Castellani, Olga

Fernandes, J. F.

Okumura, M.

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. Seio Pai.lo 8( 4):151-156, ji1lho-agôsto, 1966 UDC 616.937.3 HISTOPA THOLOGY OF THE HEART AND MUSCLES IN MICE IMMUNIZED AGAINST TRYPANOS01VIA CRUZ! J. F. FERNANDES (1), Olga CASTELLANI ( 2) and M. ÜKUMURA ( 3) SUMMAR Y Arnong mice inoculated with high nurnbers of living Trypanosoma cmzi, Y strain, cultivated for 3 days in presence of Actinomycin D, survival was 100%, no parasitemia could be detected and no parasites were found in tissues. When challenged with virulent blood forms of the sarne strain, all mice thus treated survived, while all controls died within 13 days. Parasitism was much lower in the immunized than in the control mice, but the acute inflammatory reaction was much more severe in the first group. This reaction frequently sub-sided, only a few discrete lesions being found in !ater examinations. INTRODUCTION Seveial procedures have been tried to in-duce resistance to infection with virulent T. cmzi in animais. Those that succeeded were based on inoculations of living trypanosomes from the following sources: 1) Blood forrns of a virulent strain inoculated in small dosis (HAUSCHKA et al. 6 ). 2) Culture forms of avirulent strain (H,rn-SCHKA et al. 6 ). 3) Culture forms of a vi-rulent strain attenuated by prolonged growth in artificial media (Przzr ,& PRAGER 9 ). 4) Blood forms originally virulent but attenuated by drugs ( COLLIER ") . 5) Virulent strains followed by treatment with a suppressive drug (BROWING et al.2, HAUSCHKA et al.6, Przzr et al.1°) . All these methods procluce a mild infection with low mortality, the surviving animals becoming highly resistant to infection with virulent strains. On the other hand, no pro-tection was conferrecl by injecting parasites killed by various rneans (MuNIZ et al. 7 , HAUS-CHKA et al. 6 ). These results indica te that when a-strong immunity was achieved it was the consequence of a previous infection. The séarch for ",strong immunity without a previ-011s infection" becarne the most important goal in this field. During our studies on protein and nucleic acids synthesis by T. crnzi we have found that Actinornycin D renders the flagellates unviable and devoided of multiplication capacity (FERNANDES et al. 4 ). The living but irreversibly inhibited flagellates do re-tain, however, the ability to induce resistance in mice in spite of the apparent loss of their infectivity as indicated by the negative re-This work was supported by grants from the U.S.P.H.S. (AI 05008 TMP) and Fundação de Am-paro à Pesquisa do Estado de São Paulo and performed in the Department of Histology and Em-briology of the Faculdade de Medicina da Universidade de São Paulo, Brasil Departamento de Bioquímica - Faculdad.e de Medicina da Universidade de São Paulo, São Paulo, Brasil (1) Professor Associado de Bioquímica (2) Instrutor de Bioquímica (3) Assistente do Departamento de Cirurgia, Faculdade de Medicina da Universidade de São Paulo, Brasil 151 F E;RNA NOl-:-.5, J, F .; CAST l-";1.l.ANI, O. & O:KUMURA, M. - HistOpalholugy ot lhe henrt and m usd<'S ln m ltC! l mmun lzNI ai;:;.1it\sl 1'1·m1crnv!Joma e-1·11;:i, R<w, h1~t. J1t('tl. tro7J, Selo Paulo 8: 151-15(), 19'fü. ~uh:; or (1 1hroo~lio 111 ~•t1n:+, for para:-:ih -S i o ,wimal::; inoc·ula11•d wi lh A<-linornvc-in D t rt~a1.:-d c:ultu res anel lhe-i r low p,H~sitemia anel lon:,?; sun·ival foUowin;?; leLhal closis of virulenl blood trypanosomC's tF'ERNA~OES et ai.' 1. Tlic pn':04' 111 papt•r j~ 8 11 l.'~lt'tl!-l;ÍOn or t ht'" aho\fe 1fü•u lio11("d w·o,·k io ordN to inducle pathology sludic~. íhe- marked anel con.stant lrsion~ prodtKed in the hrarl. ske leta l aml \'ÍSccral mu~·les hy i11ít•c·1 ion wi1h hlood formo!; or l lit· )' ~t rai u 8 1'(' \\'t•H kn0\\111 (ÜKL -)ll!IU d h l.g} and " 'f• proc•f"eded to ÍO\f(•sti~ate ií llw pi('turt• woul<I l,1~ modifi.:-cl in 1nice which hacl p re \· iou.sly n.'tt"ivecl the Ac·l iuo-myl'in D ln·atecl t uhurcs. METHOOS To c·11lt111·,·s conla ininf? 1.5 X IO' flagellales J>e' I' mi. Attinomyt·in D was add,-d and main-tainecl for 3 clays. Tlll' medium nnc.l 1l1t· cuhuring 1·orHlilion!-' Wt·n· ~1:- fksc·rihC'd l,y Ft:n, \'\u~s ·.S: C ,\ST~:1.1.1\\'1 1• Tlw íin:11 c·on-•·••nlral ion of Ad inomyc·io D wa~ 1 ,,g J>N mi oí nu•ciiu m. T lw <:ulturt" was then ten-Lri fuµ-NI. tlw S('climcnt washed in salinc und resu.:51>endt•d in a volume of salinc cakuh11t:d to <.·onlain in 0.2 mi lhe uumh<.·r of flagc·llalc~ lo fw injl'l-lL•tl in l'~l('h mom;(•. E.:u.·h oi 20 young aduh mice weighling urouml 20 g rtwin ·d 0.2 mi o[ lhl' l lag,·llale ::iU!'.1.H.'n:.ion iu1n;1p.-ritont·till)' Onc'l' n \\C'i'k. tl11ri11;:: :>. w,-.~k~. llw ouml11:~r o í fla.g(')la1€"~ io<-rt·a~iol:' from )Oll- in thc- la.::: t inocula1ion 10 10' anel :1 X 10),,, in th(> 2 suhS<'q11enl onc--s. Eip;hl(>en days a fkr thc.:- lasl dose,, 10 mil'<' oi this ~roup wert injc<.'h:c.l inlrapt·ritOnl'tl.11~1 \\itl, 200.000 virult·nl blood íorrns. or 1lw par,b,ih' Y ~tl'aiu 1S11.\'A t•l aLt'J . AL 1lw &lllh' limt" a <'1nl rol group of 10 dt"an mice oí the sarne-- weiglH rec:eived a similar ino. culum. Twth-t cl;.1~-s aher thr ,·irul<>nl inoc:ulation para~itemia was íl't'ôrdccl íor all anjmal!- as prr\'iou:.ly clt•!-o(·riht·c.1 (F►;lt'1,\.NOES <.' l a i.:.,. Ou llw ,wxl day lht ouly ~ur \'ÍVing M1il n·ty ill aninrnl oí llw c·onlrol (üoo prolerh'tl) ~TOUf-' anti oJH• aoimal lakt•n at random írom cath of thr olhn L\\O groups were killecl for lhe pathology stuclit...._, On the 87th anel 238111 cl..ws a íter thc last immunizi11g inoeu lalion, onc_. ;nouse was tnk<.·n 152 al rauclom from eoch of the immuni1.ed group~. haviug or 1101 n .. ·n·i \'l'd tlw \'irulent inocula1ion. aocl :;aniíic:c.:d. 1:lca rts oí tht' kille-cl an imal.:; w1.•n• c,,x·rw•I long-iludioally lo pennit examinat ion oí t lw fotrr ('t1,•i1 it·l-. Colons were opened longitucl-inally. wa$'lu~1I nnd lwi~led in spiral so that the wholf' lenght o í l hl' organ t'-Ould be i;t•t·tionrd. Fixation was in 10% n(•ull'al Jo.-. rnalin :-olution for 6 clays. T wenty :;c-c·l ioníl. 6 mina 1hkk \H're obtained from eac:h or~an and stairwd hy ha<.•malOX)'lin~t-osin. JtESULTS Afler lht' inoculalions with Ac·linomycin D-treated fhtgellates. no blood parasites \~·ere dl'l('('Lt·d eithr r hy ca rcíu] dirc·(·t exnminulion or lhrough x1.•nodiog110!'.ii;. Alro. ,,o i nl rn-('t•llula.- para.:::.ilrs or l(•~ions were íound in mice killNI for Lis:mt1 ex,1mi nation (Tahle I and Fi~. 1) . Fig. 1 - ~IOU~E' h enrt venl ri ele 13 da.y~ Mtf'r inoc-utation with 3 x 10'- rorms of 1''. rn1zí cul-ttvated during 3 days in a medium c·ontaining Actinom)·cin D (see mclhods). Ob:scn•c lhe nl.l-sence of 1mrasitl..sm us w('I) as lht normàl asJ,1<.-c..•t of tht'! OrJmt\, ll,E, SO X. t-•f;n NAN0ES, J . F. : CASTELLAN f, o . & 0KUMURA, )(. - Hlstop~1lhO)(lg}' (1( lh(' he;.H't and m uS('l('i; ín rn1('ê lr.l rntmlle<) .;, gatn~t, Tr!J),u,1()3'(Hl)(i N''ll~I. ll,81}, Itu,t . Meíl. trop. S,iv l'(lllfo 8 : 1 51-15 (), 11,)66. T AB LE 1 Protet tlve ertc.-ct o r Tl'!J1Hrn03Vm(r ('"rw:I preinc:ubnted wlth Ac:llnc)mycln agalnst a vlruJent strnln. Re~ulls reter to examinatioh mactc -On duy 3 aflcr th~ lüSt injéctlon or lhé Aclinotnycin D t rcatcd flagellatc.~ r1n(I <la>· ~13 a (tel' t he 1nocutntlon o r lhe vlru len t $lrain COnt~cntl'atton OI thc 1>art,si te in Percentagc 1 noc:u I n Uo n OI BICH:id' 1 ventrlt le º 1 Attrl('le .. S\lt'VI\IQr• • • J-\ttenualed llagdhltés (\'Ont rol) .. .. .. .. . .. . o o o 100 Atlenualcd naitct1ates + v lrulent strain .... 10 ± 6 25 18 100 V in11cnt .sl t'ô\ln . .. . .. .. .. .. ... .. .. .. .. . .. .. 501) ± 60 4 17 <IBS o • mood pnrasiles <-x1>ré':,t.Cd üs lh(' numbcr of parasite in 50 JJ'li<:rOS('Ol)ie fiClds ( .X280) ex~e))L when no J>àrtt.Site wüs foun<I when 4,000 mtcroSé<il)k net<t .íi we)·c ex;:nntnc<I. • • The 1)aras111sm ln the ventrl<;le and nurkte are expres!ted as lhe n u mber ot lelRlrnuu1lo lll(I ln the w hole m l tr(),.Cl l !de. . .. AH su r vivors were ki llCd 12 months aHc1· thé tnotulation of lhe ..,l rtllent torms, Followin~ ino(·ulatioll with virulent h')'• paooronws. hoth the- protecled anel thc.- <.'-Onlrol mite showed pa rasitc>miu whkh wn:\ h ow-(' \'er . mu('h lowcr in lh1.· )'11 group. Talilc l :-:h,rn·s l ht"~,· rt-:,:,ult:-: as \\'("li as data on lhe :,:urviving tinw. As for parasik s and le.siom; in li~uc'".5 for lhe 2 groups. results were ás follows : 1) Nlo1.1S(' having l't'ceived only lhe viru, lc·nl rhlgl'·llatcs (l he on)y survivor o[ the tontrol group on thc .. }3•ti day). H t•iH'I . ~,riülNt a nd ~moolh musclcs heavily 41:1.ra~it iu·<I. wilh le-i~hma n ia nid i in éVery (' \'Ol111 i,•t• stage-. 'When tht"Se n id i were intac:I 1 ht·rt• was no in flamma tory react ion, h ul around rupllm•d nidi an inlense., lympho--histioc·yli<· iníiltration was ohsuvcd. T hesc in flammalo ry proct-•ssrs wt~rr preíerénlially lotalizc~<l in thf• aur idf' . fo11owrcl hy the Vt'll· t ridt· hasc-> ~tnd suhepic.:ar diac rt•gion of the righl vrn lrid<'. ln Tah lc ·1 anel F'ig . 2 thc .1ht11Hlane1.· oí Ih<' myotard ium para.sit i~m can hc-' apprc.·c::iah·d. 2) i\'lic.'l· lmving r('t'(' i\'t>d inoculat ions of t lit· A1·linomyc-in Ü•lrt•á tcd Ilngd lates followc~d l ,y d1ulk ngt· wilh thc virulenl forms : a ) Au i1t1ul ki llt·d on tlw 13' h day after the vi rulcul i11on1la1 io11. T lw numher o.f leish-muniu nid i was- mrn:h lower than in the c·ontrol mouse (Tahlc ]) hut a mud, mor1· inle nse inf1ammalory proccss ,va~ sct·n OV('r the whole ventridt· aMI 1.·vc~,, in tht' auric•lí• :e. • .. ,. • ~$.i ~ • -4!. "' ,, r-· . :!.~~'\, # F ig. 2 - :O.fouse hearl vcn u ·1tl~, 1~ da)'R nftcr t he i noculn lion o r 2>00~ vh'UIC>n1 blood forms Of '1'. cnr:i. Obsc1•v é me h C:'IVY in tracellulnr f)l.ll'H· s itism wilhóut inCJamnrn101•y 1•eaction. H.E. 400X . 153 F8RNANDES . .J. f,' · CA$it-:LLANl, 0. & OKlf MUHA, M. - Hislopath ology Oí lh(' h(•arl a nel m\1$i•l~s iu m ice imnrnnizcd :'lg:lln~t Tr,nu,»os,Jt)to ,·r ,i~i. Rtv. /n.,;t. Med. trt11,. ~ti<• Pnulo a : 15 1 • l;';ll, 196<t Fip;. 3 - 1 Jcn rl vcn trldc or {l mous<.' t h::it wa,; inuc:ulu lt.'1.1 wilh 3 X 10" torm~ M '/'. (")'11.::f <'UlllVfllCd i n lhe J>rCsc:nce o f Actlnomyc:ln O, f<)IJ()w,ect IH' 11 thfl l l<'nuc dose ot 2 x 10:· \•lrulenl corm$ ($<.'C methfül!<:). T hc ani mul wus k iJled 13 dll)'$ Mtei· lh<- 1110,·ul<lth)ll óf thc vinilent forms. (J. t; , SO X . ( fif!,, :1 >. Sk.-·lc-tal n1t1$.c· le-~ ::,howl~cJ ft•vl p,,. rnsilps hui an intc~nst" myo~ili~ ( Fi~. 4> . Thc- tolon hacl fl~w lf•Í!l-hnrnoi1  nidi nnd 01ih.l myo~ilis. T lw Auerhath plcxuSt~:; ::.howf"d ir1-flmnmalion. h I Animal killf·d oo tlh' 69' h i:hir a Íl<.·r llw virulN1I inoc:ul.1Lion (on Llw 871h clay uÍIN tJw lasl immun izing ino<:ula-lion i. Thne was a r<~m,ukahlt' <·hange- from 1l1c pn.'\'ious piclurt-: 1h1~ inLratc•l1ula r para• silism wus con.spic:uously down. $incc• in all :-.l·rlions oí llu::: v<•ntridc .. only onf' l<'ishmanin uidu~ nnd a dis.c-·n·k intt-1·s1i1ial in flammnlion \\'C' l"l ' found ( Fi~. S ) . Thc- inflammntory pr01."t.':-.." was d 1iefl}' loc.;.llc-><I in Lhr suhf•pinlr· d iac· and .subendoeardia(' rf•f .doos o í 1h1· dghl VNtlt ic·k, mainly doSt.· to its hasc.•. Tht> .:-k«·l<·lal rn11~('l1· pn"S1.·ncecl a di&:rNe inílamma1 ion. Th1.·r(' wns p~u lial l1•sioo (IÍ llw 1wurons of thc-> Aucrhac·h pl1~xus And un in íl,rnunatory proec:;:; c)f Ih<' n1usc·ulal' IO)'i.'r of llw c:olon. 1· > Animnl killed 011 1lw 2201h day aÍlt-r tlw 154 I • l l ,. '·'i't -( •-•tr . ·r·\•,:-. }'.; v,"' ; ' J. ' • . ~ -Fig. 4 - Strinted m usf'le o r lhe s,vnc• rno\1~(• ,,r Fig. 3. Observe lhe ln1enJ,;(! 1nnamm:11111n wHh* ou1 Imrasite.!I. M.J•:. •IOOX. \ iru lt·11l inoculaljou (on lhf• 2~8111 chw tl Íln lh e· IH:;1 immunizing ino<:ulalion >. Th; 1·01011 aucl skdt,lul musde appean-•d normal. A clii;-c1'('l1· i11flammatory prot(>SS I lymph-hi~Liocyt i<' 1yp1~ a nd pc.·riva~rular > ~tnd some e,longaLt-d ct-Hs around u mu~·ular necrot ic ar(•il ,rnt· found in 1lw vt•nlridt> ( Fiµ-. 6 ) . O ISCUSSION Til(' high degree oí immu11i1y clt"n·lop .. ·d h)' mice· $Uhmi lle<I lo ou r pro(·c•::.:: of pro• h:('tioo wus demonstratecl liy tht• hypc·r• ~•11::l1 ivi1y ol their l issue~ lo T. cru!i mall'· riais, i.t'., hy tht> intc~nsi1 y oí inílammítltt)·y rl'·aNioos. folknn·d l,y inilammalory oeciema anel íi hN· d i::wdution in spih' oí a low inlra-ct'llular parasitÍ5-lh, The ti:;i;m_' reaetion st-em-t'd even inort" markc·d on tlw 69'11 da)' aítt"r the virul<'t1t inoculation. a p ic·lon· ::;imilar to tlw immune rc~at·tion ,lt':1=C'l'i l,1•d liy BH.11'0 1 • • FERNANDES. J. F.: CAS'f'f:l,L AN I , 0, ~t: O8.UMURA. :-,1, lllst,)l):1 tl\o)oJ!)' o( Lhe hearl AIHI m u-1dc•,,; ln Jni<•e immunizecl ag>ltnst TrJJtHutosomct c-n,::i. kev. h11tl , Jl1P1{, frui, . S ,io Paiilo il: n1-151l, m oo. ,.~1g. 5 - t-Jcart vcntricle ot a mouse on lhe 69•h rl;'ty arter thc ('hàllCngc dose of v irulent lorms or '/'. <-)•11:,(, The animttl h utl been protected h)' previous 1nMul~tUori or Actinomycin treated t1a 4 gellat~ 8$ deiwr11)e<I un<ler Fig. 3. Note lhe inflummatory re.~<:Uon w Uhout. in trncellular pa 4 rMI tes. H. E. 60 X • Mow(""\'l'I', úll tl1e inflammatory rt"attioos subsidecl anel, a fter 7 months'" the organs presentl"'d a nonna.l 1;trncture, ex<:epl for a vc•ry <li5':rNt' juxhwasçular inflnmmatory in 4 í ihr.:th•. A prohlthl\' process o f hypersensi 4 tivity .:ts des{·rihNI hy Ú K UMURA el nl / 1 has 1,tt'n found onc·t·, a<·<'ompanyjng a lesion oí tlw pul1ni:m;.1 ry ,1rh'•ry. RESUMO J I isttJJ>lltolo~ia do corü<;cfo e múscul.o.Y em camu11do11ga.t i11ur,~i:ados contra o T.-ypanow ma cruii Lolt:-s d<-- <·amurHlongos foram inocuhldos C'Ont p)ev,tdo nl1mno de r . cru:i da l'epa Y. ffogt•lados \' ivos que- 1ioham sido cuh i, nulo:,:. pôr ::S d ias cm prc-"Sf'nça de A('linom i• c·ina O. 01.:;._•rvamos nestas con<liçó<:S sobre, 1;•1g. 6 - A$ (lcscribed und('-r Jo~jg, 5, with th (' ex<:epUon that lh e nnlm:.ll was killéd 220 dnys 8rter ln0<:'Ul<.llion of lhe vlru lt>nl- f◊rm,.; . 1-:1,E. so x . v1veru:in de 100~ e ausê1u.·i;.1 clc-- parasila:-: no sat1g 111..• ,. lt<·id():; dvs animai~ inoculados4 Quando êsles anima is recehiam uma ino. <'ulação dt· formas saugui<"olas ,·irulenlas. oc:o rr ia 100% (k· sohrevivênc.:ia enquaulo mor riam Lod(r.,:. os :-(•11~ t~~mtrô lt·~ i:-to é. l o• dos os animais Cfu(• l'l"'C1•ln·nnn ('Orno iuúc.:ulo apt·1Hts os flagelados das formas &1nguí<'ol1s virult·ntas C não i mu1tizados l . Ohsf'rvamo:-: também pa ra~it ismv muilo menor entre o:-animais imuni~tt<.los (jlH,.' nos t·ontrôles. mas a reação in ílamalól'i it (~,-;1 mais intc.·n~u t'll· t re os anim;1 is imunizado~. Es1a t·t~açíio qua• se dcsapurc·c:t.>. ,x• nmlnc<·endo apt•nas io~i.f:·ni 4 fic::anks lt'SÓl~ olisi.:rvadns em ('Xames posk 4 r iores. RE F ERENCES 1. 8JtJTC), 'f. - MJO('iO'dlte "<1M J'(Jk 11 " rlQ ,,.,, 4 b<tl,, ~ tt,w p,;,i.,th),:il reld('ift) N,)11 ,1 <'""" i;o 155 FERNANDES, J. F.; CASTELLANI, O. & OKUMURA, M. muscles in mice immunized against Trypanosoma cruzi. 151-156, 1966. Histopathology of the heart and Rev. Inst. Med. trop. São Paulo 8: chagásica experimental. Tese. Faculdade de Medicina da Universidade de São Paulo, 1962. 2. BROWING, C. H.; CALVER, K. M.; LECKIE, M. W. & WALLS, L. P. - Phenanthridine compounds as chemotherapeutic agents in experimental T. cruzi infections. Nature (London) 157:263-264, 1946. 3. COLLIER, W. A. - Ueber immunitat bei der Chagas krankheit der weissen. Maus. Z. f. Hyg. 112:88-92, 1931. 4. FERNANDES, J. F.; HALSMAN, M. & CAS-TELLANI, o. - Effect of mitomycin C, actinomycin D and pyrimidine analogs on the growth rate, protein and nucleic acids synthesis and on the viability of Trypano-soma cruzi. Exp. Parasit. 18 :203-210, 1966. 5. FERNANDES, J. F.; HALSMAN, M. & CAS-TELLANI, O. - Effect of actinomycin D on the infectivity of Trypanosoma cruzi. Nature (London) 207 :1004-1005, 1965. 6. HAUSCHKA, T. S.; GOODWIN, M. B.; PALM-QUIST, J. & BROWN, H. - Immunologic relationship between seven strains of Try-panosoma cruzi and its application in the diagnosis of Chagas' disease. Amer. J. Trop. Med. 3·0:1-6, 1950. 156 7. MUNIZ, J.; NOBREGA, G. & CUNHA, M. -Ensaios de vacinação preventiva e curativa nas infecções pelo Schizotrypanum cruzi. Mem. Inst. Oswaldo Cruz 44:529-541, 1946. 8. OKUMURA, M.; FRANCA, L. C. M. & COR-RÊA Netto, A. - Comentários sôbre a pa-togenia da moléstia de Chagas; especial re-ferência à infecção experimental em camun-dongos. Rev. Hosp. Glín. Fac. Med. São Paulo 18:151-164, 1963. 9. PIZZI, T. & PRAGER, R. - Inmunidad a la sobre infección inducida mediante cultivos de Trypanosoma cruzi de virulencia atenua-da. Bol. Inform. Parasit. Chilenas 7: 20-21, 1952. 10. PIZZI, T.; RUBIO, M. & KNIERIM, F. -Immunology of Chagas' disease. Rev. Para-sito!. 15:577-592, 1954. 11. SILVA, L. H. P. & NUSSENSWEIG, V. -Sôbre uma cepa de Trypanosoma cruz·i al-tamente virulenta para o camundongo bran-co. Folia Clin. Biol. (São Paulo) 20:191-207, 1953. Recebido para publicação em 18/2/1966. 

Histopatologia doc coração e músculos em camundongos imunizados contra o Trypanosoma cruzi

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Histopathology of the heart and muscles in mice immunized against Trypanosoma cruzi

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