UNCLASSIFIED

AD NUMBER

AD843453

NEW LIMITATION CHANGE

TOApproved for public release, distributionunlimited

FROMDistribution authorized to U.S. Gov't.agencies and their contractors; ForeignGovernment Information; FEB 1968. Otherrequests shall be referred to Departmentof the Army, Fort Detrick, Attn: TechnicalRelease Branch, Attn: TID, Frederick, MD21701.

AUTHORITY

SMUFD D/A ltr, 15 Feb 1972

THIS PAGE IS UNCLASSIFIED

Nr

TRANSLATION NO. NO.-

DATE:

DDC AVAILABILITY NOTICE

Reproduction of this publication in whole or in partis prohibited. However, DDC is authorized toreproduce the publication for United StatesGovernment purposes.

STATEMENT #2 UNCLASSIFIEDThis document is Subjectcontrols and each transmittal to foreigngovernments or foreign nationals may be madeonly with prior approval of Dept. of Army,Fort Detrick, ATTNs Technical Release Branch/TID, Frederick, Maryland 21701

DPARTMENT OF:THB ARMYFort Detrick

Frederick, Maryland

-i

USE OF FLUORESCENT MICROSCOPI IN DERM TOLOGYAND VENEREOLOGY. IMMUNUAISTOCHEMICUL

FLUORESCENT ANTIBODY METHOD

Ceskoslovensks dermabologie V. Winter(Czechoslovak Dermatology)Vol. 40, No. 1, pages 51-56, 1965

In the last 20 years fluorescent microscopy has be-come the basis for immunohistochemical methods. Withthese methods one can demonstrate antigens and determinetheir localization. A substance is called an antigen ifupon injection into an animal it stimulates production ofantibodies. Antibodies have the peculiar ability to spe-cifically bind antigens forming antigen-antibody complexes,which can precipitate' The fluorescent antibody method isbased upon this principle. Antigenic material present ina tissue section will react specifically with its comple-mentary antibody. This immunological reaction will takeplace in deposits of minute amounts of antibody over thosmareas of the tissue section where antigen is Oresent. Ifconjugation of antibody with fluorescein had been prbvious-ly carried out the microdeposit of fluorescent antibodybecomes visible under a fluorescence microscope. Specifi-city of the reaction rests in selection of the specificantibody by the antigen from a solution of fluorescentproteins. Other serum protein molecules present in thefluorescent solution are washed out of the tissue sectionand the specific deposit remains in situ.

Preparation of the tissue sections must be donewitheut chemical fixation so as to avoid interference withtmmunologic activity of the antigenic material. It istherefore best to use native section prepared in a oryo-stat.

The fluorescent immunohistochemical method was in-troduced and developed by Coons, Creech and Jones (1941).

( Coons' method for localization and identification of anti-gens has in the past ten years been applied in variousareas of biology and medicine. It developed that this me-thod could be modified for diagnostic as well as basic re-search purposes such as bacterial and viral pathogenesis,collagenoses, etc.

The method has certain limitations such as non-spe-cific staining, poor contrast in some tissues which exhi-bit primary fluorescence. Experience has shown, however,that when critically utilized and interpreted the fluores-cent antibody method can to a large degree serve medicalresearch.

The first fluorochrome used for conju ation wasfluorescein isoCyanate. Its process of conjugation wasworked out in detail by Coons and Kaplan (1950). This fluo-rochrome is unstable and conjugation with it is technicallydemanding; for this reason other fluorochromes were sought.Of the many proposed materials two proved to be the mostsuitable and these are most commonly used. Riggs et al.(1958) introduced fluorescein isothiocyanate which is morestabile and produces better results than isocyanate (Mar-shall et al., 1958). Chadwick at al.(1958) used lissamine,a sulfonyl chloride derivative of rhodamine B 200. Thisfluorochrome has an orange fluorescence that is as intenseas fluorescein, which has a yellowish-green fluorescenceand has also proved useful.

The technique of demonstrating antigens can involvea direct or indirect method. In the direct method a testanimal is Rensitized to the test antigen and the resultingantihody is conjugated with the fluorescent stain. The con-jugated antibody (PL) in the tissue section is bound to thecell specifically by the antigen so that un4er a fluorescen-ce mioroecope areas where the antigen is foumd, fluoresce.This method was worked out by Coons, Creech and Jones(1941) (figure 1).

In more recent times the direct technique has beensupplemanted by various indirect methods. Thus for examplevin order to demonstrate an antibody (PL) bound to a cell,Coons et'al. (1951,1955) overlaid a tissue section with un-conjugated homologous antigen (AG). The resulting antigen-antibody complex is demonstratod by another conjugated an-tibody which is joined to the bound antigen. This is calledthe sandwich mthod.

(

.. . . . . ... . . I J . _J I . . . I N .] _ . L .I . . .I. . . . , o . _- i

ItIkFigure 1. Direct staining technique1. specific antigen2. fluorescence-marked antitody5. antigen-marked antibody complex

In order to increase the sensitivity of the reaction andeliminate cross-reactions during determination of cellbound antigens the following method is now used:

The section containing antigen (AG) is treated withunconjugated homologoits antibody (PL) and the resultingantigen-antibody complex is determined with the aid of aconjugated anti-antibody (PPL) from a different animalsource (figure 2). The indirect staining technique simpli-fies some investigations since it only requires one conju-gate for each animal type. In the direct technique a spe-cial conjugate is required for every antigen that is to bestudied. Basically the same method as for the indirecttechnique is used in fixed complement and fluorescence-con-jugated anticomplement method first introduced bv Goldwas-ser and Shephard (1958) (figure 3).

Figure 2. Indirect staining technique1. specific antigen2. unmarked antibody3. marked anti-antibody (of a different

animal species)4. antigen-antibody/marked anti-antibody

complex,

This procedure represents further simplification of themethod since conjugated anti-guinea pig serum is the onlyconjugate necessary for studying antibodies of a large num-ber of animal types. In this method antigen is overlaidwith immune serum to which is added guinea pig srum con-

* - _ " " ... .. .. .... .II....I... ... . .. L .-. .3 -. _

taining complement (the necessary presence of C1. C2 , andC4 fractions). The result of this reaction is the forma-tion of an antigen-antibody-complement complex. The guineapig complement -- and thus the original antigen -- is de-monstrated with marked rabbit globulin against guinea pigserum.

Figure 3. Characterization of antigen 7ith aid~of complement (according to Albrecht).

In the first phase antigen combines withantibody and complement ; in second phase,with marked anticomplement globulin (1).Increased intensity of fluorescence of theimmunological complex may be achieved byusing marked antibodies against globulinof antiviral serum (2).3. antiviral antibody 4. complement5. anticomplement globulin6. antiglobulin globulin

Immunohistochemical methods aided by fluorescentantibodies are analyzed in detail in the work of Meyers-bach (1956) and Coons (1958).

Since 1950 application of the fluorescent antibodytechnique spread rapidly to many fields. Coons publishedseveral extensive reviewa on this theme (1954, 1956, 1957,1958) as did ellors (1959). Considering the aim of thiswork toward dermatology and venerology we shall only brief-ly mention the application of this method in other fields.

Antibodies conjugated with fluorescein can be usedto advantage for many antigenic materials: polysaccharides,proteins and lipopolyeacoaridse. Fluorescence-marked sarawere successfully used for rapid identification of cellantigens -- bacteria, viruses, oaresites (Liu, 1956; Thos-son et al., 1961; etc.). The technique wad also applied tobasic research and to study of the pathogenesis of viralIrickettsial and baotexial infections (Cooa, 1956; Noye

L -4

and Watson, 1955; Buckley, 1956; Lebrun, 1957; etc.). Inmany works the fluorescent antibody method was used to aidin the study of foreign antigens of a nonviral nature, in-troduced into the organism by injection (Kaplan, Coons andDeane, 1950; Hill, Deane and Coons, 1950). Many studieswere devoted to investigations of the fate of foreign pro-tein antigens in an organism after parenteral introductioninto animals (Coons, Leduc and Kaplan, 1951; Mayersbach andPearse, 1956; etc.).

Marshall (1951) used fluorescence-marked serum fordetermining localization of native proteins in tissues,i.e. ACTH, enzymes (Marshall, 1954). Coons et al. (1955)and Leduc et al. (1955) introduced this method in studiesof the place and mechanism of antibody formation. Thiswork confirmed the validity of the plasmocytic theory ofthe origin of antibodies.

In recent times the fluorescent immunological tech-nique was used to investigate the location of antigens andantibodies in organs during pathological states and wasalso utilized for histopathological demonstration of auto-antibodies against antigenic tissue components. Mellors(1955), Mellors and Ortega (1956), Mayersbach et el. (1956)and others by means of this technique confirmed the immuno-allergIc pathogenesis theory of human nephritis. Vazquezand Dixon (1957) used a combination of the fluorescent me-thod and autoradiography in the study of gamma glcbulin insecondary human amyloidosis. Gitlin et al. (1957) demon-strated the basis of fibrinoid material in collaenosessuch as arthritis rheumatica, lupus erythematod.s, etc*,with the aid of conjugated antisera against human fibrin,albumin and gamma globulin. They identified tha substanceas fibrin. More recently the fluorescent technique was ex-tended to ehdie3 of hypersensitivity. For example, MeKin-non et al. (1957) investigated intravaccular precipitationof the antigen-antibody complex mad its role in anaphylaxis.Oort (1961) studied the localization and concentration offluorescent antigen in Arthus' phenomenon when iiiectod in-to actively and passively immunised guinea plga.

The works cited represent only a brief review of thepossibilities in applying the technique of fluorescence-marked antibodies. The literature is too extensive to beevaluated in more detail here, nor is this the purpose ofthis report.

As far as we could determije frou literIture avatila-ble to us, there is very little work in dervatology uNIAS

0 Ips.. ... ] .. .•' '. ... I

fluorescent entibody techniques for, diagnostic purposesor in research. This method has penetrated into derma-C tology only grsdu3a11y in reeent years. Most work isfound in tefield ofvenerology, mluhoogy and an occasion-al study of dermatoses such as collageiioses, skin tumorsaDd virus diseases. In this area we include the work ofmJcrobiologists and virologists insofar as they ae re-lated to dermatology.

Weller and Cocne 01954) used the fluorescent anti-body method to study varicella and herpes mster in tissuecultures, Iebrun (1956, 1958), using this method studiedo*1 l ocalizai~o of herpes simplex and herpes zoster.

the immunoistochemical properties of human akin aend tumorsrelated to them. Using conjugated &ntisertm against~ humanskin and human mvlignant melanoma. they demonstrated thatth~e latter is not of epidermal but of neuroepidermalorigin. Skin antibodies statned only skin and not theMelanoma While melanoma antibodies stained only aelanoma

.4elsqthepapllay mrgi o the akin snd neurilemua oZvagus tisL4on. Noye703 d par*t (1956) studied thedistrbutio *Ad localitaft -of tisim* antigens 1zn ratswith vaphos04~oma -adt found co natieAalyp-scrooa~ cells, inte5ia~leihllm bm n knotbee* anizals,.

Vallra arnd Orop(1956) in his work on lipoidnophroses also found ga-aSleulin deposits inpoirtritis '44sa, fthy defo'ie 64"a x4obuln by an in4irslot

stiiwof~i~ozs t~t rVs i -2bupund Durngmatbod w4ng antibai** rabbit ma ul n.Dd

:'efI% ",d 01013 (1955 3*foun4 fiti~ia U4 sxr~t of Zib"id.Aoco'i of arterieff

MoAV*± ee 't~r*- w'er# 'Aaaged lu tu at~&yo 0o11

riwus fi-bria.~ *0 A 4.P

-o4lwaf-

-the indirect method in their studies of the ILE phenomenon.Normal tiesue sections werm treated with IE positive serumand then fluoresc-nt an-i gamma PlobulUn was added. Spe-cific fluorescence was fjund in nuclei of cells in variousorgans. Control sections. untreated with LE positiveserum and ones treated with normml serum did not producethis res'ult. Wignal. et P-1. (1961) us3ed the fluorescentantibody method to itatinguish betweiEn desoxyribonuacleicacid of amorphoue Inclusions conta~ined in noutrophils ofpatintsaffected ,with lupue azd desoxv.,ibonucleic acid

Prom the nucleus.

Allergic reactionts were the 3ubject of intereat ofpr viously mentioneCt authors Mcrinnon Pt al. (1957) andOort et al. (1l61). R&Dpap('rt (1960) studied the antigen-antibody reaction in allergic human tiiasuo and in hiswork he pursued localizati on of reagins in human skin andtheir relationship to globulins.

Normal fluorescence microscopy and fluorebeent im-munohistochemistry is being used more and move in mycol-ogy. Vogel abd PadvLla (1958) first used indirect stain-ing reactions with fluorescent antibody for the detectionof antibodies against pathogeniic fungi. Gordon (1958)I with thie aid of fluorescent antibody carried out differ-entiation of yeasts. Mcotela-Ruiz (1961) applied thefluorescent antibody technique in the study of pityriasisroses. Gibert. The se.me derinatosis was studied with thistechniqae by Wrighit and Francis (1961). The authorstried to demonstrate a viral etiology for this disease,.Neither sei'ologica1 metbods nor the fluorescent antibodytechnique proved succousful in identi~fying the Infectious,agent. 'Sternberg and Keddie (1961) carried out immaxno-fluorescent studies ~with pityriasist versicolor. Throughtheir work -the authors attempted to clarify the pathol 'gi-Cal signific'ance of Pit roeorum orbiculare which alongWith P. ualssezia (L7Ni TiiiTrs Tou 5-03larly inpityrlsie' ;verSicolor. ran ptient sra they found high-titer ag atiiating antibodies and demonstrated a commonantigen for' P. orbiculare and the calls, It wou' 1 soenthat R. furfuir oFbioularp ara closely re~lated itnot $O4lo5gTB*afly i~renIo=*.

The study of chronic dormatites and detmtoosoccupied Dorbes (1961) and Vie? et al. (1961.). They if edsumed antibodies to ti&MMu &AtigeS &Ad ~4OUjagtod WithL

-7'J

fluorescein. They investigated skin of patients withatopic dermatitis, eczema, lupus erythem tosus, 1. ery-thematosus disseminatus, eczema nummulare, verrucae andapinocellular carcinoma. In only two patients with atopicdermatitis was there a positive reaction with affectedskin and in both patients the reaction was negative withclinically normal skin. For all the other dermatcsesthere were only non-specific reactions. No coacluaio.scan be drawn from these results except perhaps that inatopics there are strong auto-immunological reactions.

Immunohisto.hemical reactions, aided by the fluor-escent antibody method found greatest application invenerology. Here also the beet results were attainedand were worked out in detail for diagnostic purposes.

The first two applications of fluorescent anti-bodies in the field of venerology were made by Deacon,Falcone and Hazris (1957) and (1958) with lues. It wasshown that the results obtained were very specific and ex-tremely sensitive (U. S. Communicable Disease Center,1959) and more recent publications demonstrated the grow-ing significance of the fluorescent treponemal antibody(M) test for syphillis. Borel and DJrel (1959) com-pared the PTA and TPI (Treponea pallidus immobilization)test with Kolmor's card'pAn an1ie reaction andshowed that in noiaal persons and with non-specific re-actions the FTA was always negative. According Go theauthors the ftA is extremely sensitive and surpassesother methods, including TPI. This was also clearlyshown with the quantitative method where PTA was muchmore sensitive than TPK. Olansky and McCormick (1960)also consider MT an a Doon in solving serologitca prob-lems in lues. According to them the reaction is specific9% of the time. Harris, Bossack, Deocon and Bunch stud-ted other dsoaesoe in parallel with TPI, Kolmer's test andthe VDRL reaeton (Ynoreal Disease Research laboratory,US)* I was shown that MT was the most sensitive ofall the reactions even when ueed quantitatively.

D~acoc st l. (1W5, 519#, 1960) worked out de-tails o. the fluorescene test for Tr Mom vlidum.They used the ndirect reaction. Te Ats To per-lered as tollowt oneta lidus (prepared in arabbit as forthe T*- -- p a slide andI oo**z ,d with the bumn sorUn to bo studed.. The wx.plus

softs lip vashed oft with pyi~solOgicel saline oand fluor-

I. - m -I

escein-conjugated anti-human globulin 4s added as a re-action indicator. The Treponemata which had been boand totbe human antibody are also bound from the other Bide tothe fluorescent conjugate. Under the fluorescence micro-scope we then see fluorescing Treponemata. A control reac-tion is obtained~ by acting upin the organism with specific,unconjugated antiserum (e.g. rabbit serum), and after aare-ful washing, applying fluorescein-conjugated goat ariti-rab-bit globulin for' detection of any rabbit antibodi~es present.Abroad Conjugated anti-human and anti-rabbit globulins arecurrently available; in our country they are beginning tobe produced by USOL.

Among Soviet authors Ovchinnikov (1961) used FTA forthe diagnosis qf lues. According to this author the FTA re-action is P- fie, sensitive, rapid and simple. It is moresensitive -L-n both the EVWR reaction with cardiolipin anti-gen and the TPI test. It is riot suitable f or establishIngthe success of lues treatment because the test remains. posi-tive for a long period of time despite treatment. FTA is im-portant in detecting non-specific reactions.

Not long ago an application of-the fluoreszent anti-body method in identification of Neisseria &onrreae waspublished (Deacon, Peacock, et al.,15 1j9.6)-.=s~ willp certainly be of considerable value in the epidemiology ofgonorrhea and in the battle against venereal dislease. Anti-N. gonorrheae conjugates can now-be prepared and used for

1 dntMi~~aon of the organism in 16-2D hou.rs whi,'le the con-ventional culture technique requires approximately ten .days.The authors have also worked -out a delayed f1lioresezt ant1i-body method by which questionable gonococci are grown on anenrichment medium. This method is particularxly valuable forthe detection of gonorrhea in women.

Soviet authors are also using this method for diagad-~sis of gonorrhea. Migulina (1961) utilizes the direct vaotha4for -differential diagnosis of chronic uzetkhritia. Se -pro-duced axti-gonococcal and anti-stapbylooopcal. bporkioamsera and conjugated then with fluoreseein. _Th*,gxeeJof_fluorescence of gonococci depended upon fth lot of coo4ugtedilution of conjugates Ad age of culturee.

O~rchinnikoy (1961) used fl.uoroonent santibodies foridentification of gonococci and their L-fo'mlen kaaa.tbo-dies he used fluorescent anti-gonococcal sera, con1jvZatedwith fluorescein isocianuto. Xirns sera me"e obtainaed byimxmnatiac of rabbits with a polpatleat gonocomia qf&c-

'9-

77--

Cznce abd others witL a poly-valen-t gonococcus L-form~ vaccine.WiLth the help of tagged antibodies it was possible to iden-tify gonococci anx4 their L-f olms. It was not possible todemonstrate IL-ornis in acute gonorrhea. The fluorescent an-tibccty method'will1 in the future aid in clarifying thequests )n of the presence of microbial L-forms in urthr±'ispationts.

Literatuxe Cited

2. B1xridey, S.M.: Federat. Proc. 15, 509, 1956a.3- Buckley, S.M.: Arch.,ges. Vi iaforsci. 6, 388, 1956b.4. Coons, A.H., Creech,, l.J., Jones, R.N.: :Proc. Soc. Exp.

Biol. (N.Y.) 47, 200, 1941.5. Coons, A.H., Kaplan, M.H.: J. Exp. Med. 91, 2., 1950.6. Coons, AH.: Fedaration Proc. 10, 558, 1951.7. Coons, A.11., Leduc,. Bib, Kaplan,, M.H.: J. Exp. Med.

93, 173, 1951.8. Coons, A.H., ILeduc, E.H., Connoly, J.M.: J. Exp. Med.

102, 49, 1955.9. Coon.,;, A.H.:- Fluorescent antibody methods, General Cyto-cheTI;Lcal Methods, Vol. 1, 399, 1958.

10. Coon.3, A.H.: Ann. Rev. Microbiol. 8, 333, 1954.11. Coon.3, A.iI.; Iniu. Rev.. Cytol. 5, 1, 1956.12. Coon3, A.H.: Ann. N.Y. Acad. Soi. 69, 658, 1957.13. Craig, J.M., Gitlin, Dl.; Federat. Proc. 14, 401, 1955.14. Chadwick, C.S., MeEntegart, M.C., Nairn, N.C.: Immuno-

logy (Oxford) 1, 315, 1958.1.Deacon., WL.E., Falcone, V.11., Harris, A.: Proc. Soc. EBxp.

Biol. (N.Y.) 96, 477, 1957.16. Deacon, W.B.9 Falcone, V.11., Harris, A.:Ber. wiss. Biol.

125, 27, 1958.17. Deacon, W.E., Freeman, Mi.E.: 3. Invest. Biol.34, 249, 1960.18. Deacon, W.E., Peacock, it.L. Freeman, E.M., Harris, A.:

Pro0c. Soc. EXP.L Biol. Med.(1N.Y.) 101, 322, 1f95919. Deacon, W.E., Peacock, W.L., Freeman B M., HarrisA.,

.uncht W.11.: Pubi. 111th. Rep. (Wash., 75, 125, 196'L210. rerbes, J.V.: Arch. Derm. 84, 37, 1961.21. Gitlin, D., Craig, J.M., Janeway, C.A.: Amer. J. Pathol.

33v 55, 1957.22. Goldwasser, P.Ke., Z3hephard, 0.C.: J. Im~unol.80,122',5&23. Gordon, M.A.-- Proc. Soo. Exp. Biol. 97, 604&. 1958.24 LHill, A.G.Sj* Deane, H.W., Coons, A.H.:- J. Exp. Med.L

92, 35, 195'70.25. Hiramoto, R., roaman1 D.: Cancer Res. 1?, 1 .957.26. Holborow, E.J., Weir, D.M., Johnson, G.D.: BR-L ied.. JQ,

2, 732, 1957.

-10-Best Avail able Copy

27. Kaplan, M.H., Coons, A.H., Deane, H.W.: J. Exp. Med.91, 15, 1950.

28. Lebrun, J.: Virology 2, 496, 1956.29. Lebrun, J.: Bdll. Micr. Appl. 6, 94, 1953.30. Lebrun? J.: knn. Inst. Pasteur 95, 25, 1957.51. Leduc, E.H., Coons, A.H., Connoly, J.M.: J. Exp. Med.

101/102, 61, 1955.32. Liu, C.: Proc. Soc. Exp. Biol. 92, 883, 1956.33. Macotela-Ruiz, M.MoE.: Bull. Soc. Franc. Dermat. Syph.

68, 374, 1961.34, barshall, J.M.: J. Exp. Med. 94, 21, 1951.35. Marshall, J.M.: Exp. Cell Res. 6, 240, 1954.36. Marshall, J.D. Jr., Eveland, W.C., Smith, C.W.: Proc.

Soc. Exp. Biol. Med. 98, 898, 1958.37. Mayersbach, H., Pearse, A.G.E.: Brit. J. Exp. Pathol.

37, 81, 1956.38. Mayersbach, H.V. :IV. Symposium d. Arbeitsgemeinschaft

Histochemie, Mainz, 1956, Acta Histochem.. 3, 256,1957.39. McKinnon, G.E., Andrews, E.C. Jr., Heptinstall, R.H.,

Germuth, F.G. Jr.: Bull. Johns Hopk. Hosp. 101,258,195740. Mellors, R.C., Ortega, L.D.: Amer. J. Pathol. 32,

455, 1956.41..Mellors, R.C., Ortega, L. D., Holman, H.R.- J. Exp. Med.

'06, 191, 1957.42. iellors, R.C.: Fluorescent antibody methods, Analyticnl

Cytology, 2nd ed., N.Y., McGraw-Hill, 1, 1959.43. Mellors, R.C.: J. Histochem. Cytochem. 3, 284, 1955.44. Migulina, V.M.: Vestn. Dermat. Vener. 35, No 1, 54j 1961.45. Noyes, W.F., RapportM.M.: Fed. Proc. 15, 527, 1956.46. Noyes, W.F., Watson, B.K.: J. Exp. Med. 101/102, 237,1955-47. Oiansky, S., McCormick, G.E.: Arch. Derm. 81, 59, 1960.48. Oort, J., Rijssel, T.G. van: Immunology 4, 329, 1961.49. Ovchinnikov, N.M., Lurie, S.S., Byednova, V.N.: Vestn.

Derm. Vener. (Moscow) 35, No ll, 27, 1961.50. Ovchinnikov, N.M., Lurie, S.S., Byednova, V.N. : Laborat.

dyelo 12, 30, 1961.51. Rappaport, Ben Z. : J. Exp. Med. 112, 725, 1960.52. Riggs, JL., Seiwald, R.J., Burckhalter, J.H., Down8,

CM., Metcalf, T.G.: Amer. J. Pathol. 34, 1081, 1958.53. Steinberg, T.H., Keddie, F.M.. Arch. Derm. 84, 999, 1961.54. Thomason, B.M., Cherry, W.B., Davis, R.B., Pomales-Le-.

bron, A. : Bull. Wld. Hlth. Org. 25, 137, 1961.55. Vazquez, J.J(, Dixon, F.J.: J. Exp. Med. 104, 727, 1956.56. Vazquez, J.J., Dixon, F.J.: Lab. Invest. 6, 205, 1957.57. Vogel, R.A., Iadula, J.F.: Proc. Soc. Exp. Biol. 98,

135, 1958.58. Weller, T.H., Coons, A.H.: Proc. Soc. Exp. Biol. 86,

789, 1954.

Best Available Copy

Jr... 59. Wignall, N.9 Cilling, C.F.A., Vassar, P.8,: Amer. J.Clin. Pathol. 36, 469, 1961.

60. Wise, J*L. , Shames, J.M., Derbea, V.3., Hunter, P.M1.:Axrch. 1?erm. 84, 37, 1961.

61. Wright, A.D., ?xanois, R.D.8 Arch. Derm. 8'49 8?, 1961.

m12 -