergy appeared within the LCI in 1968 . There was,however, a near miss in the start-up of a possible programon clinical allergy in 1966 . In Seal's search to fillKnight's vacated positions of LCI Chief and NIAIDClinical Director, an offer was made to John H .Vaughan of the University of Rochester . Vaughan, withcredentials in allergy and clinical immunology, upon acceptanceindicated that high on his list of priorities wasthe institution of broad scope and in-depth studies onasthma . Through periodic visits to Bethesda, he alreadyhad begun to discuss programs and explore a spectrumof possibilities with Institute staff . During this process,quite suddenly, Vaughan was faced with unexpected illness. After reassessing the uncertainties of his future,Vaughan withdrew . For the time being, the positions ofLCI Chief and NIAID Clinical Director remained vacant,and the promise of an allergic disease program remainedunfilled .In building clinical immunology within the LCI, Wolffwas alert to the Institute's need to put a program inclinical allergy in place . Additionally pertinent, Davis asInstitute Director and Seal as Scientific Director from thebeginning were strongly supportive of this objective .Wolff in his search was successful in recruiting CharlesH . Kirkpatrick from the faculty of the University ofKansas . As the newly-appointed Head of a Section ofAllergy and Hypersensitivity in 1968, Kirkpatrick plannedto establish a program on the pathogenesis of asthma,extending his earlier preliminary studies on autonomicnervous system responses in patients with various typesof asthma, and to evaluate certain other allergic diseases .Upon arrival at NIAID, three items engaged hisattention .The first was an issue quite easily resolved . In order toavoid duplication of name and bypass confusion with theLI Section on Allergy and Hypersensitivity, with Stone'sagreement, the LCI's Section was renamed ClinicalAllergy and Hypersensitivity . Second, Kirkpatrick notedthat only one other scientist at NIH, Harold Baer, wasreally interested in allergic disease and allergicmechanisms . Contact was initiated and led to a Baer-Kirkpatrick collaboration in a study of synthesized pentadecylcatechols of varying structure in sensitization topoison ivy . 54The third was an unanticipated stumbling block encounteredin attempting to get an asthma programunderway-the absence of available equipment forpulmonary function studies . Some fairly sophisticated instrumentationdid exist in the National Heart and LungInstitute . However, to his chagrin, Kirkpatrick was advisedthat the NHLBI pulmonary function equipment couldnot be made available for NIAID use in a program onasthma . There were two alternatives . One, requisiteequipment could be purchased, but the necessity of convertingcritical space allotted to a research laboratory intoa pulmonary function laboratory hampered this move .The second option, in which patients were sent to theNational Naval Medical Center for pulmonary functionstudies, did not really answer the problem . As a result,the program sputtered and never really realized itspromise .94

A turning point in the direction of studies occurredabout 1970 when, on the basis of earlier observationsand experience in Kansas, the opportunity was presentedfor Kirkpatrick to establish a protocol on immunologicabnormalities in T cell functions associated with chronicmucocutaneous candidiasis . 5 z This line of investigationalso provided an opportunity for Kirkpatrick to initiatestudies on therapeutic applications of leucocyte-derivedmaterial known as transfer factor in attempting to compensatefor an immune system defect . 55Although Kirkpatrick's scientific interests were beingdiverted to problems of human deficiency disorder incell-mediated immunity, there was no loss of interest indeveloping a program on the classic allergic or atopicdiseases . Only temporarily diverted, plans for creating anew section on Allergic Diseases reached fruition . Withthe recruitment of Allan M . Kaplan from the BrighamHospitals-Harvard University Medical Center to head theSection in 1972, studies got underway on molecularmechanisms in the activation of vasoactive inflammatorymediators and syndromes of urticaria and angioedema .When Kaplan was joined in 1975 by his former Brigham-Harvard associate, Michael A . Kaliner, NIAID investigationson asthma appeared among LCI protocols withstudies on autonomic nervous system abnormalities inpatients with upper and lower respiratory tract allergicdiseases . At long last, research programs truly reflectingthe implication of the descriptor allergy in NIAID'sname had reached fruition .The Rocky Mountain Laboratory (1950-1970)In the years following the establishment of the RockyMountain Spotted Fever Laboratory in 1921 as a PublicHealth Service field station and its later incorporation intothe NIH Division of Infectious Diseases as the RockyMountain Laboratory (RML) in 1937, the primary thrustof its research program was in the area of microbiologyand infectious diseases . Thus, any immunologic relevanceof its initial endeavors was in the field of microbial immunityand, later during the World War 11 years, in thedevelopment of vaccines . Following the incorporation ofthe Division of Infectious Diseases into the newly-createdNational Microbiological Institute, RML programs wereinfluenced by the mission of its new parent Institute .In seeking new leadership to rejuvenate and expandthe RML, the Institute identified Carl M . Larson, whohad joined the NIH Division of Infectious Diseases in1939, after holding a faculty appointment at GeorgeWashington University . Appointed RML Director in1950, Larson was sensitive to the need to include immunologicresearch endeavors in redefining theLaboratory's mission . Thus, in initiating recruitmentfrom NMI staff, especially from within the Laboratoryof Infectious Diseases and the Laboratory of BiologicsControl, Larson invited Samuel B . Salvin and Edgar Ribito join him at RML . Through their combined interests,the stage was set for RML's expansion in the new directionsof immunology and cell biology .To join NIH, in 1946, all that Salvin had to do wascross the street from the US Naval Medical Center inBethesda . A background in mycology led to his recruitmentto the NIH Division of Infectious Diseases,especially to study the non-mycobacterial etiologic agentof pulmonary lesions that roentgenographicallystimulated tuberculosis . In working with Histoplasmacapsulatum Salvin was able to recover specific antigenfrom intact cultured cells that was capable of detectingcomplement-fixing antibodies . This property contrastedwith the lack of antigenic efficacy of the broth filtratepreparation histoplasmin in serologic procedures . Salvin'scontribution provided the method for correlation oflaboratory in vitro testing with clinical status . At DIDand its successor, the NMI Laboratory of InfectiousDiseases, he continued to pursue his research interests onpathogenic fungi . 56 During the thirteen years that Salvinspent at RML, he published extensively in the area offungal immunity ; his studies proved to be important inrevealing and characterizing mechanisms of delayed-typehypersensitivity and the role of cell-mediated immunity inthe control of infection . 57 In 1964, Salvin left the RMLto accept a position with CIBA Pharmaceutical Corporationas Head of its Immunology Research Division .Another important recruit was Edgar Ribi who alsoaccompanied Larson to RML . Uppsala University inSweden provided the setting for the crossing of pathsbetween Charles C . Shepard and Ribi . At that time, Ribiwas a research associate in physical chemistry ; Shepard,an NMI bacteriologist, was spending a year in residenceat the University . As a result of their interactions, Ribiwas impressed with the possible opportunities to furtherhis career at NIH and accepted a position as a fellow in1952 . After a relatively short stay, he was recruited byLarson .Ribi's subsequent studies and development of specialexpertise led to his international recognition for work indefining the composition of bacterial endotoxins and thecellular components of Mycobacterium tuberculosisresponsible for induction of immune responses to thismicrobial infectious agent . The nature of these studies onmycobacteria involved collaborative efforts with otherRML scientists, including Larson and Robert L .Anacker . 58 . 59 Another of his contributions was thetechnical development of the Ribi Cell Fractionator . Thisunique laboratory aid, which facilitated the isolation ofbacterial cell components, subsequently turned Ribi's attentionto adjuvant properties of mycobacterial cell wallsand application of these bacterial products to immunotherapyin cancer . 6° In 1980, Ribi retired from hisposition at RML and remained in Hamilton, Montana tofound Ribi Immunochem Research and head itslaboratories .As a consequence of Larson's continuing efforts tobuild immunology at RML, John J . Munoz wasrecruited to the RML staff in 1961 . Munoz was introducedto the NIAID through the proximity of RML to thenearby University of Montana where he was serving asChairman of the Department of Microbiology and PublicHealth and Director of the Stella Duncan MemorialLaboratories . His area of research interests through thattime had included studies on the fractionation, andbiochemical and immunologic properties of bacterial antigens,vaccine development, and related hypersensitivity95

mechanisms . As Chief of the RML Allergy and ImmunologySection, Munoz's studies in the field ofmicrobial immunity led to important contributions on theBordetella pertussis organism . Included in this series wereinvestigations on purification, identification andmechanisms of action of various B. pertussis protectiveantigenic fractions 61 and histamine sensitizing factors(pertussigen) . 62 From allergy's standpoint, this line of immunologicinvestigation bore direct relevance to IgEmechanisms, to the production of allergicencephalomyelitis, and to the problem of adverse reactionsto pertussis vaccine in childhood immunization .After becoming Director of RML in 1964, Herbert G .Stoenner reinforced the Laboratory's continuing interestin immunology with the appointment of John E . Coe in1965 . Coming from a fellowship in immunopathology atScripps Clinic and Research Foundation, Coe was quitefamiliar with the RML ; he had spent three years(1960-63) at the NIAID Montana facility working withSalvin on mechanisms of delayed-type hypersensitivity .Coe's early studies in his RML appointment focused onthe characterization of humoral immune responses inseveral species of rodents, especially the Syrian hamster . 63RML's historical development is an intriguing story ofthe evolution of a spectrum of microbiologic research asan outgrowth of public health concern for a singledisease entity . Additionally, it is representative ofNIAID's growth during the period of classicbacteriology's transition to present day cellular andmolecular biology and virology . To Larson, credit is duenot only for guiding RML's progress in its original,defined mission, but also for recognizing the need for animmunologic component as a vital interdisciplinary force .In retrospect, through the insights of Larson's originalgroup, full advantage was taken of unique Institute interestsand resources, which contributed in a very specialway to a noteworthy NIAID immunologic endeavormicrobialimmunity .CommentThis account of the development of immunology at theLaboratory of Hygiene/Hygienic Laboratory (1887-1930),NIH (1930-1948), NMI (1948-1955), and NIAID(1955-circa 1970) in a way presents only the beginning ofan endeavor which the Institute has just reason to viewwith pride and sense of reward . The continuing story ofevents and assessments in the years that followed belongto the current group of concerned Laboratory Chiefs,whose vibrant activities, accomplishments, and goals arevery much a part of today's intramural scene . We expectthat their tasks soliciting contributions for highlightingfrom the many made by staff and programs under theirrespective purviews will be considerably more difficultthan ours has been .It is unlikely that even the most visionary of themedical scientists, mentioned in this account of NIH'sfirst 83 years, could have envisioned the escalating paceat which basic research data have been generated-aphenomenon largely responsible for immunology'sremarkable advances and for the creation of an array ofbiotechnologies that have appeared almost explosively inso short a time . Nor is it expected that even the most insightfulclinicians of the period could have foreseen howimmunology would become so relevant to an understandingof mechanisms in a good number of diverse systemdiseases and its great potential for the application of thisunfolding new knowledge to the development of improvedmethods for their diagnosis, treatment, andprevention .Yet, in this centennial year, we would be insensitive tothe historical implications and remiss in acknowledgingappreciation to those who made it all happen ; to thosewho set the stage for future inventiveness and inquiry,and to those whose contributions to the Institute alsoprovided the foundation upon which modern-day immunologicscientists continue to build in both theoreticaland practical fashion surprising to even the keenestimagination .Note Regarding the Naming of NIAID:For reasons and events that led to the change in namefrom the National Microbiological Institute to the NationalInstitute of Allergy and Infectious Diseases proposedby concerned NIH officials, the authors aregrateful to Victor H . Haas who served as NMI andNIAID Director during that period . In providing this information,Haas recalled the following incident .b"Shortly after I began my assignment as Director ofthe Microbiological Institute, (1948) the NIH amateurtheatrical group known as the `Hamsters' presented itsannual production of skits lampooning events andpeople at NIH . One of the skits centered on theMicrobiological Institute . Its theme was that the Institutewas a sort of orphan, an amorphous creationamong the richer and more `glamorous' categorical Institutes,e .g . NCI, NHI, NIMH . The players rendereda musical plea for improving the Institute's status withthe following doggerel, among others`Lenny, please LennyWon't you categorize me tonight?Lenny, Don't you love me any,Please categorize me tonight'`Lenny' referred to Leonard Scheele, the SurgeonGeneral"96

AcknowledgmentsIn the search and gathering of material for this account,the authors took advantage of the opportunity offeredfor inquiry and discussion with several current and pastmembers of the Institute's scientific and professionalstaff . We gratefully acknowledge their thoughtful considerationand many courtesies extended to us, and thepleasantries of time spent in reflections and remembrances.We especially appreciated the availability, cordialwelcomes, and valued assistance of those to whom werepeatedly turned as special resources to amplify andclarify specific points of information and to explore leadsuncovered in various Institute files and documents ofearlier years pertaining to periods of their involvements :Dorland J . Davis for the span of his tenures in NIH intramuralpositions and as NIAID Director, Victor H .Haas for the NMI-NIAID transitional period, SanfordH . Stone for LI, Howard C . Goodman for LI and LCI,John E . Tobie for LI and LGAR, Maurice Landy forLI, Richard M . Asofsky for LMI, Baruj Benacerraf forLI, Sheldon M . Wolff for LCI, Charles H . Kirkpatrickfor LCI/Allergy, and Herbert Stoenner and John J .Munoz for RML .One of us (SGC) remembers with appreciation themany pleasant hours spent with John R . Seal during thepart of his lifetime that our respective tenures in NIAIDstaff positions overlapped . Just as in the case of currentassociation with Dorland Davis, there were spin-offbenefits in addition to the rewards of warm friendship .John Seal's reminiscences in conversation provided awealth of information on a panorama of NIH/NIAIDevents used as background material in this account . Vividmemories of associates recall the strength of judiciousleadership and support he provided in furthering thedevelopment of NIAID intramural programs in immunologyand allergy .With much appreciation, the authors also greatlyacknowledge and thank Carol K . Shapiro for valuededitorial assistance and dedication to the task of intensivepreparation of manuscript material .Sheldon G. Cohen, M.D.Dr . Cohen initially joined NIAID in 1972 as a fulltimeconsultant in a special project for the ExtramuralPrograms . The following year he moved into an ExtramuralStaff position as Chief of the Allergy and ImmunologyBranch, and with the NIAID reorganization in1977 was selected for the newly created position ofDirector of Immunology, Allergic and ImmunologicDiseases Program . Dr . Cohen was born in Pittston,Pennsylvania in 1918, graduated from Ohio State Universityin 1940, and received his M .D . from New YorkUniversity in 1943 . After internship at New York'sBellevue Hospital he served as a flight surgeon with theU .S . Army Air Force in the European Theatre, andsubsequently completed residencies in internal medicineand allergy and a research fellowship in appliedphysiology and immunology at the University of Pittsburgh. From 1952-1972 he pursued his career in theWilkes-Barre, Pennsylvania area combining clinical practiceand academic and research interests at Wilkes Collegein experimental biology and immunology and in affiliationwith the Veterans Administration Hospital . Dr .Cohen has an extensive list of publications reporting onoriginal studies in allergy and immunology, and duringmore recent years has published on subjects dealing withthe history of medicine-an interest developed during histenure as Historian of the American Academy of Allergyfrom 1963 to 1969 .William Raymond Duncan, Ph.D.Dr . Duncan joined the NIH during its Centennial yearas a Health Science Administrator in the Immunology,Allergic and Immunologic Diseases Program . Born inHarlingen, Texas in 1949, he graduated from the Universityof Texas at Austin in 1971 with a B .A . In 1976 heobtained a Ph.D . in Cell Biology from the University ofTexas Health Science Center at Dallas . There he alsoheld two postdoctoral fellowships in cancer immunologyand a faculty position in the Department of CellBiology . From 1983 to 1987 Dr . Duncan served on thefaculty of Dalhousie University in Halifax, Nova Scotia .97

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Biographical NotesANACKER, Robert Leroy (1926- ) ; b . Minneapolis, Minnesota ; Ph .D ., GOODMAN, Howard Charles (1920- ) ; b . Rochester, New York ;University of Washington, 1956 . M .D ., Johns Hopkins University, 1944 .ANDERSON, John Fleetezelle (1873-1958) ; b . Fredericksburg, Virignia ; GREEN, Ira (1926- ) ; b . New York, New York ; M .D ., State UniversityM.D ., University of Virginia, 1896 . of New York at Brooklyn, 1953 .ASOFSKY, Richard Marcy (1933- ) ; b . New York, New York ; M.D ., HABEL, Karl (1908-1981) ; b . Philadelphia, Pennsylvania ; M.D ., Jeffer-State University, New York, 1958 . son Medical College, Philadelphia, 1933 .BAER, Harold (1918- ) ; b . New York, New York ; Ph .D ., Harvard INMAN, John Keith (1928- ) ; b . St . Louis, Missouri ; Ph .D_ HarvardUniversity, 1942 . University, 1956 .BAKER, Philip John (1935- ) ; East Chicago, Indiana ; Ph .D ., Universi- KAYHOE, Donald Ellsworth (1920- ); b . Washington, D .C . ; M.D .,ty of Wisconsin, 1960 . George Washington University, 1950 .BENACERRAF, Baruj (1920- ) ; b . Caracus, Venezuela ; M.D ., Medical KIMBALL, Harry Raymond (1937- ) ; b . Los Angeles, California ;College of Virginia, 1945 . M .D ., University of Washington, 1962 .BENGTSON, Ida Albertina (1881-1952) ; b . Harvard, Nebraska ; Ph .D ., KINYOUN, Joseph James (1860-1919) ; b . East Bend, North Carolina ;University of Chicago, 1919 . M .D ., New York University, 1882 .BOZICEVICH, John (1905- ) ; b . Sunnyside, Utah ; M.A ., George KIRKPATRICK, Charles Harvey (1931- ) ; b . Topeka, Kansas ; M.D .,Washington University, 1930. University of Kansas, 1958 .CAMPBELL, Dan Hampton (1908- ) ; b . Freemont, Ohio ; Ph .D ., KNIGHT, Vernon (1917- ) ; b . Osceola, Missouri ; M .D ., HarvardWashington University, 1935 . University, 1943 .CANTOR, Harvey (1942- ) ; b . New York, New York ; M.D ., New KUVIN, Sanford Bernard (1929- ) ; b . Newark, NewYorkJersey ; M.D .,University, 1967 . Cambridge University, England, 1957 ; D .T.M .H ., University of London,1963 .COE, John Emmons (1931- ) ; b . Evanston, Illinois ; M .D ., HahnemannMedical College, 1957 . LANDY, Maurice (1913- ) ; b . Cleveland, Ohio ; Ph .D ., Ohio StateUniversity, 1940 .DAVIS, Dorland Jones (1911- ) ; b . Chicago, Illinois ; M.D ., JohnsHopkins University, 1937 ; D .P.H ., Johns Hopkins University, 1940 . LARSON, Carl Leonard (1909-1978) ; b . Butte, Montana ; M .D .,University of Minnesota, 1939 .DRAY, Sheldon (1920- ) ; b . Chicago, Illinois ; M .D ., University ofMinnesota, 1946 ; Ph .D ., University of Minnesota, 1954 . LERNER, Edwin M . (1919- ) ; b . Ottawa, Ontario ; M.D ., HarvardUniversity, 1944 .DYER, Rolla Eugene (1886-1971) ; b . Delaware County, Ohio ; M.D .,University of Texas, 1915 ; Sc .D ., Washington University, St . Louis, LIEBERMAN, Rose (1912- ) ; b . New York, New1916 .York ; M.A ., ColumbiaUniversity, 1937 .FAHEY, John Leslie (1924- ) ; b . Cleveland, Ohio ; M.D ., Harvard MAGE, Rose G . (1935- ) ; b . New York, New York ; Ph .D ., ColumbiaUniversity, 1951 . University, 1963 .FAUCI, Anthony Stephen (1940- ) ; b . Brooklyn, New York ; M .D ., MAXCY, Kenneth Fuller (1889-1966) ; b . Saco, Maine ; M.D ., JohnsCornell University, 1966 . Hopkins University, 1915 ; D .P.H ., Johns Hopkins University, 1921 .FELTON, Lloyd Derr (1885-1953) ; b . Pine Grove Mills, Pennsylvania ; McMASTER, Philip Robert Bache (1930- ) ; b . Cambridge,M.D ., Johns Hopkins University, 1916 . Massachusetts ; M.D ., Johns Hopkins University, 1956 .FRANK, Michael M . (1937- ) ; b . Brooklyn, New York ; M.D ., Harvard MUNOZ, John Joaquin (1918- ) ; b . Guatemala ; Ph .D ., University ofUniversity, 1960 . Wisconsin, 1947 .FREUND, Jules (1890-1960) ; b . Budapest, Hungary ; M.D ., University PATERSON, Philip Y . (1925- ) ; b . Minneapolis, Minnesota ; M .D .,of Budapest, 1913 .University of Minnesota,GALLIN, John Isaac (1943- ) ; b . New York, New York ; M .D ., Cornell PAUL, William Edwin (1936- ) ; b . Brooklyn, New York ; M .D ., StateUniversity, 1969 . University of New York, Downstate Medical Center, 1960 .GERMUTH, Frederick George, Jr . (1921-1984) ; b . Baltimore, REISFELD, Ralph Alfred (1926- ) ; b . Stuttgart, Germany ; Ph .D ., OhioMaryland ; M .D ., Johns Hopkins University, 1945 . State University, 1957 .GOLDBERGER, Joseph (1874-1929) ; b . Austria-Hungary ; M .D ., RIBI, Edgar (1920-1987) ; b . Zurich, Switzerland ; Ph .D ., Berne Univer-Bellevue Hospital Medical College (New York University), 1895 . sity, 1948 .100

RICH, Robert Reiger (1941- ) ; b . Newton, Kansas ; M.D ., University ofKansas, 1966 .ROOT, Richard Kay (1937- ) ; b . New York, New York ; M .D ., JohnsHopkins University, 1963 .ROSENAU, Milton Joseph (1869-1946) ; b . Philadelphia, Pennsylvania ;M .D ., University of Pennsylvania, 1889 .SALVIN, Samuel Bernard (1915- ) ; b . Boston, Massachusetts ; Ph.D .,Harvard University, 1941 .SCHULTZ, William Henry (1873-1947) ; b . Canal Fulton, Ohio ; Ph .D .,Johns Hopkins University, 1907 .SEAL, John Ridley (1912-1984) ; b . Charleston, West Virginia ; M.D .,University of Virginia, 1937 .SHEAGREN, John Newcomb (1935- ) ; b . Aurora, Illinois ; M.D ., ColumbiaUniversity, 1962 .SHEVACH, Ethan Menahem (1943- ) ; b . Brookline, Massachusetts;M .D ., Boston University, 1967 .SMADEL, Joseph E . (1907-1963) ; b . Vincennes, Indiana ; M .D .,Washington University, 1931 .STONE, Sanford Herbert (1921- ) ; b ., New York, New York ; Sc .D .,University of Paris, 1951 .STRAUSS, Arthur (1933- ) ; b . New York, New York ; M.D ., ColumbiaUniversity, 1958 .TOBIE, John Edwin (1911- ) ; b . Collison, Illinois ; Ph .D ., TulaneUniversity, 1940 .VANNIER, Wilton Emile (1924- ) ; b . Pasadena, California ; M.D .,University of California, San Francisco, 1948 ; Ph .D ., California Instituteof Technology, 1958 .VAUGHAN, John Heath (1921- ) ; b . Richmond ; Virginia, M .D, HarvardUniversity, 1945 .WILLIAMS, Alvin Curtis, Jr . (1927- ) ; b . Moorestown, New Jersey ;Ph .D ., Rutgers University, 1954 .WOLFF, Sheldon Malcolm (1930- ) ; b . Newark, New Jersey ; M.D .,Vanderbilt University, 1957 .

Laboratory of Biology of Viruses (LBV)Established : 1959Chief : Norman P . Salzman, Ph.D .1968-86Prior :Karl Habel, M.D .1959-67History :The laboratory was established in 1959 from the nucleusof the Basic Studies Section of the Laboratory of InfectiousDiseases . In 1961 the former Laboratory of CellBiology was incorporated into the structure of theLaboratory of Biology of Viruses as the Cell BiologySection . The laboratory was disbanded in 1986 when Dr .Salzman retired .1971 .Gelb, Lawrence D . ; Kohne, David E . ; and Martin,Malcolm A . Quotation on Simian Virus 40 Sequences inAfrican Green Monkey Mouse and Virus-TransformedCell Genomes . Journal Molecular Biology,57 : 129-45 .ElThis paper provided a method to quantitate thenumber of integrated DNA molecules present intransformed cells . It was a critical paper at the timethat viral transformation was first being analyzed .1971 .Sebring, Edwin D . ; Kelly, Thomas J . Jr . ; Thoren,Marilyn ; and Salzman, Norman P . Structure ofReplicating Simian Virus 40 Deoxyribonucleic AcidMolecules . Journal of Virology 8 : 478-90 .0 A description of the mechanism of replication of simianvirus 40 . It identified unique features containedin circular DNA molecules during DNA replication .1972 .Fareed, George C . ; Garon, Claude F . ; and Salzman,Norman P . Origin and Direction of Simian Virus 40Deoxyribonucleic Acid Replication . Journal of Virology10 : 484 .This study established that there was a single specificorigin for SV40 DNA replication and that DNA synthesisthen proceeded in a bi-directional way .1972 .Garon, Claude F . ; Berry, Karen W . ; and Rose, JamesA . A Unique Form of Terminal Redundancy inAdenovirus DNA Molecules . Proceedings of the NationalAcademy of Sciences, USA 69 : 2391-95 .ElThis study was the first description of inverted terminalDNA repeats that were important in themchanism of viral DNA replication .1974 .Wei, C . M ., and Moss, Bernard . Methylation of NewlySynthesized Messenger RNA by an Enzyme in VacciniaVirus . Proceedings of the National Academy of Sciences,USA 71 : 3014-18 .1-1 First in a series of papers which describe enyzmesthat cap viral and cellular messenger RNAs .1975 .Martin, S . A . ; Paoletti, E . ; and Moss, Bernard . Purificationof mRNA Guanylytranserase andmRNA(guanine-7-)methyltransferase from VacciniaVirions . Journal of Biological Chemistry 250 : 9322-29 .First isolation of capping enzyme .1975 .Wei, C . M . ; Gershowitz, A . ; and Moss, Bernard .Methylated Nucleotides Block 5' Terminus of HeLa CellMessenger RNA . Cell 4 : 379-86 .First demonstration of cap structure of cellularmRNA .1976 .Straus, Steven E . ; Sebring, Edwin D . ; and Rose, JamesA . Concatamers of Alternating Plus and Minus Strandsare Intermediates in Adenovirus-Associated Virus DNASynthesis . Proceedings of the National Academy ofSciences, USA 73 : 742 .0 The first in vivo description of the scheme of selfprimed DNA replication mechanism .

1982 .Baroudy, B . M . ; Venkatesan, S . ; and Moss, Bernard . IncompletelyBase-Paired Flip-Flop Terminal Loops Linkthe Two DNA Strands of the Vaccinia Virus Genome intoOne Uninterrupted Polynucleotide Chain . Cell 28 :315-24 .0 First demonstration of unique hairpin structure at theend of a linear DNA molecule .1983 .Smith, G . L . ; Mackett, M . ; and Moss, Bernard . InfectiousVaccinia Virus Recombinants that Express HepatitisB Virus Surface Antigen . Nature 302 : 490-5 .First demonstration of potential of vaccinia virus tomake live recombinant vaccines .1987 .Natarajan, Venkatachelos ; Madden, Mary Jane ; andSalzman, Norman P . Identification of a TranscriptionFactor which Interacts with the Distal Domain of theAdenovirus IVa2 Promoter . Journal of Virology 61 :646-52 .Gene regulation of two adjacent genes was shown tobe regulated by an identical protein binding to ashared regulatory DNA sequence .Laboratory of Cellular and Molecular Immunology(LCMI)Established : 1987Chief : Ronald H . Schwartz, M .D ., Ph .D .1987-History :The Laboratory of Cellular and Molecular Immunologywas founded in order to develop an integrated cellularand molecular approach to immunological problemsrelated to T lymphocyte function, in particular T cell differentiationand T cell activation .1978 .Schwartz, Ronald H . ; Yano, A . ; and Paul, William E .Interaction between Antigen-Presenting Cells and PrimedT Lymphocytes : An Assessment of Ir Gene Expression inthe Antigen-Presenting Cell . Immunological Reviews 40 :153-80 .Summarizes the experiments with mice whichdemonstrates that Immune Response genes of the majorhistocompatibility complex are functionally expressedin antigen--presenting cells of the immunesystem such as macrophages and dendritic cells .1985 .Schwartz, Ronald H . T Lymphocyte Recognition of Antigenin Association with Gene Products of the MajorHistocompatibility Complex . Annual Review of Immunology3 : 239-63 .El Summarizes the experiments arguing in favor of thetrimolecular complex model of T lymphocyte antigenrecognition, where antigen is postulated to physicallyinteract with the histocompatibility molecule .106

Laboratory of Clinical Investigation (LCI)Established : A clinical operation was contemplated orestablished in late 1950 . Apparently first designatedas the Clinical Investigation Branch the name of thelaboratory (LCI) was established about March 1953 .Chief : Michael M . Frank, M .D .1977-Prior:Sheldon M . Wolff, M .D . Vernon Knight, M.D .1966-77 1959-65Norman B . McCullough, Ph .D ., M.D .1952-58History :The establishment of patient facilities in the ClinicalCenter as an adjunct to the research operation broughtthe National Institutes of Health full circle, since theyhad originated in 1887 as a small research laboratory attachedto a patient facility (Marine Hospital, StatenIsland, New York) .An official organizational chart of November 22, 1950,showed the laboratory at branch level with four authorizedpositions . In August 1952, the laboratory had a staffof eight . During these early years plans were being madefor utilization of patient facilities in the new ClinicalCenter soon to be opened . A staff of 18 made the moveto the Clinical Center in July 1953 .The laboratory has grown considerably and currently itis composed of several sections in which a variety ofallergic, immunologic and infectious diseases are studied .1971 .Frank, Michael M . ; May, Joseph ; Gaither, Thelma . A . ;and Ellman, Leonard . In Vitro Studies of ComplementFunction in Sera of C4-Deficient Guinea Pigs . Journal ofExperimental Medicine 134 : 176-90 .ElThis first description of the biological properties of anew strain of guinea pigs genetically totally lacking inthe complement protein C4 . The animals were firstdescribed from NIAID by Drs . Leonard Ellman,Michael Frank and Ira Green . These were the firstanimals to be described lacking activity of theclassical complement pathway and subsequently werethe subject of hundreds of reports examiningbiochemistry, biology and molecular biology of complementproteins .1972 .Wolff, S . M . ; Dale, D . C . ; Clark, R . A . ; Root, R . K . ;and Kimball, H . R . The Chediak-Higashi Syndrome :Studies of Host Defenses . Annals of Internal Medicine76 : 293-306 .0 First review of abnormal host defense in Chediak-Higashi syndrome . Based on studies done at NIAID .1973 .Clark, R . A . ; Root, R . K . ; Kimball, H . R . ; andKirkpatrick, C . H . Defective Neutrophil Chemotaxis andCellular Immunity in a Child with Recurrent Infections .Annals of Internal Medicine 78 : 515-22 .ElFirst description of defective chemotaxis in thehyperimmunoglobulin-E recurrent infection syndrome .1973 .Gallin, J . L ; Clark, R . A . ; and Kimball, H . R .Granulocyte Chemotaxis : An Improved In Vitro AssayEmploying S 1 Cr Labeled Granulocyte . Journal of Immunology110 : 223-40 .First automated assay of leukocyte locomotion . Widelyused in 1970s and 1980s .107

1973 .Lipsky, P . E ., and Rosenthal, A . S . Macrophage-Lymphocyte Interaction . I . Characteristics of theAntigen-Independent-Binding of Guinea Pig Thymocytesand Lymphocytes to Syngeneic Macrophages . Journal ofExperimental Medicine 138 : 900-24 .11 This was the first in a series of important papersdescribing the interaction of lymphocytes withmacrophages and later the importance of macrophagesin antigen presentation and the importance ofhistocompatibility type in allowing macrophages topresent antigen to lymphocytes .1973 .Rosenthal, A . S ., and Shevach, E . M . The Function ofMacrophages in Antigen Recognition by Guinea Pig TLymphocytes . I . Requirement for HistocompatibleMacrophages and Lymphocytes . Journal of ExperimentalMedicine 138 : 1194-1212 .1973 .Rosenthal, A . S . ; Rosenstreich, D . L . ; Blake, J . T . ; Lipsky,P . E . ; and Waldron, J . A . Mechanisms of AntigenRecognition by T Lymphocytes . In F . Daguillard (Ed .),Proceedings of the Seventh Leukocyte Culture Conference.New York, Academic Press, Inc . pp . 201-206 .1973 .Rosenstreich, D . L ., and Rosenthal, A S . ThePeritoneal Exudate Lymphocytes . 11 . In vitro ProliferationAfter Brief Exposure to Antigen . Journal of Immunology110 : 934-42 .OThe research reflected in the three preceding publicationswas focused on defining the role of cell-cell interactionin macrophage-dependent activation of T-lymphocyte response to antigens ; the role and locationof immune response gene control of immunity and themechanism of antigen processing and presentation viala molecule involvement in protein antigen recognition .1974 .Gallin, J . I ., and Rosenthal, A . S . The Regulatory Roleof Divalent Cations in Human Granulocyte Chemotaxis .Evidence for an Association Between Calcium Exchangesand Microtubule Assembly . Journal of Cell Biology 62 :594-609 .ElInitial studies of calcium metabolism and microtubuleassembly in neutrophils exposed to chemoattractants .First demonstration of calcium release bychemoattractants .1975 .Gallin E . K . ; Wiederhold, M . L . ; Lipsky, P . E . ; andRosenthal, A . S . Spontaneous and Induced MembraneHyperpolarizations in Macrophages . Journal of CellularPhysiology 86 : 653-62 .L1 First study of membrane potential in macrophages .1975 .Lipksy, P . E ., and Rosenthal, A . S . Macrophage-Lymphocyte Interaction . 11 . Antigen-Mediated PhysicalInteractions Between Immune Guinea Pig Lymph NodeLymphocytes and Syngeneic Macrophages . Journal ofExperimental Medicine 141 : 138-54 .1975 .Greineder, D . K ., and Rosenthal, A . S . The Requirementfor Macrophage-Lymphocyte Interaction in T LymphocyteProliferation Induced by Generation ofAldehydes on Cell Membranes . Journal of Immunology115 : 932-38 .1976 .Frank, Michael M . ; Gelfand, Jeffrey A . ; and Atkinson,John P . Hereditary Angioedema : The Clinical SyndromeIts Management . Annals of Internal Medicine 84 : 580-93 .E This paper represents the first major clinical review ofthis syndrome and provided much of the early basisfor the rational development of therapy of thisdisorder .1976 .Gelfand, Jeffery A . ; Sherins, Richard J . ; Alling, DavidW . ; and Frank, Michael M . Treatment of HereditaryAngioedema with Danazol : Reversal of Clinical andBiochemical Abnormalities . New England Journal ofMedicine 295 : 1444-48 .0 This double blind study presented the "cure" of thisgenetically controlled disease by the use of a particularandrogen . The genetically controlled, partialdeficiency of a protein was actually reversed bytherapy and the reduced levels of protein returned tonormal in these patients . This became a mainstay oftherapy in these individuals .1976 .Kaliner, M . A . The Cholinergic Nervous System and ImmediateHypersensitivity . 1 . Eccrine Sweat Responses inAllergic Patients . Journal of Allergy and Clinical Immunology58 : 308-15 .0 This article demonstrated for the first time thatallergic subjects have hyperreactivity of thecholinergic nervous system and laid the basis forsubsequent studies which evaluated the autonomicresponsiveness of patients with allergic diseases in additionto asthma . This study was the first study todemonstrate that patients with allergic rhinitis or evenpatients who had any allergic disease had systemic abnormalitiesof autonomic responsiveness .108

1977 .Atkinson, John P . ; Waldman, Thomas A . ; Stein, SidneyF . ; Gelfand, Jeffery A . ; MacDonald, Walter J . ; Heck,Louis W . ; Cohen, Edwin, L . ; et al . Systemic CapillaryLeak Syndrome and Monoclonal IgG Gammopathy :Studies in a Sixth Patient and a Review of theLiterature . Medicine 36 : 225-39 .El This is the first clinical description of this unusualsyndrome in which patients lose the integrity of theirpost capillary venules and thus their intravascularvolume . The disease was shown to be periodic andassociated with the presence of a monoclonal immunoglobulin. This allowed for the recognition andtreatment of other patients with this syndrome whoformerly died .1977 .Frank, Michael M . ; Schreiber, Alan D . ; Atkinson, JohnP . ; and Jaffe, Charles J . Pathophysiology of ImmuneHemolytic Anemia . Annals of Internal Medicine 87 :210-22 .ElThis review summarizes a series of papers presentedin the Journal of Clinical Investigation which providedan explanation for the events that occur when animmunologically damaged erythrocyte enters the circulation. These papers provide an explanation for thepathophysiological basis of warm antibody (IgG)mediated autoimmune hemolytic anemia and cold agglutinindisease .1977 .Gallin, E . K ., and Gallin, J . 1 . Interaction of ChemotacticFactors with Human Macrophages : Induction ofTransmembrane Potential Changes . Journal of CellBiology 75 : 277-89 .0 First demonstration that chemotactic factors initiatechanges in phagocyte membrane potential .1977 .Malech, H . L . ; Root, R . K . ; and Gallin, J . I . StructuralAnalysis of Human Neutrophile Migration : Centriole,Microtubule and Micro Filament Orientation and FunctionDuring Chemotaxis . Journal of Cell Biology 75 :666-93 .Demonstration of the importance of the cytoskeletonin neutrophil motility and demonstration of thecritical role of the centriole in determining directionof cell movement .1977 .Rosenthal, A . S . ; Barcinski, M . A . ; and Blake, J . T .Determinant Selection : A Macrophage Dependent ImmuneResponse Gene Function . Nature 267 : 156-581977 .Barcinski, M . A ., and Rosenthal, A . S . ImmuneResponse Gene Control of Determinant Selection . 1 . IntramolecularMapping of the Immunogenic Sites on InsulinRecognized by Guinea Pig T and B Cells . Journalof Experimental Medicine 145 : 726-42 .11 One of the first uses of proteins with known mutationsleading to alterations in structure as antigens . Itwas possible to determine the differences between immunodominatgroups required for antibody productionas opposed to cellular responses .1978 .Stingl, G . ; Katz, S . I . ; Shevach, E . M . ; Rosenthal, A .S . ; and Green, I . Analogous Functions of Macrophagesand Langerhans Cells in the Initiation of the ImmuneResponse . Journal of Investigatory Dermatology 71 :59-64 .One of the first descriptions of the importance of theskin Langerhans cells in antigens .1978 .Platshon, L ., and Kaliner, M . The Effect of the ImmunologicRelease of Histamine Upon Human LungCyclic Nucleotide Levels and Prostaglandin Generation .Journal of Clinical Investigation 62 : 1113-21 .0 This article demonstrated that mediator release fromhuman lung mast cells had a profound effect on anumber of cell types within the human lung anddemonstrated that prostaglandin formation was asecondary event in response to human mast cellderivedmediators including histamine . This is thefirst analysis of such a response in human lung andled to a complete analysis of the role of histamine actingthrough HI and H2 receptors as well as theidentification of prostaglandin generating factor ofanaphylaxis .1979 .Frank, Michael M . ; Hamburger, Max I . ; Lawley,Thomas J . ; Kimberly, Robert P . ; and Plotz, Paul H .Defective Reticuloendothelial System Fc-Receptor Functionin Systemic Lupus Erythematosus . New EnglandJournal of Medicine 300 : 518-23 .0 Autologous erythrocytes with either IgG or complementfragments deposited on them in vitro were infusedinto the erythrocyte donor . These cells areremoved from the circulation by the action of veryspecific receptors for these molecules on the cells ofthe reticuloendothelial system . The functional statusof these cells could be determined . Patients with circulatingimmune complexes that were deposited intissues were shown to have defect in vivo function ofthe IgG Fe receptor .109

1979 .Henderson, W. R. ; Shelhamer, J. H . ; Reingold, D. B . ;Smith, L . J . ; Evans, R. ; and Kaliner, M. AlphaAdrenergic Hyperresponsiveness in Asthma-Analysis ofVascular and Puillary Responses . New England Journalof Medicine 300 : 642-47 .ElThis is the first demonstration of alpha adrenergichyperreactivity in asthmatic subjects and laid thegroundwork for a number of studies that have subsequentlygone on to analyze peripheral responses inasthmatic subjects1979 .Rosenwasser, L. J . ; Dinarello, C . A . ; and Rosenthal, A.S . Adherent Cell Function in Murine T Lymphocyte AntigenRecognition. IV . Enhancement of Murine T CellAntigen Recognition by Human Leukocytic Pyrogen .Journal of Experimental Medicine 150: 709-14 .First demonstration that endogenous pyrogens wasthe same substance as LAF (IL-1) .1981 .Lawley, Thomas J . ; Hall, Robert P. ; Fauci, Anthony S . ;Katz, Stephen I . ; Hamburger, Max I . ; and Frank, MichaelM . Defective Fc Receptor Functions Associated with theHLA-B8/DRw3 Haplotype . New England Journal ofMedicine 304 : 185-92 .11 HLA-B8/DRw3 is associated with the development ofa wide variety of autoimmune diseases . This appeardemonstrates that these individuals have a defect intheir ability to clear immune complexes from their circulationbefore they become ill .1982.Joiner, Keith A. ; Hammer, Carl H . ; Brown, E . J . ; andFrank, Michael M . Studies on the Mechanism of BacterialResistance to Complement-Mediated Killing . 11 . C8 and C9Release C5b67 from the Surface of Salmonella minnesota5218 Because the Terminal Complex Does Not Insert intothe Bacterial Outer Membrane . Journal of ExperimentalMedicine 155 : 809-919 .0 The first detailed analysis of the mechanisms thatallow a serum resistant gram negative bacterium toresist complement attack . The paper opened the doorto a series of studies that demonstrated thatmicroorganisms have evolved a series of strategies toprotect themselves from the host defenses as mediatedby antibody and complement .1983 .Gallin, J. I . ; Buescher, E . S . ; Seligmann, B. E. ; Gaither,T. ; Nath, J. ; and Katz, P . Recent Advances in ChronicGranulomatous Disease . Conference of the NIH CombinedClinical Staffs . Annals of Internal Medicine 99 :657-74 .First review pointing out that chronic granulomatousdisease of childhood is a group of disorders ofphagocyte oxidative metabolism .1983 .Joiner, Keith A. ; Goldman, Robert C . ; Hammer, CarlH . ; Lieve, Loretta ; and Frank, Michael M. Studies onthe Mechanism of Bacterial Resistance to Complement-Mediated Killing . Journal of Immunology 131 : 2563-69 .11 One of a series of papers that demonstrated that antibodydirects complement attack . The same numberof complement molecules may deposit on a bacterialsurface in the presence and absence of antibody .Those deposited in the presence and absence of antibody. Those deposited in the presence ofbactericidal antibody will kill the organism . Thosedeposited in its absence will not .1984 .Gallin, J . 1 . Neutrophil Specific Granules : A Fuse thatIgnites the Inflammatory Response . Clinical Research 32 :320-28 .El Summary of work done at NIH demonstrating thestorage of neutrophil plasma membrane receptors andmediators of inflammation within neutrophil specificgranules . Text of address to American Federation ofClinical Research upon receipt of Young InvestigatorAward .1984 .Lawley, Thomas J . ; Bierlory, Leonard ; Gascon, Pedro ;Yancey, Kim B . ; Young, Neal S . ; and Michael, FrankM. A Prospective Clinical and Immunologic Analysis ofPatients with Serum Sickness . New England Journal ofMedicine 311 : 1407-13 .The first prospective study of serum sickness in man .A new clinical sign was demonstrated and the inductionof arthritis in man was demonstrated .

Laboratory of Immunogeneties (LIG)Established : 1977Chief: Thomas J . Kindt, Ph .D .1977-History :The Laboratory of Immunogenetics was established toprovide an intramural focus for research in immunogenetics. The laboratory investigates the multigenefamilies that are involved in control of immune function .A wide range of techniques at the molecular, serologic,and functional level are used to carry out their studies .The Laboratory of Immunogenetics originated in July1977 with three investigators from Rockefeller University :Drs . John A . Sogn, John E . Coligan, and Thomas J .Kindt . The major projects then underway in thelaboratory involved the structural determination byradiochemical methods of the murine H-2Kb protein ; themolecular basis for unpredicted allotypes in laboratoryrabbits ; and studies of MHC antigens by immunochemicaltechniques . Programs of the laboratoryhave been characterized by a molecular approach toquestions in immunology and have evolved with thedevelopment of new technologies and concepts . An offshootof the protein sequence studies came with therecognition that peptide antibodies could be used to linkmolecular biological information to protein expression .Recently, the protein and peptide technology have beenincorporated into a service branch headed by Dr . Coligan,who will provide service in those areas for the entireintramural program . Dr . Sogn has taken an administrativeposition in the National Cancer Instituteleaving only Dr . Kindt from the original group .Presently, other senior investigators in LIG are Dr .Eric Long, who studies the molecular basis of HLA classII antigen function ; Dr . Ed Max, who studies regulatorcontrol of genes in B cells ; and Dr . Mary Ann Robinson,who carries genetic studies of HLA and T cellreceptor genes .1981 .Coligan, John E . ; Kindt, Thomas J . ; Uehara, H . ; Martinko,J . M . ; and Nathenson ; S . G . Primary Structure ofa Murine Transplantation Antigen . Nature 291 : 35-39 .0 This paper was listed in the 100 most cited papers of1982 because it was the first to report the completeprimary structure of a major histocompatibility complexantigen .1981 .Gates, Frederick T ., III ; Coligan, John E . ; and Kindt,Thomas J . Complete Amino Acid Sequence of Murine/3z-microglobulin : Structural Evidence for Strain-relatedPolymorphism . Proceedings of the National Academy ofSciences, USA 78: 554-58Complete sequence data were obtained byradiochemical techniques for a2microglobulin fromtwo different mouse strains showing that a singleamino acid interchange constituted the allelic differencebetween them .1981 .Yarmush, Martin L . ; Sogn, John A . ; Kern, Paul D . ; andKindt, Thomas J . Role of Immune Recognition in LatentAllotype Induction and Clearance . Evidence for anAllotypic Network . Journal of Experimental Medicine153 : 196-206 .0 It was shown that injection of anti-allotype antibodiescould induce expression of immunoglobulin allotypesnot expected from the genotype thus extending thenetwork hypothesis to include allotypes as well asidiotypes .1983 .Emorine, Laurent ; Dreher, Kevin ; Kindt, Thomas J . ;and Max, Edward E . Rabbit Immunoglobulin K Genes :Structure of a Germline b4 Allotype J-C Locus andEvidence for Several b4-Related Sequences in the RabbitGenome . Proceedings of the National Academy ofSciences, USA 80 : 5709-13 .Cloning and sequence analysis of a rabbit K immunoglobulingene revealed a cluster of five J regionsegments, a locus unique in the immunoglobulin genefamily in having only a single functional J segmentwithin the cluster .1983 .Emorine, Laurent ; Kuehl, Michael ; Weir, Lawrence ;Leder, P . ; and Max, Edward E . A Conserved Sequencein the Immunoglobulin Jk-Ck Intron : Possible EnhancerElement . Nature 304 : 447-49 .El A three species nucleotide sequence comparison of theDNA in the intron lying between the J and C regionsof the immunoglobulin kappa gene defined anisolated >,0 .3 kb conserved region that has subsequentlybeen shown to be a critical role in regulatingexpression of the gene .

1984 .Kindt, Thomas J ., and Capra, J . D . The AntibodyEnigma. New York : Plenum Press 257 p .1:1 This book traces the scientific progress leading to anunderstanding of how antibody genes are able to encodean almost unlimited potential to respond toforeign antigens .1984 .Robinson, Mary A . ; Long, Eric O . ; Johnson, A . H . ;Hartzman, R . J . ; Mach, B . ; and Kindt, Thomas J .Recombination Within the HLA-D Region . Correlationof Molecular Genotyping with Functional Data . Journalof Experimental Medicine 160 : 222-38 .D Molecular analyses of MHC genes in families withrecombinant HLA haplotypes located crossover pointsand found new recombinants that could be confirmedby cellular analyses .1984 .Tykocinski, Mark L ., and Max, Edward E . CGDinucleotide Clusters in MHC Genes and in 5'Demethylated Genes . Nucleic Acids Research 12 :4385-96 .D It was observed that the dinucleotide CpG, which isfound at a much lower frequency in mammalianDNA than statistically expected on the basis of totalC and G content, occurs with a sharply higher frequencyin the 5' regions of certain genes, particularlycertain MHC genes .1985 .Max, Edward E ., and Korsmeyer, S . J . Human J ChainGene . Structure and Expression in B Lymphoid Cells .Journal of Experimental Medicine 161 : 832-49 .0 This analysis of a cloned human J chain geneestablished the exon-intron structure of the genomicgene and the correct amino acid sequence of the protein,revealed the existence of an unexpected relatedsequence in the human genome, and provided dataon the expression of J chain mRNA at differentstages in the development of B lymphocytes .1986 .Lew, Andrew M . ; Margulies, D . H . ; Maloy, Walter L . ;Lillehoj, Erik P . ; McCluskey, James ; and Coligan, JohnE . Alternative Protein Products with Different CarboxylTermini from a Single Class I Gene, H-2Kb . Proceedingsof the National Academy of Sciences, USA 83 : 6084-88 .First protein evidence that individual major histocompatibilitycomplex class I genes can be encoded formore than one gene product suggesting that novelmechanisms may increase the class I repertoire .1986 .Lew, Andrew M . ; Pardholl, Drew M . ; Maloy, WalterL . ; Fowlkes, B . J . ; Kruisbeek, Ada ; Cheng, S .-F . ; Germain,Ronald N . ; Bluestone, Jeffery A . ; Schwartz,Ronald H . ; Coligan, John E . Characterization of T CellReceptor Gamma Chain Expression in a Subset inMurine Thymocytes . Science 234 : 1401-5 .0 First description in the thymus of a second T lymphocyteantigen receptor (termed gamma-delta) whichhas subsequently been shown to be the first receptorexpressed during ontogenesis .1986 .Marche, Patrice N ., and Kindt, Thomas J . Two DistinctT Cell Receptor-Chain Transcripts in a Rabbit T CellLine : Implications for Allelic Exclusion in T Cells . Proceedingsof the National Academy of Sciences, USA 83 :2190-94 .0 This is the first description of T cell receptor alphagenes in the rabbit and sequence analysis showed twofull-length transcripts from the same cell indicatingthat RNA production is not sufficient to stop thesynthesis of antigen receptors subject to allelicexclusion .1985 .Robinson, Mary A ., and Kindt, Thomas J . Segregationof Polymorphic T Cell Receptor Genes in HumanFamilies . Proceedings of the National Academy ofSciences, USA 82 : 3804-8 .0 This paper documents the first demonstration ofgenetic variation in genes encoding the T cell antigenreceptor in man .1985 .Tonnelle, Cecille ; DeMars ; R . ; and Long, Eric O . Doa :A New 0 Chain Gene in HLA-D with a Distinct Regulationof Expression . EMBO Journal 4 : 2839-47 .0 This paper describes the discovery of a new HLAgene and documents its highly specific pattern of expressionthat indicated that the encoded antigen maybe a marker for mature B cells .

Laboratory of Immunology (LI) History :The establishment of this laboratory was the result ofEstablished : 1957 an awareness of the extent of allergic disorders andthe increasingly important role of the discipline ofimmunology in coping with key issues in experimentalbiology and medicine . Culminating several NIH andInstitute studies and proposals during the prior year,the decision to found a separate Laboratory of Immunologywas made during the latter part of 1956 .Dr . Freund was appointed the first Chief on January2, 1957 . This new laboratory took shape during theearly part of 1957 with reassignments of personnel fromother laboratories of MAID, primarily from LTD, whichhad previously carried on basic and clinical research inImmunology .As it has evolved, the laboratory has expanded tostudy various aspects of cellular immunology, molecularChief : William E . Paul, M.D . genetics, immunogenetics, and immunochemistry, par-1971- ticulary as they bear on a fuller understanding of thefundamental mechanisms and biological significance ofPrior : the immune response .Baruj Benacerraf, M.D . Maurice Landy, Ph.D .1968-70 1962-67Jules Freund, M.D .1957-601970 .Ellman, L . ; Green, I . ; Martin, W . J . ; and Benacerraf,B . Linkage Between the Poly-L-lysine Gene and theLocus Controlling the Major Histocompatibility Complexin Strain 2 Guinea Pigs . Proceedings of the NationalAcademy of Science, USA 66 : 322-28 .ElThis work established that immune response genes inguinea pigs were linked and were part of the majorhistocompatibility complex, representing a key step inthe development of our understanding of the geneticcontrol of immune responses .1971 .Paul, W . E . ; Katz, D . H . ; and Benacerraf, B .Augmented Anti-SIII Antibody Responses to an SIII-Protein Conjugate . Journal of Immunology 107 : 685-88 .0 The demonstration that anti-polysaccharide responsescould be markedly enhanced as a result of T cell sensitizationto a protein to which the polysaccharideepitope was linked . This observation formed the basisof current strategies to increase the immunogenicityof polysaccharide vaccines by linking them to proteinsand may be of particular value in efforts to immunizeinfants .1972 .Lieberman, R . ; Paul, W . E . ; Humphrey, W . Jr . ; andStimpfling, J . H . H-2 Linked Immune Response (Ir)Genes . Independent Loci for Ir-IgG and Ir-IgA Genes .Journal of Experimental Medicine 136 : 1231-40 .0 This was the first demonstration of the existence of asecond genetic locus within the major histocompatibilitycomplex that controlled immune responsesto specific antigens . It initiated a long series ofstudies that led to the mapping of Ir gene control ofa large series of proteins .

1972 .Shevach, E . M . ; Herberman, R . ; Frank, M . M . ; andGreen, I . Receptors for Complement and Immunoglobulinon Human Leukemic Cells and on HumanLymphoblastoid Cell Lines . Journal of Clinical Investigation51 : 1933-38 .D This paper represents the initial effort to classify lymphoidtumors based on their surface markers . It initiateda large series of studies that have led to aprecise understanding of the origin of the malignantcells and have been of great value in the diagnosisand therapy of leukemias and lymphomas .1972 .Shevach, E . M . ; Paul, W . E . ; and Green, I . HistocompatibilityLinked Immune Response Gene Function inGuinea Pigs : Specific Inhibition of Antigen-InducedLymphocyte Proliferation by Alloantisera . Journal ofExperimental Medicine 136 : 1207-21 .D This paper demonstrated that class II majorhistocompatibility complex molecules were specificallyrecognized by T cells in the course of T cellmacrophageinteractions in T cell responses and thuswas the key observation in the demonstration thatclass 11 molecules were immune response geneproducts .1972 .van Boxel, J . ; Paul, W . E . ; Green, L ; and Terry, W . D .IgD Bearing Human Lymphocytes . Journal of Immunology109 : 648-54 .0 This was the first description of the fact that IgD wasbasically a cell surface receptor molecule of B cellsand thus represented the initial recognition of thefunction of this class of immunoglobulin .1973 .Rosenthal, A . S ., and Shevach, E . M . Function ofMacrophages in Antigen Recognition by Guinea Pig TLymphocytes . 1 . Requirement for HistocompatibleMacrophages and Lymphocytes . Journal of ExperimentalMedicine 138 : 1194-1212 .D This paper demonstrated that cellular interactions betweenspecific T cells and macrophages required thatthe cells be of the same major histocompatibilitytype . It opened the field of histocompatibility restrictionof cellular interactions .1976 .Shevach, E . M . ; Frank, M . M . ; and Green, 1 . Linkageof the Gene Controlling the Synthesis of the FourthComponent of Complement to the Major HistocompatibilityComplex of the Guinea Pig . Immunogenetics 3 :596-601 .D The first demonstration that the C4 gene was part ofthe major histocompatibility complex . This has hadimportant implications in understanding the geneticorganization of the MHC .1977 .Paul, W . E ., and Benacerraf, B . Functional Specificityof Typhus-Dependent Lymphocytes . A Relationship Betweenthe Specificity of T Lymphocytes and Their Functionis Proposed . Science 195 : 1293-1300 .0 A landmark review in which an influential proposalwas made that integrated immunological specificityand function of T lymphocytes .1977 .Paul, W . E . ; Shevach, E . M . ; Pickeral, S . ; Thomas, D .W . ; and Rosenthal, A . S . Independent Populations ofPrimed F I Guinea Pig T Lymphocytes Respond toAntigen-Pulsed Parental Peritoneal Exudate Cells . Journalof Experimental Medicine 145 : 618-30 .D This observation established that T cells restricted byclass 11 MHC molecules were actually co-specific forantigen and class 11 MHC molecules and representeda key step in establishing that T cells recognized acomplex class 11 and antigenic determinants .1980 .Schwartz, R . H . ; Chen, C . ; Paul, W . E . Gene Complementationin the T Lymphocyte Proliferative Responseto Poly(Glu 56Lys 35Phe 9) . Functional Evidence for aRestriction Element Coded by Both I-A and I-ESubregions European Journal of Immunology 10 : 708-14 .0 This paper provided convincing functional evidencethat IR gene products and class 11 MHC restrictionelements were identical .1980 .Sredni, B ., and Schwartz, R . H . Alloreactivity of anAntigen-Specific T Cell Clone . Nature 287 : 855-57 .D The demonstration that antigen-specific, MHCrestrictedT cells are often specific for allo-MHCdeterminants, leading to an explanation of high frequency of alloreactive T cells and providing criticalinformation toward . understanding of nature of the Tcell specificity repertoire .1980 .Sredni, B . ; Tse, H . Y . ; and Schwartz, R . H . DirectCloning and Extended Culture of Antigen-Specific MHC-Restricted, Proliferating T Lymphocytes . Nature 287 :855-57 .0 The first demonstration of one of the two majormethods for the preparation of antigen-specific clonedlong term T cell lines .1980 .Stingl, G . ; Katz, S . E . ; Green, I . ; and Shevach, E . M .The Functional Role of Langerhans Cells . Journal of InvestigativeDermatology 74 : 325-28 .0 The demonstration that epithelial Langerhans cell arecritical antigen-presenting cells and that these cellsmay have important physiologic and pathophysiologicfunctions in skin-mediated sensitization reactions .

1982 .Howard, M . ; Farrar, J . ; Hilfiker, M . ; Johnson, B . ;Takatsu, K, ; Hamaoka, T . ; and Paul, W . E . Identificationof a T Cell-Derived B Cell Growth Factor DistinctFrom Interleukin-2 . Journal of Experimental Medicine155 : 914-23 .ElThe initial description of the lymphokine now referredto as interleukin-4/B cell stimulatory factor-1 andthe demonstration of its role as a co-stimulant of Bcell growth .1982 .Paul, W . E ., and Bona, C . Regulatory Idiotypes and ImmuneNetworks : A Hypothesis . Immunology Today 3 :230-34 .0 The initial proposal of the existence of specialized setof idiotopes with regulatory function and of thespecial roles of these determinants in the immunenetwork .1983 .Heber-Katz, E . ; Hansburg, D . ; and Schwartz, R . H . Thela Molecule of the Antigen-Presenting Cell Plays aCritical Role in Immune Response Gene Regulation of TCell Activation . Journal of Molecular and Cellular Immunology1 : 3-14 .D The demonstration of unambiguous function evidencefor the existence of a trimolecular complex involvingantigen, class II MHC molecules, and T cell receptorsin the T cell activation process .1984 .Davis, M . M . ; Cohen, D . I . ; Nielsen, E . A . ; Steinmetz,M . ; Paul, W . E . ; and Hood, L . Cell-Type SpecificcDNA Probes and the Murine I Region : The Localizationand Orientation of Aud . Proceedings of the NationalAcademy of Sciences, USA 81 : 2194-98 .D The introduction of the technique of subtractivehybridization to identify tissue-specific genes and theuse of that technique to isolate the gene for the classII Au chain .1984 .Germain, R ; Norcross, M . ; and Margulies, D . FunctionalExpression of a Transfected Murine Class II MHC Gene .Nature 306 : 190-94 .D The direct demonstration by DNA-mediated genetransfer techniques that class II MHC molecules arethe co-recognition elements in T cell recognition .1984 .Hedrick, S . M . ; Cohen, D . I . ; Nielsen, E . A . ; andDavis, M . M . Isolation of cDNA Clones Encoding TCell-Specific Membrane Associated Protein's . Nature 308 :149-53 .D The first molecular cloning of the gene for the achain of the T cell receptor for antigen . This paperopened the field of study of the molecular biology ofT cell receptors and of the molecular and cellularmechanisms for the generation of the T cell repertoire .1985 .Germain, R . N . ; Bentley, D . M . ; and Quill, H . Influenceof Allelic Polymorphism on the Assembly and SurfaceExpression of Class 11 MHC (Ia) Molecules . Cell 43 :233-42 .El The first demonstration that pairing a class II a and(3 chains displays allelic specificity providing evidencefor the specificity of this pairing function .1985 .Lamoyi, E ., and Mage, R . G . The Lack of Klb9 LightChains in Basilea Rabbits is Probably Due to a Mutationin an Acceptor Site for mRNA Splicing . Journal of ExperimentalMedicine 162 : 1149-60 .El A demonstration of the molecular basis of the failureto produce an immunoglobulin light chain isotype .Has importance in understanding thecular mechanismsthat lead to other instances of immunodeficiency .1985 .Ohara, J ., and Paul, W . E . B Cell Stimulatory Factor(BSF)-1 : Production of a Monoclonal Antibody andMolecular Characterization . Nature 315 : 333-36 .El The purification of interleukin-4/B cell stimulatoryfactor-1 and production of a monoclonal antibody . Thispaper represents a key step in the molecular andfunctional characterization of this importantlymphokine .1985 .Samelson, L . E . ; Lindsten, T . ; Fowlkes, B . J . ; van denElsen, P . ; Terhorst, C . ; Davis, M . M . The Expression ofGenes of the T Cell Antigen Receptor Complex inPrecursor Thymocytes . Nature 315 : 765-68 .O The initial description of the molecular eventsassociated with T cell maturation in the thymus asthey relate to the expression of the T cell receptorand associated molecules .1985 .Vitetta, E . S . ; Ohara, J . ; Myers, C . ; Layton, J . ; Krammer,P . H . ; and Paul, W . E . Serologic, Biochemical andFunctional Identity of B Cell Stimulatory Factor-1 and BCell Differentiation Factor for IgGI . Journal of ExperimentalMedicine 162 : 1726-31 .D This paper establishes that interleukin-4/B cellstimulatory factor-1 .1986 .Coffman, R . L . ; Ohara, J . ; Bond, M . W . ; Carty, J . ;Zlotnik, A . ; and Paul, W . E . B Cell StimulatoryFactor-1 Enhances the IgE Response of Lipopolysaccharide-Activated T Cells . Journal of Immunology 136 : 4538-41 .D Interleukin-4/B cell stimulatory factor-1 is the IgEswitch factor .

1986 .Finkelman, F . D . ; Katona, I . ; Urban, J . ; Snapper, C . ;Ohara, J . ; and Paul, W . E . Suppression of In VivoSuppression of In Vivo Polyclonal IgE Responses byMonoclonal Antibody to the Lymphokine BSF-1 . Proceedingsof the National Academy of Sciences, USA 83 :9675-78 .0 The demonstration that interleukin 4/B cellstimulatory factor-1 is the principle in vivo regulatorif IgE synthesis in a model of parasite infection . Acritical observation in the design of strategies to controlIgE synthesis in parasite infections in allergicresponses .1986 .Kroczek, R . A . ; Gunther, K . C . ; Germain, R . N . ; andShevach, E . M . Thy 1 Functions as a Signal TransductionMolecule in T-Lymphocytes and Transfected B Lymphocytes. Nature 181-84 .0 The direct demonstration by DNA-mediated genetransfer techniques that Thy 1 is capable of transducingbiochemical growth-inducing signals inlymphocytes .1986 .Lechler, R . I . ; Ronchese, F . ; Braunstein, N . S . ; andGermain, R . N . I-A Restriction Antigen-Recognition :Analysis of the Roles of Au and A0 using DNA-Mediated Gene Transfer . Journal of ExperimentalMedicine 163 : 678-96 .0 The analysis of structure function relationships in therole of class II MHC molecules in antigen-recognitionby T cell receptors .1986 .Margulies, D . H . ; Ramsey, A . L . ; Boyd, L . F . ; andMcCluskey, J . Genetic Engineering of an H-2Dd/QlobChimeric Histocompatibility Antigen : Purification ofSoluble Protein from Transformant Cell Supernatants .Proceedings of the National Academy of Sciences, USA83 : 5252-56 .0 The development of a general technique to producelarge quantities of secreted, soluble forms of class IMHC molecules . This approach should be of greatvalue in structural studies of class I molecules and inthe study of T cell receptor-binding to class Imolecules .1986 .Saito, T . ; Weiss, A . ; Miller, J . ; Norcross, M . A . ; andGermain, R . N . Specific Antigen-la Activation ofTransfected Human T Cells Expressing Murine Ti a,Q-Human T3 Complexes . Nature 325 : 125-29 .1- 1 Through the use of DNA-mediated transfer of genesfor the T cell receptor a and (3 chains, it wasestablished that a single receptor is responsible forgenetic restriction and for antigen recognition .1987 .Paul, W . E . Between Two Centuries . Specificity andRegulation in the Immune Response . Journal of Immunology139 : 1-6 .0 American Association of Immunologist presidentialaddress describing the importance of the developmentof a modern science of immunoregulation and ofunderstanding the in vivo physiology of immuneresponse .1987 .Snapper, C . M ., and Paul, W . E . Interferon Gammaand B Cell Stimulatory Factor-1 Reciprocally Regulate IgIsotype Production . Science 236 : 944-47 .0 The demonstration that interferon gamma controlsswitching to IgG2a and the proposal of a hypothesisfor the T cell mediated control of Ig isotypes basedon their relative protective value in viral and parasiticinfections .1987 .Stingl, G . ; Koning, F . ; Yamada, H . ; Yokoyama, W . M . ;Tschachler, E . ; Bluestone, J . A . ; Steiner, G . ; Samelson,L . E . ; Lew, A . M . ; Coligan, J . E . ; and Shevach, E . M .Thy-1 + Dendritic Epithelial Cells Express T3 Antigenand the T Cell Receptor Gamma Chain . Proceedings ofthe National Academy of Sciences, USA 84 : 2430-34 .0 A demonstration that a population of epidermal Tcells have a heightened expression of a subset of Tcells that use the T cell receptor T chain, probablytogether with the S chain, in the formation of theirreceptor . This work provides an approach to thestudy of the function of TS-cells and to the isolationof the T cell receptor 8 chain for chemical studiesand for cloning .1986 .Mosmann, T . R . ; Bond, M . W . ; Coffman, R . L . ;Ohara, J . ; and Paul, W . E . T Cell and Mast Cell LinesRespond to B Cell Stimulatory Factor-1 . Proceedings ofthe National Academy of Sciences, USA 83 : 5654-58 .0 The initial recognition that interleukin-4/B cellstimulatory factor-1 has activity on a wide range ofhematopoietic lineage cells and is not limited to Bcells in its action .

Laboratory of Immunopathology (LIP)Laboratory of Immunoregulation (LIR)Established : 1985Established : 1980Chief : Herbert C . Morse, III, M .D .1985-History :The Laboratory of Immunopathology conducts researchon the biology of virus infections in relation to thedevelopment and function of the hematopoeitic system .It was established in May 1985 from the Laboratory ofViral Diseases .1985 .Davidson, Wendy F . ; Holmes, Kevin L . ; Roths, JohnB . ; and Morse, Herbert C ., III . Immunologic Abnormalitiesof Mice Bearing the gld Mutation Suggest aCommon Pathway for Murine Nonmalignant LymphoproliferativeDisorders with Autoimmunity . Proceedingsof the National Academy of Sciences, USA 821 :1219-23 .LlStudies of mice mutant for the autosomal recessivemutations lpr and gld indicated that these defectsmay alter different enzymes in a common metabolicpathway associated with the development of autoimmunedisease .1985 .Lewis, Andrew M ., Jr ., and Cook, James L . A NewRole for DNA Virus Early Proteins in Viral Carcinogenesis. Science 227 : 15-20 .El This study contributes to the continuing pursuit ofhow tumor virus genes alter normal cells to produceneoplastic cells that may or may not be tumorigenicby describing previously unrecognized functions ofDNA tumor virus early genes-namely their ability toinduce in transformed rodent cells specific susceptibilitiesor resistances to lysis by natural killer cellsor activated macrophages-that correlate with theirtumor inducing capacities in a quantitative model oftransformed cell tumorigenicity .Chief : Anthony S . Fauci, M.D .1980-History :This laboratory was established to apply new knowledgein immunology to the clinical diagnosis and treatment ofpatients with immunological disorders .1974 .Fauci, Anthony S ., and Dale, David C . The Effect of InVivo Hydrocortisone on Subpopulations of Human Lymphocytes. Journal of Clinical Investigation 53 : 240-46 .0 This paper demonstrated that corticosteroid administrationto man results in a profound, but transientselective depletion of T lymphocytes from thecirculation . This was the first observation that singledoses of corticosteroids could have a significant effecton the circulatory capabilities of human lymphocytesand caused depletion not by lympholysis, but by alteringcirculatory kinetics .1976 .Fauci, Anthony S ., and Pratt, Karen R . Polyclonal Activationof Bone-Marrow-Derived Lymphocytes FromHuman Peripheral Blood Measured by a Direct Plaque-Forming Cell Assay . Proceedings of the NationalAcademy of Sciences, USA 73 : 3676-79 .El This study represents the development of the firstassay in the human system for the induction andmeasurement of antibody production by human lymphocytesat the single cell level .1976.Fauci, A . S . ; Dale, D . C . ; and Balow, J . E . GlucosorticosteroidTherapy : Mechanism of Action and ClinicalConsiderations . Annals of Internal Medicine 84 : 305-15 .El This paper is considered the classic paper in delineationof the mechanisms of action of corticosteroids inhumans . It received the Citation Classic designationfrom Current Contents .

1978 .Fauci, A . S . ; Haynes, B . F . ; and Katz, P . The Spectrumof Vasculitis : Clinical Immunologic and TherapeuticConsideration . Annals of Internal Medicine 89 : 660-76 .0 This paper established the classification scheme forthe vasculitic syndromes which is currently usedworldwide and which has become the classic in thefield .1979 .Fauci, A . S . ; Katz, P ; Haynes, B . F . ; and Wolff, S . M .Cyclophosphamide Therapy of Severe Systemic NecrotizingVasculitis . New England Journal of Medicine 301 :235-38 .El This was the first paper to demonstrate the efficacyof cyclophosphamide in inducing long-term remissionsin systemic vasculitis .1979 .Hayes, Barton F . ; Eisenbarth, George S . ; and Fauci, AnthonyS . Human Lymphocyte Antigens : Productions of aMonoclonal Antibody that Defends Functional Thymus-Derived Lymphocyte Subsets . Proceedings of the NationalAcademy of Sciences, USA 76 : 5929-33 .0 This paper reported the development of a monoclonalantibody against a distinct subset of human T cellsand helped ultimately to map intrathymic developmentof T cell repertoire .1981 .Lane, Clifford H . ; Volkman, David J . ; Whalen, Gail ;and Fauci, Anthony S . In Vitro Antigen-Induced,Antigen-Specific Antibody Production in Man . Specificand Polyclonal Components, Kinetics, and Cellular Requirements. Journal of Experimental Medicine 154 :1043-57 .0 This study was the first to demonstrate antigeninduced,antigen-specific antibody production inhuman peripheral blood . It has since laid theframework for the study of the specificity of thehuman immune response to soluble antigens .1982 .Lane, Clifford H . ; Shelmaner, James H . ; Mostowski,Howard S . ; and Fauci, Anthony S . Human MonoclonalAnti-Keyhole Limpet Hemocyanin Antibody-SecretingHybridoma Produced from Peripheral Blood B Lymphocytesof a Keyhold Limpet Hemocyanin-Immune Individual. Journal of Experimental Medicine 155 : 333-38 .0 This paper describes the first hybridoma developedfrom human peripheral blood which secretesmonoclonal antibody to a soluble antigen againstwhich the subject was immunized . It proved thefeasibility of developing human monoclonal antibodiesagainst predetermined antigenic specificities .1983 .Fauci, Anthony S . ; Haynes Barton, F . ; Katz, Paul ; andWolff, Sheldon M . Wegener's Granulomatosis : ProspectiveClinical and Therapeutic Experience with 85 Patientsfor 21 Years . Annals of Internal Medicine 98 : 76-85 .El This paper is the classic work in the field of therapyfor Wegener's granulomatosis and the vasculitic syndromes. It describes the cumulative experience over 2decades with the use of cyclophosphamide therapy inthe cure of this previously fatal disease . It is one ofseveral papers over the years reporting on thesepioneering studies .1985 .Lane, Clifford H . ; Depper, Joel, M . ; Greene, WarnerC . ; Whalen, Gail ; Waldmann, Thomas A . ; and Fauci,Anthony S . Qualitative Analysis of Immune Function inPatients with the Acquired Immunodeficiency Syndrome .Evidence for a Selective Defect in Soluble AntigenRecognition . New England Journal of Medicine 313 :79-84 .0 This paper describes the selectivity of the immunologicdefect in AIDS . It establishes the basis forthe dissection of the immunopathogenic mechanismsrelated to infection with the human immunodeficiencyvirus .1985 .Ambrus, Julian L ., Jr . ; Jurgensen, Cynthia H . ; Brown,Eric J . ; and Fauci, Anthony S . Purification toHomogenicity of a High Molecular Weight Human BCell Growth Factor ; Demonstration of Specific Bindingto Activated B Cells ; and Development of a MonoclonalAntibody to the Factor . Journal of ExperimentalMedicine 162 : 1319-35 .0 This paper describes the purification of a highmolecular weight human B cell growth factor and thedemonstration of its specific binding to activated Bcells . It is one of a series of papers from theLaboratory of Immunoregulation which havedelineated the mechanisms of regulation of the humanB cell cycle .1986 .Folks, T . ; Powell, D . M . ; Lightfoote, M . M . ; Berm, S . ;Martin, M . A . ; and Fauci, A . S . Induction of HTLV-III/LAV Expression from a Nonvirus-Producing T CellLine : Implications for Latent Infection in Man . Science231 : 600-020 This study established a model system for the studyof latency of infection with the AIDS virus and themechanisms of conversion of a latent infection to aproductive infection . It has assumed major importance in our understanding of the immunopathogenesisof infection with the human immunodeficiencyvirus (HIV) .

Laboratory of Infectious Diseases (LID) History :This laboratory is the most direct descendant of theEstablished : 1948 with the formation of NMI . Existed in original Marine Hospital Service research laboratoryother forms since August 1887 .established in August 1887 at the Marine Hospital onStaten Island, New York . At that time its function wasto assist the Service in diagnosing infectious diseasesamong passengers on incoming ships . Detached from thehospital and moved to the Butler Building inWashington, D.C . in 1891, this "Laboratory ofHygiene" became known as the Hygienic Laboratory .Authorized in 1901 by Congress to investigate "infectiousand contagious diseases and matters pertaining to thepublic health," the research of the Hygienic Laboratoryfocused primarily on bacteriology and pathology, the twomajor fields of nineteenth century medical interest . In1902 a reorganization of the Service divided theLaboratory into four divisions ; infectious disease researchwas located in the Division of Pathology andChief : Robert M . Chanock, M .D . Bacteriology .1968- On February 1, 1937, the Division was renamed theDivision of Infectious Diseases, becoming one of thePrior :eight Divisions and one Office (of Cooperative Studies)defined in the National Institute of Health reorganizationof that date . At this time, the Division was assignedadministrative jurisdiction over the Rocky MountainLaboratory, and the Division incidentally carried on theheart and dental research of the Public Health Service .In the reorganization of November 1948 that createdthe (plural) National Institutes of Health, the Divisionbecame the Laboratory of Infectious Diseases, one of thefour original components of the National MicrobiologicalInstitute (NMI), also established at that time . The RockyMountain Laboratory was assigned equal administrativestatus as part of the new NMI, and heart and dental activitieswere placed in their own newly created Institutes .Robert J . Huebner, M .D . Dorland J . Davis, M.D .1956-67 1954-56Karl Habel, M.D . Charles Armstrong, M.D .1948-54 1942-481972Kapikian, A . Z . ; Wyatt, Richard G . ; Dolin, R . ; Thornhill,T .S . ; Kalica, Anthony R . ; and Chanock, RobertM . Visualization by Immune Electron Microscopy of a2 .7 nm Particle Associated with Acute Infectious NonbacterialGastroenteritis . Journal of Virology 10 :1075-81 .ElFirst recognition of non A, non B,hepatitis whichnow constitutes the major form of post-transfusionhepatitis as well as a common form of sporadic viralhepatitis .1975 .Purcell, Robert H ., and Gerin, J . L . Hepatitis B SubunitVaccine : A Preliminary Report of Safety and EfficacyTests in Chimpanzees . American Journal of MedicalScience 270 : 395-99 .ElFirst demonstration that purified hepatitis B virus(HBV) surface antigen induces resistance to infectionby HBV in the chimpanzee . This observation wascritical to the development of the HBV vaccine .

1980 .Lamb, R . A . ; Choppin, P . W . ; Chanock, Robert M . ;and Lai, Ching-J . Mapping of the Overlapping Genes forPolypeptides NS F and NS Z on RNA Segment 8 of the InfluenzaVirus Genome . Proceedings of the NationalAcademy of Science, USA 77: 1857-61 .ElFirst description of RNA splicing of transcripts froman RNA genome .1980 .Wong, Doris C . ; Purcell, Robert H . ; Sreenivasan, M .A . ; Prasad, S . R . ; and Pavri, K . M . Epidemic andEndemic Hepatitis in India : Evidence for a Non-A Non-B Hepatitis Virus Etiology . Lancet 2 : 876-79 .0 First evidence for the existence of a second form ofnon A, non B hepatitis virus that is responsible forlarge common source (usually water borne) epidemicsof hepatitis .1980 .Rizzetto, M . ; Canese, M . G . ; Gerin, J . L . ; London,William T . ; Sly, L . D . ; and Purcell, Robert H .Transmisson of the Hepatitis B Virus-Associated DeltaAntigen to Chimpanzees . Journal of Infectious Diseases141 : 590-602 .El Demonstration that the delta antigen was atransmissible agent which required simultaneous infectionby hepatitis B virus for its replication . Thus,delta was shown to be a defective, transmissible viruswhich required for its replication functions providedby its helper virus, hepatitis B virus . Subsequentstudies established an important role for delta virus inthe etiology of unusually severe, often fatal viralhepatitis .1982 .Murphy, Brian R . ; Sly, D . L . ; Tierney, Eveline L . ;Hosier, N . T . ; Massicot, J . G . ; London, William T . ;Chanock, Robert M . ; Webster, R . G . ; and Hinshaw, V .S . Reassortant Virus Derived from Avian and HumanInfluenza A Viruses is Attenuated and Immunogenic inMonkeys . Science 218 : 1330-32 .0 Substitution of genes coding for the internal viral andnon-structural proteins of an avian influenza A virusfor the corresponding genes of a human influenza Avirus by gene reassortment attenuates human influenzaA virus .1986 .Kapikan, A . Z . ; Flores, Jorge ; Hoshino, Yatsutaka ;Glass, Roger I . ; Midthun, Karen ; Gorziglia, Mario ; andChanock, Robert M . Rotavirus : The Major EtiologicAgent of Severe Infantile Diarrhea May be Controllableby a "Jennerian" Approach to Vaccination . Journal ofInfectious Diseases 153 : 815-22 .ElDescription of "Jennerian" approach to preventionof severe rotavirus diarrhea of infancy and earlychildhood employing a rotavirus of simian originwhich is attenuated . but immunogenic in humans .1986 .Olmstead, Robert A . ; Elango, Narayansamu ; Prince,Gregory A . ; Murphy, Brian R . ; Johnson, Philip R . ;Moss, Bernard ; Chanock, Robert M . ; and Collins, P . L .Expression of the F Glycoprotein of Respiratory SyncytialVirus by a Recombinant Vaccinia Virus : Comparisonof the Individual Contributions of the F and GGlycoproteins to Host Immunity . Proceedings of the NationalAcademy of Sciences, USA 83 : 7462-66 .ElConstruction of vaccinia virus-respiratory syncytialvirus (RSV) glycoprotein gene recombinants which expressauthentic RSV F or G glycoproteins and whichinduce significant resistance to RSV in the lungs ofimmunized cotton rats . First description of a successfulexperimental strategy for immunoprophylaxisof RSV which may have application to the control ofthis virus which is the most important respiratorytract viral pathogen of infancy and early childhood .120

Laboratory of Microbial Immunity (LMI)Established : 1969Chief : Richard Asofsky, M.D .1972-Prior : (Laboratory of Germfree Animal Research until 1969)John E . Tobie, Ph.D . Walter L . Newton, Ph.D .1964-72 1959-1964History :The Laboratory of Microbial Immunity was formallyestablished in 1969 . Its investigations are concernedwith the elucidation of basic mechanism involved inboth the cellular and humoral responses to microbialand tissue antigens . This laboratory developed fromthe earlier Laboratory of Germfree Animal Research,(LGAR) which was established in 1959 .1970 .Baker, Philip J . ; Stashak, Philip W . ; Amsbaugh, D .F . ; Prescott, B . ; and Barth, R . F . Evidence for theExistence of Two Functionally Distinct Types of Cellswhich Regulate the Antibody Response to Type 111Pneumococcal Polysaccharide . Journal of Immunology105 : 1581-83 .E This was one of the earliest papers to show theexistence of "suppressor" T lymphocytes . It wasalso the first to suggest that "T-independent"responses are regulated by T lymphocytes whichamplify or suppress the response .lw1971 .Pierce, C . W . ; Solliday, S . M . ; and Asofsky, Richard .Immune Responses In Vitro . IV . Suppression of PrimaryyM, yG, and yA Plaque-Forming Cell Response inMouse Spleen Cell Cultures by Class-Specific Antibodyto Mouse Immunoglobulins . Journal of ExperimentalMedicine 135 : 675-97 .Paper showed that anti-IgM blocked antibodyresponses of all isotypes in vitro, and that theblock was specific for B cells, indicating that IgMwas the major (or only) antigen-specific receptoron B lymphocytes (see Lawton reference) .1972 .Cantor, H ., and Asofsky, Richard . Synergy AmongLymphoid Cells Mediating the Graft- Versus-HostResponse . III . Evidence for Interaction Between TwoTypes of Thymus-Derived Cells . Journal of ExperimentalMedicine 135 : 764-79 .1972 .Tigelaar, R . E ., and Asofsky, Richard . Synergy AmongLymphoid Cells Mediating the Graft- Versus-HostResponse . IV . Synergy in the GVH Reaction Quantitatedby a Mortality Assay in Sublethally Irradiated Recipients .Journal of Experimental Medicine 135 : 1059-70 .These two papers provided important evidence forfunctional heterogeneity among T lymphocytes, andwere the first to show that some T cells wouldamplify the activities of others .1972 .Lawton, A . R . III ; Asofsky, Richard ; Hylton, M . B . ;and Cooper, M . D . Suppression of ImmunoglobulinClass Synthesis in Mice . 1 . Effects of Treatment withAntibody to P-Chain . Journal of Experimental Medicine135 : 277-97 .Purified anti-IgM given to mice from birth suppressedcompletely the development of B lymphocytes andblocked development of antibody responses of allisotypes . The work indicated that all B lymphocytespass through a stage in which membrane-bound IgMwas expressed . Animals treated in this way also providea useful model of B-lymphocyte deficiency .1972 .Amsbaugh, Diane F . ; Hansen, C . T . ; Prescott, B . ;Stashak, Philip W . ; Barthold, D . R . ; and Baker, .PhilipJ . Genetic Control of the Antibody Response to Type IIIPneumococcal Polysaccharide in Mice . Evidence that anX-Linked Gene Plays a Decisive Role in DeterminingResponsiveness . Journal of Experimental Medicine 136 :931-49 .0 The first description of an X-linked immunodeficiencyin strain CBA/N mice, now a standard model forstudying such deficiencies .

1979 .Pasanen, V . J . ; Asofsky, Richard ; and Baker, Philip J .Synthesis of Two Classes of Antibody, yM, and -yG, oryG and yA by Identical Cells . Amplification of the AntibodyResponse to Pneumococcal Polysaccharide Type111 . Journal of Experimental Medicine 149 : 1227-37 .El Demonstration of large numbers cells secreting twoisotypes of antibody to the same antigensimultaneously ("double-secretors"), providing, directevidence for an isotype switch in individual cells duringdevelopment of an antibody response .1984 .Fowlkes, B . J . Characterization and Differentiation ofThymic Lymphocytes in the Mouse . Ph.D . Thesis .Washington, DC : George Washington UniversityGraduate School of Arts and Sciences .1985 .Fowlkes, B . J . ; Edison, L . ; Mathieson, B . J . ; and Chused,Thomas M . Early T Lymphocytes : Differentiation InVivo of Adult Intrathymic Precursor Cells . Journal ofExperimental Medicine 162 : 802 .0 These two works described the isolation of a smallsubpopulation of thymocytes almost lacking expressionof differentiation antigens of T lymphocytes, andshowed that all other types of T-cell could be derivedfrom it .1985 .Baker, Philip J . ; Bailey, D . W . ; Fauntleroy, M . B . ;Stashak, Philip W . ; Caldes, G . ; and Prescott, B . Genesin Different Chromosomes Influence the AntibodyResponse to Bacterial Antigens . Immunogenetics 22 :269-76 .0 Paper localized genetic factors controlling antibodyresponses to polysaccharide antigens to at least 5 differentmouse chromosomes, none closely linked to theMHC .Laboratory of Molecular Microbiology (LMM)Established : 19811Chief: Malcolm A . Martin, M .D .1981-History :The Laboratory of Molecular Microbiology wasestablished in 1981 to exploit new methods in recombinantDNA technology and to study other molecularareas thus expanding the Institute's interests in bothbacterial and viral pathogenesis and virulence .1971 .Gelb, L . D . ; Kohne, D . E . ; and Martin, Malcolm A .1971 . Quantitation of SV-40 Sequences in Green MonkeyMouse and Virus Transformed Cell Genomes . Journal ofMolecular Biology 57 : 29-45 .0 This paper was the first to quantitate the number ofviral genomes integrated into the chromosome of aviral-induced tumor cell .1971 .Gelb, L . D . ; Aaronson, S . A . ; and Martin, Malcolm A .Heterogeneity of Murine Leukemia Virus In Vitro DNA :Detection of Viral DNA in Mammalian Cells . Science172 : 1353-55 .1-1 This paper was first to demonstrate that normalmammalian cells (in this case, mouse) contain copiesof retroviral sequences .1972 .Khoury, G ., and Martin, Malcolm A . A Comparison ofSV40 DNA Transcription In Vivo and In Vitro . Nature238 : 4-6 .0 This paper was the first to show that both strands ofa viral DNA can be used to program the synthesis ofviral messenger RNA and proteins .122

1973 .Khoury, G . ; Martin, Malcolm A . ; Lee, N . H . ; Danna,K . J . ; and Nathans, D . A Map of SV40 TranscriptionSites Expressed in Productively Infected Cells . Journal ofMolecular Biology 78 : 377-89 .El This paper describes the first use of restriction enzymesto prepare transcriptional maps of genes orgenomes .1975 .Howley, P . M . ; Mullarkey, M . F . ; Takemoto, K . K . ;and Martin, Malcolm A . Characterization of HumanPapovavirus BK DNA . Journal of Virology 15 : 173-81 .1975 .Howley, P . M . ; Khoury, G . ; Byrne, J . ; Takemoto, K .K . ; and Martin, Malcolm A . Physical Map of the BKVirus Genome . Journal of Virology 16 : 959-73 .11 These two papers describe, for the first time, thestructure of the human papovavirus, BKV .1979 .Israel, M . A . ; Chan, H . W . ; Rowe, W . P . ; and Martin,Malcolm, A . Molecular Cloning of Polyoma Virus DNAin E. coli . I . Plasmic Vector System . Science 203 : 883-87 .1979 .Chan, H . W . ; Israel, M . A . ; Garen, Claude F . ; Rowe,Wallace P . ; and Martin, Malcolm, A . Molecular Cloningof Polyoma Virus DNA in E. coli . 11 . Lambda PhageVector System . Science 203 : 887-92 .1979 .Israel, M . A . ; Chan, H . W . ; Martin, Malcolm A . ; andRowe, W . P . Molecular Cloning of Polyoma Virus DNAin E. coli: Oncogenicity Testing in Hamsters . Science205 : 1140-42 .D These three papers describe "risk assessment" experimentsto evaluate potential hazards associatedwith recombinant DNA research . Conclusion : no apparentdanger .1981 .Martin, Malcolm A . ; Bryan, T . ; Tasheed, S . ; and Khan,Arifa S . Identification and Cloning of EndogenousRetroviral Sequences Present in Human DNA . Proceedingsof the National Academy of Sciences, USA 78 :4892-96 .D This paper showed that human DNA contains multiplecopies of endogenous retroviral sequences .1985 .Rabson, A . B . ; Daugherty, D . F . ; Venkatesan, S . ;Boulukos, K . E . ; Berm, S . I . ; Folks, T . M . ; Feorino, P . ;and Martin, M . A . Transcription of Novel Open ReadingFrames of AIDS Retrovirus During Infection of Lymphocytes. Science 229 : 1388-90 .El This Science paper describes, for the first time all ofthe human immunodeficiency virus encodedmessenger RNAs .1986 .Koenig, S . ; Gendelman, Howard ; Orenstein, J . ; DalCanto, M . ; Pezeshkopour, G . ; Yungbluth, M . ; Janotta,F . ; Aksamit, A . ; Martin, Malcolm; and Fauci, Anthony .Detection of AIDS Virus in Macrophages in Brain TissueFrom AIDS Patients with Encephalopathy . Science 233 :1089-93 .El This paper was the first to show that HIV resides inmacrophages, not in nerve cells, in the brains ofAIDS patients with neurological symptoms .1986 .Gendelman, Howard ; Phelps, W . ; Feigenbaum, L . ;Ostrove, Jeffrey ; Adachi, A . ; Howley, P . ; Khoury, G . ;Ginsberg, H . ; and Martin, Malcolm . Trans-Activation ofthe Human Immunodeficiency Virus Long TerminalRepeat Sequence by DNA Viruses . Proceedings of theNational Academy of Sciences, USA 83 : 9759-63 .El This paper was the first to show that virusesunrelated to HIV have the capcity to activate a dormant(inactive) copy of the AIDS virus .123

Laboratory of Parasitic Diseases (LPD)Laboratory of Tropical VirologyEstablished : 1959 . This laboratory existed in many differentforms from its founding in 1902 .Alexis 1 . Shelokov, M.D .1959-64Chief : Franklin A . Neva, M.D .1969-Prior :Laboratory of Tropical Diseases(1949-59) ,Division of Tropical Diseases(1947-49)Division of Zoology(1902-47)Paul P . Weinstein, Sc .D . Leon Jacobs, Ph .D .1965-68 1959-65IPrevious organization :Willard H . Wright, Maurice C . Hall,Ph.D ., D.V .M . Ph .D ., D.V .M .Laboratory of Parasite Chemotherapy 1938-58 1936-38Geoffrey M . Jeffery, J . Robert Coatney, Charles Wardell Stiles, Ph.D .Sc.D., M .P.H . Ph.D ., Sc .D . 1902-19311965-69 1959-65124

History :This laboratory's origins can be traced to the Division ofZoology created in 1902 in the Hygienic Laboratory . Dr .Stiles headed this Division from 1902 until his retirementin 1931, and for the next five years the position remainedvacant . In 1936 Dr . Hall was named director ; he serveduntil his sudden death in 1938 . Under Dr . Wright,who served from 1938 to 1958, the Division of Zoologywas renamed the Division of Tropical Diseases (1947)and then the Laboratory of Tropical Diseases (LTD,1949) . When he retired, a reorganization of LTD occurred. Four new laboratories were formed : 1) Laboratoryof Parasitic Diseases (LPD) ; 2) Laboratory of ParasiteChemotherapy (LPC) ; 3) Laboratory of GermfreeAnimal Research (LGAR), which had started as a LTDproject in 1951 to perform studies on parasites ; 4)Laboratory of Tropical Virology (LTV), which was comprisedof the Arbo-borne Virus Section of LTD and theMiddle American research Unit in the Panama CanalZone . In a reorganization during the late 1960's, LTVwas disbanded, LGAR was transformed into theLaboratory of Microbial Immunity (LMI), and LPC wasintegrated with LPD into a single Laboratory of ParasiticDiseases under Dr . Neva .1971 .Gelderman, A . H . ; Keister, D . B . ; Bartgis, 1 . L . ; andDiamond, L . S . Characterization of the DeoxyribonucleicAcid of Representative Strains of E . histolytica, E.histolytica-Like Amebae, and E. moshkovski. Journal ofParasitology 57 : 906-11 .ElOne of the earliest examples of use of molecularbiology to demonstrate important differences inparasites .1975Dvorak, J .A . ; Miller, L .H . ; Whitehouse, W .C . ; andShiroishi, T . Invasionof Erythrocytes by MalarialMerozoites Science 187 : 748-50 .O Use of low light level video techniques permittedobservation that invasion of red cell first involved attachmentvia specific orientation and later invasionand provided basis for receptor paper .1975 .Miller, L . H . ; Mason, S . J . ; Dvorak, J . A . ; McGinnis,M . H . ; and Rothman, 1 . K . Erythrocyte Receptors for(P . knowlesi) Malaria : Duffy Blood Group Determinants .Science 189 : 561-563 .ElFirst demonstration of receptor mediated invasion ofRBCs and explanation for resistance to P . vivax inmany blacks .1981 .Udeinya, 1 . J . ; Schmidt, J . A . ; Aikawa, M . ; Miller, L .H . ; and Green, 1 . Falciparum Malaria-InfectedErythrocytes Specifically Bind to Cultured Human EndothelialCells . Science 213 : 555-57 .ElProvided an in-vitro method to study the most importantpathophysiologic process in Falciparummalaria .1984 .Sacks, D . L ., and Perkins, P . V . Identification of an InfectiveStage of Leishmania Promastigotes . Science 223 :1417-19 .11 First clear cut demonstration that infective parasiteswere in stationary instead of log phase of growth,and could be differentiated from noninfectiveorganisms .1984 .Dame, J . B . ; Williams, .1 . L . ; McCutchan, T . F . ; Weber,J . L . : Wirtz, R . A . ; Hockmeyer, W . T . ; Maloy, W . L . ;Haynes, J . D . ; Schneider, I . ; Roberts, D . ; Sanders, G .S . ; Reddy, E . P . ; Diggs, C . L . ; and Miller, L . H . Structureof the Gene Encoding the Immunodominant SurfaceAntigen on the Sporozoite of the Human MalariaParasite, P. falciparum . Science 225 : 593-99 .11 This paper-along with work of the Nussenzweig'swasthe first gene cloning of the circumsporozoiteprotein of a human malaria parasite .125

Laboratory of Viral Diseases (LVD)Established : 1967Chief : Bernard Moss, M.D ., Ph.D .1984-Prior :Wallace P . Rowe, M .D . Robert J . Huebner, M .D .1968-83 1967-68History :In the 1967 reorganization of intramural research activity,this laboratory was established for Dr . Huebner, whohad previously been Chief of the Laboratory of InfectiousDiseases . In 1968 Dr . Rowe became Chief of LVDwhen Dr . Heubner transferred to NCI . In 1984, afterDr . Rowe's death, Dr . Moss left the Laboratory ofBiology of Viruses to become Chief of LVD .1971 .Lowy, Douglas R, ; Rowe, Wallace P . ; Teich, N . ; andHartley, Janet W . Murine Leukemia Virus : High-Frequency Activation In Vitro by 5-Iododeoxyuridineand 5-Bromodeoxyuridine . Science 174 : 155-56 .LlThis study indicated that the complete MuLV genomeis present in unexpressed form in all cells of the AKRmouse and provided a powerful technique for activationof C-type retrovirus genomes in other systems .1971 .Pincus, Theodore ; Hartley, Janet W . ; and Rowe,Wallace P . A Major Genetic Locus Affecting Resistanceto Infection with Murine Leukemia Virus . I . TissueCulture Studies of Naturally Occurring Viruses . Journalof Experimental Medicine 133 : 1219-33 .El These studies established that sensitivity of differentlaboratory mouse strains to either of the two classesof naturally occurring ecotropic MuLVs is geneticallyspecified and that alleles of the Fv-1" gene represent amajor determinant of virus spread .1972 .Rowe, Wallace P . Studies of Genetic Transmission ofMurine Leukemia Virus by AKR Mice . 1 . Crosses withFv-1° Strains of Mice . Journal of Experimental Medicine136 : 1272-85 .0 Application of Mendelian genetics to demonstratethat the AKR mouse contains two unliked, autosomalchromosomal loci either of which suffices to induceinfectious virus in Fv-1" progeny .1973 .Lewis, Andrew M . ; Levine, A . S . ; Crumpacker, C . S . ;Levin, M . J . ; Samaha, R . J . ; and Henry, P . H . Studiesof Non-Defective Adenovirus 2-Simian Virus 40 HybridViruses . V . Isolation of Additional Hybrids which Differin Their Simian Virus 40-Specific Biological Properties .Journal of Virology 11 : 655-64 .ElBy foretelling future developments, this unique studyintroduced the complexities of the recombinant DNAera by describing the first successful cloning and expressionof foreign viral DNAs (in this case overlappingsegments of the tumor-inducing region of thegenome of SV40) in adenovirus type 2 vectors whichwere themselves capable of independent replication .1973 .Kelly, Thomas J ., Sr ., and Lewis, Andrew M ., Jr . Useof Nondefective Adenovirus-Simian Virus 40 Hybrids forMapping the Simian Virus 40 Genome . Journal ofVirology 12 : 643-52 .El This study demonstrated unequivocally, by electronmicroscopic study of artificially duplexed viral DNAs,that the series of nondefective adenovirus 2- SV40hybrids described by Lewis et al . 1973 did, in fact,contain overlapping segments of the tumor-inducingregion of the genome of SV40 .126

1974 .Chattopadhyay, Sisir K . ; Lowy, Douglas R . ; Teich,Natalie M . ; Levine, Arthur S . ; and Rowe, Wallace P .Evidence that the AKR Murine-Leukemia-Virus Genomeis Complete in DNA of the High-Virus AKR Mouse andIncomplete in the DNA of the "Virus-Negative" NIHMouse . Proceedings of the National Academy ofSciences, USA 71 : 167-71 .D This paper represents the first step in establishing themolecular basis of endogenous MuLVs and their expression,establishing that the complete MuLVgenome is present in AKR cells as DNA, and thatmajor differences can be detected between high-virusand virus-negative strains of mice .1976 .Rowe, Wallace P . Leukemia Virus Genomes in theChromosomal DNA of the Mouse . Lecture DeliveredMarch 18, 1976 Harvey Lectures Series 71, 1978,Academic Press .D The concept of endogenous murine leukemia virusesis developed biologically and molecularly, anddiscussed in the context of the synthesis of theories ofgenetic and viral etiology of murine leukemia .1977 .Hartley, Janet, W . ; Wolford, Nancy K . ; Old, Lloyd J . ;and Rowe, Wallace P . A New Class of MurineLeukemia Virus Associated with Development of SpontaneousLymphomas . Proceedings of the NationalAcademy of Sciences, USA 74 : 783-92 .D This paper describes the isolation of naturally occurringdual-tropic MuLVs-MCF, or mink cell focusinducingviruses, characterizing a biologically importantnew class of MuLV and suggesting its recombinantorigin and relationship to murinelymphomagenesis .1985 .Yewdell, J . W . ; Bennink, J . R . ; Smith, G . L . ; andMoss, Bernard . Influenza A Virus Nucleoprotein is aMajor Target for Cross-Reactive Anti-Influenza VirusCytotoxic T Lymphocytes . Proceedings of the NationalAcademy of Sciences, USA 82 : 1785-89 .D This study illustrated the power of using recombinantvaccinia viruses for determination of cytotoxic T celltargets and was one of the first to demonstrate thatinternal viral proteins are recognized .1986 .Rohrman, G . ; Yuen, L . ; and Moss, B . Transcription ofVaccinia Virus Early Genes by Enzymes Isolated fromVaccinia Virions Terminates Downstream of aRegulatory Sequence . Cell 46 : 1029-35 .D The development of a faithful transcription systemopened the way to defining the nucleotide sequences,enzymes and factors involved in initiation and terminationof RNA synthesis .1980 .Rowe, Wallace P ., and Kozak, C . A . Germ Line Insertionsof AKR Murine Leukemia Virus Genomes inAKV-1 Congenic Mice . Proceedings of the NationalAcademy of Sciences, USA 77 : 4871-74 .D This report established that although geneticallytransferred MuLV loci are stable, during inbreedingof mice congenic for these loci new chromosomal insertionscan become established in the germ line .127

Rocky Mountain Laboratories (RML)Laboratory of Persistent Viral DiseasesEstablished : Reorganized in 1979 . Attained laboratorystatus as one of the four components of NMI onNovember 1, 1948 . Prior existence in other forms from1902, 1921, 1930-31 .Directors, RML before 1979 :t'Bruce W . Chesebro, M.D .1979-Epidemiology BranchHerbert G . Stoenner, D .V .M. Cornelius B . Philip, Ph .D .1964-1979 1962-64Willy Burgdorfer, Ph .D . Robert N . Philip, M.D1982-85 (Acting Chief) 1979-81 (Acting Chief)Laboratory of PathobiologyCarl Larson, M .D . Ralph R . Parker, Ph.D .1950-62 1921-1949Laboratory Chiefs Since 1979 :Laboratory of Microbial Structure and FunctionClaude F . Garon, Ph.D .1985- (Acting Chief)John L . Swanson, M.D .1979-128

History :The Public Health and Marine Hospital Service in 1902first became interested in Rocky Mountain spotted feverwhen it assigned one person from the HygienicLaboratory (Dr . Julius O . Cobb) to work with the MontanaState Board of Health in the Bitterroot Valley ofMontana . In the following year, Dr . John F . Andersonwas assigned to the study, and in 1904 the group wasjoined by Dr . Charles Wardell Stiles, who later identifiedthe tick Dermacentor andersoni . By 1922 the state hadthe laboratory in a two-story ex-school building inHamilton, and it was a recognized field station of thePHS . In 1931 Congress authorized the purchase of thefacilities from the state of Montana and the constructionof additional buildings . The laboratory continued as afield station under the Division of Scientific Research (asit had from 1922) until 1935 when it was officially namedthe "Rocky Mountain Laboratory" still under the sameadministrative jurisdiction . In February 1937, when theDivision of Scientific Research merged into the NationalInstitute of Health, the Rocky Mountain Laboratory wasmade administratively a part of the Division of InfectiousDiseases, although retaining its full name . Almosttwelve years later, when the National Microbiological Institutewas formed, the Rocky Mountain Laboratory attainedequal laboratory status with the Institute's otherthree original components (LID, LTD, DBC) . In 1979the Rocky Mountain Laboratory was reorganized and itsname made plural . Dr . Swanson was named Chief of theLaboratory of Microbial Structure and Function and Dr .Chesebro of the Laboratory of Persistent Viral Diseases .No permanent Chief of the Epidemiological Branch wasnamed, but Drs . Philip and Burgdorfer served as ActingChiefs . Similarly, Dr . Garon has served as Acting Chiefof the Laboratory of Pathobiology .Laboratory of Microbial Structure and Function1981 .Hackstadt, Ted, and Williams, Jim C . BiochemicalStratagem for Obligate Parasitism of Eukaryotic Cells byCoxiella burnetii. Proceedings of the National Academyof Sciences, USA 78 : 3240-44 .D Hackstadt and Williams show that metabolic activityof Coxiella burnetii is dependent on acidic conditions,like those in host cell's phagolylosomes ; suchdependency correlates with this obligate intracellularparasite's survival in a host cell compartment which isusually destructive to bacteria .1982 .Barbour, A . G . ; Tessier, S . L . ; and Stoenner, H . G .Variable Major Proteins of Borrelia hermsii . Journal ofExperimental Medicine 156 : 1312-24 .D Identifies outer membrane proteins that are responsiblefor the antigenic variation found among relapsingfever causing borreliae .1985 .Nano, Francis E ., and Caldwell, Harlan D . Expressionof Chlamydial Genus-Specific Lipopolysaccharide Epitopein Escherichia coli . Science 228 : 742-44 .ElNano and Caldwell clone a gene that encodes an enzymeresponsible for synthesis of a chlamydia genusspecificLipopolysaccharide antigen .1986 .Swanson, John ; Bergstrom, Sven ; Robbins, Kenneth ;Barrera, Osmar ; Corwin, Dan ; and Koomey, Michael J .Gene Conversion Involving the Pilin Structural GeneCorrelates with Pilus +- -- Pilus - Changes in Neisseriagonorrhoeae . Cell 47:267-76 .Shows that similar gene conversion events whichutilize different, silent pilin gene sequences accountfor both on/off switching and antigenic changes ofpili by Neisseria gonorrhoeae .1986 .Watkins, Nancy G . ; Hadlow, William J . ; Moos, AbbieB . ; and Caldwell, Harland D . Ocular Delayed Hypersensitivity: A Pathogenic Mechanism of Chlamydial Conjunctivitisin Guinea Pigs . Proceedings of the NationalAcademy of Sciences, USA 83 : 7480-84 .1:1 Explores a guinea pig model of chlamydia-associatedconjunctivitis ; guinea pigs that spontaneously recoverfrom primary chlamydial infection, though proctectedagainst reinfection by the same agent, exhibit markedocular hypersensitivity to a common chlamydialantigen .1987 .MacDonald, Gregory A . ; Anacker, Robert, L . ; and Garjian,Kareen . Cloned Gene of Rickettsia rickettsi SurfaceAntigen : Candidate Vaccine for Rocky Mountain SpottedFever . Science 235 : 83-85 .D Reports the cloning of a gene from Rickettsia rickettsii; the encoded product constitutes a protective vaccinein guinea pigs against Rocky Mountain spottedfever .Laboratory of Persistent Viral Diseases1974 .Chesebro, Bruce ; Wehrly, Kathy ; Stimpfling, Jack . HostGenetic Control of Recovery from Friend LeukemiaVirus-Induced Splenomegaly : Mapping of a Gene Withinthe Major Histocompatability Complex . Journal of ExperimentalMedicine 140 : 1457-67 .D This paper was the first to identify the importance ofthe D region of the H-2 complex in spontaneousrecovery from retrovirus-induced leukemia .

1977 .Prusiner, Stanley B . ; Hadlow, William J . ; Eklund, CarlM . ; and Race, Richard E . Sedimentation Properties ofthe Scrapie Agent . Proceedings of the National Academyof Sciences, USA 74 : 4656-60 .ElThis paper defined conditions for sedimentation ofscrapie agent infectivity and this suggested scrapiewas a discrete infectious particle .1981 .Coe, John E . ; Margossian, Sarkis S . ; Slayter, Henry S . ;and Sogn, John A . Hamster Female Protein : A NewPentraxin Structually and Functionally Similar to C-Reactive Protein and Amyloid P Component . Journal ofExperimental Medicine 153 : 977-91 .11 This paper showed that hamster female protein, ahamster homologue of the acute phase serum component,C-reactive protein, was a pentraxin under hormonalcontrol .1983 .Lodmell, Donald L . Genetic Control of Resistance toStreet Rabies Virus in Mice . Journal of ExperimentalMedicine 157 : 451-60 .L1This paper demonstrated that recovery from a rabiesvirus infection was under genetic control in mice andsuggested that it might be possible to intervene in thisusually fatal infection .1985 .Bloom, Marshall E ., et al . Analysis of Aleutian DiseaseVirus Infection In Vitro and In Vivo : Demonstration ofAleutian Disease Virus DNA in Tissue of InfectedMinks . Journal of Virology 53 : 696-703 .0 This paper was the first to demonstrate replicativeforms of viral DNA of the Aleutian disease of minkparvovirus in tissues of infected mink .1985 .Chesebro, Bruce, et al. Identification of Scrapie PrionProtein-Specific mRNA in Scrapie-Infected andUninfected Brain . Nature 315 : 331-33 .0 This paper demonstrated that a functional gene forthe scrapie associated prion protein occurs in normalas well as scrapie infected animals and that the prionmRNA was not specific for scrapie infection .1985 .Evans, Leonard, and Lloyd, Miles . Friend and MoloneyMurine Leukemia Viruses Specifically Recombine withDifferent Endogenous Retroviral Sequences to GenerateMink Cell Focus Forming Viruses . Proceedings of theNational Academy of Sciences, USA 82 : 459-63 .0 This paper indicated that polytropic retroviruses inmice arise by recombination with distinct endogenousviral genes .1987 .Portis, John ., et al. Horizontal Transmission of MurineRetroviruses . Journal of Virology 61 : 1037-44 .0 This paper demonstrated that an AIDS-like retroviruscould be transmitted venereally between mice of thesame or different sexes .Laboratory of Pathobiology1972 .Garon, Claude F . ; Berry, K . W . ; and Rose, James A . AUnique Form of Terminal Redundancy in AdenovirusDNA Molecules . Proceedings of the National Academyof Sciences, USA 69 : 2391-95 .El This paper was the first description of a unique andimportant sequence arrangement in viruses . Subsequentlythis type of terminal redundancy was foundin a variety of animal virus DNA's and appears to bean important structural feature of many, if not all,linear, duplex genomes .1981 .Arai, Hideo, and Munoz, John J . Crystallization ofPetussigen From Bordetella Pertussis. Infection and Immunity31 : 495-99 .0 This paper reported, for the first time, the crystallizationof pertussigen (pertussis toxin) from Bordetellapertussis .1981 .Munoz, John J . ; Arai, Hideo ; and Cole, Robert L .Mouse-Protecting and Histamine-Sensitizing Activities ofPertussigen and Fimbrial Hemagglutinin from Bordetellapertussis . Infection and Immunity 32 : 243-50 .0 This paper demonstrated conclusively that pertussigen(pertussis toxin) is the main mouse protecting antigenin pertussis vaccine .1981 .Munoz, John J . ; Arai, Hideo ; Bergman, Robert K . ; andSadowski, Peter L . Biological Activities of CrystallinePertussigen from Bordetella pertussis . Infection and Immunity33 : 820-26 .0 This paper demonstrated conclusively that a simplecrystalline toxin from Bordetella pertussis has most ofthe biological activities found in pertussis vaccine .130

1982 .Burgdorfer, Willy ; Barbour, Alan G . ; Hayes, Stanley F . ;Benach, Jorge L . ; Grundwaldt, Edgar ; and Davis, JefferyP . Lyme Disease-A Tick-borne Spirochetosis?Science 216 : 1317-1319 .ElThis article reports the discovery in and isolationfrom Ixodes dammini of the hitherto unknownetiologic agent of Lyme disease in the United States .It also refers to the detection of similarmicroorganisms (sprochetes) in I. pacificus fromCalifornia and in I. ricinus from Switzerland-areaswhere Lyme disease and related disorders are common. The discovery of Lyme disease spirochete, laternamed Borrelia burgdorferi, has led to intensiveepidemiological, clinical, bacteriological, andparasitological investigations of chronic borreliosesthroughout the world, but especially in NorthAmerica and Europe .

The National Institute of Allergy and InfectiousDiseases is grateful to the committee of former investigatorsof NIAID or its predecessors, who assistedthe Director, Intramural Research Program and internalstaff members in planning and executing this document .Suggestions also came from many other people, toonumerous to list, but whose assistance enhanced thedocument significantly .Thanks also are extended to the History of MedicineDivision, National Library of Medicine, and especially toLucy Keister, Jan Lazarus, and Peter Hirtle, for theirhelp in locating photographs and information . Dr .Ramunas Kondratas of the National Museum ofAmerican History, Smithsonian Institution, also kindlyshared information and material .132

General BibliographyBarry, Jeanette, comp . Notable Contributions to MedicalResearch by Public Health Scientists : A Bibliography to1940 . Washington : PHS Publication No . 752, 1960 .Bloomfield, Arthur L . A Bibliography of InternalMedicine: Communicable Disease . Chicago : University ofChicago Press, 1958 .Davis, Dorland J ., "Recollections of NIH During WorldWar 11," NIH Alumni Association Newsletter, October/November1981, pp . 9-11 .Dyer, R . Eugene, "Medical Research in the UnitedStates Public Health Service," Bulletin of the Society ofMedical History of Chicago 6 (1948) : 58-68 .Furman, Bess . A Profile of the United States PublicHealth Service, 1798-1948 . Washington, D.C . : U.S .Department of Health, Education, and Welfare, DHEWPublication No . (NIH) 73-369 .Harden, Victoria A . Inventing the NIH. FederalBiomedical Research Policy, 1887-1937. Baltimore : JohnsHopkins University Press, 1986 .National Institute of Allergy and Infectious Diseases .History and Fact Book . NIAID internal publication,1963 .O'Hearn, Elizabeth . Profiles of Pioneer Women Scientists. Washington, D.C . : Acropolis Books, 1986 .Schmeckebier, Laurence F . The Public Health Service,Its History, Activities, and Organization . Baltimore :Johns Hopkins Press, 1923 .Stetten, DeWitt and Carrigan, W . T ., eds ., NIH : An Accountof Research in Its Laboratories and Clinics . NewYork : Academic Press, 1984 .Stimson, Arthur H ., "A Brief History of BacteriologicalInvestigations of the U .S . Public Health Service," SupplementNo . 141 to Public Health Reports, 1938 .Williams, Ralph C . The United States Public HealthService, 1798-1950 . Washington, D.C . : CommissionedOfficers Association of the United States Public HealthService, 1951 .133

Laboratory of HygieneEstablished in August 1887,Staten Island, New York• Bacteriological methodsintroduced into the fight againstepidemic disease.NIAIDChronologyChartHygienic LaboratoryMoved to Washington, D.C. , 1891• During the next two decades,additional fields of research werelaunched in allergy, zoology, andbiologics control.National Institute of HealthEstablished in 1930• Major subunits included theDivision of Infectious Diseases,Biology, and Biologic Control. TheRocky Mountain Laboratory wasmade a part of the Division ofInfectious Diseases.National MicrobiologicalInstituteEstablished in 1948 as one of theNational Institutes of Health• Historic research fields weregiven institute status.National Institute of Allergyand Infectious DiseasesEstablished in 1955• Categorical name adopted;immunology joined older fields as amajor area for research.