0014-4894/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.09.023
Contents lists available at ScienceDirect
Experimental Parasitology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y e x p r
1. Intro duc tion
Try pan o soma cru zi, the caus a tive agent of Cha gas dis ease, is char ac ter ised by a com plex bio log i cal cycle involv ing an inver te-brate vec tor and mam ma lian hosts. The pro to zoan exists in at least three mor pho log i cally dis tinct stages. Epim astig otes pro lif er ate in the inver te brate host and are released as meta cy clic try pom astig-otes (MT) in the fae ces. In the ver te brate host, am astig astig-otes and blood try pom astig otes (BT) are the intra cel lu lar devel op men tal and infec tive forms, respec tively (And rade and Andrews, 2005).
Trans mis sion mech a nisms include vec to rial, trans fu sional and oral infec tion, con gen i tal pro cesses and lab o ra tory acci dents (Pra ta, 2001). Vec to rial infec tion is the main and most fre quent form of trans mis sion and occurs when mucous mem branes or abraded skin are exposed to MT-infected fae ces of tri ato mine insects. In
the domes tic cycle of Cha gas dis ease, Tria tom a in fe stans and Rhod-nius pro lix us are the prin ci pal inver te brate vec tors (Kol lien and Schaub, 2000). Although the ver te brate host can develop a cel lu lar and humoral immune response to the par a site, chronic infec tion is main tained by a reduced num ber of cir cu lat ing BT forms. The pres ence of BT forms in the blood thus gen er ates a fur ther pub lic health prob lem, and trans mis sion of the par a site via blood trans-fu sion has become the sec ond most fre quent route of infec tion (Coura et al., 2007). Trans mis sion of T. cru zi result ing from con gen-i tal pro cesses, oral gen-infec tgen-ion or lab o ra tory accgen-i dents may be med gen- i-ated by either MT or BT forms (San dler et al., 2003).
Dur ing the acute phase of infec tion, the destruc tion of try pom-astig otes is depen dent on the pro duc tion of nitric oxide (NO), a pro cess that is cat a lysed by induc ible NO syn thase (iNOS) (Ves pa et al., 1994; Ali berti et al., 1996; Hol scher et al., 1998). Stud ies have shown that gly co syl phos pha ti dyl in o si tol-anchored mucin-like gly-co pro teins puri fied from BT forms (tGPI muc ins) are potent elic i tors of pro in flam ma tory responses (i.e. cyto kine and NO pro duc tion) by IFN-c primed murine mac ro phages. In con trast, the cor re spond ing gly co pro teins derived from MT forms (mGPI muc ins) are reported
Trypanosoma cruzi
: Serum levels of nitric oxide and expression of inducible nitric
oxide synthase in myocardium and spleen of dogs in the acute stage of infection
with metacyclic or blood trypomastigotes
Paula Melo de Abreu Vieira
a, Amanda Fortes Francisco
a, Sheler Martins de Souza
a,
Luiz Cosme Cotta Malaquias
b, Alexandre Barbosa Reis
a,d, Rodolfo Cordeiro Giunchetti
a,c,
Vanja Maria Veloso
a,d, Marta de Lana
a,d, Washington Luiz Tafuri
a, Cláudia Martins Carneiro
a,d,*aNúcle o de Pes qui sas em Ci ên cias Bi ol óg i cas (NU PEB), In sti tu to de Ci ên cias Exa tas e Bi ol óg i cas (ICEB), Uni ver sid ade Fed eral de Ouro Preto (UFOP), Cam pus Uni ver sitá rio Mor ro do
Cru zeiro, 35 400-000 Ouro Preto, MG, Bra zil
bNúcle o de Pes qu isa em Imun o lo gia, Blo co F13, Cam pus Antô nio Ro dri gues Coel ho, 35020-220, Uni ver sid ade Vale do Rio Doce (UNI VALE), Gov ern ador Valad ares, MG, Bra zil cDe parta men to de Ci ên cias Bi ol óg i cas, ICEB, UFOP, Cam pus Uni ver sitá rio Mor ro do Cru zeiro, 35 400-000 Ouro Preto, MG, Bra zil
dDe parta men to de Aná lis es Clín i cas, Es co la de Farmá cia, UFOP, 35 400-000 Ouro Preto, MG, Bra zil
a r t i c l e i n f o a b s t r a c t
Article history:
Received 14 April 2008
Received in revised form 25 September 2008
Accepted 30 September 2008 Available online 18 October 2008
The par tic i pa tion of nitric oxide (NO) in the con trol of blood par a si te mia and par a sit ism dur ing the acute phase of infec tion in dogs inoc u lated with blood try pom astig otes (BT) or meta cy clic try pom astig otes (MT group) of Bere nice-78 Try pan o soma cru zi strain has been eval u ated. Ani mals of the MT group (n = 4) pre sented increased lev els of serum NO through out the infec tion when com pared with the BT (n = 4) or con trol (n = 4) groups, and a delay in par a si te mia peak com pared with the BT group. In spleen frag ments, tis sue par a sit ism was not observed but the MT group pre sented larger areas asso ci ated with induc ible NO syn thase (iNOS) in rela tion to BT and con trol groups. Heart frag ments of MT-infected ani mals exhib ited com par a tively low tis sue par a sit ism and high iNOS expres sion, while ani mals of the BT group pre sented high inflam ma tory infil trate, high tis sue par a sit ism and low iNOS expres sion. These results indi cate that the source of inoc u lum can inter-fere with the devel op ment of the acute phase of Cha gas dis ease, and may also trig ger a dis tinct par a site–host inter ac tion dur ing this phase.
© 2008 Elsevier Inc. All rights reserved.
Key words:
Cha gas dis ease
Try pan o soma cru zi
Blood try pom astig otes (BT) Meta cy clic try pom astig otes (MT) Nitric oxide (NO)
Induc ible nitric oxide syn thase (iNOS)
* Cor re spond ing author. Address: Núcle o de Pes qui sas em Ci ên cias Bi ol óg i cas
(NU PEB), In sti tu to de Ci ên cias Exa tas e Bi ol óg i cas (ICEB), Uni ver sid ade Fed eral de Ouro Preto (UFOP), Cam pus Uni ver sitá rio Mor ro do Cru zeiro, 35 400-000 Ouro Preto, MG, Bra zil. Fax: +55 31 3559 1680, +55 31 3559 1694.
to be at least 100–1000-fold less active than tGPI muc ins in the induc tion of NO by murine mac ro phages (Cam ar go et al., 1997a,b; Almeida et al., 2000). Recent stud ies from our group have revealed that BT and MT infec tions are asso ci ated with dis tinct par a si to log-i cal and sero log log-i cal fea tures, together wlog-ith log-intrlog-in slog-ic and log-inoc u lum source-spe cific changes in cir cu lat ing leu ko cytes (Car nei ro et al., 2007).
The canine model is cur rently con sid ered to pro vide an appro-pri ate exper i men tal sys tem with which to inves ti gate var i ous aspects of Cha gas dis ease, since Canis fa mili aris dis plays high sus-cep ti bil ity to infec tion and shows con sid er able sim i lar i ties in the man i fes ta tion of the acute and chronic phases that are rep re sen-ta tive of the human dis ease. More over, it is pos si ble to repro duce in this model the symp tom atic acute phase and the chronic phase in both the unde ter mined and heart forms of the dis ease (Lana et al., 1988, 1992; Ba hi a et al., 2002). Using such a model sys tem, we have recently dem on strated that MT forms of the Be-78 strain of
T. cru zi (a strain iso lated in 1978 from Bere nice patient, in whom Cha gas dis ease was first described) are more vir u lent than the BT forms (Ba hi a et al., 2002).
On the basis of the above, the canine model has been employed to inves ti gate aspects of the par tic i pa tion of NO dur-ing the acute phase of exper i men tal infec tion by MT forms, sim u lat ing vec to rial trans mis sion, and by BT forms, sim u lat ing trans fu sion trans mis sion (or, indeed, any trans mis sion mech a-nism involv ing BT forms) of Bere nice-78 (Be-78) T. cru zi strain. The objec tive of the study was to obtain a bet ter under stand ing of the mech a nisms related to the path o gen e sis of Cha gas dis ease in the canine model.
2. Mate ri als and meth ods
Details of the pro ject were sub mit ted to and approved by the Eth i cal Com mit tee on Ani mal Research of the Uni ver sid ade Fed eral de Ouro Preto. All pro ce dures were car ried out in com pli ance with cur rent Bra zil ian reg u la tions relat ing to Exper i men tal Biol ogy and Med i cine as described in the guide lines issued by the Colé gio Bra-sile iro de Ex per i men tação Ani mal (CO BEA, 2006). Study ani mals were main tained in the cen tral ani mal facil ity at the Uni ver sid ade Fed eral de Ouro Preto (UFOP), Min as Ge rais, Bra zil.
2.1. Par a sites, ani mals and exper i men tal infec tion
Two groups of four dogs were inoc u lated intra per i to ne ally with either MT or BT forms of strain Be-78 T. cru zi (2000 forms per kg body weight), obtained from nymphs of Tria tom a in fe stans and Swiss mice, respec tively. Four dogs were main tained unin fected as the con trol group.
2.2. Par a si to log i cal param e ters
From days 1–35, sam ples (5 lL) of blood were col lected daily from infected dogs by vein-punc ture of ear veins. The num bers of par a sites in the blood sam ples were deter mined under the opti cal micro scope accord ing to the method of Bren er (1962), and par a si-te mia curves were plot si-ted using the daily mean num bers of par a-sites per group of four ani mals.
2.3. Serum lev els of NO
Periph e ral blood (5 mL) was col lected from the brachial cephalic veins of exper i men tal ani mals prior to inoc u la tion and weekly there af ter (on days 7, 14, 21, 28 and 35), and serum was sep a-rated and stored fro zen (¡70 °C) until required for anal y sis. Lev els of endog e nously syn thes ised NO in the serum were assessed by reduc ing nitrate to nitrite with nitrate reduc tase (1 U/mL) fol lowed
by the Gri ess reac tion (Green et al., 1982). Nitrite con cen tra tions were deter mined by extrap o la tion from stan dard curves obtained using var i ous con cen tra tions of sodium nitrite, and the results were expressed in lM.
2.4. His to pa thol o gical exam in a tions and immu no his to chem i cal anal y ses
Exper i men tal ani mals were sub mit ted to nec ropsy dur ing the acute phase of infec tion, and frag ments of the heart and spleen were re sec tioned, fixed in 10% buf fered for ma lin (pH 7.2) and embed ded in par affi n. Sec tions (5 lm thick) were mounted on poly-L-lysine-coated glass slides and were either stained with
Hae-ma tox y lin–Eosin (HE) for stan dard his to log i cal pro ce dures, or sub-mit ted to immu no his to chem i cal anal y sis.
2.4.1. Expres sion of iNOS
Endog e nous per ox ide was blocked by incu bat ing embed ded tis-sue sec tions with 3% hydro gen per ox ide (H2O2) in meth a nol.
Sec-tions were then heated in a micro wave oven (700 W) and cooled to room tem per a ture. After this, the sec tions were fur ther blocked with nor mal horse serum (Vec tor Lab o ra to ries Bur lin game, CA, USA), and then incu bated over night at 4 °C with the primary anti-body against iNOS diluted 1:200 (Cat. No. sc-651; Santa Cruz Bio-tech nol ogy Inc., Santa Cruz, CA, USA). The sec tions were incu bated with the sec ond ary anti body con ju gated with bio tin (Elite ABC Kit, Vec tor Lab o ra to ries), and then incu bated with strep ta vi din–per ox-i dase com plex. The reac tox-ion prod ucts of per ox ox-i dase were vox-is u al-ised by incu ba tion with 3.39-di am inobenzi dine (DAB). Finally the sec tions were lightly coun ter stained with Har ris’s hae ma tox y lin solu tion. Neg a tive con trol slides were prepared in the same con di-tions but in the absence of the primary anti body.
2.4.2. Deter mi na tion of tis sue par a sit ism
Embed ded tis sue sec tions were incu bated over night at 4 °C with poly clonal anti-T. cru zi serum (obtained from a rab bit that had been im mun ised with T. cru zi Y strain) diluted 1:1000. Sub-se quently, Sub-sec tions were incu bated with Sub-sec ond ary anti body anti-rab bit IgG and per ox i dase–anti-per ox i dase com plex, and the label was detected by incu ba tion with DAB. Sec tions obtained from murine acute myo car di tis, that were rich in amas ti gote nests, were used as positive con trols. Finally the sec tions were stained for nuclei with diluted Har ris’s hae ma tox y lin solu tion. Neg a tive con trol slides were prepared in which the primary anti se rum was substi tuted by PBS.
2.5. Mor pho met ric stud ies
Mor pho met ric stud ies of iNOS expres sion, inflammation and tis sue par a sit ism were per formed by ana lys ing images of 20 ran-domly-selected fields (total area 1.5 £ 106lm2) of tis sue frag ment sec tions on a sin gle slide per ani mal. Inflam ma tory infil tra tion in the heart was quan ti fied by count ing the cell nuclei pres ent in sec-tions of heart frag ments, while iNOS and T. cru zi immu no re ac tive areas were mea sured in sec tions of heart and spleen frag ments. All anal y ses were per formed using a 40X objec tive, and images were ana lysed with the aid of Le ica QWin soft ware (Le ica Mi cro sys tems, Wetz lar, Ger many).
2.6. Sta tis ti cal anal y ses
anal y sis of var i ance (ANOVA) and Tu key post-tests. Stu dent’s t-test was employed to deter mine the sig nifi cance of dif fer ences in tis-sue par a sit ism. In all cases, dif fer ences were con sid ered sta tis ti-cally sig nifi cant when P val ues were <0.05.
3. Results
3.1. Pro duc tion of NO and lev els of par a si te mia
The dynam ics of the lev els of NO in serum col lected from groups of exper i men tal ani mals within the period com menc ing imme di ately before inoc u la tion and for the fol low ing 35 days are pre sented in Fig. 1. Ani mals of the MT group showed sig nifi cantly higher lev els of NO when com pared with those of the BT and con-trol groups at days 14 and 28 after infec tion. In the MT group, enhanced pro duc tion of NO began at day 7 but dimin ished after day 28, while in the BT and con trol groups the lev els of NO were sim i lar through out the 35 days. No sig nifi cant dif fer ences in serum NO lev els were observed in the lon gi tu di nal anal y ses. Although no sig nifi cant dif fer ences were observed between groups MT and BT with respect to the areas under the par a si te mia curves (Fig. 1), the early pro duc tion of NO in MT-infected ani mals may be related to the later peak of par a si te mia observed in this group.
3.2. Heart inflam ma tory infil tra tion
The results of the mor pho met ric anal y sis of inflam ma tory infil tra tion in the heart are pre sented in Fig. 2A. Sta tis ti cally sig nifi cant increases were observed in the num bers of cell nuclei pres ent in the heart frag ments of MT- and BT-infected ani mals in com par i son with those of the con trol group. More over, the num bers
of inflam ma tory cells were greater in ani mals of the BT group com pared with the MT group (P < 0.05). In infected ani mals, the inflam ma tory infil trate com prised pre dom i nantly mono nu clear cells (Fig. 3B and C), the major ity of which exhib ited the mor phol-ogy of lym pho cytes. How ever, ani mals of the MT group exhib ited focal inflam ma tory infil trates while in those of the BT group the inflam ma tion was dif fuse.
3.3. Par a si to log i cal param e ters
The results of the quan ti ta tive eval u a tion of tis sue par a sit ism in heart frag ments derived from MT- and BT-infected ani mals are shown in Fig. 2B. Tis sue par a sit ism was observed to be dis creet in all infected ani mals, and mor pho met ric anal y sis of dif fer ent areas of heart tis sue revealed no sig nifi cant dif fer ences in par a sit ism between the two groups (Fig. 2B and insert Fig. 3B and C). No par a-sit ism in spleen tis sue was observed in ani mals of either group.
3.4. iNOS expres sion in heart and spleen
Immu no his to chem i cal stain ing for iNOS was car ried out after nec ropsy dur ing the acute phase of infec tion. Results of the quan-ti fi ca quan-tion of areas of sec quan-tions of heart and spleen exhib it ing iNOS immu no re ac tiv ity are shown, respec tively, in Fig. 2C and D, while the his to pa thol o gical aspects are dis played in Fig. 3. Heart frag-ments from ani mals of the MT group pre sented sig nifi cantly larger iNOS positive areas com pared with the con trol group. With respect to the BT group, how ever, the iNOS positive areas were not sta tis-ti cally dif fer ent in size com pared with either the MT or the con trol groups. In com par i son with con trol ani mals, sig nifi cant stain ing was detected in the arter ies and arte ri oles in the heart frag ments of infected ani mals (inserts to Fig. 3D–F). More over, a blush of iNOS immu no re ac tiv ity through out the myo car dium was observed in all infected ani mals, and was most intense in ani mals of the MT group (Fig. 3D–F).
With respect to spleen frag ments, the pattern of iNOS expres sion was very sim i lar to that estab lished for lev els of serum NO, i.e. MT-infected ani mals pre sented a sig nifi cantly larger area of iNOS immu no re ac tiv ity com pared with both the BT and the con-trol groups (Fig. 2D). The white pulp was the region in which has focused most of the marked area for iNOS in the three exper i men-tal groups. How ever, the ani mals of MT group had a strong mark just below the cap sule that was not observed in the other groups. Only in the BT group we observed a marked area for iNOS in the fol li cles.
4. Dis cus sion
The aim of the pres ent study was to eval u ate serum NO lev-els, blood par a si te mia, expres sion of iNOS and tis sue par a sit ism in spleen and heart of dogs infected with MT or BT forms of Be-78
T. cru zi strain. Pre vi ous results from our lab o ra to ries have indi cated that infec tion, via the intra peri to neal route, with MT forms of the Be-78 strain leads to a higher par a si te mia peak com pared with ani mals infected with BT forms (Ba hi a et al., 2002). This appar ently con tro ver sial data may rep re sent an age related phe nom e non con-sid er ing that the dogs included in the pre vi ous inves ti ga tion were much youn ger (aver age age 60 days) than those pres ently eval u-ated (aver age age 120 days). Major changes in the immu no log i-cal sta tus of young pup pies, involv ing mat u ra tion of the immune response, could be the puta tive key ele ment con trib ut ing to the con trol of par a si te mia lev els in older ani mals.
The two infec tive forms of the par a site are known to exhibit dis tinct char ac ter is tics that induce dif fer ent immune responses in vitro and in vivo (Bren er and Gaz zi nel li, 1997; Car nei ro et al., 2007). Infec tion med i ated by meta cy clic forms in vitro can, in part, mimic
Fig. 1. Dynam ics of the lev els of nitric oxide (nitrite + nitrate) in canine serum ver sus
that caused by vec tors in vivo. On the other hand, in vitro infec tion with blood forms imi tates blood trans mis sion or even lab o ra tory acci dents. It is worth men tion ing that the major ity of exper i men-tal infec tions are con ducted using blood try pom astig otes because these forms are eas ier to obtain and main tain in the lab o ra tory.
The results obtained in the pres ent study showed that dogs infected with MT forms of the par a site exhib ited ele vated expres-sion of iNOS and higher serum lev els of NO in com par i son with BT-infected ani mals (p < 0.05).
Although iNOS is not con sid ered essen tial for the con trol of
T. cru zi infec tion (Cum mings and Tarl eton, 2004), the elim i na tion of BT forms in the acute phase is depen dent on a num ber of fac-tors (Pas cutti et al., 2003; Car dil lo et al., 2007), one of which is the pro duc tion of NO cat a lysed by iNOS (Ves pa et al., 1994; Pe tray et al., 1995; Ali berti et al., 1996; Hol scher et al., 1998). Thus mac ro-phages infected with T. cru zi pro duce IL-12, a pow er ful cyto kine that induces INF-c syn the sis by NK cells. INF-c plays an impor tant role in the acti va tion of mac ro phages that pro duce high lev els of NO and effec tively con trol par a site rep li ca tion (Car dil lo et al., 1996; Sard in ha et al., 2006).
The increases in serum lev els of NO observed in the MT group could be related to the high expres sion of iNOS detected in dif-fer ent tis sues (i.e. heart and spleen) that have direct con tact with blood. In this man ner, such com part ments could con trib ute to the sys temic increase in NO in the MT group. On the other hand, the dif fer ences in pro file of serum NO observed between the MT and BT groups could arise from the par a site–host inter ac tion ini tially
estab lished by the two infec tive forms, since towards the end of the acute phase the dif fer en tial effect had dis ap peared.
It was dem on strated that, in a murine model, tGPI-muc ins of BT forms are potent elic i tors of the immune sys tem and pro duce large amounts of NO (Cam ar go et al., 1997a,b). The dis par ity between these two sets of find ings could be due to the exper i men tal model used (i.e. dogs ver sus mice) or to the type of exper i ment (i.e. in vitro ver sus in vivo) car ried out. Addi tion ally, the ear lier work focused on a sin gle com po nent of the par a site form whereas in the pres ent inves ti ga tion all com po nents were involved.
Par a site clear ance in periph e ral blood was observed simul ta-neously in MT- and BT-infected ani mals on day 28 fol low ing inoc-u la tion, and woinoc-uld accoinoc-unt for the observed sim i lar i ties between the MT and BT infec tion pro files. This sug gests, there fore, that the major inoc u lum-depen dent immu no log i cal events take place early in the infec tion pro cess. This was dem on strated in this study, because from the 28th day after infec tion, the serum lev els of NO in ani mals of the MT group showed a decline, and at day 35 were sim-i lar to those of the BT and con trol groups. These fsim-ind sim-ings are con-sis tent with those reported by other author (Saef tel et al., 2001), who dem on strated that in the murine model NO was impor tant for the con trol of infec tion in the first 3 weeks fol low ing infec tion, but declined in level there af ter.
Con sid er ing pre vi ous results of Car nei ro et al. (2007) con cern-ing the phe no typ cern-ing of periph e ral blood, a positive cor re la tion between serum lev els of NO and the fre quency of CD5+ T lym
the MT group (data not shown). This sug gests that these cell types could be par tic i pat ing directly or indi rectly in the pro duc tion of NO in this group. CD4+ T cells could gen er ate NO directly through
the action of iNOS, as has been observed by Tay lor-Rob in son et al. (1994). Con versely, CD4+ T lym pho cytes may pro duce INF-c, which
in turn acti vates mac ro phages to pro duce NO by the action of iNOS (Rus so et al., 1988; Ara ujo, 1989; Rot ten berg et al., 1993, 1995).
Addi tion ally, Car nei ro et al. (2007) observed that, in the experi men tal dogs employed experin the pres ent study, there was a sexperig nexperifexperi -cant decrease of CD4+ cells in the periph e ral blood of MT-infected
ani mals on day 28 in com par i son with those of the BT group. Such
a find ing may indi cate a greater migra tion of CD4+ cells to the sites
of inflam ma tion in the MT group, and this could be respon si ble for the increased pro duc tion of NO in the tis sues of MT-infected ani-mals. Numer ous attempts have been made by our research group to char ac ter ise the T cells (CD4+ and CD8+) in heart and spleen
sam-ples, both fro zen and embed ded in par affi n, but cur rently with out suc cess.
Park et al. (2000) detected the pres ence of iNOS mRNA in cells from nor mal human heart, imply ing that heart mus cles express iNOS con sti tu tively under base line con di tions. This pos-si bil ity would explain the expres pos-sion of iNOS in dif fer ent areas
Fig. 3. Pho to mi cro graphs of heart or spleen sec tions dur ing acute canine infec tion with meta cy clic (MT) or blood try pom astig otes (BT) of Bere nice-78 Try pan o soma cru zi
of the heart of con trol ani mals as observed in the pres ent work. Immu no his to chem i cal stud ies by Chandr ase kar et al. (1998)
revealed high expres sion of iNOS in the arter ies and arte ri oles of rats com menc ing 36 h after infec tion but reduc ing by the 15th day. In con trast, iNOS expres sion was intense in cardio myo cytes on the 15th day after infec tion. The same pattern was observed in the pres ent study, with strong expres sion in the arter ies and espe cially in cardio myo cytes.
Ani mals of the BT group showed greater inflam ma tory pro cess com pared with the con trol and MT groups, but the areas asso ci-ated with iNOS were, in gen eral, smaller. This fact could be relci-ated to the pres ence of anti-inflam ma tory cyto kines such as IL-10 and TGF-b (Oswald et al., 1992; Sher et al., 1992), which can dis able the mac ro phages and thus pre vent expres sion of iNOS.
No par a sit ism was detected in spleen tis sue frag ments derived from any of the exper i men tal dogs, a find ing that could be explained by the myot ro pism observed in Be-78 T. cru zi strain.
Since BT and am astig otes, but not MT and epim astig otes, are able to pro mote the syn the sis of cyto kines in mac ro phages and to acti vate dif fer ent com part ments of the immune sys tem (Cam ar go et al., 1997a; Dos Reis et al., 2002), we hypoth e sized that, ini tially, MT may infect host cells in a silent man ner. How ever, appar ently, inoc u la tion of ani mals with MT forms induces an effec tive ini tial response that lim its par a si te mia and subsequent tis sue par a sit ism, result ing in a lower inflam ma tory response and only minor tis sue dam age. In con trast, infec tion with BT forms appears not to induce an early immune response that is suffi ciently elab o rate to achieve the rapid con trol of the infec tion, and this per mits the devel op-ment of inten sive his to log i cal changes, espe cially in the heart. Will be per formed by our group, in vitro stud ies to under stand how the dif fer ent infec tive forms change the pattern of expres sion of cyto-kines in cardio myo cytes and mac ro phages, try ing to under stand how the inter ac tion between infec tive forms-host cells pro duces dif fer ent immu no log i cal answers.
The results pre sented herein rein force those obtained by
Car nei ro et al. (2007), who found that the source of inoc u lum could inter fere with the devel op ment of the acute phase of Cha gas dis ease in respect of par a si tol ogy, serol ogy and phe no type of the periph e ral blood cells. It is pos si ble that the intrin sic pattern of par a site–host cell inter ac tions trig gered dur ing the acute phase by dif fer ent inoc u lum sources may influ ence var i ous immu no log i cal aspects of Cha gas dis ease.
Our find ings empha sise the impor tance of tak ing into account the inoc u lum source of T. cru zi when attempt ing to trans fer infor ma tion from exper i men tal mod els to human infec tion, since dif fer ent inoc u lum sources may trig ger dis tinct par a site–host inter ac tions dur ing the acute phase that may influ ence rel e vant bio log i cal aspects of chronic Cha gas dis ease.
Acknowl edg ments
The authors wish to express their appre ci a tion of the hard work car ried out by the ken nel staff of the Fed eral Uni ver sity of Ouro Preto, and for their spe cial ded i ca tion dur ing the exe cu tion of this pro ject. The authors are also grate ful to Rede Mine ira de Bi ot er ismo, Fun dação de Am par o à Pes qu isa do Es ta do de Min as Ge rais and Con se lho Nac-ion al de De sen volvi men to Cient ífi co e Tec nológ i co (CNPq), Bra zil.
Ref er ences
Ali berti, J.C., Car doso, M.A.G., Mar tins, G.A., Gaz zi nel li, R.T., Vie ira, L.Q., Silva, J.S., 1996. IL-12 medi ates resis tance to Try pan o soma cru zi infec tion in mice and is pro duced by nor mal murine mac ro phage in response to live tryp o mas ti gote. Infec tion and Immu nity 64, 1961–1967.
Almeida, I.C., Cam ar go, M.M., Proco pio, D.O., Silva, L.S., Me hl ert, A., Trav as sos, L.R., Gaz zi nel li, R.T., Fer gu son, M.A.J., 2000. Highly puri fied gly co syl phos phat idyli-nos i tols from Try pan o soma cru zi are potent pro in flam ma tory agents. EMBO Jour nal 19, 1476–1485.
And rade, L.O., Andrews, N.W., 2005. The Try pan o soma cru zi-host cell inter play: loca tion, inva sion, reten tion. Nature Reviews: Micro bi ol ogy 3, 819–823. Ara ujo, F.G., 1989. Devel op ment of resis tance to Try pan o soma cru zi in mice depends
on a via ble pop u la tion of L3T4+ (CD4+) T lym pho cytes. Infec tion and Immu nity 57, 2246–2248.
Ba hi a, M.T., Ta fu ri, W.L., Ca liar i, M.V., Ve los o, V.M., Car nei ro, C.M., George, L.L.M.C., Lana, M., 2002. Com par i son of Try pan o soma cru zi infec tion in dogs inoc u lated with blood or meta cy clic try pom astig otes of Bere nice-62 and Bere nice-78 strains via intra peri to neal and con junc ti val routes. Re vi sta da So cied ade de Med i ci na Trop i cal 35, 339–345.
Bren er, Z., 1962. Ther a peu tic activ ity and cri te rion of cure in mice exper i men tally infected with Try pan o soma cru zi. Re vi sta do In sti tu to de Med i ci na Trop i cal de São Paulo 4, 389–396.
Bren er, Z., Gaz zi nel li, R.T., 1997. Immu no log i cal con trol of Try pan o soma cru zi infec-tion and path o gen e sis of Cha gas’ dis ease. Inter na infec-tional Archives of Allergy and Immu nol ogy 114, 103–110.
Cam ar go, M.M., Almeida, I.C., Pere ira, M.E., Fer gun son, M.A., Trav as sos, L.R., Gaz zi-nel li, R.T., 1997a. Gly co syl phos phat idyl i nis i tol anchored mucin-like gly co pro-teins iso lated from Try pan o soma cru zi try pom astig otes ini ti ate the syn the sis of pro in flam ma tory cyto kines by mac ro phages. Jour nal of Immu nol ogy 158, 5890–5901.
Cam ar go, M.M., And rade, A.C., Almeida, I.C., Trav as sos, L.R., Gaz zi nel li, R.T., 1997b. Gly co con ju gates iso lated from Try pan o soma cru zi but not from Leish mania spe-cies mem branes trig ger nitric oxide syn the sis as well as micro bi ci dal activ ity in IFN-gamma-primed mac ro phages. Jour nal of Immu nol ogy 159, 6131–6139. Car dil lo, F., Vol tar el li, J.C., Reed, S.G., Silva, J.S., 1996. Reg u la tion of Try pan o soma
cru zi infec tion in mice by gamma inter feron and inter leu kin-10: role of NK cells. Infec tion and Immu nity 64, 128–134.
Car dil lo, F., Pos tol, E., Ni hei, J., Aroe ira, L.S., Nom i zo, A., Men gel, J., 2007. B cells mod-u late T cells so as to favomod-ur T helper type 1 and CD8+ T-cell responses in the acute phase of Try pan o soma cru zi infec tion. Immu nol ogy 122, 584–595. Car nei ro, C.M., Mar tins-Fil ho, O.A.M., Reis, A.B., Ve los o, V.M., Ara újo, F.M.G., Ba hi a,
M.T., Lana, M., Mach ado-Coel ho, G.L.L., Gaz zi nel li, G., Cor rea-Oli veira, R., Ta fu ri, W.L., 2007. Dif fer en tial impact of meta cy clic and blood try pom astig otes on par-a si to log i cpar-al, sero log i cpar-al par-and phe no typic fepar-a tures trig gered dur ing par-acute Try pan-o span-oma cru zi infec tion in dogs. Acta Tro pica 101, 120–129.
Chandr ase kar, B., Mel by, P.C., Tro yer, D.A., Col ston, J.T., Free man, G.L., 1998. Tem-po ral expres sion of pro-inflam ma tory cyto kines and induc ible nitric oxide syn thase in exper i men tal acute chaga sic car dio my op a thy. Amer i can Jour nal of Pathol ogy 152, 925–934.
COBEA, 2006. Colé gio Bra sile iro de Ex per i men tação Ani mal. Leg is lação e éti ca. Avail able from: <http://www.co bea.org.br/>.
Co nov er, W.J., 1980. Practical Non para met ric Sta tis tics, 3rd ed. Wiley, New York. Coura, J.R., Jun que ira, A.C.V., Carv alho-More ira, C.J., Bor ges-Pere ira, J., Al ba jar, P.V.,
2007. Uma visão sistê mi ca da ende mia chagá si ca. In: Sil ve ira, A.C. (Ed.), La En ferme dad de Cha gas a la Pu erta de los 100 Años del Co noc im i en to de Una Ende mia Amer i cana Ances tral. Or ga ni za ción Pan amer i ca na de la Sa lud y Fun-da ción Mun do Sano, Bue nos Aires, pp. 25–35.
Cum mings, K.L., Tarl eton, R.L., 2004. Induc ible nitric oxide syn thase is not essen tial for con trol of Try pan o soma cru zi infec tion in mice. Infec tion and Immu nity 72, 4081–4089.
Dos Reis, G.A., Pe ça nha, L.M., Bel lio, M., Prev ia to, J.O., Men do nça-Prev ia to, L., 2002. Gly coi nos i tol phos pho lip ids from Try pan o soma cru zi trans mit sig nals to the cells of the host immune sys tem through both cer a mide and gly can chains. Microbes and Infec tion. 4, 1007–1013.
Green, L.C., Wag ner, D.A., Glo glow ski, J., Skip per, P.L., Wish nok, J.S., Tan nen baum, S.R., 1982. Anal y sis of nitrate, nitrite, and (15N) nitrate in bio log i cal flu ids. Ana-lyt i cal Bio chem is try 126, 131–138.
Hol scher, C., Koh ler, G., Muller, U., Moss mann, H., Schaub, G.A., Bromb ach er, F., 1998. Defec tive nitric oxide effec tor func tions lead to extreme sus cep ti bil ity of Try-pan o soma cru zi-infected mice defi cient in gamma inter feron recep tor or induc-ible nitric oxide syn thase. Infec tion and Immu nity 66, 1208–1215.
Kol lien, A.H., Schaub, G.A., 2000. The devel op ment of Try pan o soma cru zi in tri atom-i nae. Par a satom-i tol ogy Today 16, 381–387.
Lana, M., Chiar i, E., Ta fu ri, W.L., 1992. Exper i men tal Cha gas’s dis ease in dogs. Memó-rias do In sti tu to Oswaldo Cruz 87, 59–71.
Lana, M., Ta fu ri, W.L., Ca liar i, M.V., Bamb ir ra, E.A., Chiar i, C.A., Rios Le ite, V.H., Barb-osa, A.J.A., Toledo, M.J.O., Chiar i, E., 1988. Fase crô nica cardí aca fi bros an te da trip anos somí ase cru zi exper i men tal no cão. Re vi sta da So cied ade Bra sile ira de Med i ci na Trop i cal 21, 113–121.
Oswald, I.P., Gaz zi nel li, R.T., Sher, A., James, S.L., 1992. IL-10 syn er giz es with IL-4 and trans form ing growth fac tor-beta to inhibit mac ro phage cyto toxic activ ity. Jour nal of Immu nol ogy 148, 3578–3582.
Park, C.S., Krishna, G., Ahn, M., Kang, J., Chung, W., Kim, D., Hwang, H., Lee, J., Paik, S., Cha, Y., 2000. Dif fer en tial and con sti tu tive expres sion of neu ro nal, induc ible, and endo the lial nitric oxide syn thase mRNAs and pro teins in path o log i cally nor mal human tis sues. Nitric Oxide: Biol ogy and Chem is try 4, 459–471. Pas cutti, M.F., Bott as so, O.A., Hour ques cos, M.C., Wiet zer bin, J., Re vel li, S., 2003.
Age-related increase in resis tance to acute Try pan o soma cru zi infec tion in rats is asso ci ated with an appro pri ate anti body response. Scandinavian Jour nal of Immu nol ogy 58, 173–179.
Pe tray, P., Cast anos-Velez, E., Grin stein, S., Orn, A., Rot ten berg, M.E., 1995. Role of nitric oxide in resis tance and his to pa thol ogy dur ing exper i men tal infec tion with Try pan o soma cru zi. Immu no log i cal Let ters 47, 121–126.
Rot ten berg, M.E., Bakh i et, M., Ols son, T., 1993. Dif fer en tial sus cep ti bil i ties of mice ge nom i cal ly deleted of CD4 and CD8 to infec tions with Try pan o soma cru zi or
Try pan o soma bru cei. Infec tion and Immu nity 61, 5129–5133.
Rot ten berg, M.E., Ri ar te, A., Spor rong, L., 1995. Out come of infec tion with dif fer ent strains of Try pan o soma cru zi in mice lack ing CD4 and/or CD8. Immu no log i cal Let ters 45, 53–60.
Rus so, M., Mi no pri o, P., Cout in ho, A., Hon te bey rie-Jos kowicz, M., 1988. Deple tion of L3T4+ T lym pho cytes dur ing acute Try pan o soma cru zi infec tion abol ishes mac ro phage and B lym pho cyte acti va tion but not tis sue inflam ma tory reac tion. Memó rias do In sti tu to Oswaldo Cruz 83, 527–538.
Saef tel, M., Flei scher, B., Hoe rauf, A., 2001. Stage-depen dent role of nitric oxide in the con trol of Try pan o soma cru zi infec tion. Infec tion and Immu nity 69, 2252– 2259.
San dler, S.G., Yu, H., Ras sai, N., 2003. Risks of blood trans fu sion and their pre ven-tion. Clin i cal Advances in Hema tol ogy and Oncol ogy 1, 307–313.
Sard in ha, L.R., Elias, R.M., Mo sca, T., 2006. Con tri bu tion of NK, NK T, gam madelta T, and al phab e ta T cells to the gamma inter feron response required for liver pro-tec tion against Try pan o soma cru zi. Infec tion and Immu nity 74, 2031–2042. Sher, A., Gaz zi nel li, R.T., Oswald, I.P., Cler i ci, M., Kul berg, M., Pe arce, E.J.,
Ber zof sky, J.A., Mos mann, T.M., James, S.L., Mor ce, H.C., Shearer, G., 1992. Role of T-cell derived cyto kines in the down reg u la tion of immune responses in par a sitic and ret ro vi ral infec tion. Immu no log i cal Reviews 127, 183–204.
Tay lor-Rob in son, A.W., Liew, F.Y., Se vern, A., Xu, D., McSor ley, S.J., Gar side, P., Pa dron, J., Phil lips, R.S., 1994. Reg u la tion of the immune response by nitric oxide dif fer-en tially pro duced by T helper type 1 and T helper type 2 cells. Euro pean Jour nal of Immu nol ogy 24, 980–984.
Ves pa, G.N., Cunha, F.Q., Silva, J.S., 1994. Nitric oxide is involved in the con trol of
Try pan o soma cru zi-induced par a si te mia and directly kills the par a site in vitro. Infec tion and Immu nity 62, 5177–5182.
