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SIALYLATION PROTECTS TRYPANOSOMA CRUZI TRYPOMASTIGOTES AGAINST LYTIC ANTIBODIES IN HUMAN CHAGAS' DISEASE

Pereira-Chioccola, V. L.^1,2 ; Rodrigues, M.M.¹; Travassos, L.R.¹; Schenkman, S.¹

1- Dep. de Microbiologia, Imunologia e Parasitologia -UNIFESP - Escola Paulista de Medicina - R. Botucatu, 862-CEP 04023-062, São Paulo, Brasil.

2-Laboratório de Xenodiagnóstico- Inst. Dante Pazzanese de Cardiologia- Av.Dante Pazzanese, 500-CEP 04012-909

We and others have reported that lytic anti-anti-a galactosyl antibodies (anti-a-Gal) purified from chronic Chagas' disease patients recognize anti-a-galactosyl epitopes of O-linked oligosaccharide chains of mucin-like glycoproteins of trypomastigotes. These antibodies strongly agglutinate and destroy trypomastigotes independent of complement. By scanning electron microscopy we found that parasite membrane was dramatically affected by the presence of anti-a-Gal. We have also found previously that most of Chagasic patients present antibodies that inhibit trans-sialidase (TS) activity in vitro and prevent the sialylation of live parasites. As the sialic acid acceptors are the same molecules that contain the a galactosyl epitopes, we studied how the presence of anti-TS antibodies affect the lysis promoted by anti-a galactosyl antibodies. Trypomastigotes obtained from infected cells in culture in the absence of sialic acid sources were lysed at much lower anti-anti-a-Gal concentrations when compared with sialylated parasites. When desialylated parasites were incubated with anti-TS obtained from mice immunized with a recombinant TS, which prevents parasite sialylation, the lytic effect of anti-a Gal was again obtained at very low antibody concentration. The anti-TS alone was unable to induce lysis or agglutination. This antibody synergism was observed at very low dilution of anti-TS (up to 10000), but not when the parasites were first incubated with serum or other sialic acid sources, indicating that it was dependent of parasite sialylation. The anti-TS effect was not obtained with mAb anti-TS unable to inhibit the enzymatic activity. Therefore, the presence of anti-TS antibodies, preventing parasite sialylation in human Chagasic patients may contribute to the elimination of blood trypomastigotes induced by the anti-a-Gal antibodies.

Supported by FAPESP, CNPq, PADCT, FINEP.

BIODEGRADABLE MICROSPHERES AS ADJUVANT IN IMMUNIZATION AGAINST LEISHMANIA MAJOR INFECTION

Lima, K.M¹, Dias, O.J.G.², Affonso, L.C.C.² & Rodrigues Jr, J.M¹

¹Laboratório de Tecnologia Farmacêutica, Faculdade de Farmácia, UFMG, Av. Olegário Maciel, 2360, CEP 30.180-112. Belo Horizonte (MG) Brazil. Tele/fax: [phone]DECBI/ICEB, UFOP, Ouro Preto (MG) Brazil.

Successful application of the next generation of vaccines will require that protection be induced with a minimal number of administrations and that effective immune response be elicited to eliminate the pathogen. Biodegradable poly (lactide-co-glycolide) microespheres have been widely used in several immunizations studies due to its attractive features: (i) controlled antigen release; (ii) targeting to antigen-presenting cells; (iii) safety, and, (iv) protection of the antigen in storage. For these reasons, we have focused our studies in the development of soluble Leishmania antigens-containing microspheres. To obtain a soluble antigen fraction, promastigotes of Leishmania major were cultured, washed and suspended in buffered saline. Parasites were disrupted by means of a sonifier and then centrifuged. The supernatant was recovered and filtered in a 0.22 mm membrane. PLGA microspheres were obtained by the water-in-oil-in-water solvent evaporation technique and freeze-dried. The medium size of microspheres prepared from PLA, PLGA 75:25 and PLGA 50:50 were 13mm, 13 mm and 9 mm respectively. The content of protein loading was 3,4 mg/mg of polymer when we used a 1,28mg/ml antigen solution, and 10,6 mg/mg of polymer when we used a 20 mg/ml antigen solution. BALB/c mice were immunized with soluble L. major antigens, either dissolved in PBS or entrapped in PLGA microspheres. To determine the kind of immune response we assessed cytokine production by popliteal lymph node cells, inguinal lymph nodes cells and spleen cells 3 and 10 days respectively after footpad injection with L.major antigen in solution or entrapped in microspheres. Mice immunized with entrapped antigen (3,4 mg/mg of polymer) showed an increased IL-4 production 3 days after immunization when compared to animals immunized with soluble antigen alone. Ten days after immunization, IL-4 production decreased in all groups. No significant IFN-g production was observed at both time points. Apparently, IL-4 production was induced by and proportional to the amount of microsphere injected. Unstimulated cells from animals inoculated with lower doses of the polymer produced less IL-4 than those from animals injected with higher doses. Our results indicate that a higher antigen:polymer ratio may be necessary for successful immunization.

Supported by FAPEMIG

IMMUNE RESPONSE TO RECOMBINANT AND NATIVE LEISHMANIA ANTIGENS IN MICE VACCINATED WITH GP-63 GENE FROM LEISHMANIA MAJOR.

Carvalho, F.A.A.,1,4 Toledo, V.P.C.P.,2,3 Fran‡a, R.C.S.,1 Nascimento, E.,2 Costa, F.T.M.,5 Gazzinelli, R.T.1 and Tavares, C.A.P.1

1Departamento Bioqu¡mica e Imunologia, 2Departamento de Parasitologia e 3Faculdade de Farm cia, Universidade Federal de Minas Gerais, Belo Horizonte, MG; 4Departamento de Bioqu¡mica e Farmacologia, Universidade Federal do Piaui, Teresina, PI; e 5Departamento de Parasitologia, Microbiologia e Imunologia, Universidade Federal de Sao Paulo, SÆo Paulo, SP, Brasil.

Nucleic acid vaccines have been show to induce a strong cellular and humoral immune responses, without the risk of live attenuated vaccines. Susceptible mice (BALB/c) were vaccinated with a plasmid (pcDNA3) containing the gene of GP-63, a major surface antigen found in different species of Leishmania. Mice were immunized with intra-muscular injection of 50 æg of plasmids in the external face of tibial muscle from each posterior member. Each animal received five doses, with 30 days intervals. The antibody response against recombinant GP-63 was evaluated by an ELISA, 2-4 weeks after each immunization. We observed a gradual increase in the titters of antibodies against GP-63, mainly after the fourth immunization. In order, to evaluate the type of immune response induce by our vaccination protocol, we measured the levels of antibody isotypes (i.e. Ig1 and IgG2a) against GP-63 as well as the level of cytokines (i.e. IFN-g and IL-4)

produced by splenocytes in response to Leishmania extracts. Our results show that naked-DNA vaccination, elicited mainly a Type 1 cytokine response. Thus, the vaccinated animals had a higher levels of IgG2a than IgG1 parasite specific circulating antibodies. In agreement with these data, we also observed that spleen cells from animals vaccinated with GP-63 gene produced high levels of IFN-g but not IL-4 when stimulated with Leishmania antigens. Finally, we challenged the vaccinated and control animals with 1 x105 stationary promastigotes, in the foot pad. So far, our preliminary results, at six weeks post-infection, show that as compared to the control animals vaccination with GP-63 gene partially inhibited the development of foot pad lesion.

Supported by PADCT/CNPq, CAPES and PRONEX.

THE LIKE RESPONSE ASSOCIATED WITH IMMUNOTHERAPY OF AMERICAN CUTANEOUS LEISHMANIASIS (ACL).

Cabrera, M.¹, Shaw, MA.², Castes, M.¹, Trujillo, D.¹, Convit, J.¹, Blackwell, J.M.²

¹ Instituto de Biomedicina, UCV, apartado 4043, Caracas 1010A, Venezuela; ²University of Cambridge, Addenbrooke´s Hospital, Department of Medicine, level 5, Hill Rd. Cambridge, England, CB2 2QQ.

In Venezuela Convit et al. have developed a successful immunotherapy (IMT) to treat ACL. This immunotherapy had proved to be as effective as the conventional chemotherapy in resolving LCL infections. They obtained more than 90 % clinical cures using both treatments. However, the immunological basis of this IMT have not been examined until now. This work shows an analysis of the T cell responses of ACL patients before and after treatment with three doses of live BCG plus heat killed L.mexicana amazonensis (L.m.a.) promastigotes. Proliferative and cytokine (IFN-g and IL-5) responses were measured to crude and defined antigenic preparations (L.major rGP63 and mycobacterial heat shock proteins: M. bovis rHSP65kD and M. tuberculosis rHSP70kD. T cell epitopes were predicted using the Tsites computer package and a panel of Leishmania-specific and cross-reactive peptides were synthesised. The study was carried out on a total of 49 ACL patients and 10 healthy volunteers. The patients were diagnosed at the "Instituto de Biomedicina" by established clinical, epidemiological and histopathological criteria as either localized cutaneous leishmaniasis (LCL n= 23) and mucocutaneous leishmaniasis (MCL n=26).

MCL and LCL patients showed low proliferation and high IFN-g production to BCG, in contrast to endemic controls. The IMT elicited a TH1 like response in LCL patients with an increase in mean IFN-g response and a decrease in the responders by IL-5 to all crude antigens (BCG, PPD, L.m.a., L.m.m). An increase IFN-g responsiveness to rGP63, rHSP65 and to peptides HSP70 residues 111-120 and 455-468 (both Leishmania-specific) was observed following IMT. MCL patients receiving IMT plus Glucantime also showed an increased IFN-g response to all crude antigens except to L.m.a. These patients showed an increased proliferation to rHSP70 and to two leishmanial-specific peptides (amino acids 90-101 and 111-120 of HSP70), with and decreased IFN-g response again to the aa 111-120 and to the conserved residue 474-484 after treatment. Overall results suggest that IMT may induce a protective TH1-like response particularly in LCL patients with a no associated self reactivity. Therefore, BCG plus promastigotes is a safe alternative therapy to treat ACL patients. Supported by UNDP/World Bank/WHO/TDR, and Venezuelan MSAS-World Bank project No 55.

PHASE 2 TRIAL WITH THE BIOBRAS VACCINE AGAINST LEISHMANIASIS.

De Luca, PM, Mendonça, SCF, Santiago, MA, Coutinho, SG, Pinto, J, Genaro, O, Costa, CA, Toledo, VP & Mayrink, W.

Instituto Oswaldo Cruz, Fiocruz, Caixa Postal 926, Rio de Janeiro, 21045-900, RJ. Instituto de Ciências Biológicas. Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.

The vaccine used was industrially produced under conditions of Good Manufacturing Practice (GMP) by Biobras (Montes Claros, MG) using an Leishmania. amazonensis strain. The objective of this randomized, double-blind, placebo-controlled study was to evaluate the immunogenicity of various vaccination schemes and to select one of them for further efficacy trials (phase 3) in the field. The study subjects were Montenegro skin test (MST)-negative healthy volunteers allocated into eight groups. Six groups received either two or three deep IM injections of vaccine/placebo in the following doses: 180, 360 and 540 mg protein nitrogen ml^-1. The two remaining groups received either two or three similar injections of placebo. The time interval between the vaccine/placebo applications was always 21 days. MST and proliferative response assays (PRA) of peripheral blood mononuclear cells stimulated with Leishmania sonicated promastigotes were performed two days before and 20 days after vaccination. While none of the subjects who had received placebo had positive reactions to the second MST, the majority of subjects who had received vaccine converted the skin test (89,5%). The dose of 360 mg ml^-1 was more efficient in inducing MST conversion than the other doses (p<0.05, Fisher's Exact Test). With regard to PRA, there was a significant increase in the stimulation indexes (SI) after vaccination as compared to those before vaccination in all groups, including those who took placebo (p<0.05 at least, Wilcoxon's Test). The MST results indicate that this vaccine is immunogenic in all doses used and indicate that the dose of choice should be 360 mg ml^-1 (the same that has been used in previous studies). Regarding the PRA, the relatively unexpected finding of a significant increase of SI after vaccination also in the placebo groups could be explained by the possible sensitization provided by the first MST. These results also suggest that MST may be a more reliable method to evaluate the immunogenicity of such vaccines than PRA, which might be too sensitive. Financial support: UNDP/World Bank/WHO TDR and CNPq.

VACCINATION OF BALB/C MICE WITH GP36 ANTIGEN OF L. DONOVANI AND SAPONIN.

Paraguai de Souza, E., Santana, DM. & Palatnik de Sousa, CB.

Instituto de Microbiologia "Prof. Paulo de Góes", Universidade Federal do Rio de Janeiro, Caixa Postal 68040, Rio de Janeiro, 21941-590, RJ, Brasil.

The GP36 glycoprotein, identified by rabbit hyperimmune, human Kala-azar and mouse monoclonal antibodies is the main species-speciffic component of the FML. The GP36 was isolated from FML through SDS-PAGE and chemical elution+ sonication. An hyperimmune serum obtained in rabbit, specifically recognized a band of 36kDa in total promastigote lysate of L. donovani (stationary phase) confirming that GP36 is indeed present in the living parasite. A band of higher molecular weight was also detected in both promastigote and amastigote lysate. Sera of Balb/c females immunized with total promastigote f/t lysate either strongly reacted with FML and lymphocytes from popliteal ganglia proliferated in contact with GP36 after 24,48 and 96h (p<0.001) in the sc group and after 24 and 96h (p<0.001) in the ip group. Balb/c mices were imunized with 16ug of purified GP36 and Riedel De Haen saponin, by the sc route. A strong and specific response was observed in: anti-FML antibodies, DTH response (p<0.001), popliteal cell proliferation with GP36 (0.15 e 0.6 ug) and a 68.1% of significant (p<0.025) and specific (p<0.05) reduction in liver parasitic burden. Protection was also evident in the increase of IgG1, IgG2a and IgG2b anti-FML antibodies (log2 titers: 17:19:15). Our results suggest that GP36 is also a major immunoprotective component of the Fucose mannose ligand antigen of L. donovani.

Support: PNUD-FNS, PRONEX, CNPQ, RHAE-CNPQ, FINEP, CAPES, FUJB-CEPG-UFRJ.

VACCINATION AGAINST EXPERIMENTAL CANINE KALA-AZAR WITH THE FML ANTIGEN OF LEISHMANIA DONOVANI AND THE QUIL-A SAPONIN.

Borja-Cabrera GP¹, Bernardo,RR², Paraguai de Souza,E¹,Palatnik M³. & Palatnik de Sousa, CB¹.

Instituto de Microbiologia "Prof. Paulo de Góes"¹, Núcleo de Pesquisas de Produtos Naturais ², Fac. Medicina-HUCFFo, Universidade Federal do Rio de Janeiro, Caixa Postal 68040, RJ, 21941-590, RJ, Brasil.

In this work we initiated the vaccination of 4 month old mongrel dogs with FML+ Quil-A saponin. One group receive 3 subcutaneous saline injections. The second and third groups were treated with 1.5mg FML in combination with 0.5mg or 1mg QuilA saponin (three dosis, 20 days interval). Animals were challenged with an endovenous injection of 10⁸ amastigotes of L. donovani. Anti-FML antibodies were significantly increased in sera soon after the first vaccine dosis and maintained up to 30 days after the infection, in 3/3 dogs from the 0.5mg and 1mg QuilA vaccinated animals while no reaction was detected in the saline group. Also 30 days after infection, the DTH response to 200ug of promastigote lysate protein (>5mm) was positive in 2/3 animals of both vaccinated groups and negative in control animals which started to be reactive only by the 90th day. Furthermore, 150 days after infection, 2/3 dogs from the 0.5mg QuilA group were reactive as well as 1/3 animals from the 1mg QuilA and saline control group. In vitro mononuclear blood cell proliferation to the GP36 glycoprotein and the FML antigens was positive in 3/3 animals of all groups. At the same time (120 days after infection) no haematocryte alteration but lymphocytosis was detected in all individuals. Total leukocytes, purified mononuclear cells and platelets were decreased in 2/3 saline and QuilA 1mg treated animals while the 0.5mg QuilA group remained normal. Taken together, these preliminary results show that when compared to control animals, both QuilA+FML formulations induce a protective response against experimental Visceral leishmaniasis (x² p<0.005 for 0.5 and 1mg saponin). Protection against canine Kala-azar in this vaccination experiment will be monitored during 24 months in order to obtain final conclusions.

Support: PNUD-FNS, PRONEX, CNPQ, RHAE-CNPQ, FINEP, CAPES, FUJB-CEPG-UFRJ.

VACCINATION AGAINST CANINE KALA-AZAR WITH THE FML ANTIGEN AND SAPONIN IN AN ENDEMIC AREA (SÃO GONÇALO DO AMARANTE, RN)

da Silva , VO, Borja-Cabrera GP, de Azevedo Pereira, R, & Palatnik de Sousa, CB.

Instituto de Microbiologia "Prof. Paulo de Góes", Universidade Federal do Rio de Janeiro, Caixa Postal 68040, Rio de Janeiro, 21941-590, RJ, Brasil.

Prevention of canine zoonotic visceral leishmaniasis appears to be the best approach for interrupting the domestic cycle of the disease, reducing the human cases of Kala-azar. In previous work, using the FML antigen of Leishmania donovani and the Riedel De Haënsaponin, we were able to achieve an 87.7% (p<0.01) and 84% (p<0.001) average protection against Kala-azar, in the isogenic CB hamster and Balb/c mouse models. In this work we initiated the vaccination with FML+saponin in dogs of a Kala-azar endemic area, São Gonçalo do Amarante, RN. Only domiciliary dogs were included in this trial. A first serological screening was performed with the FML-ELISA assay. The seronegative individuals were divided into two groups: one treated with saline and the second with three subcutaneous doses of FMC (1.5mg) and saponin (0.5mg). Forty five days after the last immunization the seroreactivity to FML and DTH response to L. donovani promastigote lysate was recorded. A significant increase of the |FML-seropositive animals was observed in the vaccinated group (19/48) over the control (6/53). Seropositivity was highly correlated to FML vaccination (c2= 10.802, p<0.005). The IDR (24h was also significantly increased in the vaccinated group (28/48) over the saline control (8/53) (c²= 20.529 p<0.005). The FML-ELISA and IDR reactive dogs in the saline group bear probably recent infections. The protective effect of the FML+saponin vaccine is strongly support by the detection of 4 obits due to kala-azar in dogs of the saline control group while no death was detected in the group of FML+saponin vaccinated animals. Protection against canine Kala-azar in this vaccination experiment will be monitored during 24 months in order to obtain final conclusions

Support: PNUD-FNS, PRONEX, CNPQ, RHAE-CNPQ, FINEP, CAPES, FUJB-CEPG-UFRJ.

VACCINATION AGAINST EXPERIMENTAL KALA-AZAR WITH SAPONINS FROM ALBIZIA SAMAN (PsAsu & PsAgle) AND THE FML ANTIGEN OF LEISHMANIA DONOVANI

Bernardo, RR¹, Santos, WR², Parente, JP¹ &Palatnik de Sousa, CB².

Núcleo de Pesquisas de Produtos Naturais¹ &Instituto de Microbiologia "Prof. Paulo de Góes", Universidade Federal do Rio de Janeiro, Caixa Postal 68040, Rio de Janeiro, 21941-590, RJ, Brasil.

In previous work, five different triterpenoidic fractions isolated from A. saman were compared regarding their adjuvant activity on FML antigen (IgG, IgM titers, DTH, letality, pain and sugar moieties on the triterpenoid ring). Two saponins were selected for further analysis. The PsAgle fraction induced the best humoral response (IgG2a and IgG2b) and the minor glicidic moiety. The PsAsu saponin was the only one inducing a signifficant DTH response against promastigote lysate (p<0.005). In this work, vaccination of Balb/c mice was performed with three dosis of FML (150 mg) in combination with either Ps Agle or Ps Asu saponins (200mg) by the subcutaneous route. Controls treated with only saline or saponin were included. Fifteen days after vaccination, animals were challenged with 2x 10⁷ amastigotes of L. donovani by the endovenous route. The levels of total IgG anti-FML antibodies were signifficantly and speciffically enhanced over the controls for both saponins before infection and only for PsAsu saponin (IgG and IgM response) after infection. The IgG1, IgG2a, IgG2b and IgG3 scores were: 6:5:5:5 for saline control; 15:6:6:7 for PsAsu+FML and 15:8:8:7 for PsAgle+FML, before infection and 10:9:8:8 for PsAsu+FML and 11:10:7:6 for PsAgle+FML after infection. IgG2a and IgG2b are subtype of IgG immunoglobulins related to protection. Indeed, a signifficant protection was detected in reduction of liver parasitic burden of animals vaccinated with the two formulations: PsAgle+FML (93.9% p<0.05) and PsAsu+FML (84.4%, p<0.05). Saponin control animals were signifficantly different from animals that receive saponin+FML (p<0.005 and p<0.025, respectively). The characterization of the detailed structure these saponins is underway.

Support: PRONEX, FNS-PNUD, CNPQ, FINEP, RHAE-CNPQ, CEPG-UFRJ, FUJB-UFRJ.

ASSESSMENT OF THE STABILITY OF A CUTANEOUS LEISHMANIASIS VACCINE USING IMMUNOLOGICAL PARAMETERS

Costa, T.C¹.; Toledo, V.P.C.P².; Costa, C.A.da²; Genaro, O.¹ & Mayrink, W.¹

¹Depto. de Parasitologia, Instituto de Ciências Biológicas, ²Depto. de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais. Av. Antonio Carlos 6627, CP 486, CEP 31270-901, Belo Horizonte, MG

Studies have shown that the vaccine against cutaneous leishmaniasis described by MAYRINK et al. (1979) and manufactured by BIOBRAS S.A., suffer proteolitic degeneration process during storage. The stability of different vaccine formulations (original vaccine, autoclaved and lyophilized) freshly prepared and after 12 months of storage at 4^o C, was assessed in C57BL/10 mice. Groups of 20 animals were immunized with 100 µg/dose of vaccine, each containing 250 µg of Corynebacterium parvum as adjuvant. The vaccines were administered subcutaneously in two doses with 7-day intervals. Thirty five days after the second dose, the animals received a 10 µg booster dose without adjuvant. On day 42, cellular immunity was evaluated by lymphoproliferative assay using Leishmania amazonensis antigen and splenic cells from immunized mice.

Lymphoproliferative response was detected with all vaccine preparations except the 12 month-old autoclaved vaccine. Similar results were obtained with the footpad swelling tests. In the same way, high levels of g-IFN and IL-2 were detected in the supernatant of simulated splenic cells. IL-4 was detected in lower levels. Ten mice of each group were challenged with 10⁵ infective promastigotes of L. amazonensis in their tail base. Six months after challenge it was observed that the protection conferred by all vaccine preparations was about 50% and 30% for the 12 month-old autoclaved. We conclude that, with the exception of autoclaved vaccine, all other preparations conserve their immunogenic properties after 12 months of storage at 4^oC.

Supported by CNPq

HISTOPATHOLOGICAL FEATURES OF EXPERIMENTAL LEISHMANIASIS USING PENTOXIFYLLINE ( POF ) AS TNF-a INHIBITOR

-CUPOLILO,SMN & GONÇALVES DA COSTA,SC.-Lab.- Imunomodulação, Dpto.protozoologia- FIOCRUZ-RJ-Brasil.

Recently, some studies demonstrated that TNFa plays a protective role in experimental murine cutaneous leishmaniasis, by its ability to activate macrophages to kill intracellular leishmanial parasites, either alone or in combination with IFN-g. Leishmaniasis is a wide spectrum disease and its course depends on the genetic background of the host and the kind of cell mediate immune response. The infection resolution is associated with a predominance of TH1 lynphocytes and, in susceptible hosts, a preferencial expansion of TH2 occurs. In Diffuse Cutaneous Leishmaniasis (DCL), an anergy is observed when specific leishmanial antigen are employed to elicit delayed type hypersensitivity. Diffuse and massive infiltrate of macrophages heavily parasitised is demonstrated in tissues sections. Six to eigth weeks old female mice of different strains (Balb/c, C57Bl6 and C57Bl10) were treated IP with 2 mg of Pentoxifylline ( 3,7dimetil-1-(5-oxo-hexil)xantine ), that is able to suppress the TNFa synthesis, one hour before being challenged with 10⁴ Leishmania amazonensis amastigotes, subcutaneouslly (SC), in the left footpad. The kinetics of infection has been evaluated by footpad swelling measures and histopathological studies were carried out Limiting Dilution Analysis were done to estimate parasites load, 45, 60, 75 and 90 days after infection. An ulcerative progressive lesions with severe inflammatory infiltrate composed by large number of parasitised macrophages, variable number of lymphocytes and necrosis have been observed. Lesions were also progressive but in a lower degree in resistent and non-treated mice, suggesting that TNFa can be protetive and plays a central role in the resistence.TNF-a levels has been determined by ELISA assay, at different intervals (2, 6, 24 hours, 7, 15 and 30^th days), in sera specimes. Mice were challenged with LPS 100mg IP or 10⁴ amastigotes (SC) and groups of both were treated with POF prior to challenge, to further characterize the action of POF on TNF-a synthesis in leishmaniasis compared to LPS-induced and controls.

IMMUNOGLOBULIN MEDIATED LESION IN VISCERAL LEISHMANIASIS IN HAMSTER

^1,2Goto, H.**, ¹Mathias, R., ¹Lindén C. & ³Nose, M.

¹Dep. Preventive Medicine and ²Dep. Pathology, Faculdade de Medicina da Universidade de São Paulo, 01246-903, SP, Brazil, ³Dep. Immunopathology, Ehime University Medical School, Japan.

The pathogenesis of interstitial inflammation in mesenchimal organs, one of the main pathological alterations found in visceral leishmaniasis (VL), is still unknown. Immunocomplex-mediated mechanism has been suggested in glomerulonephritis in VL but recently a novel mechanism of lesion due to ingestion of IgG by endothelial cells has been described in Lupus nephritis (Nose et al., submitted). In this study we searched for similar mechanism in hamsters infected with Leishmania (Leishmania) chagasi. In the frozen sections of these organs C3 and immunoglobulin deposits were searched as well as the in vitro ingestion of hamster serum IgG by endothelial (HUVEC) cells.

Immunoglobulin deposits were found on cells of glomeruli and around tubuli in the kidney, scattered on cells in the liver and in the lung. C3 deposits were found in lesser degree. The immunoglobulin deposits were more pronounced at 15 days of infection.

When sera from infected animals were incubated in vitro for 2 hours with HUVEC cells more pronounced ingestion was observed upon immunostaining with samples taken at 15 days of infection, still present at 30 days but absent at 90 days of infection.

We thus suggest this high ingestion of IgG by endothelial cells as possible pathogenic mechanism of interstitial inflammatory processes observed in visceral leishmaniasis.

Supported by: LIM-50 (HC-FMUSP) and Japan International Cooperation Agency

SAND FLY SALIVA AND MAXADILAN: DISSOCIATION BETWEEN VASODILATATION AND LEISHMANIA INFECTION ENHANCING EFFECTS.

dos-Santos WLC, Paranhos-Silva M, Sherlock I, Paixão MS, Sales LA, Pontes-de-Carvalho LC

LIMC - Centro de Pesquisas Gonçalo Moniz - FIOCRUZ, Rua Waldemar Falcão 121, Brotas, Salvador 40295-001, Bahia, Brasil

Sand fly saliva has been shown to enhance Leishmania infection in mice (Science 239:1306, 1988; Infect Immu 59:1592, 1991). Such effect is attributed to maxadilan, a polypeptide that also produces vasodilatation and presumably, inhibits the killing of Leishmania by macrophage in vitro. In this study, we tested the ability of maxadilan to increase the susceptibility of CBA mice to L. major infection.

Groups of 6 and 18 CBA mice were used in two separate experiments. Two different batches of recombinant maxadilan were kindly supplied by Dr. John David (Harvard Univ., USA). As expected, each batch was able to produce diarrhoea in mice and/or cutaneous hyperaemia in rabbit. Salivary glands were isolated from L. longipalpis and the lysates prepared with these glands produced hyperaemia in rabbit skin. The animals were infected with 10⁵ fourth in vitro passage, stationary phase, promastigotes of L. major in 1) phosphate buffered saline containing 1% bovine serum albumin (PBS-0.1%BSA) alone, or 2) PBS-0.1%BSA containing half acinus of salivary gland of L. longipalpis, or 3) PBS-0.1%BSA containing maxadilan (the dose of maxadilan was adjusted according to its ability to produce cutaneous hyperaemia in rabbit). The animals were followed-up for 9 weeks (first experiment) or 14 weeks (second experiment), with measurements of size of the lesion, parasite burden in the site of infection and in the regional lymph node (as determined by limiting dilution), and parasite dissemination to spleen, liver and lung. The infectivity and virulence of the parasite were tested in a parallel experiment using groups of five Balb/c mice.

The infection with L. major led to a small increase in the thickness of CBA mouse footpad in presence or absence of maxadilan or salivary glands of L. longipalpis. No difference was observed in the size of the lesion, parasite burden or dissemination of L. major in presence or absence of maxadilan or salivary gland lysate, during 9 or 14 weeks of observation. The data presented herein show that the hyperaemia caused by salivary gland lysate of L. longipalpis or maxadilan is not associated with their ability to enhance Leishmania infection. They also suggest that the enhancing effect of maxadilan or salivary gland lysate on Leishmania infection is not always observed, and requires further studies.

TARGETED DISRUPTION OF THE TRANSCRIPTION FACTOR NFAT1 RESULTS IN A SUSCEPTIBILITY TO LEISHMANIA MAJOR INFECTION

Viola, J.P.B.^1,2; Kiani, A.¹ and Rao, A.¹

¹The Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA, USA, 02115; ²Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brasil, 21949-900.

The newly-identified NFAT (Nuclear Factor of Activated T Cells) family of transcription factors is thought to play a critical role in immune response. NFAT DNA-binding activity has been detected in nuclear extracts of antigen-stimulated T cells, B cells, mast cells and NK cells, and NFAT binding sites have been identified in the promoter/enhancer regions of many genes encoding immunoregulatory cytokines such as IL-2, TNF-a, IL-3, GM-CSF, IL-4 and IL-5. The NFAT family comprises several structurally related proteins that are encoded by at least four distinct genes, named NFAT1, NFATc, NFAT3 and NFAT4. The differential expression of NFAT proteins in tissues, and the differences in their binding preferences for recognition sites in cytokine genes, suggest that each NFAT protein may control the expression of a distinct set of genes in vivo. To study the unique functions of NFAT1 in vivo, we generated mutant mice carrying a disrupted NFAT1 allele by gene targeting. The targeting vector was designed to delete most of an exon encoding amino acids near the amino-terminus of the DNA-binding domain. Protein immunoblot and gel mobility shift assay demonstrated that the homozygous mutant mice possessed a null phenotype for NFAT1. Here we show that NFAT1 selectively downregulates the late phase of IL-4 gene transcription, thus potentially inhibiting the development of the Th2 responses in normal mice. Whereas T cells from wild-type littermates show a transient increase and then a rapid decline in the steady-state levels of mRNA, in an in vitro stimulation with anti-CD3, the levels of IL-4 gene transcripts in NFAT1-deficient T cells are maintained at high levels under the same conditions. Consistent with this in vitro phenotype, NFAT1-/- mice are more susceptible to Leishmania major infection than are their littermates, despite a C57BL/6 genetic background. Furthermore, when NFAT1-/- mice were treated with anti-IL4 neutralizing antibody there was a reduction of the lesion size that was comparable with the wild-type and anti-IL4 treated-Balb/c mice. We conclude that NFAT1 uniquely regulates IL-4 expression and T helper subset differentiation by facilitating a late feedback inhibitory mechanism that downregulates IL-4 gene transcription in normal T cells.

This work was supported by NIH grants.

REGULATION OF TRYPANOSOMA CRUZI-INDUCED CHEMOKINE mRNA EXPRESSION DURING INFECTION IN MICE.

Aliberti, J.C.S.¹; Talvani, A.^2,3; Ribeiro, C.S.²; Vieira, L.Q.³; Gazzinelli, R.T.^2,3 & Silva, J.S.¹.

¹Departamento de Parasitologia, Microbiologia e Imunologia, FMRP-USP, Ribeirão Preto, SP; ²Laboratório de Chagas, CPqRR, FIOCRUZ; ³Laboratório de Imunoparasitologia, Departamento de Bioquímica e Imunologia, ICB, UFMG, Belo Horizonte, MG, Brazil.

Chemokines (CK) are a family of cytokines, whose most prominent biological feature is their ability to act as chemotact factors. As during infection with the virulent protozoa T. cruzi, a strong inflammatory reaction occurs, either at the inoculation site and, later, in the myocardium. To address the role of these molecules in T. cruzi-induced inflammation, we first studied the CK mRNA expression triggered by the interaction of T. cruzi trypomastigotes with macrophages. We also investigated the role of cytokines as modulators of T. cruzi-induced CK expression in macrophages. Our results show that IFN-g increases expression Mig, MIP1-b, JE and Crg-2. In contrast, KC and MIP-1a expression was completely blocked. We also investigated the role of other pro-inflammatory or regulatory cytokines which are induced by macrophage exposure to T. cruzi trypomastigotes. The pro-inflammatory cytokines, IL-1b and TNF-a, potentate the expression of the CKs KC, Crg-2, MIP-1a and JE. The regulatory cytokines, IL-10 and TGF-b inhibited the expression of almost all CK tested. The only exception observed was the induction of KC in macrophages by IL-10. To investigate the regulatory role of IFN-g and TNF-a in vivo, we used IFN-g- (GKO) or TNFRp55-deficient (p55-/-) mice following ip infection with 5.000 trypomastigote forms of T. cruzi (Y or CL strain). The cellular infiltrates observed in the inoculation site in control animals (wt) consisted primarily of neutrophils at the first day pi, and mainly lymphocytes at the seventh day pi. The CK expression pattern correlated with the cell type observed. Thus, when neutrophils predominated, the expression of CK that attract these cells (KC) was detected and, seven days pi, the expression of CKs Mig, Crg-2 and RANTES (lymphocytes chemoattractants) was predominant. A distinct inflammatory response was observed in the GKO mice. In these, a predominant neutrophilic and a discrete lymphocytic infiltrate were observed only after five days pi (peaking at seven days pi). The infected GKO mice exhibited strong expression of KC and MIP-2, which may explain the neutrophil migration, RANTES expression at 5 days pi and only a basal expression of Mig and Crg-2. Similar results were found in the cardiac tissue of GKO mice acutely infected with T. cruzi. Interestingly, p55-/- mice did not presented neither neutrophilic infiltrate, or expression of CKs that attract neutrophis during acute infection. In contrast, a large lymphocytic infiltrate was observed in these animals at second day pi. The early appearance of lymphocytes was associated with high level expression of Crg-2 mRNA. Finally, spleen cells of infected mice exhibited a CK expression pattern similar to that observed in the peritoneal cavity cells. In the latter, lymphotactin, SDF-1a and -b were detected after 5 days pi. Altogether, these results suggest that IFN-g, TNF-a and CKs, play a crucial role in the modulation of the inflammatory response observed during T. cruzi infection and lead us to speculate that a modulation of cytokines and CKs could result in susceptibility or resistance to the parasite.

Supported by CAPES, CNPq, FAPESP, FAPEMIG and PAPES-FIOCRUZ.

INCREASED IN SITU PRODUCTION OF IFNg AND IL-12 IN NK-DEPLETED LEISHMANIA (VIANNIA) PANAMENSIS INFECTED MICE.

¹Laurenti,M.D.**, ⁴Sunnemark,D.; ^1,4Gidlund,M.; ¹Ura,D.M., ¹Corbett,C.E.P., ³Sinhorini,I.L.^1,2Goto,H.

¹Dep. Patologia FMUSP-Brazil, ²Dep. Med. Preventiva FMUSP, ³Dep. Patologia FMVZ-USP, ⁴MTC Karolinska Institute-Sweden.

NK cells has been considered as an important source of IFNg in the initial phase of Leishmania infection with the potential to trigger the Th-1 type of immune response in cutaneous leishmaniasis. Since it is known that IFNg significantly inhibits synthesis of C3 by inflammatory cells (Volanakis, Annu.Rev.Immunol.13:277-305,1995) we have been studying the role of NK cells in conjunction with complement that is important for the evasion of the parasite (Laurenti, et al. - Int.J.Exp.Pathol.77:15-24,1996). In the previous study, in NK-depleted and Leishmania (Viannia) panamensis-infected mice we observed data suggestive of link between NK depletion, decrease in IFNg production and consequent increase in complement activity that could be responsible for higher number of parasites found in the skin.

In this work we analyzed the presence of IFNg and IL-12 at the inoculation site of parasite in the skin by immunohistochemistry in BALB/c and C57BL/6 mice depleted in NK cells. Comparing the mouse strains, both cytokines were present in higher amount in C57BL/6 mice than in BALB/c mice. When depleted in NK cells we observed higher amount of IFN-g and IL-12 at 24 hours and 7 days of infection than in respective non-depleted control mice.

Our data suggest the existence of different mechanisms of control for systemic and local production of IFNg.

Supported by CAPES, FAPESP, LIM/50 (HC-FMUSP)

DHFR-TS'DOUBLE KNOCKOUT LEISHMANIA MAJOR MUTANT CROSSREACTS WITH LEISHMANIA AMAZONENSIS

Veras, PST*, Brodskyn, CI**, Balestieri, F.**, Ramos, APS*, Querioz, ARP**, Barral, A, Beverly, S***, and Barral-Netto, M****

*Laboratório de Patologia e Biologia Celular - Centro de Pesquisas Gonçalo Moniz - FIOCRUZ, Bahia, Brazil, 'Serviço de Imunologia - Hospital Univ. Prof. Edgard Santos - Universidade Federal da Bahia, Bahia, Brazil, *Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 , §Laboratório de Integrado de Microbiologia e Imunoregulação - Centro de Pesquisas Gonçalo Moniz - FIOCRUZ, Bahia, Brazil

E1O-A3 is a dhfr-ts- Leishmania major double knockout auxotrophic mutant that induces substantial protection against virulent L. major infection in BALB/c and CBA mice. ln the present report we investigated the capacity of EIO-A3 to protect against heterologous infection by L amazonensis. First, we determinei that stationary phase promastigotes of auxotrophic L. major induced lymphoproliferative responses of healthy donors and of cutaneous leishmaniasis patients, in the same magnitude as L. amazonensis promastigotes. These lymphocytes produced high levels of IFN-,Y and non detectable levels of IL-5, suggesting that L. major dhfr-ts- induce a Thl type of response upon in vitro stimulation of human lymphocytes. Additionally, we evaluated the degree of protection compared by immunization of BALB/c or C57BU6 mice with EIO-A3 either by SC or IV inoculation against L. amazonensis challenge. Independent of immunization route, susceptible and resistant mice displayed a partial degree of protection against L. amazonensis challenge infection. Subcutaneously immunized BA-LB/c mice presented lesions from 40 to 65% smaller than non immunized mice. IV immunization lead to lesions 12 to 75% smaller than controls. The resistant C57BU6 mice displayed comparable degrees of protection, 57% or 49% by SC or IV routes, respectively. The degree of protection conferred by SC immunization is promising in a notorious difficult route for leishmania immunization.

CBA MICE DEVELOP REISTANT OR SUSCEPTIBLE PATTERNS OF TISSULAR AND IMMUNE RESPONSES AFTER INFECTIONS WITH DIFFERENT SPECIES OF LEISHMANIA

Souza, V.L.; Souza, J.ª; correia, T.; Moreno, M.L.V.; Veras, P.S.T.; Freitas, L.A.R.

Laboratório de Patologia e Biologia Celular, Centro de Pesquisas Gonçalo Moniz (FIOCRUZ) R.Valdemar Falcão 121, Brotas 40295-001.

Most of the knowledge on the immune-inflammatory response in experimental murine leishmaniasis comes from studies performed using of strains of mice with different genetic background.. Some strains of mice however are resistant to one prototype of Leishmania and susceptible to another. Using these strains may provide a better understanding for the role of different species of Leishmania in the outcome of the infection and help in further design of vaccines

ln this work, we show that CBA mice, resistant to infection with L. major (Lm), are unable to control infection with L. amazonensis (La). When infected with La, CBA mice develop a mixed- mononuclear cells infiltrate with scattered parasitized macrophages. The number of lymphocytes increased progressively in the footpad, and fibrosis, fibrinoid necrosis foci and granulomas were observed. In mice infected with La there was a monotonous, "virchowian" infiltrate of heavily parasitized macrophages. Necrosis, micro-abscess and skin ulceration were seen later in the course of infection. The profile of cytokine production was distinct in both groups. At 2 weeks of infection, La infected mice produced 3OO-fold less EFN-,Y than Lm infected mice. At 10 weeks of infection, the amount of IFN-,Y produced by La infected mice was 13-fold less than Lm infected mice. IL-4 was produced in small amount in both group of mice at the 2nd week of infection. On the 4th and I Oth weeks however the levels of IL-4 was more then 50 times higher in the group infected with La than in the group infected with L.m.

The pattems of tissular reaction and cytokine production presented herein, strongly suggest that, at least in the CBA mice, factors related with Leishmania species drive immune response to a predominately Th I or Th2 pattem.

CNPq-522305/96.2 & Fiocruz (PAPES 97)

INVOLVEMENT OF GLUTATHIONE AND THE RESISTANCE OF LEISHMANIA AMAZONENSIS TO THE CYTOTOXIC EFFECT OF NITRIC OXIDE.

Morais, R.H.²; Romão, P.R.T.¹; Fonseca, S.G.¹; Lima, H.C.³; Hothersall, J.S.⁴; Noronha-Dutra, A.⁴. & Cunha, F. Q².

Dept. of Immunology¹ and Pharmacology², Faculty of Medicine Ribeirão Preto, USP-SP. Dept of Microbiology and Parasitology³, University of Santa Catarina, Florianópolis-SC; Institute of Urology and Nefrology⁴, University Colllege London-UK.

Leishmaniasis ranges from self-healing cutaneous and uncontrolled destructive muco-cutaneous froms, to a fatal systemic visceral diseases. In leishmaniasis it is well documented that the macrophage activation and the induction of nitric oxide (NO) determine the outcome of the disease. We have recently reported that glutathione (GSH), which in tripanosomatids is found as a glutathione-spermidine conjugate - tripanothione, is involved in the protection of mammalian cells and protozoan parasites against cytotoxic effect of NO (Mem.Inst.Osw.Cruz vol.91 Suppl. 1996). In present work, we extended these observations by comparing the GSH levels and NO sensibility of different Leishmania species (amazonensis, braziliensis and major). Total glutathione was measured on promastigotes' lysates of Leishmania spp using the Tietze method. (Anal. Biochem., 27:502-522, 1969). The direct leishmanicidal effect of SNAP (a NO source) was evaluated through ³[H]thymidine incorporation by alive parasites. The GSH concentration expressed as mM GSH/5 x 10⁶ parasites were 31.8 ± 1.7, 58.9 ± 3.9 and 85.8 ± 0.9 in L. braziliensis, L. major and L. amazonensis respectively. The survival percentage of these species above treated with 100mM of SNAP were 4, 48 and 68% and at 30mM were 68, 86 and 85% respectively. These results suggest that the sensibility of distinct species of Leishmania to SNAP was inversely correlated with their GSH concentration. L. amazonensis, which is the most resistant to NO killing effect has higher GSH levels than the other species. We are currently investigating the GSH levels and susceptibility of axenically grown amastigote forms these species to the others NO donors and others microbicidal mediators such as peroxynitrite.

Supported by CNPq, CAPES and FAPESP.

ANALYSIS OF INTRACELLULAR CYTOKINES IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH AMERICAN TEGUMENTARY LEISHMANIASIS (ATL) BY FLOW CYTOMETRY

Santiago, MA, De Luca, PM, Bertho, AL, Azeredo-Coutinho, RBG & Coutinho, SG.

Instituto Oswaldo Cruz, FIOCRUZ, Caixa Postal 926, Rio de Janeiro, 21045-900, RJ, Brasil.

The importance of cellular immune response and T cell cytokine profiles in leishmaniasis is well established. The production of cytokines by activated peripheral blood cells has been extensively studied, although, the methodologies currently used do not permit the analysis of the T cell cytokine profile at a single cell level. In this context, a flow cytometric method was described by Andersson et al. (J.Immunol.Methods 112: 139-142, 1988) for detection of cytokines at a single cell level. In this work we show our preliminary results of a flow cytometric method to detect intracellular IL-2, IL-4 and IFN-g produced by T cells derived from the blood of ATL patients upon in vitro stimulation with Leishmania braziliensis antigens (LbAg).

The peripheral blood mononuclear cells were culture in the absence or in the presence of LbAg and harvested, at different time points, for intracellular staining using a protocol which consisted of cell fixation with paraformaldehyde, permeabilization with saponin and staining with monoclonal antibodies anti-IL-2, anti-IL-4 and anti-IFNg conjugated with FITC. The cells were analyzed by flow cytometry and the percentage of positive cells were obtained using the Immuno-4 histogram analysis (Coulter).

The kinetic analysis of intracellular IL-2, IL-4 and IFN-g production suggests that the best time point to detect these cytokines is 16 hours after cell stimulation with LbAg in culture. In order to assure that these cytokines producing cells were specific to L.braziliensis, we have performed cultures of unstimulated cells, and these results were subtracted from those of the stimulated cultures using the Immuno-4 analysis.

The method described above is a rapid, easy and semiquantitative assay and permits the simultaneous determination of cytokine production and cell phenotype.

Financial support: EEC

COMPARISON OF THE T-CELL MEDIATED IMMUNE RESPONSES FROM CUTANEOUS AND MUCOCUTANEOUS LEISHMANIASIS PATIENTS: A SIX MONTH FOLLOW-UP.

Da-Cruz, A.M.¹; Paes-Oliveira, M.²; Santiago, M.A.¹; Bertho, A.L.¹; Silva-Lima, V.P.¹; Dantas-Souza, T.¹ & Coutinho, S.G.¹.

¹Lab. Imunidade Celular e Humoral¹-Dept. Protozoologia; ²Hospital Evandro Chagas, Instituto Oswaldo Cruz, FIOCRUZ, RJ

BACKGROUND AND GOALS: The cell mediated immune responses play a pivotal role in the mechanisms leading to a mild or severe disease in leishmaniasis. The phenotypes and IFN-g production of L. braziliensis reactive T-cells derived from cutaneous leishmaniasis (CL) or mucocutaneous leishmaniasis (MCL) patients were then studied.

METHODS: Patients suffering from CL or MCL were studied before therapy (BT), at the end of therapy (ET) and 6 month after the end of therapy (6M-ET). Assays of lymphocyte proliferative response of PBMC induced in vitro by L. braziliensis promastigotes antigens (Lb) were performed. Five day cultures Lb-reactive blast-cells were also separated in a Percoll (Sigma) gradient for CD4⁺ and CD8⁺ phenotypic analysis by flow cytometry. The supernatants were harvested for cytokine quantification (IFN-g) by using ELISA kits (RD Systems).

RESULTS: In CL patients the mean percentages of Lb-reactive CD8⁺ blast T-cells were BT=23%±13%, increased to ET = 45%±21% (P=0.007) and then 6M-ET=35%±13% (P=0.14). The mean percentages of Lb-reactive CD4⁺ blast T-cells decreased, although not quite significantly, during the study: 60%±22% (BT), 43%±18% (ET) and 35%±26% (6M-ET). In MCL patients a pattern of higher mean percentages of Lb-reactive CD4⁺ blast T-cells (BT=53%±19%) than CD8⁺ blast T-cells (BT=20%±9%) was maintained during the whole period of study. The mean levels of IFN-g in the cell culture supernatants were higher in MCL patients (3929 pg/ml±742pg/ml) than in LCL (2075 pg/ml±1120pg/ml) patients during the active disease. A similar pattern was maintained ET and 6M-ET.

CONCLUSIONS: In CL patients the healing process seems to be associated with increased proportions of Lb-reactive CD8⁺ blast T-cells and decreased proportions of Lb-reactive CD4⁺ blast T-cells. This CD4⁺-CD8⁺ switch was not observed in MCL patients that displayed a pattern of higher proportions of CD4⁺ than CD8⁺ T-cells during the whole period (BT, ET and 6M-ET). The levels of IFN-g were higher in MCL patients than in CL patients. These results could be associated with the severity of the MCL disease, and a tendence to a mild CL disease.

Supported by CNPq and Economic European Community (EEC).

DOES THE FAS-FASL PATHWAY PLAY A ROLE IN THE HEALING OF LEISHMANIASIS?

Fatima Conceição-Silva, Michael Hanne, Jacques Louis & Jürg Tschopp

Oswaldo Cruz Institute, Rio de Janeiro Brazil; WHO-IRTC and Institute of Biochemistry, University of Lausanne, Switzerland

In the last few years, several studies have pointed out the Fas/Apo-1 cytotoxic pathway as playing an important role in the regulation of peripheral immunity. Recent publications suggest that this regulatory function operates through deletion of activated B and T cells, and probably also macrophages, by CD4+ T cells expressing FasL. In the leishmaniasis, the predominance of either Th1 and Th2 subsets of CD4+ T cells can profoundly alter the evolution of the disease, at least in the murine model.

In order to verify the possible role of the Fas-FasL pathway in the resolution of murine leishmaniasis, we compared the L.major infection between C57BL/6 mice and mice from the same background but presenting a mutation in the FasL (B6-GLD) or Fas (B6-LPR) genes. In contrast to the wild type, both B6-GLD and B6-LPR mice develop a chronic and non-healing lesion at the site of subcutaneous infection. Studies of T cell function performed by in vitro culture in the presence of L.major antigens or ConA showed a similar Th1-type response in all three groups. A T-cell specific proliferation was followed by a consistent secretion of IFN-g (up to 1000 U/ml) and IL-2 (up to a 4 U/ml) and almost no IL-4. The macrophage function, verified by the capacity to secrete NO and the upregulation of the expression of class II molecules was also similar in the three groups studied. The capacity of T cells and macrophages to undergo apoptosis in vitro in the B6-GLD was equivalent to that observed in the wild type only after restoring the Fas-FasL pathway Finally, the in vivo treatment of B6-GLD with a FasL was able to decrease both lesion size and parasite burden.

It is already known that IFN-g and TNF-a increase Fas and FasL expression on the surface of activated immune cells. The observation that B6-GLD and B6-LPR were not able to control and heal their lesions, despite the presence of a Th1-type response indicate that the Fas-FasL pathway might be crucial in the process of healing of cutaneous lesions produced by L.major infection. One may hypotesize that the inability to limit the immune response observed in GLD or LPR mice mice would result in the failure to eliminate lymphocytes and macrophages that are no longer needed. The consequence would be an uncontrolled inflammatory reaction at the site of the lesions.

Supported by the National Suisse Fund.

INHIBITION OF THE PRODUCTION OF NITRIC OXIDE IMPAIRS CYTOTOXICITY OF MACROPHAGES TO LEISHMANIA AMAZONENSIS.

Jardim, IS*, Santos, JL*, Horta, MFM* & Ramalho-Pinto, FJ^§.

*Departamento de Bioquímica-Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, Belo Horizonte, 30161-970, MG and ^§Departamento de Parasitologia, Microbiologia e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, 14040-900, SP, Brazil.

The production of nitric oxide (NO), important for the cytotoxicity of macrophages, is controlled by the inducible nitric oxide sinthase (iNOS), which can be activated by LPS and some cytokines such as interferon-gama (IFN-g). Although NO has been shown to play a role in controlling the infection by L. major, no information is available for L. amazonensis. In this work, we show that inhibition of NO decreases the cytotoxicity of macrophages infected with L. amazonensis. To inhibit the production of NO by macrophages we have used a proteasome inhibitor, N-benzyloxycarbonyl-Ile-Glu-(o-t-butyl)-Ala-Leucinal. By activating the transcription factor NF-kB, proteasomes, multicatalytic protein complexes, activate the expression of iNOS and consequently the production of NO. We have also used L-N^G-arginine methyl ester (L-NAME), a competitive inhibitor of the NOS. Peritoneal macrophages from BALB/c and C57BL/6 mice, susceptible and resistant to L. amazonensis, respectively, were plated in chamber slides overnight. Macrophages were then treated or not with different concentrations of the inhibitors and with 0.1 ng/ml LPS plus 60 U/ml IFN-g. The cells were infected with promastigotes of L. amazonensis and maintained under stimulation with LPS and IFN-g. After 72 hours, the amount of NO was determined in the supernatants of the cultures using the Griess reaction. The slides were stained with May-Grünwald-Giemsa and the number of parasites inside the macrophages was determined. We have shown that, in the absence of the inhibitors, the production of NO by macrophages as well as their ability to eliminate the parasites is very low in the susceptible BALB/c mice. On the other hand, macrophages from resistant C57BL/6 mice produce high levels of NO and eliminate L. amazonensis amastigotes more efficiently than susceptible mice. Both inhibitors cause a dose-dependent decrease in the production of NO by C57BL/6 mice macrophages and in their cytotoxicity to the parasites. These results show a correlation between the production of NO and the citotoxicity of macrophages, indicating that NO is involved in the elimination of L. amazonensis amastigotes.

AN EXAMINATION OF THE DIRECT BINDING OF LEISHMANIA MAJOR TO MURINE MACROPHAGES

Tafuri, Wg L¹, Brittingham, A², Carroll, Mc³, Mosser, D.²

¹Dept of General Pathology, ICB, Federal University of Minas Gerais; Belo Horizonte, 31270-901, MG, Brazil; ²Dept of Microbiology and Immunology, School of Medicine, Temple University, Philadelphia, 19140, Pa, USA. ³Dept of Pathology, Harvard Medical School, Boston, 02115, Ma, USA.

In this study we have reexamined the interaction of Leishmania promastigotes with murine macrophages, as well as the role of local opsonization in the binding of promastigotes to their target cells. Mac-1 (CR3) is considered to be crucial for the interaction of serum opsonized promastigotes with human and murine macrophages. Several groups have reported a direct interaction (without opsonic serum) of Leishmania spp promastigotes as well as zymosan particles to macrophage Mac-1 ("local opsonization"). Using macrophages derived from mice rendered deficient in the complement protein C3 via targeted gene disruption (C3-/-) we have demonstrated a role for Mac-1 in the binding of promastigotes to macrophages in the absence of exogenous or macrophage secreted complement components. Increasing concentrations of radiolabeled Leishmania major promastigotes (MHOM/IL/80/ Friedlin) were added to monolayers of murine bone marrow-derived macrophages from control (C57BL/6) or C3 -/- animals, in either the presence or absence of 5% C5 deficient mouse serum (C5D) for 1 hour at 35 ºC. After a 1h- incubation, plastic plates (24 wells) were washed extensively to remove unbound parasites and the amount of radiolabel associated with cell lysates was determined. In parallel, bodipy labeled zymosan particles were added to macrophages monolayers at a 100/1 ratio of particles/cell. The number of particles per 100 macrophages were visualized and quantified by immunofluorescence microscope . To inhibit promastigotes or zymosan binding to Mac-1, macrophages monolayers were preincubated with mAbs (M1/70) at a final concentration 10 µg/ml for 15 minutes at 35ºC. The in vitro binding of these two particles to macrophages isolated from either C3-/- or C57BL/6 was compared. Macrophages from C3-/- mice bound Leishmania or zymosan as well as did macrophages from C57BL/6 mice. As expected, particles bound better to macrophages when the assays were performed in the presence of serum, and on both macrophages populations this binding was inhibited by mAbs to Mac-1. In the absence of serum, both populations of macrophages exhibited a comparable reduction in particle binding, relative to the observed in the presence of serum, and a significant portion of this binding was also inhibited by the addition of mAb to Mac-1. This inhibition of binding by mAb to Mac-1 was not dependent on the ability of macrophages to secret C3. These results indicate that the local opsonization by macrohages-derived C3 plays only minor role, if any, in the binding of L.major or zymosan to macrophages.

EXPERIMENTAL MURINE VISCERAL LEISHMANIASIS: MARKED EXPANSION AND ACTIVATION IN VIVO, BUT FUNCTIONAL ANERGY OF SPLENIC CD4⁺ T CELLS IN VITRO

Gomes, N.A., Barreto de Souza, V. & Dos Reis, G.A.

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21944-970, RJ, Brasil

Cellular immune responses play a central role in visceral forms of Leishmaniasis. Leishmania chagasi (infantum)-induced infection is characterized by heavy visceral parasitism, hepatosplenomegaly and polyclonal B- and T- cell activation. We investigated the functional status of T-accessory cell interactions after a prolonged (2 months) infection with Leishmania chagasi using a murine model. BALB/c mice were inoculated i.v. with 2x10⁷ amastigote forms. Flow cytometry analysis of splenic cells from infection, demonstrated an increase in both absolute cellularity and relative percentage of CD4⁺ T cells (32%), compared to controls (13%). We also observed an expansion of in vivo activated CD4⁺ T cells (17% CD25⁺), compared to controls (2% CD25⁺). Moreover, the number of CD4⁺ T cells expressing a memory/activated cell phenotype (CD45RB low) doubled in infected mice. Despite absolute and relative expansion, and expression of activation markers, CD4⁺ T cells showed decreased mitogenic responses to anti-CD3 antibody in vitro. This decrease in the mitogenic response was not due to activation-induced cell death, since viability was comparable to controls after 20h in culture. In cultures containing accessory cells, activation of control CD4⁺ T cells increased cell viability, and blockage of B7.1 molecule did not affect the increase. However, activation of CD4⁺ T cells from infected mice, resulted in only marginal increase in viability. Blockage of B7.1 markedly increased viability upon activation. The results suggest that infection with L.chagasi drives host CD4⁺ T cells to a refractory state regulated by the B7.1 pathway.

Supported by CNPq, FINEP, PADCT-CNPq, RHAE-CNPq, PRONEX-MCT

PRIMING IN VITRO STIMULATION OF CBA MICE SPLENIC CELLS WITH LEISHMANIA AMAZONENSIS OR LEISHMANIA MAJOR

Santana, CD de; Gomes, IN; Freitas, LAR de & Veras, PST

Laboratório de Patologia e Biologia Celular - Centro de Pesquisa Gonçalo Moniz (FIOCRUZ)

CBA n-iice develop a susceptible pattem of immune inflammatory response when infected with L. amazonensis or a resistant profile when infected with L. major. This finding suggests that, at least in CBA niice, factors of the parasite can modulate the immune response leading to a Thl or Th2 predorrúnance. Several studies have demonstrated that the course of murine cutaneous leishmaniasis is detem-iined by events in the early phase of infection. In order to identify factors potentially involved in the differential immune response of CBA to both species of leishmania, we used PIV stimulation of CBA splenic cells with L. major or L. amazonensis. The levels of IFN-@Y and H,-4 production by CBA spleen cells primed in vitro with L. major or L. amazonensis, were measured using an ELISA technique. Supematants were assayed for the presence of these cytokines from the first to the seventh day after priming.

The levels of IFN-m production by splenic cells primed with L. amazonensis were 9 times higher than those produced by cells stimulated with L. major. On the other hand, the levels of IL4 were 3 times higher when the cells were primed with L. major than with L. amazonensis. The data presented herein is in apparent contrast what is reported in litterature which shows that IFNm is associated with resistance and IL-4 with higher susceptibility to leishmania infection. Our results, however, may represent cytokine profile in the early phase of the disease. The role of other cytokines responsible for the subsequent definition of a Thl or Th2 profile of immune response are under investigation in our laboratory.

Financing: CNPQ - 522305/96-2, PAPES (FIOCRUZ).

PRODUCTION OF NITRIC OXIDE (NO) BY HUMAN MONOCYTE-DERIVED MACROPHAGES STIMULATED WITH L. MEXICANA GP63 AND FIXED PROMASTIGOTES OF L. MEXICANA.

Alfonzo, M. ⁽¹⁾ , Santaella C. ⁽²⁾ and Cabrera, M. ⁽²⁾ . (1) Cátedra de Fisiología, Escuela J.M. Vargas, UCV; ⁽²⁾ Instituto de Biomedicina, U.C.V., Apartado 4043, Caracas 1010A , Venezuela.

The resistance to leishmanial infections depends on the killing of Leishmania parasite by infected macrophages. Several studies in mice have shown that the production of NO is important for the killing of these parasites by macrophages. In contrast, the role of NO in the anti-leishmanial activity of human macrophages is controversial. The major Leishmania glycoprotein gp63 is one of the most important molecules which has been implicated in the process of invasion of host macrophages and in development of an immune response. In addition, gp63 may be responsible for immunomodulation during the early non-specific response, since it is able to inhibit the respiratory burst of macrophages. This study is an evaluation of the effect of fixed Leishmania mexicana and gp63 on NO production by human monocytes/ macrophages . The study was carried out on a total of 10 healthy volunteers from the DF blood bank (Caracas-Vzla). Mononuclear cells (MNC) were isolated from heparinized peripheral blood from controls by centrifugation over histopaque and the monocytes were obtained after plastic adherence of MNC. Monocyte and monocyte-derived macrophages at day 3, 5 and 7 of differentiation were cultured with: fixed L. mexicana, native gp63, LPS, IFN-g, LPS+IFN-g or medium. Supernatants were collected after 2, 4, 24 and 48 hrs of stimulation and NO-₂ concentration was measured using the Griess reagent. Comparisions between the different culture conditions were made using an unpaired Student´s t test. We found significant differences in monocyte-derived macrophages at day 7 of differentiation. An increase in the NO levels in macrophages stimulated with fixed L.mexicana and gp63 was detected, compared with unstimulated cultures (P<0.05). In contrast, in the macrophages stimulated with LPS, IFNg or LPS plus IFNg no differences were observed in the NO levels. Overall results suggest that the microbicidal mechanism mediated by NO may be important in the killing of Leishmania parasites by human macrophages as has been demonstrated in mice. The leishmanial gp63 could trigger the production of NO.

Supported by CONICIT project BTS59, MSAS-World Bank , project No 55.

IS THE GLUTATHIONE AN IMPORTANT VIRULENT FACTOR IN LEISHMANIASIS ?

Romão, P.R.T¹; Antoniazi, S.A²; Lima, H.C³; Cruz, A. K² & Cunha F.Q¹.

Departamento de Farmacologia¹ e Departamento de Bioquímica² da Faculdade de Medicina de Ribeirão Preto - USP - Ribeirão Preto - SP, Brazil. Departamento de Microbiologia e Parasitologia³ da Universidade Federal de Santa Catarina-SC

We previously reported that glutathione (GSH), which in tripanosomatids is conjugated with the polyamine spermidine to originate trypanothione, is an essential component of protective mechanism against the leishmanicidal effect of Nitric Oxide (NO). The possible involvement of GSH as an important virulence factor was not investigated yet. We examined this issue using different species of Leishmania (amazonensis, braziliensis, donovani) and different clones of L. major (CC1, LV39, D2 and D6) showing distinct degrees of virulence in vivo. These clones are transfectants from an avirulent line of L. major (MHMO/IR/83/LT 252) that contain approximately 100 repeats of the miniexon gene. The cytosolic glutathione concentration was analyzed on promastigotes' lysates of Leishmania spp. The promastigotes sensibility to NO source (SNAP) was evaluated by thymidine incorporation. Here we show that GSH levels on parasites is inversely correlated with its sensibility to SNAP. The order of sensibility to NO killing was L. braziliensis > L. major > L. amazonensis. Nevertheless, when we compared the GSH levels and NO sensibility of CC1, LV39, D2 and D6 clones of L. major, we did not find significant differences among the clones. Furthermore, the NO production and leishmanicidal activity of BALB/c macrophages infected with these clones and stimulated with IFN-g (1, 10 or 100 U/ml)/LPS (10 ng/ml) were the same, regardless the infected clone. This results indicate that GSH is not involved in the virulence levels observed on the L. major clones. Further analyses are necessary to understand such findings.

Supported by CNPq, CAPES and FAPESP.

EVALUATION OF TISSULAR RESPONSE IN THE FIRST HOURS OF THE INFECTION OF CBA WITH L. AMAZONENSIS

Moreno MLV; Correia, T; Freitas, LAR

Laboratório de Patologia e Biologia Celular, Centro de Pesquisa Gonçalo Moniz (FIOCRUZ) ,R.Valdemar Falcão l2l,Brotas, Salvador-Bahia 40295-001

The initial phases of infection with Leishmaniaare decisive in the outcome of the disease. lmmune response may be modulated within the first 48-72 hours after infection. Morphological studies have demonstrated that there is correlation between the tissular and inunune pattems of response related to resistance or susceptibility. At present, there are few publications describing morphological studies of the initial phase of Leishmania infection. Therefore, we decided to investigate the tissular response of CBA mice infected with L.amazonensis (La), at both the site of inoculation and in the draining lymph node, within 48 hours after infection. lnbred CBA mice were inoculated with La in the rear footpad and sacrified I, 3, 6, 12, 18, 24 and 48 hours after infection. Infected footpad and draining lymph node were subn-útted for optical and electronic n-úcroscopic studies. Footpads, draining lymph nodes, fragments of spleen, liver and lung were macerated and cultured, and immunohistological techniques employing antlLeishmania antibody were used to detect parasites in tissue sections.

In the first hour after infection, tissular response was characterized by acute inflammation, with edema and vascular congestion. Infiltration of neutrophils, eosinophils, mast cells, and several macrophages was also observed. Parasites, both intact and phagocytized by neutrophils and macrophages, were observed in the free interstitial space. After that time, infiammatory infiltrate became increasingly composed of parasitized macrophages, with fewer granulocytes observed. Changes in the draining lymph node followed sin-iilar kinetics: subcapsular sinuses were infiltrated by granulocytes and macrophages in the first hours. One hour after infection, intact and fragmentei parasites were observed inside phagocytic cells (mainly neutrophils) in the subcapsular sinus of draining lymph nodes. This finding was confirmei by immunohistochen-ústry and electron microcopic observation. Culture demonstrated that parasites were viable in the draining lymph node of all mice beginning one hour after infection. Our results suggest that parasite antigens come in contact with the immune system very early in the course of leishmania infection. Furthermore, these antigens appear to initiate an inflammatory response which bring antigen presenting cells (APCS) to the lymph node. These observations lnitiate new perspectives for investigating the biological significance of this early phenomenon.

Financiamento:CNPQ -522395/96-2, FIOCRUZ: PAPES 97

THE ROLE OF THE MICROBIOTA ON THE OUTCOME OF LEISHMANIA MAJOR INFECTION IN SWISS OUTBRED MICE

Oliveira, M.R., Tafuri, W.L., Afonso, L.C.C., Nicoli, J.R., Vieira, E.C., Melo, M.N. & Vieira, L.Q.

Depts.of Biochemistry and Immunology, Parasitology and Microbiology, ICB, UFMG, Belo Horizonte; Dept. Biochemistry, ICEB, UFOP, Ouro Preto, MG, Brazil.

The presence of microbiota can play an important role in the immune response of a host during parasite infections. In order to investigate the importance of the microbiota on the development of murine leishmaniasis, we compared the course of infection and some immune parameters in outbred swiss mice in the presence and absence of their microbiota. After 13 weeks of infection with Leishmania major, all of the conventional animals had resolved their lesions, demonstrating resistance to the infection. However, during the same period of time germfree mice were unable to resolve their lesions, exhibiting a large individual variation in the lesion sizes. This observation suggests that the microbiota is modulating the response to infection by L. major, rendering the germfree mice more susceptible than their corresponding conventional counterparts. In the conventional murine model, resistance to L. major is associated with the development of Th1 CD4+ lymphocytes, which produce high levels of IFN-g. Susceptibility to the infection, on the other hand, is associated with Th2 lymphocytes, which produce IL-4. The initial production of IL-12 by macrophages induces NK cells to produce INF-g, a fundamental signal to initiate the development of Th1 cells. Our results show that the initial producton of IL-12 and IFN-g was similar between the conventional and germfree mice, suggesting that the absence of microbiota does not influence the potential of these animals to develop a Th1 phenotype when infected by L. major. In accordance with these results, we subsequentely verified that the germfree mice produce elevated levels of IFN-g throughout the course of infection (after 2, 6 e 13 weeks of infection), comparable to the levels observed in the conventional animals. This observation demonstraties that both groups develop a Th1-type response when faced wich this infection. In the conventional murine model, IFN-g and TNF-a activate macrophages to produce NO which is responsible for the death of intracellular forms of the parasite. We observed that the production of NO by intraperitoneal macrophages infected "in vitro" by L.major amastigotes was similar for conventional and germfree mice. The production of TNF-a is being analyzed, and the results will hopefully help the understanding of the reasons for the greater susceptibility to L. major observed in the germfree mice.

IMMUNOLOGICAL EVALUATION OF PATIENTS WITH CUTANEOUS LEISHMANIASIS SUBMITTED TO IMMUNOCHEMOTHERAPY

Toledo, V.P.C.P.¹, Pinto, J.A.², Costa, C.A.³, Genaro, O^.4, Mayrink, W.⁴ & Afonso, L.C.C.⁵

¹Depto. Análises Clínicas, Faculdade de Farmácia, UFMG, ²Depto. de Pediatria, Escola de Medicina, UFMG, ³Biobrás S.A., ⁴Depto. de Parasitologia, ICB, UFMG, ⁵Depto. Ciências Biológicas / NUPEB, ICEB, Morro do Cruzeiro, 35400 - Ouro Preto, MG.

In this study we evaluated some aspects of the immune response of patients with Cutaneous Leishmaniasis (CL) from the Rio Doce Valley, Caratinga, MG submitted to different treatment protocols. Patients were either treated with regular chemotherapy with Glucantime® or with an association of the antimonial with subcutaneous injections of one of two different vaccine preparations. One vaccine formulation was made using 5 different Leishmania strains (Polyvalent) (Mayrink, W. et al.,1979,Trans.R.Soc.Trop.Med.Hyg. 73: 385-387) while the other was prepared using only the PH8 strain of L. amazonensis (Monovalent). Inasmuch the different treatments did not differ in their efficacy, an interesting observation could be made regarding the levels of IFN-g produced by stimulated PBMC from patients before and after treatment. Patients that received the association of Glucantime® + Monovalent showed a marked decrease in IFN-g production after treatment when compared to their production before treatment. IFN-g production from patients from the other two groups did not change during treatment, although a tendency of decreased cytokine production after treatment was observed in cells from patients receiving the Glucantime® + Polyvalent association . As to the levels of other cytokines, we were not able to detect significative IL-12 (24 hr stimulation) or IL-4 (72 hr stimulation) production in stimulated PBMCs from patients from any of the groups at any time point before or during treatment. Also, no differences were detected amongst the three groups regarding lymphocyte proliferation stimulated by Leishmania antigens. These results may suggest a different mechanism for the cure induced by the immunochemotherapeutic approach.

Financial Support: Biobrás S.A., CNPq and WHO TDR proj id 940876

AMERICAN TEGUMENTARY LEISHMANIASIS: ASSOCIATION WITH HIV INFECTION

Mattos M, Silva-Gonçalves AJ, Andrade TCB, Lima RB, Pirmez C & Oliveira Neto MP

Hospital Evandro Chagas & Dept. Biochemistry & Molecular Biology

Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil.

AIDS epidemics are in expansion in developing countries. Initially restricted to urbanized areas of major cities, the epidemics began to reach periurban and rural locations where American tegumentary leishmaniasis (ATL) and other tropical diseases occur. To date, 23 cases of ATL/AIDS association were mentioned in the literature, 19 of these in Brazil.

We report here four new cases of such an association. All cases had positive ELISA and immunofluorescent tests for HIV. Three cases were Group IV (cases 1, 2 and 4) and one case was Group II (case 3) according to the CDC classification. Clinically, all cases presented multiple skin lesions (more than ten) and, in two of them (cases 2 and 4), nasal and/or oral destruction was also observed. Parasites were demonstrated in smears, histopathological sections and/or culture in all cases. L. (V.) braziliensis was identified in case 1 and in all other cases the PCR product obtained from lesions hybridized with a Viannia probe. Although treatment with Glucantime was efficient in healing, all of them relapsed a few months after. Peripheral blood CD4/CD8 ratio was low in all cases, CD4⁺ levels being under 500/mm³. Immunohistochemical analysis of the lesions showed that in all cases CD8⁺ T cells (45 to 60%) predominate over CD4⁺ T cells (20% of total inflammatory cells). A positive response of peripheral blood mononuclear cells against Leishmania antigens or concanavalin A was detected in three cases. One patient, who presented the lowest level of CD4⁺ cells (23/mm³), did not respond in vitro or to the Montenegro skin test. He died soon after, and necropsy show no evidence of visceralization of Leishmania.

The unusual clinical presentation of multiple lesions, often associated with mucosal destruction, combined with resistance to antimonial therapy, suggest that an investigation of HIV infection should be carried out in such cases. In addition, the frequent relapses indicates that the parasite persists within the host and that T-cells, particularly CD4⁺, have an important role in resolution of the infection.

Supported by FAPERJ.

LEISHMANIASIS RECIDIVA CUTIS IN THE NEW WORLD

Oliveira Neto, MP, Mattos, M, Da Costa LMC, Este MGM, Fernandes, O & Pirmez, C.

Hospital Evandro Chagas, Dept. Tropical Medicine and Dept. Biochemistry & Molecular Biology

Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil.

Leishmaniasis recidiva cutis is well recognized in Old World cutaneous leishmaniasis and was first described by Berlin in cases of Oriental sore as small papules located at the borders of leishmanial scars. The lesions bear a striking clinical resemblance to lupus vulgaris and, characteristically, few or no parasites are detectable. Subsequent workers tried to assimilate these lesions with the lupoid variant of cutaneous leishmaniasis.

Concerning leishmaniasis of the New World, only seven cases have been described so far. We present here four new cases that after healing the primary lesions, showed small papules located at the periphery of the old scar after a variable period of time. Parasitological diagnosis of initial lesions was positive in all cases either by inprints, histopathological sections and/or cultures. Parasites were identified as L. (V.) braziliensis. Although these standard methods were consistently negative in recidivant lesions, the PCR reaction was able to demonstrate parasite DNA in all of them.

Since parasite DNA has been demonstrated in clinically healed Leishmania lesions, our conclusion is that these recidivant cases most likely represent a reactivation of persistent parasites in scarred tissue. However, the designation of leishmaniasis recidiva cutis should be maintained in order to designate this particular clinical presentation of recurrence occurring at the border of an old scar of cutaneous leishmaniasis and thus avoiding the confusion with the lupoid form of the disease.

Supported by FAPERJ

HEMATOGENIC DISSEMINATION OF LEISHMANIA (VIANNIA) BRAZILIENSIS

Camera PO, Oliveira Neto MP, Junger J, Trajano VS, Degrave W, Fernandes, O & Pirmez C.

Hospital Evandro Chagas, Dept. Tropical Medicine and Dept. Biochemistry & Molecular Biology

Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil.

American tegumentary leishmaniasis (ATL) caused by L. (V.) braziliensis has the possible hazard of late development of mucosal lesions. Hematogenic dissemination of the parasite is the most likely mechanism to explain this occurrence. The presence of L. (V.) braziliensis in blood samples has been occasionaly demonstrated by haemoculture (Trans R Soc Trop Med 86:392, 1992). In order to better evaluate the presence of Leishmania in blood, we performed the PCR reaction on DNA isolated from blood of patients with localized cutaneous leishmaniasis (LCL) or patients with mucosal disease (MCL), both clinical forms in activity. In addition, DNA from 24 individuals cured by antimonial treatment and three individuals without past history of leishmaniasis but with a positive Montenegro skin test, was also analysed. All individuals live in endemic areas of Rio de Janeiro, Brazil, where only L. (V.) braziliensis has been described. Whole blood was taken by venopuncuture using vacutainer tubes containing EDTA. DNA was extracted from 500 ml of whole blood by column cromatography (Genomic DNA isolation kit, Pharmacia) following the manufacture's instructions. The hot start PCR was performed with a pair of oligonucleotides that amplify the conserved region of the minicircle molecule of Leishmania. Reaction was positive in 9/43 LCL (21%), 4/12 MCL (33%), 5/24 clinically cured individuals (21%) and 1/3 positive skin test individuals. Hybridization of all products with a PCR amplified conserved region of L. (V.) braziliensis (MHOM/BR/75/2903) as a molecular probe showed that only the products visualized by agarose gel electrophoresis were positive, thus identifying the samples as belonging to the Viannia subgenus. These results confirm data observed in Venezuela (Lancet 341:1341, 1993; Clin Diag Lab Immunol 1:385, 1994) and suggest that persistence of parasites within the host may be lifelong and important to maintain the protective response.

Supported by FAPERJ

COMPARATIVE STUDY OF TWO ANTIMONIAL THERAPY SCHEDULES FOR TREATING CUTANEOUS LEISHMANIASIS

Azeredo Coutinho, RB & Mendonça, SCF.

Instituto Oswaldo Cruz, Fiocruz, Caixa Postal 926, Rio de Janeiro, 21045-900, RJ, Brasil.

The Brazilian Ministry of Health´s latest recommendation for the antimonial therapy of cutaneous leishmaniasis (CL) is: 10 to 20mg of Sb/[Kg.day] for 20 days, repeating this regimen if complete healing of lesions is not obtained within two weeks after its end. Before the publication of this recommendation, the most widely used antimonial regimen for treating cutaneous leishmaniasis in Brazil consisted of similar daily doses given in cycles with rest intervals between. The objective of this study was to compare these two therapeutic regimens with regard to adherence to therapy, proportion of therapeutic failure (need of additional treatment), side effects and mean time period for healing of lesions. Patients from an area where CL is due exclusively to Leishmania braziliensis were allocated into two groups with similar composition with regard to age, sex, number of lesions and period of disease progression before the onset of treatment. A group of 59 patients received the continuous schedule of 20 days and the other group of 33 patients received the intermittent treatment with three ten day cycles with ten day intervals between. In both groups the daily doses of antimonial (Meglumina antimoniato, provided by the Brazilian Ministery of Health) were given by deep IM injections. All patients were evaluated weekly by the same medical personnel at oupatient units of the Municipality of Rio de Janeiro´s Health Secretariat. In the group receiving the continuous schedule, 18,6% of the patients spontaneously interrupted the therapy, therapeutic failure occurred in 21% and side effects were observed in 28% of the cases. In the group undergoing the intermittent regimen only one patient spontaneously interrupted the treatment (3%), there was 9% of therapeutic failure and side effects were observed in 15,2% of the subjects. There was a statistically significant difference between the groups with regard to adherence to therapy (p<0,05, Fisher´s exact test). The mean time for the complete healing of lesions was similar: 97,4 ± 94,2 days for the continuous and 110,8 ± 61,1 days for the intermittent schedule. Our results show that, in our experience, the adherence to therapy is better with the intermittent schedule than with the continuous schedule of antimonial therapy. Financial support: CNPq.

EXPERIMENTAL INFECTIONS OF LEISHMANIA (LEISHMANIA) AMAZONENSIS IN CEBUS APELLA (PRIMATES: CEBIDAE) : EVALUATION OF HUMORAL RESPONSES BY THE ELISA TECHNIQUE USING HOMOLOGOUS AND HETEROLOGOUS ANTIGENS.

Gomes, P.A.F.¹; Ramos, P.K.¹; Garcez, L.M.¹ ; Silveira, F.T.¹; ; Brigido, M.C.²; Muniz, J.P.C. ² & Shaw, J.J. ^{1 & 2}

¹ Programa de leishmaniose, Instituto Evandro Chagas/FNS, Av. Almirante Barroso, 492, 66090-000 Belém, Brazil - email < [e-mail] >; ² Centro Nacional de Primatas/FNS; ³ Depto de Parasitologia, Instituto de Ciências Biomédicas, USP.

The present study is part of a project aimed at determining the immunogenicity and effectiveness of candidate leishmanial vaccines in a non-human primate model. The principal objectives of the present work were to confirm the infectivity of a single inoculum of 1.6 x 10⁶ Leishmania (Leishmania) amazonensis promastigotes in 7 laboratory bred monkeys (Cebus apella) and to determine their humoral response during different phases of the infection by the enzyme linked immune assay (ELISA) using heterologous and homologous leishmanial antigens. Promastigotes from 12 day old cultures, that represented the 2nd sub-passage from an infected hamster, were washed and suspended in a saline/glucose solution to give a final concentration of 1.6 x 10⁶ organisms per 0.1 ml. Each of the 7 animals was inoculated intradermally in a shaved area of the upper surface of the tail with 0.1 ml this solution. Heparanised blood samples were taken at the time of the inoculation and thereafter at 15 day intervals. The plasma samples thus obtained from each animal were then frozen. ELISA antigens of L. (L.) amazonensis and L. (Viannia) braziliensis were prepared by freezing (-182^OC) and thawing (37^OC) washed cultures 10 times. Optimum antigen concentrations were determined in a block tritration using different antigen and antibody concentrations. A human anti-IgG/peroxidase conjugate was used with an OPD substrate and reactions were read at 492nm. The different titters were transformed into neperian log values and analysed using the analysis of variance. Significant titers were found at 60 days (p > 0.5) and between 82 - 91 days (p > 0.5). The titers for these two periods were used to look for differences between the two antigens. All the animals developed erythematous nodules 15-20 days post inoculation (p.i.) which persisted for approximately 3 months. The mean diameter of the lesions was 8.7 mm with maximum and minimum diameters of 13mm to 3 mm at 2 months p.i. Parasites were seen in the stained smears of 4 animals. The titers during these primary infections varied from 40 to 320, reaching their maximums 2 months p.i., with the homologous antigen. Titers with the L. (V) braziliensis heterologous antigen varied from 40 to 80, peaking 3 months p.i. The differences between the two antigens were significant and indicate that the homologous antigen should be used to follow the humoral response of C. apella to experimental infections of L. (L.) amazonensis. It was also confirmed that an inoculum of 1.6 x 10⁶ promastigotes is infective and provokes an antibody responses that is comparable to that seen in man naturally infected with the same parasite indicating that an inoculum of this concentration is suitable for testing the immune state of monkeys that have been vaccinated with candidate vaccines.

Financial Support: Instituto Evandro Chagas/FNS, Centro Nacional de Primatas/FNS, CNPq., Programa PCMAM/FNS.

Key words: L. (L.) amazonensis; Cebus apella; humoral response

IMMUNOGENIC STUDY OF MICE STRAIN INFECTED WITH LEISHMANIA AMAZONENSIS.

Rosa, M.S.S.; Maia, H.S. & Gonçalves da Costa, S. C. Fundação Oswaldo Cruz, Dept°. Protozoologia, Lab. de Imunomodulação - Av. Brasil, 4365, Manguinhos. CEP.21045-900, Rio de Janeiro-Brasil.

In New World cutaneous leishmaniasis the delayed-hypersensitivity (DTH) level present a strong correlation with the different stages of the spectrum of the disease. Exarcebated specific DTH reaction occur chiefly in muco-cutaneous form of the disease in which antibody levels is lower than those observed in patients with localized lesions. This is correlated with the results reported in experiments employing artificial immunizations using sheep red blood cell (Lagrange & Mackaness, 1975; J. Exp. Med. 141: 82-96). Diffuse cutaneous leishmaniasis on the other polar position of the spectrum, shows a limited expression of cell-mediated immunity (CMI) including protection and DTH to specific antigen. The present data deal with the induction of DTH to leishmania antigens in different inbred strains of mice , which can reproduce a similar spectrum of leishmaniasis.

When 1x10⁴ amatigotes were used in C57BL/6 (intermediated), Balb/c (susceptible) and C3H.He/N relatively resistance mice. A significant increase in footpad swelling was elicited in BCG-pretreated mice by Riboleish; in only Balb/c mice.

In a second protocol were investigated the kinetics of DTH reaction to virulent amastigotes cells of L. amazonensis in outbred mice. Comparative kinetics between Riboleish vaccine and Montenegro antigen in footpad skin test were made through out the course of infection. Doses of 4x10⁵ /ml , 4x 10⁶/ml and 4x10⁷/ml of Montenegro were inoculated in groups of OF1 mice on days 20, 30, 60 and 90 days after infection the 1x10⁴ of L.amazonensis amastigotes (H21 strain). Simultaneously, groups of mice were inoculated with 10 mg of Riboleish (microsomal fraction) for comparison.

The present data shows that the microsomal fraction induce stronger DTH when compared with Montenegro reaction.Levels of DTH developed in response to different schedules of Riboleish vaccine, by normal mice, mice sensitized with 10mg of Riboleish and mice inoculated subcutaneously with 10⁶ BCG 21 days before sensitization. Footpad tests for DTH were performed 6 days after sensitization. Means of 6± SEM.

EFFECTS OF SEB ADMINISTRATION ON PROTECTIVE IMMUNITY AGAINST TRYPANOSOMA CRUZI

Ribeiro, LJ¹, Paiva, CN¹, Soares, MBP¹, Gonçalves R¹, Bozza, M² and Gattass, CR¹.

¹ Depto de Imunologia - Instituto de Biofísica - Universidade Federal do Rio de Janeiro

² Instituto Oswaldo Cruz - Fundação Oswaldo Cruz

Chagas' disease presents an acute phase, characterized by high levels of parasitemia and severe immunosuppression. Nevertheless, immunity against T. cruzi develops in this period, leading to the control of patent parasitemia that characterizes the end of the acute phase. Many studies have demonstrated the role of CD4⁺ and CD8⁺ lymphocytes in this immunity, but TCR nature of antigen-activated cells remains unknown. Recently, the participation of Vb6⁺ lymphocytes in the natural resistance to parasitemia development exhibited by Xid BALB/c mice was demonstrated. Although alterations of TCR Vb-usage were described in BALB/c infected with CL and Tulahuen strains of T. cruzi, the possible role played by specific lymphocyte subsets in the immunity exhibited by these mice against T. cruzi is far from being elucidated. To assess the role of Vb8⁺ T cells in the immunity developed by normal BALB/c mice against T. cruzi, we administrated staphylococcal enterotoxin B (SEB), a superantigen that initially activates this subset to produce cytokines and expand, and then deletes or anergizes them. Inoculation of SEB 3 days or 12 weeks before infection with T. cruzi CL strain severely impaired natural immunity presented by BALB/c mice, as revealed by delayed control of patent parasitemia at the end of acute phase. Next, we investigated the effects of SEB administration to mice previously infected with T. cruzi. Inoculation of low doses of SEB killed all acutely infected mice within 3 hours after inoculation, while all normal mice survived. In this period, mice developed the classic clinical symptoms of toxic shock syndrome, such as piloerection, prostration and diarrhea. Analysis of cytokine levels present in the serum of acutely infected mice 1 hour after SEB administration revealed high levels of IFN-g and TNF-a when compared to normal, SEB-treated mice, confirming the occurrence of lethal toxic shock. Taken together, our data demonstrate the participation of Vb8 T cells in the natural control of patent parasitemia exhibited by non-treated BALB/c mice. Moreover, our results suggest that activation of Vb8 T cells from T. cruzi infected mice leads to IFN-g and TNF-a production.

Supported by: CNPq, CAPES, FINEP, FUJB, FAPERJ, PRONEX and WHO.

IL-2 BUT NOT IL-12 IS THE MAJOR CYTOKINE RELATED TO THE HIGH LEVELS OF IFN-g PRODUCTION IN PATIENTS WITH THE CARDIAC FORM OF CHAGAS' DISEASE

Bahia-Oliveira, L.M.G.¹,Gomes, J.A.S.²; Rocha, M.O.C.³,Afonso,L.C.C ⁴, Pereira, P ⁵ & Correa-Oliveira, R⁶.

1-UENF, CBB, LBR, Campos dos Goytacazes, RJ; 2- Deparatamento de Bioquímica e Imunologia, ICB-UFMG, Belo Horizonte, MG;3- Hospital das Clínicas, UFMG, Belo horizonte, MG; 4- Laboratório de Imunoparasitologia, ICEB, Ouro Preto, MG; 5- laboratório de Hanseníase, FIOCRUZ, Rio de Janeiro, RJ; 6- Centro de Pesquisas René Rachou- FIOCRUZ, Belo Horizonte, MG.

We have shown that IFN-g production by Peripheral Blood Mononuclear Cells (PBMC) from chagasic patients can be related to the cardiac form of Chagas'disease. We found that the frequency of PBMC from chagasic patients secreting high levels of IFN-g is different in Cardiac (C) and Indeterminate (I) patients. Eighty -tree percent of C patients were found to be high IFN-g producers while fifty-five percent of I patients were high procuders of this cytokine. We have also demonstrated that the IFN-g production by PBMC from chagasic patients is dependent on the IL-2 and IL-12 production. In this paper we evaluated the kinetics of IL-2 and IL-12 secretion and the correlation between these cytokines and the clinical form of Chagas' disease. Our data demonstrate that the peak of IL-2 production is at day four after in vitro stimulation with antigens derived from Trypanosoma cruzi and that C patients produced significantly higher amounts of IL-12 when compared with I patients. No difference was detected between C and I patients when we evaluated the amount of IL-12 produced. The peak of IL-12 secretion occurred between the third and the fourth day of culture. We conclude that both IFN-g and IL-2 are import cytokines for the specific immune response in Chagas' disease and that these factors are probably enhancing the inflammatory pathogenic process in patients chronically infected with T. cruzi.

Support by: CNPq , NIH- A126505 and PAPES-FIOCRUZ.

IS IL-12/IFN-g CYTOKINE CASCADE A CHECKPOINT IN THE PROGRESSION TOWARDS CHRONIC CHAGASIC CARDIOMYOPATHY?

¹Abel LCJ, ²Rizzo LV, ¹Ianni B, ¹Mady C, ¹Portugal, K.G., ³Gazzinelli R, ⁴Almeida IC, ⁴Travassos L, ¹Kalil J and ¹Cunha-Neto E. - ¹Instituto do Coração, HC-FMUSP, São Paulo, ³Instituto do Ciencias Biomédicas-UFMG, Belo Horizonte, MG; ⁴Disciplina de Biologia Celular, EPM-UNIFESP São Paulo, Brazil and ²NEI/NIH, Bethesda, Maryland, USA.

Our group recently demonstrated that heart infiltrating T cells from Chronic Chagas'disease Cardiomyopathy (CCC) patients produce IFN-g- and TNF-a in the absence of IL-4, suggesting that inflamatory Th1 cytokines may play a pathogenic role in CCC. In order to investigate this finding, we studied the cytokine production by PBMC from CCC, asymptomatic Chagas'disease patients (ASY) and normal (N) individuals in response to B13 T. cruzi antigen and PHA. Supernatants from heart infiltrating T cell lines of CCC patients stimulated in the presence of T. cruzi trypomastigote mucin were also assayed. Culture supernatants (collected after 12 and 48 h incubation) were assayed by ELISA for cytokines. Results for B13-induced cytokine production indicate a clear dichotomy: 53% (9/17, 5.04 ng/ml) of the Chagasic group (CCC + ASY patients) produce IFN-g- and only 12% (2/17, 0.137 ng/ml) produce IL-4. Among N individuals, we observed the opposite picture: 63% (5/8; x=0.44 ng/ml) produce IL-4 and only 13% (1/8; x=0.077 ng/ml) produce IFN-g. The production was always reciprocal, and there was no difference between CCC and ASY groups. Results for PHA-induced cytokine production indicate that average IFN-g production was 18-fold higher among CCC+ ASY than N individuals (9.16 ng/ml vs 0.491 ng/ml, respectively) but the frequency of responders was similar (11/17, 64% and 5/8, 62%, respectively). IL-12 production by non-stimulated PBMC was comparable between CCC+ ASY and N groups (3.5 and 1.93 ng/ml, respectively). This may suggest that B13-specific T cells, and maybe the peripheral T cell pool of Chagasic individuals, have a predominant T₁ profile with a suppressed T₂ profile. As it is known that mucin-like glycoconjugates from T. cruzi are potent stimulators of IL-12 production by monocytes, we studied the role of T. cruzi mucin in the induction of IL-12 by heart infiltrating T cell lines from Chagasic patients. We found that IL-12 production is potentiated by activated T cells; this potentiation is dependent on CD40-CD40L interaction and IFN-g. It is possible that the shift to T₁ profile observed in chronically T. cruzi-infected patients may be a long-term effect of IL-12 production driven by T. cruzi mucin.

Suypported by FAPESP, CNPq, HHMI

ENDOGENOUSLY PRODUCED IL-10 AND PROSTAGLANDINS REGULATE PERIPHERAL BLOOD MONONUCLEAR CELLS PROLIFERATION TO TRYPANOSOMA CRUZI ANTIGEN

de Barros-Mazon¹, S., Guariento², M. E. & Abrahamsohn ³ , I. A.

¹ Departamento de Patologia Clínica and ² Departamento de Clínica Médica da Faculdade de Ciências Médicas, UNICAMP, C. Postal 6111, Campinas, 13083-970 SP, Brasil and ³ Departamento de Imunologia, Instituto de Ciências Biomédicas, USP, 05508-900, SP, Brasil. e-mail: [e-mail]

Introduction: Decreased IL-2 production and/ or lower expression of IL-2 receptors have been reported as mechanisms underlying the suppression of peripheral blood mononuclear cells (PBMC) lymphoproliferative responses to mitogens and antigens described in patients with chronic Chagas' disease. In this context, we have recently shown that in vitro IL-12 treatment significantly increased T.cruzi-antigen (T-Ag)-specific proliferation by PBMC from Chagas' Disease patients ( Immunology Letters 57: 39-45, 1997 ). Freshly isolated PBMC from chronic-phase patients also have increased IL-10, IL-13, IFN-g and IL-5 message levels (Dutra et al., Scand. J. Immunol. 45: 74-80, 1997). Lymphocyte activation and proliferation can be regulated by prostaglandins (PG), reactive oxygen and nitrogen intermediates (ROI and RNI) and by several cytokine-mediated mechanisms. We investigated the roles of PG, ROI, RNI and of the cytokines: IL-10, IFN-g, IL-13 and IL-4 on T-Ag-specific responses by PBMC from chronic Chagas'disease patients. Materials and Methods: The in vitro proliferative responses to disrupted tissue culture trypomastigote antigen (T-Ag) by PBMC from chronic chagasic patients (cardiac, indeterminate and cardiac + digestive forms) and from healthy donors were analyzed in cultures treated with: a) neutralizing monoclonal Abs to IL-10, IFN-g, IL-13 and IL-4; b) Indomethacin as a COX inhibitor of PG production; c) NMLA as a competitive inhibitor of NO and d) Glutathione-peroxidase as inhibitor of ROI.. Results:. T-Ag-specific proliferation was significantly increased by treatment with anti-IL-10 or with Indomethacin; the other anti-cytokine mAbs tested and inhibitors of RNI or ROI did not modify these responses. Among the patients , those with indeterminate or cardiac forms had their PBMC responses to T-Ag augmented by Indomethacin treatment but anti-IL-10 mAb treatment increased only the responses from cardiac patients. Conclusion: These results indicate that parasite-Ag-stimulated T cell responses in chronic chagasic patients are under negative regulation by prostaglandins and suggest that endogenous IL-10 exerts negative regulation on T cell responses in symptomatic Chagas' disease patients but not in asymptomatic (indeterminate) chagasic patients. Stimulation of in vitro cytokine production by these protocols is currently under investigation.

Supported by FAPESP, CNPq and FAEP. The neutralizing anti-human cytokines mAbs were a kind gift from Dr. Robert Coffman, DNAX.

POLYMORPHISMS IN TUMOR NECROSIS FACTOR GENES (TNF-a & TNF-b) AND THEIR POSSIBLE ASSOCIATION WITH THE CARDIAC FORM OF CHAGAS' DISEASE.

Ribeiro-Rodrigues, R.^1,2 , Oliveira, K.L.¹, Rocha, M.O.³, Correa-Oliveira, R⁴., & Carter, C.E.²

¹- Núcleo de Doenças Infecciosas & Depto de Patologia, Universidade Federal do Espírito Santo, Vitória, ES, BRAZIL;

²- Dept. of Biology, Vanderbilt University, Nashville, TN, USA;

³- Faculdade de Medicina, UFMG, Belo Horizonte, MG, BRAZIL;

⁴- Centro de Pesquisas René Rachou, Belo Horizonte, MG, BRAZIL.

Tumor necrosis factor a and TNF-b are produced by activated macrophages and lymphocytes and are pleiotropic, mediating a wide range of inflammatory and immunological responses. However, if the inciting stimulus is not removed or the cytokine production becomes dysregulated, prolonged or excessive levels of TNF result in significant pathology. Increased TNF levels have been demonstrated in the sera of patients with bacterial, viral and parasitic infections. High serum levels of TNF-a have been correlated with disease severity in several parasitic diseases, such as African trypanosomiasis, Chagas'disease, cerebral malaria, mucocutaneous and visceral leishmaniasis. The genes for TNF-a and TNF-b are tandemly arranged in the central region of the MHC, between the HLA-B and HLA-D locus, on the short arm of chromosome 6. Polymorphisms in the promotor regions of these genes have been correlated with the levels of TNF produced by the individual.

In the present work, we analyzed polymorphisms in the promoter region of TNF-a and TNF-b genes from patients with either chronic cardiac (CCC, n=28) or asymptomatic (IND, n= 15) Chagas' disease, and from healthy individuals (n= 25). A polymorphism at position _308bp in the promoter region of TNF-a (TNFa Nco), and two at positions +252 (TNFB Nco) and +368bp (TNFB Asp H) in the TNF-b gene were studied by PCR and RFLP. A correlation was found between the presence of certain polymorphisms and the cardiac form of Chagas'disease. Among CCC patients, 43% were homozygous for TNFa Nco allele 1, heterozygous for TNFB Nco and TNFB AspH (Haplotype TNF-1), and 25% were homozygous for TNFa Nco allele 1, heterozygous for TNFB Nco and homozygous for TNFB Asp H allele 2 (Haplotype TNF-2). Among IND and NORMAL individuals, the haplotypes were randomly distributed and not significant. Our results demonstrated a higher risk of CCC in individuals with either TNF-1 (RR=2.85) or TNF-2 (RR=2.33) haplotypes, and suggest a possible correlation between the presence of these polymorphisms and CCC.

IL-12 REGULATION OF IFN-g SYNTHESIS IN THE ACUTE PHASE OF TRYPANOSOMA CRUZI INFECTION.

Galvão da Silva, A. P. and Abrahamsohn, I. A.

Departmento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900, SP, Brasil. FAX: [phone].

Control of the acute phase of murine T.cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing. Both susceptible and resistant mouse strains show increased IFN-g secretion by spleen cells (SC) and treatment of infected mice with cytokine-neutralizing mAbs confirmed the major role of IFN-g in resistance to infection. IL-12 has been shown to stimulate IFN-g production by NK and T cells. Recent results have demonstrated that IL-12 has an important role in the innate and acquired resistance to T. cruzi infection (Abrahamsohn & Coffman, 1996). We are investigating the role of endogenous IL-12 production in stimulating IFN-g synthesis at different phases of the infection. Parasite-Ag-stimulated SC cultures from infected mice were treated with neutralizing mAbs: anti-IL-12 (C17.8 and policlonal); anti-CD4⁺(GK1.5) and anti-CD8⁺ (TIB105). IFN-g and IL-12 (p40) production were measured by ELISA. Our results show that until day 7 of infection 70 % of IFN-g synthesis is IL-12 dependent and 60-90 % is CD4⁺ -activation dependent. Later in the infection (day 12), IFN-g production becomes independent of endogenously produced IL-12 and completely CD4⁺ -activation dependent. Progressive decrease in IL-12 production by SC cultures was observed from day 7 of infection. Blocking of the CD8⁺-dependent activation did not modify IFN-g production levels by SC at any time of infection. Taken together, our results show that endogenous IL-12 is a major stimulant of IFN-g production by CD4⁺-activation-dependent T cells and by CD4⁺-activation-independent cells (possibly NK cells) in the early phase of acute T.cruzi infection.

Supported by FAPESP and CNPq.

THE ROLE OF IL-12 ON LYMPHOCYTE PROLIFERATION OF SPLEEN CELLS FROM TRYPANOSOMA CRUZI INFECTED MICE.

Galvão da Silva, A.P., Costa, V. M. A. & Abrahamsohn, I.A.

Depto. de Imunologia, Inst. de Ciências Biomédicas, USP, São Paulo, SP.

During the acute phase of T. cruzi infection, the lymphoproliferative responses are highly suppressed. IL-12 is a cytokine produced by phagocytic cells that has been described as a growth-stimulating factor of activated T and NK cells. Our previously results showed that: a) rIL-12 added to spleen cell culture from normal (NSC) or from T. cruzi C57Bl/6 infected mice (ISC), at day 12 of infection, enhanced Con A-induced cell proliferation; b) parasite Ag-specific proliferation was only enhanced by rIL-12 when NO production was inhibited by NMLA; c) the production of IL-12 (p40) increased in the first days of infection (days 3 and 5) and decreased as infection progressed (days 7 to 12 of infection). We decided then to study the proliferative response of ISC on different days of infection and the action of endogenously synthesized IL-12 on maintaining cell proliferation. ISC from days 5, 9 and 12 of infection, were treated with mAbs anti-IL-12 (C17.8) and stimulated in vitro with Con A or T-Ag (frozen-thawed trypomastigote antigen) in presence or not of NMLA. Suppression of cell proliferation was gradual with a period of pre-suppression (day 5), when the responses were still preserved, followed by a period of partial suppression (days 7 and 9) and a intense suppression from day 12. Neutralization of IL-12 inhibited 50% of the T-Ag-stimulated ISC proliferation only on day 5. Con A-induced proliferation of ISC was enhanced by rIL-12 addition: 50% on day 5, and 100% on days 9 and 12. Ag-specific proliferation was only enhanced by rIL-12 in ISC on days 9 and 12 provided NO production was inhibited. Increased proliferation promoted by rIL-12 was independent of endogenous IL-2 production. These results suggest that: a) endogenous IL-12 exerts a significant role in regulating antigen-specific proliferation on day 5 but not later in the course of infection; b)the effect of rIL-12 on cell proliferation is maximal when the production of this cytokine is lower during the infection.

Supported by FAPESP

INTERLEUKIN-12 ENHANCES IN VIVO PARASITICIDAL EFFECT OF A NITROHETEROCYCLIC DERIVATIVE DURING ACUTE EXPERIMENTAL INFECTION WITH A NATURALLY-RESISTANT STRAIN OF TRYPANOSOMA CRUZI

Michailowsky, V., Carvalho-Oliveira, L., Murta, S.M.F, Pereira, M.E.S., Ferreira, L., Romanha, A J., Brener, Z. & Gazzinelli, R.T. Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima, 1715, 30190-002; Departamento de Bioquímica e Imunologia, UFMG, Av. Antônio Carlos, 6627, 31270-010, Belo Horizonte, MG, Brasil

In this study we evaluated the role of IFNg and IL-12 in mediating and/or enhancing the in vivo trypanosomicidal activity of a nitroheterocyclic derivative, named Benznidazole (Bz), during early stages of experimental Chagas' disease. Our study shows that treatment with anticytokine mAbs had no apparent effect when optimal dose (100 mg/kg) of Bz was used. In contrast, treatment with anti-IL-12 or anti-IFNg mAbs enhanced the level of blood parasitemia and accelerated mortality of mice treated with the suboptimal dose of Bz (25 mg/kg). Simultaneous treatment with suboptimal dose of Bz and rIL-12 enhanced the efficacy of drug treatment in terms of blood parasitemia and animal survival. Interestingly, we found that drug resistant T. cruzi strains are poor inducers of IL-12, both in vitro and in vivo, as compared to strains of T. cruzi which are susceptible or partially resistant to Bz treatment. These results suggest that early activation of cellular compartment from immune system by IL-12 may favors in vivo Bz activity against T. cruzi. In order to test this hypothesis we treated mice infected with the Colombiana strain (drug-resistant) of T. cruzi with 100 mg/kg of Bz plus different concentrations of rIL-12. Our results, based on PCR, serological and parasitological methods show that mice receiving Bz therapy combined with rIL-12 have a higher percentage of cure, when compared to control group treated with the same dose of Bz alone.

Supported by FIOCRUZ, MG.

CHARACTERIZATION OF CYTOKINE AND ANTIBODY PRODUCTION IN MICE VACCINATED AGAINST TRYPANOSOMA CRUZI

Soares, MBP¹, Gonçalves, R¹, Pyrrho, AS^1,2, Costa, DA¹ and Gattass, CR¹.

¹ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brasil

² Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, RJ, Brasil

We have previously shown that mice inoculated with CL-14, a non-infective clone of Trypanosoma cruzi obtained from CL strain, develop a protective immune response against lethal challenge with virulent strains CL and Y. To investigate the mechanisms of protection induced by CL-14, we have analyzed the cytokine and antibody production in mice inoculated with CL-14 and infected with CL strain. Infection with trypomastigotes of CL strain induced an elevation in the serum levels of IFN-g. Two weeks after challenge with CL strain, sera from vaccinated mice contained 4 fold less IFN-g than non-treated mice. Likewise, mitogen-stimulated splenocytes from vaccinated mice challenged with CL trypomastigotes produced 3 to 5 fold less IFN-g than non-vaccinated mice. Mitogen-stimulated splenocytes from CL-14 inoculated or non-infected mice showed similar production of IL-2, whereas culture supernatants of splenocytes from CL infected mice produced 5 fold less IL-2. Moreover, CL-14 vaccination partially prevented the suppression of IL-2 production caused by CL infection. No significant differences were found in IL-4 production by mitogen-stimulated splenocytes from non-treated, CL-14-vaccinated, CL-14-vaccinated mice challenged with CL strain, and CL infected mice. Production of IgG1 and IgG2a isotypes was also assessed. After challenge with CL strain, vaccinated mice developed higher titers of IgG1 (5 fold) and IgG2a (2 fold) than non-treated mice. However, vaccination prevented the total IgG2a increase induced by CL infection. These results suggest a role for parasite-specific IgG1 in the control of infection and show that high levels of IFN-g may be detrimental to the establishment of a protective response, possibly by inhibiting the development of this IgG1 and favoring the production of nonspecific IgG2a.

Work supported by: CNPq, FAPERJ, PRONEX, FUJB, FINEP and WHO

EXPERIMENTAL CHAGA'S DISEASE IN MICE SELECTED FOR MAXIMAL (AIRMAX) OR MINIMAL (AIRMIN) ACUTE INFLAMMATORY REACTION.

Starobinas, N.; Ribeiro,O. G.; Vorraro, F.; De Franco,M.; Ramalho, A.; Ibañez, O.M. Laboratório De Imunogenética, Instituto Butantan, São Paulo, 05503-900,SP, Brasil.

The inflammatory reaction has been shown to play an important role for the host's capacity to control parasite multiplication in Trypanosoma cruzi (T.cruzi) infection. Two lines of mice, AIRmax and AIRmin, were produced by bidirectional selection based on the capacity to mount a strong or weak acute inflammatory reaction (AIR) to foreign material (polyacrilamide beads) measured by the leukocyte and protein content of the 48 hour-subcutaneous exudate. The selective breedings were initiated from a highly polymorphic population (F0) obtained by equipoised intercrossing of 8 inbred strains of mice: A/J, BALB/c/J, CBA/J, C57BL/6/J, DBA2/J, SJL/J, SWR/J and P/J. At the selection limit, reached after 12 generations of selection, mice of the two lines AIRmax and AIRmin are considered homozygous at the relevant loci. These lines of mice offer an appropriate model for the investigation of an association between the genetic factors acting in the intensity of inflammatory response and resistance to infection. AIRmax and AIRmin mice were infected with different doses (10², 10³ and 10⁴ parasites) of the CL strain of T.cruzi, and the number of circulating parasites was analysed, as well as the mortality of mice. In both lines an important difference was observed between male and female concerning susceptibility to T.cruzi infection. Mortality reached 100% in males and 50% in females. Comparing the lines, we noticed that AIRmin mice were more susceptible to infection with lower doses of parasites, presenting higher mortality and lower survival time, than AIRmax mice. Similar curves of circulating parasites were observed in male mice of both lines. AIRmin females showed high parasitemia and death while in AIRmax the number of parasites decreased around 20 days being indetectable after 32 days of infection. These results suggest that the modifications occured in AIRmax and AIRmin mice due to selection, repercute in the natural resistance to acute T.cruzi infection.

Supported by FAPESP and CNPq.

CHEMOKINE EXPRESSION IN HEART TISSUE FROM MICE EXPERIMENTALLY INFECTED WITH TRYPANOSOMA CRUZI

Talvani, A.^1,2; Ribeiro, C. S.²; Aliberti, J.C.S.³;Alves-dos-Santos, P.V.⁴; Farber, J.⁵, Lannes-Vieira, J.⁴; Silva, J. S.³ & Gazzinelli, R.T.^1,2

¹Laboratório de Chagas, C PqRR - FIOCRUZ; ² Departamento de Bioquímica e Imunologia, UFMG, Belo Horizonte, MG; ³Departamento de Parasitologia, Microbiologia e Imunologia, FM-USP, Ribeirão Preto, SP; and ⁴ Departamento de Imunologia, IOC - FIOCRUZ, RJ; and Belo Horizonte, MG, Brazil; ⁵Laboratory of Clinical Investigation, NIAID, National Institutes Health, Bethesda, USA.

Chemokines are a group of multifunctional cytokines, produced by different type of cells and involved on recruitment of leukocytes to the site of inflammation. In order to investigate the participation of chemokines in the development of chronic chagasic cardiopathy we infected outbred Swiss or inbred C57Bl/6 mice with 50 blood trypomastigotes from Colombiana strain of Trypanosoma cruzi. Animals were sacrificed in days 0, 15, 25, 30, 60, 120 and 240 days after infection, and heart tissue collected for analyzing the tissue parasitism, inflammatory infiltrate, fibrosis, and expression of different cytokines. Expression of T cell derived cytokines (IFN-g, IL-2, IL-4 and IL-5), monokines (IL-1, IL-10, TNF-a and IL-12) and chemokines (MIP-2, MIP-1a, MIP-1b, JE, RANTES, MIG, IP-10, KC, SDF-1a, SDF-1b, TCA-3, EOTAXIN, LIX) were analyzed by RT-PCR. Our data show that parasites were in the cardiac tissue of all infected animals by day 15 post-infection. The peak of cardiac tissue parasitism was around days 25 to 30 after infection. An inflammatory process accompanied the heart tissue parasitism. We observed both diffuse and focal inflammatory process that were followed by gradual increase of fibrosis from 60 days post-infection. Our preliminary results indicate a dominance of CD4⁺ T cells over CD8⁺ T lymphocytes during the initial phase of infection. We also observed an inversion of CD4⁺ T to CD8⁺ T cells dominance in the inflammatory infiltrate along with parasitism increase and appearance of amastigote nests in the cardiac tissue at 25 days post-infection. The dominance of CD8⁺ T lymphocytes persisted at latest phase of infection and was associated with a gradual and considerable enhancement of mononuclear cells expressing MAC-1 receptors. We are at the moment using this model, to determine the influx of polymorphonuclear cells during different stages of chagasic cartiopathy. The RT-PCR data showed a different pattern of cytokine expression during different stages of infection with T. cruzi. From T-cell derived cytokine mRNAs we observed a dominance of IFN-g at different stages of infection. Similar pattern of expression was observed for the monokines TNF-a and IL-10 mRNAs. Consistent with this observation the IFN-g induced chemokines MIG, IP-10 and RANTES were all expressed in high levels in the cardiac tissue of infected animals. Similar pattern of expression was observed by the chemokine JE. The monokines IL-1 and IL-12 as well as the chemokines MIP-1a, MIP-1b and MIP -2 were expressed mainly at initial phase, before 30 days of infection. Finally, the cytokines involved in allergic processes such as IL-4 and IL-5 were poorly expressed in the cardiac tissue of mice infected with T. cruzi at any time of infection. Consistent with this data we were unable to detect any expression of EOTAXIN and LIX which are involved on activation and/or recruitment of eosinophils and mast cells. We were also unable to detect any expression of SDF-1a and SDF-1b, TCA-3 mRNAs during infection with T. cruzi. Interestingly, inflammatory macrophages activated in vitro with T. cruzi trypomastigotes or tGPI-mucins (trypomastigote derived glycosylphosphatidylinositol anchored mucin-like glycoproteins) produced similar pattern of monokine and chemokine expression as the one observed in the heart tissue of mice infected with T. cruzi. This findings along with the histochemistry data indicate that macrophage maybe a major source of chemokines during chronic chagasic cardipathy.

Supported by CNPQ, FAPEMIG, FAPESP and PAPES-FIOCRUZ.

REGULATION OF TRYPANOSOMA CRUZY -INDUCED CHEMOKINE mRNA EXPRESSION DURING INFECTION IN MICE

Aliberti, JCS¹; Talvani, A^2,3,; Ribeiro, CS²; Vieira, LQ³; Gazzineli, RT^2,3 & Silva, JS¹

IDepartamento de Parasitologia, Microbiologia e Imunologia, FMRP-USP, Ribeirão Preto, Sp; 2Laboratório de CRUZ; 3Laboratório de Imunoparasitologia, Departamento de Bioquímica e Imunologia, ICB, UFMG, Belo Horizonte, MG, Brazil.

Chemokines (CK) are a family of cytokines, whose most prominent biological feature is their ability to act as chemotact factors. As during infection with the virulent protozoa T cruzi, a strong inflammatory reaction occurs, either at the inoculation site and, later, in the myocardium. To address the role ofthese molecules in T cruzi-induced inflammation, we first studied the CK MRNA expression triggered by the interaction of T cruzi trypomastigotes with macrophages. We also investigated the role of cytokines as modulators of T cruzi-induced CK expression in macrophages. Our results show that IFN-g increases expression Mig, MIPI-P, JE and Crg-2. ln contrast, KC and MIP- I (x expression was completely blocked. We also investigated the role of other pro-infiammatory or regulatory cytokines which are induced by macrophage exposure to T cruzi tr'ypomastigotes. The pro-inflammatory cytokines, IL-IP and TNF-a, potentate the expression of the CKs KC, Crg-2, MIP- I ct and JE. The regulatory cytokines, IL- I O and TGF-g inhibited the expression of almost all CK tested. The only exception observed was the induction of KC in macrophages by IL- I 0. To investigate the regulatory role of IFN-g and TNF-a in vivo, we used IFN-a- (GKO) or TNFRp55-deficient (p55-/-) mi—e following ip infection with 5.000 trypomastigote forms of T cruzi (Y or CL strain). The cellular infiltrates observed in the inoculation site in control animais (wt) consisted primarily of neutrophils at the first day pi, and mainly lymphocytes at the seventh day pi. The CK expression pattem correlated with the cell type observed. Thus, when neutrophils predominated, the expression of CK that attract these cells (KC) was detected and, seven days pi, the expression of CKs Mig, Crg-2 and RANTES (lymphocytes chemoattractants) was predominant. A distinct infiammatory response was observed in the GKO mice. ln these, a predominant neutrophilic and a discrete lymphocytic infiltrate were observed only after five days pi (peaking at seven days pi). The infected GKO mice exhibited strong expression of KC and MIP-2, which may explain the neutrophil migration, RANTES expression at 5 days pi and only a basal expression of Mig and Crg-2. Similar results were found in the cardiac tissue of GKO mice acutely infected with T cruzi. lnterestingly, p55-/- mice did not presented neither neutrophilic inflltrate, or expression of CKs that attract neutrophis during acute infection. ln contrast, a large lymphocytic infiltrate was observed in these animais at second day pi. The early appearance of lymphocytes was associated with high level expression of Crg-2 MRNA. Finally, spleen cells of infected mice exhibited a CK expression pattem similar to that observed in the peritoneal cavity cells. In the latter, lymphotactin, SDF- I (i and -o were detected after 5 days pi. Altogether, these results suggest that IFN-g, TNF-a. and CKs, play a crucial role in the modulation of the inflammatory response observed during T cruzi infection and lead us to speculate that a modulation of cytokines and CKs could result in susceptibility or resistance to the parasite.

Supported by CAPES, CNPQ, FAPESP, FAPEMIG and PAPES-FIOCRUZ.

MOUSE MACROPHAGES DERIVED FROM BLOOD MONOCYTES CANNOT KILL TRYPANOSOMA CRUZI OR PRODUCE NITRIC OXIDE WHEN TREATED WITH INF-gAND LPS.

DaMatta, R.A.^1&2 , Seabra, S.H.¹, Manhães DS, L.¹ & De Souza, W^{1&2 - 1}Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, UENF and ²Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil.

It has been shown that mice macrophages (peritoneal) activated 24 hours in advance with interferon-g (INF-g) and lipopolyssacharide (LPS) produce nitric oxide and it are also able to kill Trypanosoma cruzi. Trying to understand the production of nitric oxide by macrophages derived from blood monocytes and its relation to the survival of Trypanosoma cruzi, the production of this radical was measured after the interaction with this parasite. Bloodstream trypomastigote of the Y strain of Trypanosoma cruzi was purified from blood harvested from mice on the seventh day post infection. Mouse macrophages were obtained by blood monocytes purification on a Percoll cushion and cultivation for 5 days with DMEM supplemented with 10 % inactivated fetal bovine serum. Macrophages were washed with DMEM and infected with the parasites in a 10 to 1 parasite/macrophage ratio. After 2 hours of interaction the macrophages were washed with DMEM and new medium containing fetal bovine serum supplemented or not with INF-g (50 U/ml) and LPS (1µg/ml) was added. After 2, 24 and 48 hours the supernatants of the cultures were collected and assayed with the Griess reagent for the presence of NO₂^-. Mouse macrophages derived from blood monocytes treated or not with INF-g and LPS could not kill Trypanosoma cruzi and nitric oxide production could not be detected in the supernatant of these cells after 2, 24 and 48 hours. As expected, resident mouse peritoneal macrophages that received the same treatment as the macrophages derived from monocytes did produce nitric oxide. These results suggest that macrophages derived from monocytes cannot become activated with INF-g and LPS, allowing the survival of this parasite. Different activation protocols are in progress to better characterize the mouse macrophages derived from blood monocytes.

Supported by FINEP, FENORTE, PRONEX and CNPq.

THE COURSE OF TRYPANOSOMA CRUZI INFECTION IN PERFORIN KNOCK-OUT MICE REVEALS THAT PERFORIN-MEDIATED CYTOTOXIC MECHANISMS ARE NOT THE MAIN BUT ARE RELEVANT COMPONENTS OF THE PROTECTIVE IMMUNE RESPONSE

Andrea Henriques Pons¹, Alexandre F.S.Correa¹, Marcos M.Batista¹, Cleber M. Silva¹, Pedro M. Persechini² , Rodrigo Bissagio^2, Chau-Ching Liu³,Vinicius Cotta-de-Almeida¹,Claudia M.L.M.Coutinho^1,4 & Tania C.Araújo-Jorge¹

¹Lab. de Biologia Celular -DUBC-Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro; ²Lab. de Imunobiofísica -Inst. de Biofísica Carlos Chagas Filho -UFRJ, Rio de Janeiro; ³Lab. of Cellular Physiology and Immunology, Rockfeller University, New York; ⁴Depto. Biologia Celular e Molecular, Instituto de Biologia, UFF, Niterói, RJ

The activity of cytotoxic cells are decisive to control infections by intracellular microorganisms. NK, CD4 and CD8 T cells are involved in the control of the ascendent phase of parasitaemia in mice infected by T. cruzi, but the precise mechanisms and relative contributions of each cell subtype are not fully understood. The two major mechanisms of cytotoxicity of the activated lymphocytes are mediated by the secretion of perforin and proteinases which follows TCR-MHC interaction, leading to target cell lysis and apoptosis, and the induction of apoptosis by the recognition of FasLigand of the cytotoxic cells and Fas antigen in the target. In the present study we used perforin knock-out mice (-/-, P0) (Walsh et al. PNAS USA 91: 10854, 1994), and their respective +/+ homozigous controls (P2) to address the relative role of this mechanism in the resistance of mice to infection with an intraperitoneal inoculum of 10⁴ trypomastigote forms of the T. cruzi Y strain. P2 and P0 mice with 8-10 weeks old showed identical kinectic curves of parasitaemia. Circulating trypomastigotes were first detected 6-7 days post infection (dpi), showed a peak in the 8th dpi (mean of 2,09 x 10⁴ parasites/ml) and decreased by the 10th dpi. P0 mice were than able to control parasitaemia, which CD8 or CD4 knock-out or depleted mice are unable to do. However, the peak level of circulating parasites were two times higher in P0 than in P2, indicating that perforin mediated cytotoxicity is at least partially important at that time. Only about 10-20 % of the infected P0 mice survided the acute infection and chronified for longer periods. These chronic mice were always females, which showed higher resistance to T. cruzi infection than males, just as P2 mice also do. No difference in the parasitaemia of the animals was observed relative to their ages at the time of the infection. Splenomegalia and thymic atrophy were noted in both P0 and P2. The collection of histopathological data is in course, and will be presented.

INO-SINTHASE AND NO MODULATION IN CALOMYS CALLOSUS AND SWISS MICE INFECTED WITH TRYPANOSOMA CRUZI. EFFECT OF IN VIVO ADMINISTRATION OF L-NITRO-ARGININE.

Oliveira, L.C.^1,2 ; Leal, M. P.¹; Assreuy, J.³; Koetzel, J.K.^1,4 1-Inst.de Medicina Tropical de S.Paulo, Av. Eneas de Carvalho Aguiar, 470- S.Paulo, 05403-000, S.Paulo, Brazil. 2-Lab. Inst. da Criança, HC-FMUSP; 3-Univ. Federal de Sta. Catarina, Florianopolis; 4-Dept. Parasitologia, ICB-USP.

NO₂ may participate in the effector mechanism of intracellular death of pathogens, including T.cruzi in macrophages, although they may also sustain their development. Previously we compared the kinetics of NO production by macrophages of C. callosus, a silvatic rodent, reservoir of Chagas disease and Swiss mice during the course of infection with T.cruzi strains M226 (a silvatic strain originally isolated from a C.callosus) and strain F, up to the 40^th day of infection. Activated peritoneal macrophages of C.callosus did not produce NO, while those of Swiss mice did. However, NO₂+NO₃ (NOx), was found in the sera of both animal species, probably originating from other cells. In the present paper we investigate the presence of iNOS (Induced NO sinthase) in macrophages of C.callosus and Swiss mice, as well as the effect of an analogue of arginine, L-Nitro-Arginine, inhibitor of NO, on serum NOx levels and on T.cruzi infection. 30 days old Swiss mice and C.callosus were inoculated s.c. with 4x10³ trypomastigotes, strain M226 or F. On the 21rst day, when maximum macrophage activation was observed by H₂O₂ levels, iNOS was determined in explanted macrophages, by the conversion of arginine to citruline. No iNOS activity was found in C.callosus cells, while it was present in those of mice (11.00pmol citruline/10⁸ cells/min). F strain inoculated animals were treated or not with a daily dose of 50 mg/kg L-Nitro-Arginine. In C. callosus no statistically significant difference of parasitemia was observed between the two groups of animals in spite of approximately 9 times reduction of NO_x levels (108.64 uM to 12.00uM). However, in Swiss mice the parasitemia was higher (around 3 fold) in animals inoculated with L-Nitro-Arginine with 80% mortality as compared with 20% in infected controls without drug administraion. This finding suggests that in C. callosus NO does not play an important role in the control of Chagasic infection, but it seems to be important in Swiss mice. It also shows the complexity involved in the control of Chagasic infection.

Financed by CNPq, FAPESP and LIM49-HC

NITRIC OXIDE PRODUCTION BY SPLENOCYTES DURING EXPERIMENTAL RODENT MALARIA AND ITS RELATIONSHIP WITH PARASITEMIA DEVELOPMENT

Garnica, MR & Andrade Jr., HF

Lab. Protozoologia - Instituto de Medicina Tropical de São Paulo-FMUSPAv. Dr. E. C. Aguiar, 470, CEP 05403-000, São Paulo, SP, BRAZU,

Malaria immunology studies are niainly devoted to protectivc immunity by vaccines. with feiv stadies on the mechanism of lhe control of installed parasitemia. ln the unúeated infected mice infected mith Pchabaudi AS, relatively high parasitemia was rapid and effectively controllcd in two or three days, with the appearance of "crisis" forms. The spleen appcars to be essential for this process as splenectonúzed mouse maintains parasitenúa during all his life. Most reports in this area ascribe this control to free radicais, as oxygen and nitrogen reactive intermediates, like peroxinitrite or nitric oxide, with the latter proposed as the main effector svstem. We studied the production ofnitric oxide during rodent malaria, as we already detected a clear increase in serum nitrite concentration in our rodent malaria models, wíthout adequate timing with parasitemia. ln order to explain those confiicting data, we study the production of nitric oxide by splenocytes during models of rodent malaria. We used four models, two isogenic strains ofnúce, Balb/C and C57Bl/6j and two malana, P berghei ANKA(Iethal) and P chabaudi CR(non-lethal), looking for nitrite production on supernatant of dissociated splenocytes, as detected by Gness reaction. Groups of 12 mice were inoculated i.p. with 10⁶ erythrocytic parasitos, and parasitemia was assessed regularly by tail blood films, stained by Giemsa. Spleen of mice from several periods ivas collected, and its cells mechanically separated, with erithrocyte elimination by NH4Cl lysis. After viability determination, Hanks BSS washed splenocytes were seed, 2xlO⁵ viable cells, in 96 micronvell plates, in HEPES buffered RPNU 1640 w/lO% FCS. After 1 h, 37^oC, 5% CO₂, the supernatant media Nvas collected and fresh media added to cell layer for 24 h collection. The NO production in spleens clearlv correlated with parasitemía in Pchabaudi models, differing from serum data that shows progressivo increase that was maintained after clearing of the parasitenúa. P.berghei infccted models show a less efficient NO production, despite its correlation with parasitemia, rnore evideiit in Balb/C mice, the less effective NO production association. Both collections(lh and 24 hs) resulted in sinúlar NO curves. The malntained serum concentration of NO. after parasite clearing, in P.chabaudi models, could be ascribed to extrasplenic NO production. This fact could be also occurring in the early stages of Pberghei infection, with immune response modulation, but this fact needs much more clarifícation. Splenic NO production correlates with the control of parasitenúa in both models, suggcsting that NO could be the main effector molecule in this system.

This work was partially supported by CNPq (PIBlC MRG), FAPESP and LIMHCFMUSP 49

IMMUNOLOGIC MECHANISMS INVOLVED IN THE REACTIVATION OF THE CHRONIC INFECTION WITH TRYPANOSOMA CRUZI

Pereira, M.E.S., Melo, U.B.*, Martins-Filho, O.A., Brener, Z. & Gazzinelli, R.T.**

Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima, 1715, 30190-002; *Instituto de Ciências Biológicas e da Saúde (PUC-MG); **Departamento de Bioquímica /UFMG, Av. Antônio Carlos, 6627, 31270-010, Belo Horizonte, MG, Brasil.

Chronic Chagas' disease is characterized by an apparent steady biological equilibrium between the host and the parasite. However, recrudescence of the parasitism in the chronic phase may occur upon administration of immunosuppressive agents or infection with HIV. To investigate the role of different components from the immune response controlling T. cruzi expansion during chronic infection, groups of female C57Bl/6 mice were inoculated with Buriti strain and kept in the laboratory for 90 to 120 days. Chronically infected animals were then treated with either weekly doses of cyclophosphamide (Cy) (175 mg/kg, i.p.), mAb anti-CD4 (0,5 mg/A, i.p.) or silica (3 mg/A, i.v.). Before treatment the persistence of ongoing T. cruzi infection was confirmed by hemocultures. The ratio of T and B cells in peripheral blood were analysed before and during the treatment by flow cytometry (Becton Dickinson FACScan). The surface markers analysed were CD4, CD8, B220 and MAC-1. Mice immunosuppressed with Cy showed the decrease on the percentage of B cells (96%) and normal levels of CD4+ lymphocytes. Animals treated with anti-CD4 had 92% of their T lymphocyte depleted for 8 weeks. The number of bloodstream forms in the treated and control mice was daily determined according to Brener (1962), during the entire experiment. During the experiments some mice were sacrificed at days 24, 27, 32, 40 and 60 days after initating the various immunossupressive treatments. The serum collected was frozen (-70ºC) and either antibodies level or cytokines evaluated by ELISA. Different organs (i.e. heart, spleen, intestine and brain) were isolated and processed for histopathology and immunohistochemical analysis. The parasitemia levels of experimental groups compared with the control values were used as criteria for considering the infection recrudescence. Patent parasitemia was detected in 71% (17/24) of chronically infected mice immunosuppressed by Cy (treated for 30 days), an immunosupressor that blocks specifically the expansion of B cells. Patent parasitemia was not detected in infected mice treated with either anti-CD4 (treated as long as 60 days) or silica (treated as long as 30 days), nor in nontreated infected mice (group control). The histopathological and immunohistochemical analysis are been carried. We are now treating animals with antibodies specific to IFNg, depleting CD4+ and CD8+ lymphocytes, as well as using different immunossupressive drugs in a effort to determine the immunologic mechanisms involved in the parasite control during chronic Chagas'disease.

Supported by FAPEMIG, FIOCRUZ

THE ACTIVATION PATTERN OF THE IMMUNE SYSTEM IN THE LATE CHRONIC PHASE OF MURINE T.CRUZI INFECTION IS INFLUENCED BY THE PARASITE LOAD DURING THE ACUTE PHASE.

Marinho C.R.F., D'Império Lima M.R., Grisotto M.G. & Alvarez J.M.

Deptartment of Immunology, USP, São Paulo, SP, Brasil.

We have shown that the parasite load on the acute phase of murine T.cruzi infection directly influences the parasitaemia level at the chronic phase. In the present work, we have examined the long-term impact of the parasite load in the size and activation state of the immune system. A/J mice were infected with 10³ or 10⁵T.cruzi blood parasites (Y strain), treated with Benzonidazol, and their blood and spleens studied one year later. When infected groups were compared, we observed that: 1) mice infected with the higher inoculum had higher levels of chronic parasitaemia; 2) mice infected with 10⁵ parasites showed, among CD4⁺ cells, a shift towards a CD45RB^low phenotype (from52.8% to 58.3% in the 10³ and 10⁵ groups; controls 52.0%) and higher frequencies of CD4⁺ blasts of both CD45RB^high and CD45RB^low phenotypes; 3) Ig producing cells were higher in the 10⁵ parasites group, except for IFNg-dependent isotypes, IgG2a and IgG3, which did not differed among the infected groups (IgG2a/IgG1 ratios were 5.8 and 3.7 respectively for 10³ and 10⁵ groups; controls 2.7); and 4) mice infected with 10⁵ parasites produced higher amounts of IL-4 and IFNg than those injected with the lower parasite inoculum. Compared to control animals, IFNg levels were augmented in animals infected with 10⁵ parasites and decreased in the low dose group (167.7 ng/ml and 29.5 ng/ml, respectively; controls 111.7 ng/ml). IL-4 production was also suppressed in the later group, but not in the former (0,28 ng/ml and 0,44 ng/ml, respectively; controls 0,45 ng/ml). IL-10 was similarly produced by all groups (5,16 ngs/ml for controls; 4,57 and 5,81 ng/ml for the low and high dose groups, respectively). IL-2 secretion was suppressed in both infected groups (0.3 ng/ml and 0.2 ng/ml; controls 1.3 ng/ml).

Conclusions: Parasite load level in the acute phase determines different activation patterns of the immune system at the late chronic phase. These patterns, which could result from chronic levels of parasitemia, do not completely accommodate into a predominance of TH1 or TH2.

This work was supported by grants from CNPq and FAPESP.

THE CELLULAR IMMUNE RESPONSE AFTER BALB/c INFECTION WITH STRAINS TRYPANOSOMA CRUZI Y, SC38 AND PF.

Silva Filho, H.H.; Carobrez, S.G.; Silva, J.S.**; Sousa, M.A.*; Mineo, J.R.* Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, SC, Brasil. *Departamento de Patologia, Universidade Federal de Uberlândia, MG, Brasil. **Departamento de Microbiologia, Parasitologia e Imunologia, Faculdade de Medicina de Ribeirão Preto - USP, SP, Brasil.

Considering not much knowledge regarding T. cruzi infection with median and low virulence have been produced, we decided to observe the cellular immune responses among strains with different level of virulence. To perform theses experiments, we employed 6 groups of BALB\c females, 10 - 12 week-old, which were inoculated intraperitoneally with T. cruzi strains. Two group with 100 blood forms parasites of the Y strain (high virulence), two group with 5 x 10³ blood forms parasites of the SC38 strain (median virulence), two group with 2 x 10⁷ culture forms parasites of the PF strain (non virulent) were set. Seven days later, three groups of mice, which were previously inoculated with each strain of T. cruzi, were also inoculated with 2,5 x 10⁸ SRBC to evaluated the heterologous antibodies production. We also set two control groups. One was inoculates with physiological solution and another with SRBC.The sera were collected at 7, 15, 30 45, 60, 90 day and the levels of heterologous antibodies against SRBC was determined by hemaglutiation. We also studied the IL-4, IL-10 and IFN-g cytokines (ELISA method) and nitric oxide (Griess method) production by spleen cells of the mice inoculated with different strains wich were sacrificed 7, 15, 30, 45, 60 and 90 day after initial challenge.The results with heterologous antibodies showed reduction in antibody production which was correlated with the degree of virulence of the parasites. This may be related with some particular biological feature of each strain. High levels of IL-10 and IL-4 were observed in the supernatants from spleen cells from animals infected with SC38 strain. We also detected early production of IFN-g (days 7 and 15) by unstimulated spleen cells in the animals infected with the Y and PF strains of T. cruzi. The production of this cytokine in the SC38 strain infected group was present much later. It was detected only by day 30. Moreover, the levels of IFN-g secretion increased steadly until day 90.The nitric oxide production followed the same pattern as the one found for IFN-g production. Taken together, the results obtained in the present study showed reduction in the production of heterologous antibodies against SRBC that follows the T. cruzi infection. This alteration is closely related to the level of nitric oxide production and may be associated to the biological feature of each strain. Also, the IL-10 and IFN-g may be acting antagonistically in this process of regulation which affects heterologous antibodies production against SRBC. This may be associated to the differential activation of Th1 and Th2 clones.

Supported by CNPq

IMMUNOBLOGULIN PRODUCTION BY TRYPANOSOMA CRUZI-INFECTED MICE: THE ROLE OF B LYMPHOCYTE-NATURAL KILLER (NK) CELL INTERACTION.

Arruda-Hinds, LB*, Previato, JO⁺, Nunes, MP**, Mendonça-Previato, L⁺. & Peçanha, LMT*.

Depto de Imunologia* e de Microbiologia Geral⁺, Inst. de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro and Depto de Protozoologia**, FIOCRUZ, Rio de Janeiro, RJ, Brasil.

We have recently shown that T cells obtained from chronic T. cruzi-infected mice induce an exacerbated immunoglobulin (Ig) secretion when these cells are incubated with B cells obtained from normal mice. Previous studies have shown that NK cells cytotoxic activity is enhanced during experimental T. cruzi infection and that those cells are important for resistance to infection. Based on the findings that NK cells stimulates Ig production, in this study we investigated to what extent NK cells can modulate Ig secretion by B cells from infected mice. 10⁵ chemically-induced Dm28c strain trypomastigotes were i.p. injected. Splenic murine B cells were purified after different periods of infection. A SCID mice-derived NK cell line, selected based upon its ability to support Ig production in response to polysaccharide antigens was used. This line was maintained by stimulation with IL-2. Purified B cells stimulated by bacterial lipopolysaccharide were induced to secrete Ig and this response was increased by coculture with the NK cell line. We observed that B cells obtained after 45 to 55 days of T. cruzi infection produced four times higher amounts of both IgM and IgG2a in the presence of the NK cell line when compared to normal B cells. Both low- and high-density B cells obtained from infected mice showed higher secretory responses. The increased Ig production was absent if B cells were obtained either earlier (15 days) or later (70 days) during infection. We also investigated whether the NK line could be activated by T. cruzi-derived products. Glycoinositolphospholipids purified from T. cruzi increased the interleukin 2-induced proliferative response of the NK line. Our data suggest that, besides T cells, NK cells may be one cell population supporting B cell activation during T. cruzi infection and that T. cruzi-derived molecules provide one of the mechanisms by which NK cells are activated during infection.

Supported by CNPq (RHAE, PADCT), FIN EP, PRONEX, FAPERJ, UFRJ (CEPG e FUJB).

IMMUNO- AND MORPHOLOGICAL ALTERATIONS OF SPLENIC CELLS AFTER IN VIVO TREATMENT WITH GLYCOINOSITOLPHOSPHOLIPIDS (GIPLs) PURIFIED FROM TRYPANOSOMA CRUZI.

Bilate, AMB*, de Brito Gitirana, L**, Previato, JO⁺, Cunha, J.M.T.*, Mendonça-Previato, L⁺ & Peçanha, LMT*.

Depto de Imunologia* and Microbiologia Geral⁺, Inst. de Microbiologia Prof. Paulo de Góes and Depto Histologia e Embriologia**, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

We have previously observed that GIPL purified from T. cruzi (G strain) is a potent in vitro murine B cell activator (Bento et al., J. Immunol, 157: 4996, 1996). This glycoconjugate stimulates, by itself, detectable immunoglobulin (Ig) production and potentiates the response induced by surface Ig (sIg) ligation or cytokines. Those results suggested that this glycolipid could be a potent immunostimulator. In this study we have tested if the purified T. cruzi GIPL would stimulate B cells in vivo. Mice were injected i.v.with GIPL and spleens were obtained at different time points. The spleens were either used as a source of purified B cells or processed for histological analysis. Sera IgM levels were also analyzed and a slight increase was detected 7 days after GIPL injection. Following culture, control B cells did not respond to either sIg ligation or IL 5. However, cells obtained 3 days after GIPL injection secreted at least 100 times higher levels of IgM. In addition, B cells from injected mice secrete 250 times more IgM than control B cells in response to LPS. Spleens from GIPL-treated mice showed white pulp hypertrophy and a consequent displacement of the red pulp. The later was restricted to the periphery of the organ. This structural disarrangement was prominent after 7 and 14 days of injection, although it could be detected even 21 days after treatment. A conspicuous high number of giant cells was observed throughout the red pulp after 14 or 21 days of injection. Despite the morphological alterations, spleen cellularity and B cell numbers were not modified. These data suggest that the GIPL purified from T. cruzi has a potent in vivo stimulatory activity and this parasite glycolipid could be, at least in part, responsible for T. cruzi infection-associated immunological changes.

Supported by CNPq (RHAE, PADCT), FINEP, PRONEX, FAPERJ, UFRJ (CEPG e FUJB).

PROGRAMMED CELL DEATH IN HUMAN CHRONIC CHAGAS' DISEASE CARDIOMYOPATHY.

Granja, C.B., Oshiro, S., Monteiro, S., Cunha-Neto, E., Kalil, J. and Coelho, V.

Laboratório de Imunologia de Transplantes, Instituto do Coração, HC- FMUSP- Universidade de São Paulo, São Paulo.

Programmed cell death has been implicated as a mechanism of immunosupression in experimental Chagas' disease. In addition, a previous report has shown an increase in CD28 negative cells, a phenotype prone to undergo apoptosis, in chronic human disease. However, the role of apoptosis in the pathogenesis of Chronic Chagasic Cardiomyopathy (CCC) has not been elucidated. In this study, we investigated the presence of spontaneous and induced apoptosis in peripheral lymphocytes of 10 patients with CCC compared to 13 normal individuals. PBMC were studied, both freshly separated with Ficoll-Hypaque, and cultured (5 X 10⁶/ml) in vitro for 24 and 48 hours. Cells were also cultured in the presence of PHA (5µg/ml), anti-CD3 monoclonal antibody (200 ng/ml) or IL-2(10 U/ml). Apoptosis was evaluated by flow cytometry and incorporation of 7- amino-actimomycin D (7-AAD), a method previously described, which allows simultaneous measurement of apoptotic cells and surface phenotype characterization with either fluorescein-isothiocyonate (FITC) or phycoerythrine (PE). Apoptosis was also confirmed in some samples by DNA fragmentation in 2% agarose gel electrophoresis. We found no difference in the percentage of apoptosis in fresh cells between CCC patients and controls as well as in the other conditions. At 24 h, the percentage of apoptosis found in cells cultured without IL-2) was slightly higher among patients (mean=11.9, SD=9.7) than in the controls (mean=6.36, SD=5.7), but showed no statistic significance (p=0.09, Mann-Whitney Sum Test). Typical DNA ladder of 180 bp was seen in patients samples cultured with and without IL-2, for 48 h. Thus, contrary to the experimental model where apoptosis of CD4⁺ cells was reported, our preliminary data suggest that in CCC patients, this phenomenon is not predominant in the periphery. However, we cannot rule out the occurrence of apoptosis among heart-infiltrating T cells.

Supported by CNPq.

TNF-ALPHA RECEPTOR (P55-/-) AND IFN-GAMMA (IFN-G-/-) KNOCKOUT MICE SHOW REDUCED APOPTOSIS CELL DEATH IN THE ACUTE PHASE OF TRYPANOSOMA CRUZI INFECTION.

Martins, G. A.¹; Vieira, L. Q.², Gazzinelli, R. T. ² Machado, F. S. and Silva, J. S.¹. ¹Dept. of Immunology, FMRP, USP, Ribeirão Preto, SP, Brazil. ²Dept. of Biochemistry and Immunology, ICB, UFMG, BH, MG, Brazil.

The cytokines IFN-g and TNF-a participate in the regulation of immune response in mice acutely infected by T. cruzi, and induce nitric oxide (NO) production, which is important in parasite killing. In addition to its role in the trypanocidal macrophage activity, NO inhibits lymphoproliferative response to specific antigens and to mitogens in T. cruzi infected mice. Furthermore, we have previously shown that NO is involved as a mediator of apoptosis induction in splenocytes of mice acutely infected by T. cruzi. In an effort to better understand the mechanisms of apoptosis regulation during T. cruzi infection, and to evaluate the role of TNF-a and IFN-g in apoptosis modulation, C57Bl/6 (wild type, TNFRp55-/- or IFN-g-/-) mice were infected with T. cruzi (Y strain) and, on day 9 after infection, splenocytes were harvested and assayed for apoptosis, before or after 48 hours culture. In addition, NO production was measured in the culture supernatants. Flow cytometry (FACs) analysis using 7-AAD incorporation showed that while the percent of apoptosis was 16.3% in ex vivo infected wild type (wt) splenocytes (3 folds higher than wt normal mice), it was 9% and 8% in IFN-g-/- and TNFRp55-/- infected mice, respectively. Likewise, in splenocytes cultured for 48 hours the percentages of apoptosis was 54%, 19% and 24% in wt, IFN-g-/- and TNFRp55-/- infected mice, respectively. The control cultured splenocytes from normal wt mice showed 20% of apoptotic cells. NO production was significantly reduced in supernatants from infected knockout mice in comparison to supernatants of splenocytes from wt mice. This reduction was greater in cultures of splenocytes from IFN-g-/- than splenocytes from TNFRp55-/- infected mice. As reported previously, when splenocytes obtained from wt infected mice were cultivated in the presence of an NO synthesis inhibitor (LNMMA), there was significant reduction in apoptosis levels, although, it was still less pronounced then the reduction observed in splenocytes from both knockout mice. Together, these results suggest that, besides mediating apoptosis induction by stimulating NO production, IFN-g and TNF-a could themselves induce apoptosis cell death during the acute phase of experimental T. cruzi infection.

Supported by FAPESPand CNPq.

MODULATION OF CD4⁺ T CELL DEATH BY TYPE 1 CITOKINES AND TRYPANOSOMA CRUZI GLYCOINOSITOLPHOSPHOLIPID DURING EXPERIMENTAL CHAGAS' DISEASE.

Nascimento, D.O.; Lopes, M.F; Decoté-Ricardo, D.; Previato, J.O.; Mendonça-Previato, L.; DosReis, G.A.

Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, and Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ.

Activation-induced T cell death by apoptosis has been described in the course of experimental Chagas'disease induced by metacyclic forms of Trypanosoma cruzi, and has been indentified as a cause of immunosupression. However, modulation of apoptosis by parasite molecules and host citokines has not been investigated. The effects of T.cruzi -derived glicoinositolphospholipid (GIPL) and cytokines produced early in infection were investigated on CD4⁺ T cells isolated from infected mice. The addition of GIPL to CD4⁺ T cells from infected, but not control mice, resulted in cell death in culture. The cytokine rTNF-a had no direct effect on CD4⁺ T cell viability in culture, but completely prevented CD4⁺ T -cell activation-induced cell death, in the presence of TCR/CD3 crosslinking. Contrasting with these results, rIL-10 and rIL-1b had no effect on cell viability, and rINF-g induced cell death in T cells from infected, but not control mice, in the absence of TCR/CD3 ligation. Interestingly, cells stimulated with anti-CD3 and rTNF-a had no increase in proliferative response, compared to CD3-stimulated T cells only, suggesting that rescue from ativation-induced cell death (AICD) was followed by anergy induction.For comparison, cells stimulated with anti-CD3 and anti-FasL mAb had a marked increase in proliferation, besides a blockade of AICD. These data identify several mechanisms contributing to immunosupression observed in acute phase of Chagas'disease. The role of citokines and GIPL is being further investigated.

Supported by: PADCT / CNPq, PRONEX-MCT, CNPq, FINEP.

HOST MACROPHAGE APOPTOSIS SIGNALED BY TRYPANOSOMA CRUZI GLICO-INOSITOLPHOSPHOLIPID (GIPL) CERAMIDE DOMAIN.

Freire de Lima, C. G.;^1,2 Magarinos-Torres, R.;¹ Soares, M.;¹ Côrte-Real, S.;³ Meirelles, M. N.;³ Previato, J. O.;² Mendonça-Previato, L.² & DosReis, G. A.¹

¹Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho. ²Instituto de Microbiologia, Universidade Federal do Rio de Janeiro. ³DUBC-Instituto Oswaldo Cruz-FIOCRUZ, Rio de janeiro, RJ.

We have investigated the effects of Trypanosoma cruzi GIPL and its isolated carbohydrate and lipid moieties on murine peritoneal macrophages and J774 cell line. Purified GIPLs induced increased spreading and proliferation on the J774 cell line. Increased proliferation could be induced by the isolated GIPL oligosaccharide-PI, but only the isolated lipid moiety induced a dramatic increase in macrophage endocytic activity, further caracterized as fluid phase macropynocytosis. When added together with the cytokine rINF-g, the GIPL lipid moiety induced an intense cell death response. Identical results were obtained with rGM-CSF, but not with rIL-4, rIL-1b, rTNF-a or LPS/INF-g. Cell death was due to apoptosis, as assessed by transmission eletronic microscopy and DNA fragmentation analysis in agarose gels. The lipid moiety from T. cruzi GIPLs contains mainly N-lignoceroydihidrosphingosine. Synthetic C2-dihydrosphingosine in combination with rINF-g, also induced macrophage apoptosis. Both GIPL and the isolated oligosaccharide-PI induced limited IL-6, but not TNF-a secretion by macrophages. Only purified GIPL induced NO production. Isolated GIPL ceramide failed to elicit IL-6, TNF-a and NO secretion. These results caracterize a NO-independent signaling pathway of macrophage apoptosis triggered by a synergism between dihydroceramides and INF-g. This mechanism could be involved in parasite evasion from cellular response mediated by cytokine INF-g.

Supported by: PADCT/CNPq, PRONEX-MCT, CNPq, FINEP and FAPERJ.

PROSTAGLANDINS MEDIATE SUPPRESSION OF LYMPHOPROLIFERATION AND CYTOKINE SYNTHESIS DURING THE EARLY PHASE OF TRYPANOSOMA CRUZI INFECTION

Pinge Filho, P.¹, Tadokoro, C. E.², da Silva, U. R.² & Abrahamsohn, I. A.²

¹Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina, PR & ²Departamento de Imunologia, Instituto de Ciências Biomédicas, USP, Cidade Universitária, São Paulo, SP, 05508-900, Brasil.

Suppression of host lymphoproliferative responses to mitogens and antigens is one of the hallmarks of infection with the protozoan organism Trypanosoma cruzi. The cellular basis for immunosupression includes the generation of suppressor macrophages that down regulate T cell proliferative allied to insufficient IL-2 production. NO synthesized by splenic macrophages has been shown to be involved in macrophage-mediated suppression of lymphocyte proliferation in mice infected with T. cruzi. Since splenic macrophages from infected animals display activation characteristics, we have asked whether products of activated cells, specifically prostaglandins (PGE₂), may mediated the immunosupression observed. We examined here the role of prostaglandins and TNF as mediators of immunosuppression at different times of infection. Spleen cells cultures (SC) from infected mice were treated with: indomethacin (INDO - inhibitor of COX), anti-TNF or anti-IFN-g mAbs, NMLA (inhibitor of NO production) or combinations thereof. Cytokines in the supernatants were measured by two site sandwich ELISA assays. We found that days 6/7 post infection (PI) and on day 22 PI, but not on day 14 PI (maximum suppression phase), Ag-specific lymphocyte proliferative responses were increased by INDO treatment. However, on day 14 PI, increased proliferation inhibiting PG synthesis could be observed provided NO production was also concomitantly inhibited by NMLA. The combined addiction of INDO to anti-TNF or to NMLA further augmented Ag-specific proliferation early in the infection. Increase of IFN-g, IL-12 and IL-10 production was observed in INDO - or NMLA - treated SC from infected mice (6/7 and 22 PI). However, on day 14 PI, IL-2 production was not increased by either treatments. IL-12 levels were not modified by INDO or NMLA treatment. Treatment with COX inhibitor enhanced mortality rates of infected mice. Taken together, the results indicates that PG a important mediator of immunosuppression during the early and acute phases of infection and can also play a role in the resistance of infected mice. Thus, PG and TNF together with IFN-g, and NO are part of a complex circuit that controls lymphoproliferative responses in T. cruzi infections. Supported by FAPESP, CNPq and UEL.

IN VITRO STIMULATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM CHRONIC CHAGASIC PATIENTS WITH TRYPANOSOMA CRUZI-DERIVED ANTIGENS LEADS TO PREFERENTIAL EXPANSION OF T CELLS EXPRESSING TCR-Vb5REGIONS

Dutra, W.O.¹, Costa, R.P.², Gollob, K.J.³, Chaves, A.C.L.^2,3, Rocha, M.O.⁴, Colley, D.G⁵., Gazzinelli, G.² , Correa-Oliveira, R.², Carvalho-Parra, J.C.²

¹Depto. Morfologia, ICB, UFMG, CP 2486, Belo Horizonte, 30161-000, MG, Brazil; ²Centro de Pesquisas René Rachou, FIOCRUZ, CP 1743, Belo Horizonte, 30190-002, MG, Brazil; ³Depto. Bioquímica-Imunologia, ICB, UFMG, CP 2486, Belo Horizonte, 30161-000, MG, Brazil; ⁴Hospital das Clínicas, CTR-DIP, UFMG, Belo Horizonte, 30000, MG, Brazil; ⁵Centers for Disease Control, NCID, Atlanta, GA, USA.

Stimulation with dominant antigens or superantigens leads to preferential expantion of T cells bearing restricted Vb regions followed by a deletion of the chronic stimulated T cells. It has been shown that experimental T. cruzi infection leads to preferential expantion of polyclonally activated Vb8+ and Vb14+ T cells during the acute phase. In this work we analysed the usage of TCR-Vb regions by CD4 and CD8+ T cells from chronic chagasic patients using flow cytometry. We determined the Vb expression by T cells freshly isolated from patients and after in vitro stimulation with T. cruzi-derived antigens. Analysis of TCR-Vb region expression by T cells freshly isolated from the patients did not show any preferential usage or deletion of Vb regions as compared to normals. Chronic nature of the disease and the presence of other immune responses in the host can lead to "dilution" of the T. cruzi specific T cells. Thus, to reveal any possible skewed TCR-Vb region usage in response to T. cruzi antigens, we cultured PBMC from patients with epimastigote (EPI) or trypomastigote (TRP)-derived antigens. As a positive control, PBMC were cultured with S. aureus enterotoxin B (SEB). As expected, blast cells from indeterminate or cardiac chagasic patients cultured in presence of SEB showed a preferential expansion of Vb5 and Vb17-TCR regions as compared to control cultures. Noteably, analysis of TCR-Vb region usage by T cells stimulated in culture with EPI or TRP antigens showed a dramatic increase of Vb5 expressing T cells. However, no expansion was detected of T cells expressing Vb2, 3.1, 8 and 17. These data show that parasite-specific antigens stimulate preferentially a portion of the T cell repertoire with restrict use of the TCR-Vb5 region suggesting the presence of either a dominate peptide or of superantigenic activity in these parasite antigens. Supported by CNPq, NIH, FIOCRUZ.

T.CRUZI IMMUNODOMINANT T CELL PEPTIDE IS TAKEN UP BY HUMAN MONOCYTES / MACROPHAGES AND DRIVEN TO MIIC VESICLES ACCORDING TO ITS DIFFERENTIATION AND ACTIVATION STATE.

Ribeiro Gomes, F L¹, Carvalho, TMU²,Kipnis, TL¹ & Arnholdt, ACV¹.

¹Lab. Biol. do Reconhecere ² Lab de Biol. Cel. e Tecidual, CBB-UENF, Campos dos Goytacazes/RJ, Brazil.

A defined 33-mer peptide (P214) from the central domain of major T.cruzi cysteinyl proteinase (cruzipain, cruzain), within which a great sequence polymorphism has been observed, is capable of triggering the secretion of IFN-g from activated CD4+ human lymphocytes. We showed at least 2 patterns of recognition of this peptide by human T cells, usually connected to 14-15 a.a. long peptides from the N-term or C-term of P214, suggesting that this peptide could be taken up by antigen presenting cells and processed from 33-mer to small peptides and then associated to MHC molecules. In this report, we seek to investigate the pathways of uptake, processing and presentation of P214 by human macrophages in vitro. Confocal analysis showed that biotinilated P214 is taken up by 8 days culture, fully differentiated macrophages, and after a 3 hours pulse can be found inside cells, in MIIC vesicles, as well at cell surface, in association to MHC class II molecules. Preliminary results showed that at 1h already the MIIC pattern is observed, with vesicles around nucleus. A 2 h pulse with Brefeldin A does not alter this, suggesting that the association of P214 occur with newly synthesized MHC class II molecules and not resulting of cell surface recycling. Flow citometry analysis reinforce these data, since macrophages do not express P214 at cell surface, besides high MHC class II density through out the entire population. In contrast, 24h in vitro cultured monocytes bind P214 at cell surface and two subpopulation can be observed (CD14⁺HLA^-P214⁺ and CD14⁺HLA⁺P214^-), suggesting a distinct pattern of internalization according to their differentiation and cell activation state, as demonstrated by HLA-DR,DQ,DR expression. Early macropinocytosis/endocytosis kinetics are under investigation.

Supported by CNPq, FENORTE and PRONEX.

FLOW CYTOMETRY ANALYSIS OF PERIPHERAL BLOOD FROM CHAGASIC PATIENTS TREATED DURING THE ACUTE PHASE OF CHAGAS'DISEASE

Gomes, J.A.S.¹; Bahia-Oliveira, L.M.G.²; Lemos, E.M.¹; Luz, Z.M.P.³; Pereira, M.E.S.³;Gazzinelli,G.³; Cançado, J.R.⁴; Moreira, M.C.V.⁴ &Correa-Oliveira,R³.

1-Deparatamento de Bioquímica e Imunologia, ICB-UFMG, Belo Horizonte, MG; 2- UENF, CBB, LBR, Campos dos Goytacazes, RJ; 3- Centro de Pesquisas René Rachou- FIOCRUZ, Belo Horizonte, MG; 4- Faculdade de Medicina, UFMG, Belo Horizonte, MG.

Chagas' disease, caused by Trypanosoma cruzi, is a major public health problem in Latin America. It is estimated that treatment during the acute phase leads to about 50-70% of cure, however, if the treatment is applied during the chronic phase the percentage of cure decreases to about 7%. We examined the Peripheral blood mononuclear cells (PBMC) phenotype of 26 patients treated 15 years ago during the acute phase of the infection with nitroheterocyclic drug (RochaganÒ) by flow cytometry before (ex vivo) and after antigenic stimulation with antigens derived from Trypanosoma cruzi. According to the efficacy of the treatment the patients were classified as cured (C) when parasitological (hemoculture) and serological (conventional and lysis mediated by complement - LMC) tests were negative for Chagas' disease, as Dissociated (D) when parasitological and serological conventional tests were negative but LMC was positive for Chagas' disease, as Not Cured (NC) when parasitological and serological tests were positive for Chagas' disease. The ex vivo analysis demonstrated that C, D and NC patients have similar percentages of total CD4+, CD8+ and CD19+ cells with to Non infected normal individuals (NI). However, the percentage of CD3+/HLA-DR+ lymphocytes was significantly higher among NC patients when compared with C, D and NI groups. These data are in accordance with those demonstrating that chronically infected chagasic patients have significantly higher percentage of HLA-DR+ cells (Dutra et al 1995). After antigenic stimulation we observed that C, D and NC groups had small lymphocytes expressing significantly higher levels of HLA-DR than NI group. CD8+/HLA-DR+ cells were higher in the gate of Lymphoblast from NC when compared to C and D patients. Our data suggest that the specific immune response of lymphocytes from NC individuals are dependent on cytotoxic T cells, however, the role of these cells in the context of Chagas' disease still needs further investigation.

Supported by: CNPq , NIH A126505 and PAPES-FIOCRUZ.

FLOW CYTOMETRY ANALISIS OF CORD BLOOD FROM NEWBORNS OF CHAGASIC MOTHERS SHOW AN INCREASE IN CD4+/CD45 R0 + CELLS

Suzane Figueiredo Neves*, Roger Ramos**,Simone Rigueirinho**,Giovanni Gazzinelli*, Silvana Elói-Santos** e Rodrigo Correa-Oliveira*

* Centro de Pesquisas René Rachou- FIOCRUZ,Belo Horizonte, MG

** Faculdade de Medicina da UFMG, Belo Horizonte, MG

Chagas Disease is an infection caused by the protozoan Trypanosoma cruzi and is one of the major health problems in Latin America. Previous studies concerning newborns of chagasic mothers showed the occurrence of antigenic and idiotypic sensitization in utero. It has been postulated that the available idiotypic repertoire of an adult is a result of influences from early signals that have reached the immune system as it develops. These signals comprise auto-antigens, external antigens that incidentally cross the placental barrier and regulatory idiotypes expressed on maternal imunoglobulins. These signals would deviate initial connections leading to different available repertoires in each individual. Based on this hypothesis, we investigated the phenotypic characteristics of lymphocytes from cord blood of newborns of chagasic and non-infected mothers with the objective to identify differences on cell activation. Cord blood cells were analyzed by flow cytometry using monoclonal antibodies to different surface markers ( CD3, CD19, CD4, CD8), and state of cell activation ( HLA-DR, CD5, CD45RA e CD45R0 ). Our results show a significant increase in the CD4+/CD45R0+ sub-population (p<0,01 ) in newborns of chagasic mothers when compared to those born of non- infected mothers. Other findings include a decrease on CD3+ cells and increase on the expression of HLA-DR on the CD8+ population. Since congenital infection did not occur in these newborns, we suhggesy that the observed alterations are a consequence of maternal influences during pregnancy.

Supported by: CNPq, PAPES/FIOCRUZ and NIH AI26505

FLOW CYTOMETRY ANALYSIS OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH PROLONGED ACUTE PHASE OF TOXOPLASMOSIS.

Araujo L.C.¹, Martins-Filho, O.A. ², Kanashiro, M.M.¹, Gama, L.M.¹, Kipnis, T.L. ¹, Gazzinell,G.³;Artiles, N.F.¹, Correa-Oliveira, R. ³ & Bahia-Oliveira, L.M.G¹.

1-UENF, CBB, LBR, Campos dos Goytacazes, RJ; 2- Deparatamento de Bioquímica e Imunologia, ICB-UFMG, Belo Horizonte, MG;3- Centro de Pesquisas René Rachou- FIOCRUZ, Belo Horizonte, MG.

Flow cytometry analysis of Peripheral Blood Mononuclear Cells (PBMC)from patients with acute phase of toxoplasmosis in patients from Campos dos Goytacazes (north of Rio de Janeiro state) was performed. Our data demonstrate that in general the percentage of cell surface markers expressed by PBMC from these patients are comparable to those of Non-infected normal individuals (NI). The only difference observed was on the percentage of T cells expressing the gd TCR, we observed a higher percentage of gd TCR T cells in Acute patients (AC) with an average of 20 times over that of NI. Patients with persistent high levels of IgM in their sera and with lymphoadenopathy (individuals with prolonged acute phase) had higher percentages of gd T cells which has been significantly and consistently elevated as observed in evaluations at 6 and 15 months after chemotherapy. The functional characterization of gd T cells is being investigated to clarify the role of these cells on the acute symptomathology of the patients.

Support by: FENORTE, CNPq/ FINEP- 1282/94 (PRONEX).

IMMUNE - CELL FACTORS OF THE SEVERE CHRONIC CHAGASIC HEART DISEASE

Cabral, H.R.A. & Novak I.T.C.

Histology I y II, Faculty of Medicine, National University of Cordoba, Agency Postal 4, ZIP Code 5000, Cordoba, Argentina. Supported by CONICOR and SECyT, UNC, Argentina.

In studies on hearts of patients with chronic chagasic heart disease (ChHD) we identified three factors. They are: a)PAS+ T-lymphocytes (whose presence is prevalent between the intracardiac infiltrate cells); b)Mastocytes, whose number is increased in the chronic chagasic hearts; c)Microvessels with high endothelium like that exists, normally, in the lymphatic tissues (Cabral HRA, Novak ITC y Robert GB, Rev Argentina Cardiol, 61 (5): 463-465, 1993. Cabral HRA and Novak ITC, Mem Inst Oswaldo Cruz, Rio de Janeiro, vol 89, Suppl 1, p 132, 1994). The constant occurrence and the respective distribution associated with lesions suggest that they are important in the pathogenesis of ChHD. We have studied such aspects in 14 cases of patients who died of severe ChHD. Sections of heart samples were submitted to histochemical and immunohistochemical procedures with monoclonal antibodies for lymphocytes characterization and for vascular endothelium. Results: The PAS+ lymphocytes reacted with monoclonal for CD4 receptor and for activated T lymphocytes. They were found in sites of active damage of cardiac myocytes and, also, in surrounding neural intracardiac structures (which were recognized morphologically and by its positivity for neuron specific enolase monoclonal antibodies); and in areas of collagen increased deposition. Their number was greater than 65 % of the intracardiac infiltrated cells. With respect to the mastocytes these cells were found in number of 2 to 8 per high power field at 400 x, that is, four to ten fold over the number of cardiac mastocytes in normal hearts. They were found contacting cardiac muscle fibers and microphotographs were obtained showing secretion of substances of the mastocytes toward the cardiomyocytes. Besides, mastocytes were present in areas of active deposition of collagen, near of fibroblasts. High endothelium microvessels were seen near of cardiomyocytes in some collagen areas. They reacted with CD31 antibodies and contained T-lymphocytes which reacted with their respective monoclonal antibodies. A functional relationship between the above described cellular and microvascular factors in the production of lesions of chagasic heart disease is postulated.

PRESENCE OF ANTIBODIES AGAINST CENTRAL NERVOUS SYSTEM COMPNENT DURING EXPERIMENTAL CHAGAS'INFECTION

Silva, AA, Roffê, E, Marino, APMP, Alves-dos-Santos, PV, Torres, RA & lannes-Vieria

Department of lmmunology, Institute Oswaldo Cruz - Fiocruz, Av. Brasil, 4365, 21045-900, Rio de Janeiro, Brazil.

During Chagas' infection damage in central nervous system (CNS) is more severe in children under 2-years and immunosuppressed patients. Rare amastigote forms of the T cruzi and inflammatory infiltrates with variable intensity and irregular distribution are localizei within the CNS during the acute phase of the disease. Also, lymphocytes and clrculating antibodles specific for neurons and myelin basic protein (NMP) have been detected in humans and experimental animais suggesting the participation of immune system in the genesis of CNS lesions. However, some questions regarding the participation of antibodies against CNS components in the tissue damage during Chagas' disease remain unsolved. To shed light on these questions experimental Chagas' infection was induced in C3H/He mice using Colombian strain of T. cruzi. These animais exhibited peak of parasitemia 42 days post infection and showed several pathological alterations in CNS such as edema, enlargement of perivascular spaces and inflammatory infiltrates during the acute phase. These inflammatory lesions were not related to the presence of T cruzi antlgens, that were immunohistochemically characterized as isolated positive cells scattered throughtout the parenchyma. Hjgh levels of circulating antibodies recognizing NMP and its encephalitogenic fragment comprising the 4-14 aminoacid sequence were detected 28 days post-infection, contrasting to low levels of antibodies against T criízi antigens and without evidence of polyclonal B cell activation. During the late acute phase and the chronic infection the levels of anti-MBP antibodies remained high and paralleled the levels of total lgG and antibodies against T cruzi. lnterestingly, when sera of infected mice were incubed with normal brain sections we noticed that the white matter was specifically recognized. Taken together these results suggest an important role of antibodies recognlzing CNS components ln the development of pathological alterations observed during acute infection. This possibility is presente under investigation.

Sponsored by: CNPQ, Capes, IOC-Fiocruz.

[e-mail]

ENDOTHELIAL CELL INTERACTIONS WITH TRYPANOSOMA CRUZI CYSTEINE PROTEINASES: POSSIBLE ROLE IN MICROVASCULAR PATHOLOGY

Schmitz,V., Morrot, A., Morandi, V., Colonese, A., Leal, L.R. and Scharfstein, J. Instituto de Biofísica Carlos Chagas Filho-UFRJ e UERJ, Dept. Biologia Celular.

In the past few years, the introduction of sensitive immunohistochemical methods in the analysis of myocardial specimens has validated the concept that parasite factors may play a role in the pathogenesis of chronic chagasic myocardiopathy. In this context, the finding of antigenic depots of extracellular cruzipain (the major cysteine proteinase of T. cruzi) in cardiac inflammatory foci (1) merits further investigation. Encoded by a multiple polymorphic genes, cruzipain (and related isoforms) generally induce potent humoral and cellular immune responses in chagasic patients (2). The amastigote-cruzipain(s) are likely released into interstitial spaces when the parasitized myofibers rupture. Stable and active at a broad pH range, these multifunctional proteinases may promote myocardial damage because they (i) degrade ECM components (submitted) (ii) stimulate TH1 CD4 cells, causing release of inflammatory cytokines (2) (iii) process kininogens (precursor of bradykinin), thereby releasing pro-inflammatory kinins (3) and (iii) promote drastic increases in the permeability of post-capillary venules(4). We now report that cruzipain(s) antigens can bind tightly and selectively to the surface of endothelial cells (HUVEC) in vitro, this interaction being promptly followed by host cell sensitization with anti-cruzipain IgG (human) antibodies. Our studies suggest that soluble cruzipain can be efficiently focused to the luminal surfaces of vascular endothelial cells, thus rendering the microvasculature sensitive to antibody-dependent mechanisms of cytotoxicity. Direct evidences for the participation of Fc-dependent mechanisms of tissue injury are being presently sought.

1. Morrot et al., 1997. Intern. Immunol., vol 9, n^o 6, pp. 825-834.

2. Arnhodlt et al., 1993. J. Immunol., 151:3171.

3. Del Nery et al., 1997. J. Biol. Chemistry (in press)

4. Svensjo et al., 1997. Microvascular Research (in press)

Supported by CNPQ.

Abstract not received.

ANTI-HUMAN ENDOTHELIAL CELL ANTIBODIES IN CHAGAS DISEASE

Carlos Aita, Lucia C. J. Abel, Mônica Spadafora-Ferreira, Verônica Coelho, Edécio Cunha-Neto and Jorge Kalil

Laboratory of Transplantation Immunology, Heart Institute, Faculty of Medicine, University of São Paulo

Autoimmunity and microvascular lesions have been incriminated as possible pathogenic factors for chronic Chagas' disease cardiomiopathy. Antibodies detectable by immunofluorescence against rodent endothelial cells, with specificity to the a1,3-galactosyl epitope, have been previously described in sera from Chagasic patients (so-called EVI antibodies). However, such antibodies did not bind detectably to human endothelial cells, which are devoid of the a1,3-galactosyl epitope due to lack of the functional enzyme (a1,3-galactosyl-transferase). We have studied plasma samples from CCC, ASY and normal individuals for reactivity to human umbilical vein endothelial cells (HUVEC) by flow cytometry, with FITC conjugated anti-human polyvalent Ig, or with anti-hIgG FITC + anti-hIgM PE. All CCC and ASY samples were reactive, but none of the controls. Reactivity to endothelial cells was not due to anti-HLA antibodies, as sera were not reactive to T lymphocytes from the same donors from HUVEC. Inhibition assay was performed with a-D-galactopyranoside, N-acethyl-D-galactosamine, a-methyl-mannopyranoside and L-galactose (at a final concentration of 25 mM). Results showed that the endothelial cell reactivity of plasma samples from chagasic patients and affinity purified anti-Gal antibodies obtained from pooled chagasic sera, were completely inhibited with a-D-galactopyranoside, but not with a-methyl-mannopyranoside and L-galactose. Surprisingly, affinity purified anti-Gal antibodies obtained from pooled normal sera were not reactive to endothelial cells. A polyreactive anti-HLA serum was reactive, but not inhibited by any of the above monossacharides. Results seem to indicate that an epitope present in human endothelial cells, in molecular mimicry with a1,3-galactosyl epitopes, is recognized by low affinity antibodies present in all chagasic sera, irrespective of the clinical form. This may indicate that the generation of anti-endothelial cell antibodies is associated with T. cruzi infection per se rather than to a possible microvascular damage in CCC. We are currently working on the characterization of the antigens recognized by these antibodies.

CHRONIC CHAGASIC PATIENTS' ANTIBODIES DISPLAY MULTIPLE MECHANISMS OF ACTION IN ISOLATED RABBIT HEARTS

Costa, PCS, Oliveria, SF, Almeida NAS, Pedrosa, RC, Masuda & Campos de Carvalho, AC

Chagasic patients' sera contain antibodies that recognize muscarinic and adrenergic receptors in the heart. We recently reported that sera from a group of these patients, those with complex cardiac arrhythmias, block AV conduction and decrease beat rate in a model of isolated rabbit heart (Farias de Oliveira et ai., Circulation 1997), and we atributed this effect to activation of cardiac muscarininc receptors by the antibodles. To further examine this question we have tested sera from 17 chronic chagasic patients in isolated rabbit hearts in the presence of the muscarinic receptor antagonist atropine. Hearts were perfused with normal Tyrode solution containing 10-6 M atropine, followed by perfusion with the same solution containing serum from the patients diluted I:IOO (v:v) and then a return to the initial solution. ECGs were monitored throughout the perfusion periods, each of which lasted for 30 min. Analysis of the ECGs of the atropinized hearts showed that beat rate was increased by 4 sera, decreased by 5 sera and not altered by the remaining 8 sera. AV conduction was improved by I serum, prolonged or blocked by 9 sera and not altered by 7 sera. We conclude that chagasic sera are indeed able to activate muscarinc receptors ln the heart as evidenced by the lack of effect of about half the sera in atropinized hearts (these sera were previously shown to reduce beat rate and block AV conduction), but this is not the sole mechanism by which the sera exert their effect, since about 30% of sera still decreased beat rate and 50% blocked AV conduction in the presence of atropine. Additionnally, atropinization unmasked activation of adrenergic recepetors by a small percentage of the sera.

Financial Support: FAPERJ, CNPQ, Finep, Pronex

GENERATION OF ANTIBODIES WITH ADRENERGIC STIMULATING ACTIVITY IN CRONIC CHAGAS HEART DISEASE.

Ines Ferrari^*, Dan Kaplan^*, Evelina Mahler^*, Pablo Lopez Bergami^*, Sergio Ghio^*, Francisco Quintana^*, Gabriela Levitus^*,Pablo Chiale^†, Johan Hoebeke^‡, Marc H.V. Van Regenmortel^§ and Mariano J. Levin^*.

*Lab. de Biología Molecular de la Enfermedad de Chagas, INGEBI-CONICET, FCEN-UBA and ^†División Cardiología, Htal. Ramos Mejía, Buenos Aires, Argentina; ^‡Lab. d'Enzymologie et de Chimie de Protéins, URA1334 CNRS, Tours, and ^§Institut de Biologie Moléculaire et Cellulaire CNRS, Strasbourg, France.

[e-mail]

We have previously suggested that antibodies (Ab) directed against the ribosomal P0 protein of Trypanosoma cruzi cross-react with the b1 adrenergic receptor (b1AR) (Ferrari et al, J. Exp. Med., 1995, 182: 59-65 and Elies et al, J. Immunol., 1996, 157: 4230-4211). The target of this cross-reaction was mapped to a region possessing a short stretch of negatively charged residues present in the C-terminal end of the parasite protein (AESEE) and in the second extracellular loop of the b1AR (AESDE).

In this report, we analyze the functional autoreactive properties of Ab directed against T. cruzi ribosomal P1 and P2 proteins in chronic Chagas heart disease (cChHD). These Ab, known as anti-P Ab, present in sera from patients with cChHD recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the T. cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the C-terminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P auto Ab in systemic lupus erythematosus (SLE), and with the acidic epitope, of the b1AR. Anti-P Ab from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P auto Ab from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi quantitative estimation of the binding of cChHD anti-P Ab to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P Ab bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the b1AR. Anti-P Ab isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-b1AR Ab isolated from the same patient. In contrast, SLE anti-P auto Ab have no functional effect. These results suggest that the adrenergic stimulating activity of anti-P Ab may be implicated in the pathogenesis of myocardial impairments observed in cChHD.

ANTIBODIES WITH CHOLINERGIC EFFECT IN CHAGASIC AND IDIOPATHIC SINUS NODE DYSFUNCTION

Evelyn Mahler*, Inés Ferrari*^¶, Marcelo V. Elizari^¶, Mauricio B. Rosenbaum^¶, Pablo A. Chiale^¶, Mariano J. Levin*.

*laboratorio de Biología Molecular de na Enfermedad de Chagas INGEBI-CONICET, FCEN-UBA and ^¶División Cardiología, Hospital Ramos Mejía, Buenos Aires, Argentina. [e-mail]

Functionally active antibodies directed against membrane cell receptors play a main role in the pathogenesis of several diseases. As sera from patients with sinus node dysfunction may react with the membrane of the human sinus node cells, it was hypothesised that the sinus node might be the target of an immune response. The aim of our study was to comparatively assess the prevalence of antibodies with cholinergic stimulating effect in patients with and without sinus node dysfunction.

The chronotropic activity of IgG (alone and after addition of atropine) from 9 patients with sinus node dysfunction (7 with Chagas´ heart disease and 2 with structurally normal heart), 12 patients with normal sinus node function (7 with Chagas´ heart disease and 5 with idiopathic dilated cardiomyopathy), and 5 healthy individuals was studied on spontaneously beating cultured neonatal rat cardiomyocytes.

The prevalence of positive functional tests for cholinergic receptor stimulating antibodies was significantly higher in patients with sinus node dysfunction compared with those having a normal sinus node function (7/9: 77,7% Vs 2/17: 11,7%; p < 0.01). A consistent and ostensible negative chronotropic action which was antagonised by atropine was observed in 5 out of the 9 patients with sinus node dysfunction (3 chagasic and 2 idiopathic cases), and was never seen in patients with normal sinus node function. Furthermore, IgG from patients with normal sinus node function, either chagasic or idiopathic dilated ones, had a positive chronotropic action (11/12 cases) caused by anti-b-adrenergic receptor antibodies.

Our findings show for the first time a strong link between chagasic or idiopathic sinus node dysfunction and antibodies with cholinergic stimulating effect. This raises the possibility that these antibodies may play a role in the pathogenesis of clinical sinus node dysfunction through their potential depressing effect on sinus node activity.

EFFECTS OF SERUM OF CHRONIC CHAGASIC PATIENTS ON TENSION DEVELOPMENT BY RABBIT ATRIUM MYOCARDIUM

*Leite, C.M.; #Pedrosa, R.; ‡Campos de Carvalho, A.C. & ‡Masuda, M.O. *Depto de C. Fisiológicas, CBM, UFES, Vitória, ES. # Hospital Universitário Clementino Fraga Filho, CCS, UFRJ, Riode Janeiro, RJ. ‡Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro, RJ.

Autoimmune mechanisms have long been considered to contribute to the cardiac damage occuring in the chronic phase of Chagas' disease. Some authors suggest that IgG is involved in the generation of cardiac electrical disturbances observed in a number of chronic chagasic patients by interacting with muscarinic receptors. In order to investigate the possible involvement of antibodies in cardiac mechanical disturbances that are also observed in some chagasic patients, we decided, as a first approach, to test the effects of the total serum (TS) from 6 patients with chagasic myocardiopathy, 3 with complex cardiac arrhythmias (ChAI), detected by standard electrocardiography, and 3 without arrhythmias (ChI), on tension development by isolated atrium myocardium. The results were compared with the effects of the TS from 3 non-infected individuals without cardiovascular dysfunctions (NI). Left atrium strips from New Zeland rabbits were superfused with Tyrode solution at 30-32 ºC, aerated with a mixture of 95% O₂ + 5% CO₂ and stimulated with suprathreshold square pulses (8 V, 8 ms) at 1 Hz. Cummulative dose-response curves (0,03% to 1,0% , v/v) were obtained for TS of the 3 ChAI . The TS of these individuals was shown to induce atropine inhibitable AV blockade (AVB) in isolated rabbit heart and we observed that they were also able to reduce the tension developed by the atrial myocardium in a dose-dependent manner. Atropine (10 mM) added to the superfusion medium did not abolished the tension reduction. The serum of one ChI also reduced tension development. The other 2 ChI, as well the TS of the 3 NI, did not significantly change the contractile force. In conclusion, serum from chronic chagasic patients presenting cardiac arrhythmias produce a negative inotropic effect in the isolated myocardium in addition to inducing AVB. The mechanisms of these two effects are probably diverse since atropine did not prevent the negative inotropic effect while prevented the AVB.

CALCIUM CURRENNT ALTERATIONS IN ISOLATED RABBIT CARDIAC MYOCYTES TREATED WITH SERA FROM CHRONIC CHAGASIC PATIENTS

Barcellos, LC; Pedrosa, RC; Campos de Carvalho, AC & Masuda, MO

Instituto de Biofisica Carlos Chagas Filho. UFRJ, RJ.

lnteraction between sera of chronic chagasic patients and muscarinic and O adrenergic receptors has been demonstrated by several laboratories. This interaction may be due to molecular mimicry between parasite ribosomal proteins and the second extracelular loop of G-protein coupled receptors. Recently Farias de Oliveira et.al. (Circulation, 1997), demonstrated that sera from chronic chagasic patients, can induced conduction block and decrease in heart rate in isolated rabbit hearts and this effect was abolished by atropine. Activation of muscarinic receptor by these sera should result, among other things, in a reduction in calcium currents through the cardiac myocyte's sarcolema. To test this we dissociated ventricular myocytes from rabbit hearts and analyzed them by whole cell patch clamp technique. To isolate the calcium currents we used Tyrode solution containing (mM) NaCl 150.8, KCI 2.7, MgCl O.5, Glucose 6.0, HEPES 10.0, CaCl2 2.7 with the potassium blocker 4- Aminopyridine (2 mM), and a pipette solution containing (mM), CsCl 120. NaCl, 20, CaCl2 O.5, HEPES 10, TEA-CI 20. lsoproterenol (IO -9 M) was added to the bath to increase the current. After control records in Tyrode we perfused the cells with the same solution containing sera from chronic chagasic patients at a dilution of I:IOO (v:v), for a period of 5 min before returning to tyrode. Our results show marked reduction in calcium currents amplitude (l221 ± 131 pA to 809±137 pA, n=4, p<O.OOl) during perfusion with the sera. Perfusion with normal sera did not effect the calcium current. This date are compatible with the effect of sera from chronic chagasic patients being mediated through activation of muscarinic receptors.

Financial Supports by CNPQ, finep, faperj, Pronex

PRESENCE OF ANTIBODIES AGAINST HUMAN AND MOUSE LYMPHOCYTES IN THE SERA OF PATIENTS WITH CHRONIC CHAGAS DISEASE.

Fernandez-Ferreira, E.*, Soares R.O.A.*, Pirmez, C** & Ribeiro-dos-Santos, R*.

* Applied Pharmacology, Far-Manguinhos-FIOCRUZ, Sizenando Nabuco 100, CEP 21041-250, Rio de Janeiro, RJ; ** Immunopathology Laboratory, DBBM, IOC - FIOCRUZ

Patients with Chagas'disease develop antibodies against several mouse tissues and cells such as blood vessels, endocardium, peripheral nerves and lymphocytes. We have previously shown that sera from chagasic patients recognise 70% of CD4⁺ and 20% of CD8⁺ mouse T- cells subpopulations.

Material and Results: Sera from chagasic and normal subjects were absorbed with antigens obtained from normal mouse tissues (liver and cardiac muscle), and antigens from T. cruzi epimastigote forms. After this procedure, total mouse spleenocytes were incubated with sera from chagasic or normal individuals pre-absorbed or not. The specific biding of those sera to the different T-cell populations were analysed by FACS. Our results demonstrated that sera from patients absorbed with both antigens recognised 52% of CD4+ and 17% of CD8+ T-cells, while pre-absorbed control sera did not recognised these cells. Next, we evaluate the sera reactivity against soluble antigens from mouse spleen or human peripheral T-cells. Analysis by immunoblotting demonstrated that patients' sera react with several bands present in soluble antigens from mouse spleen. The approximated molecular weight were 37, 42, 45, 50 and 55 kDa. These sera also recognised bands with mw of 45 and 55 kDa from soluble antigens of human peripheral T-cells. Taken together, these results suggest that sera from chagasic patients specifically reacts with conserved epitopes present in mouse and human T-cells.

CHRONIC CHAGASIC MYOCARDITIS INDUCED BY TRYPANOSOMA CRUZI ANTIGEN HOLD IN SILICONE CAPSULES.

Barreto, JR; Castro, DA; Leite, HG; Sadigursky, M.

Faculdade de Medicina -UFBa. and CPGM-FIOCRUZ. Salvador-Bahia.

Many different mechanisms have been offered for the pathogenesis of chronic Chagasic myocarditis. Carlos Chagas was the first that suspected of an immunologic mechanism as the responsible for the inflammatory process. Nowadays many evidences point out for an autoimmune mechanism as the responsible for the myocarditis that occur in the cronic form of Chagas's disease. One of the possible mechanism that trigger an autoimmunity, the cross-reactive antigens have been related. An experimental demonstration of a cronic myocarditis induced by the antigens in the absence of parasites would add subsidies for the autoimmune hypothesis. This work had the objective of checking the truthfull of the hypotesis, exposing mice to T. Cruzi antigens trap in capsules of slow elimination of antigens.

It was used 10 test mice and 10 control mice of AKR strain. In test group it was implanted silicone capsules with T. Cruzi antigens, and in control group it was implanted silicone capsules with saline. All the animals were monitorized with electrocardiographic tracings before the implantation of the capsules and periodically after. After 6 months the mice were sacrificed and segments of their main organs were processed for hystophatological study

The hystophatological study showed that 12,5% of the animals had generalized multifocal myocarditis with a large area of infiltration in the left ventricle and 87,5% showed a mild myocarditis in sparse focus. The control animals did not have electrocardiographic and histologic alterations.

The results showed that is possible to induce the myocarditis only with the parasite antigens, helping the autoimmunity theory. We describe also an excellent experimental model of autoimmune disease.

CHAGAS' DISEASE: ADOPTIVE TRANSFER OF THE DAMAGE INDUCED BY CRUZIPAIN. CHARACTERIZATION OF A CROSSREACTIVE SELF-ANTIGEN.

Giordanengo L, *Rivarola W, Motran C, **Iosa D, Vottero-Cima E, Gea S. Inmunología, Fac. C. Químicas, U.N.C.*Física Biomédica, Fac. C. Médicas, U.N.C.**Centro Priv. de Med., Córdoba, ARGENTINA. Fax [phone].

Previously, we demonstrated that the inmunization of mice with cruzipain is able to induce autoimmune response against self-antigens as skeletal muscle (SM) and heart (H). Moreover, electromiographics and electrocardiographics alterations were also detected. The goal of the current study was to investigate whether the adoptive transfer of spleen mononuclear cells (SMC) from cruzipain immunized mice to naive receptors is able to induce the electrocardiographic alterations observed in the donors. The characterization of the SM antigens recognized by the anti-cruzipain antibodies was also done. BALB/c mice were i.d. immunized with cruzipain on days 0, 14 and 28 in complete Freund adjuvant (CFA). Control mice were injected with CFA. The adoptive transfer studies were performed as follows: SMC were obtained from immune and control mice on day 12 after the last injection and they were in vitro activated with cruzipain during 96 hours. Three naive mice received 1.3 x 107 activated cells from immune donors and two animals received the same number of cells from the controls. Besides, two receptors were injected with 0.6 x 107 immune cells without activation. Both, the activated and non-activated immune cells were capable to transfer the electrocardiographics alterations, registered on day 8 and 27 after the transfer. The alterations more frequently detected were bradycardia, intraventricular conduction disturbances, and in some cases, left anterior hemiblock. These results suggest that the cruzipain immune cells can play a role in the pathogenesis of Chagas' disease.

For the.characterization of the SM antigens recognized by the anti-cruzipain antibodies, sera from immune mice obtained 30 days after the last immunization were assayed by western blot against myosin rich soluble fraction of mouse skeletal muscle. A band of 210 kDa was observed. The band was identified as the myosin heavy chain when the reactivity of immune sera was assayed using myosin from rabbit skeletal muscle.

PRESENCE OF CD4+ CD69+ T CELLS IN HEART MONONUCLEAR INFLAMMATORY CELLS FROM BALB/C MICE CHRONICALLY INFECTED WITH T. CRUZI.

Soares, R.O.A*; Mengel, J.O **; Henriques, M.G.M*& Ribeiro-dos-Santos, R*

* Applied Pharmacology, Far-Manguinhos-FIOCRUZ, Applied Pharmacology, Far-Manguinhos-FIOCRUZ, Rua Sizenando Nabuco 100, CEP 21041-250, Rio de Janeiro, RJ; ** Department of Immunology, ICB, USP

In order to evaluate the presence of different cell phenotypes in the heart lesions of chronically T. cruzi infected mice, we developed a new technique. Chronically infected (after 8 months of infection with 100 parasites of Colombian strain) BALB/c mice were killed and their hearts were extensively perfused with PBS to remove the remaining blood. Hearts were cut in small fragments and passed through a metal mesh. Single cell suspensions were resuspended in 80% Percoll, followed by the addition of a second layer of 40% Percoll. The gradients were centrifuged and cells obtained from the 40-80% Percoll interface were stained with different combinations of monoclonal antibodies to be used for further FACS analysis (FacsScalibur B&D). The results showed that the majority of the cells presented in the heart inflammatory infiltrates are ab⁺ T cells (48.1%). The relative proportion of CD4 and CD8 inside the ab+ T cell population was 29 and 54%, respectively. The relative proportion of double negative (CD4- CD8-) inside the ab⁺ T cells was, approximately, 15%. Of more importance was the fact that 22.6% of the CD4⁺ population and 41.7% of the CD8⁺ T cells were CD69⁺. IgM⁺ cells correspond to 16.4% of the total lymphocyte gated population. This cell preparation also contained 33% of MAC1⁺ IA⁺ cells and 3.6% of gd⁺ T lymphocytes. We conclude that primarily T cells of both, CD4 and CD8 phenotype compose the heart inflammatory infiltrate found during the chronic phase of the T. cruzi infection, with the Colombian strain. Furthermore, there are strong indications that a significant part of these T cells are recently activated in the heart, but not in peripheral lymphoid tissues, since CD4⁺ and CD8⁺ T cells from blood, spleen and lymphonodes from infected animals are virtually negative for the CD69 marker. Altogether these data suggest that a significant proportion of CD4⁺ and CD8⁺ T cells are activated in the heart by antigens other then the ones derived from T. cruzi, possibly by heart tissue-specific self antigens.

Partial Support by CNPq

BALB/C MICE CHRONICALLY INFECTED WITH T. CRUZI SHOW A MARKEDLY INCREASE OF XENOGENEIC AND HALOGENEIC SPECIFIC RESPONSE AGAINST HEART.

Ribeiro-dos-Santos,R.; Soares, R.O.A; Violante,F.A.; Haussmann, M.E.F.; Gibaldi, D. & Henriques, M.G.M.

Applied Pharmacology, Far-Manguinhos-FIOCRUZ, Rua Sizenando Nabuco 100, CEP 21041-250, Rio de Janeiro, RJ, Brasil.

In the present study we characterize xenogenic and halogeneic responses of T lymphocytes from BALB/c mice chronically infected with T. cruzi (Colombian strain-100 parasites). Primary cultures of heart myoblast, skin and liver fibroblasts obtained from newborn Wistar rats or fetal C57Bl/B6 mice (ED-18) were co-cultivated with total spleen T lymphocytes, CD4⁺ or CD8⁺ T cells of BALB/c mice chronically infected with T. cruzi. As antigens presenting cells we used irradiated spleenocytes from normal BALB/c mice. These co-cultures were accomplished for 5 days, when it was made the tymidin incorporation [metil-3H] during12 hours. Culture cells were collected with cell harvester (Filtermate 196-Packard) and the reading was performed in equipment Beta-Matrix 9600 (Packard). As control we used lymphocytes from normal non infected BALB/c mice. Furthermore, mixed culture of lymphocytes from normal or chronically infected BALB/c was achieved in the presence of C57BL/B6 or Wistar rats lymphocytes. The results show that total T or CD4⁺ lymphocytes from chronically infected animals co-cultivated in presence of halogeneic or xenogeneic heart presented as much as 5 to 6 times more tymidin incorporation than in the presence of halogeneic or xenogeneic fibroblasts from skin or liver. Total lymphocytes obtained from normal BALB/c mice or CD8⁺ T cells from chronically infected mice did not present significant differences in the halogeneic or xenogeneic responses to primary culture of heart myoblasts or to fibroblasts. Finally, the mixed culture of lymphocytes also did not show significant differences between the groups of chronically infected and normal mice. The results suggest that, in our model, autoreactivity against heart antigens in chronic chagasic animals is dependent of CD4⁺ T cells, and is direct to epitopes preserved in different strains and species.

Partial Support by CNPq.

ABSENCE OF TRYPANOSOMA CRUZI IN REJECTED SYNGENIC HEART TRANSPLANTS OF CHRONICALLY INFECTED MICE.

Bozza, M., Soares, R.O.A., Fernandez-Ferreira, E., Stutz, C., & Ribeiro-dos-Santos, R.

Applied Pharmacology, Far-Manguinhos, FIOCRUZ, Rua Sizenado Nabuco 100, CEP 21041-250, Rio de Janeiro, RJ, Brasil.

We have previously shown that mice chronically infected with T. cruzi (Colombiana strain) reject syngenic newborn heart transplants into the pinna of the ear (Ribeiro-dos-Santos et al., J. Exp.Med 175:29, 1992). Rejection was dependent on autoreactive CD4⁺ T cells, since in vivo treatement with anti-CD4 but not anti-CD8 mAb abrogated rejection, and transfer of spleen CD4⁺ but not CD8⁺ T cells from chronically infected mice induced graft rejection on normal recipient mice. Contrary to our results, it has been recently demonstrated long term engraft of syngenic heart transplants into mice chronically infected with Sylvio or Y strains of T. cruzi (Tarleton et al., Proc. Natl. Acad. Sci. USA 94:3932, 1997). In this study, parasitization of heart tissues was nescessary and sufficient for the induction of inflammatory response, tissue damage and graft rejection. In order to characterize the presence of parasites in the model of rejection of mice chronically infected with Colombiana strain, we used a PCR technique to amplify T. cruzi kDNA, followed by southern blot hybridization using kDNA as a probe. As expected, normal syngenic newborn heart transplants engrafted on normal DBA-2j mice (beating after 1 week), but were rejected on chronically infected mice. No PCR amplified product were detected in the hole transplant tissues from normal or chronically infected mice. Furthermore, no specific PCR product was detected on 10⁵ spleen T cells from chronically infected mice, despite the ability of those cells to transfer rejection of transplanted heart tissues into normal mice. It should be mentioned that the PCR technique used was capable of detecting up to one parasite in the same number of cells. These results strongly suggest that in the model of chronic infection with the Colombiana strain rejection of syngenic heart transplants is independent of the presence of parasites.

Partial Support by CNPq

CYTOTOXIC T CELL ACTIVATION AND CHAGASIC MYOCARDITIS DEVELOPMENT

Marques, C.S., Henriques Pons, A. & Araújo-Jorge, T.C.

Lab. Biologia Celular, DUBC, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.

Chagas' disease or American trypanossomiasis is caused by the hemoflagellate Trypanosoma cruzi. In the acute phase of this disease many organs are heavily parasitized particularly the heart. A massive inflammatory infiltrate is locally found in the myocardium which plays an important role in the pathogenesis of heart failure in chronic chagasic disease. However the cell subtypes and mechanisms responsible for this myocytolytic necrosis are poorly understood. Some evidences suggest that this pathology is based on cellular immune responses.

In the present study we have used cytotoxicity assays to address the kinetics of T cell activation in the spleen of acutely infected mice C57/Bl6 and the correlation with chagasic myocarditis development in vivo after 8, 15, 22 days of infection. Spleenic T cell obtained from mice infected with 10⁴ trypomastigote forms from T. cruzi Y strain were used for the cytotoxicity assays. The cell were placed in the presence of syngeneic infected or non infected myocyte culture for 16 hours. Concomitantly the serum of the mice were collected for posterior follow up of the myocarditis damage. The experimental readings of both in vitro and in vivo approaches were based in the relative activity of creatine kinase (CK) released from the myocytes.

Our preliminary data suggest that, although there is a detectable myocarditis necrosis on the 8^th day post infection, there is a significant T cell population specifically activated against T. cruzi epitopes in the spleen, and that this T cell compartment migrates from this lymphoid tissue before 15 days of infection. Probably the infected heart is one of the major target organs of this immune response. In addition, we are collecting tissue specimens for hystopathological analysis and comparison with our data about myocarditis development based in CK activity.

Supported by CNPq and FIOCRUZ

USE OF A RECOMBINANT L. (L.) AMAZONENSIS AMASTIGOTES ANTIGEN IN LYMPHOPROLIFERATIVE ASSAYS AND ACTIVE IMMUNIZATION OF BALB/c MICE.

Beyrodt, C.G.P., Silveira, J.F. & Barbiéri, C.L.

Disciplina de Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, Caixa Postal 20.342, São Paulo, 04023-062, SP, Brasil

Immunization by use of Leishmania purified antigens has been performed with susceptible and resistant mice resulting in significative protection. The protection conferred by recombinant antigens has also been tested and mice immunized with Salmonella expressing gp63 were partially protected against L. (L.) major challenge (Yang et al. 1990, J. Immunol. 145, 2281.). It was also demonstrated that native and recombinant gp63 induce proliferation of lymphocytes from patients with cutaneous leishmaniasis (Mendonça et al. 1991, Clin. Exp. Immunol. 83, 472). Vaccinia virus and BCG expressing genes encoding L. (L.) amazonensis proteins have also successfully used in vaccination schedules of mice against homologous infection (Connell et al. 1993, Proc. Natl. Acad. Sci. 90, 11473; McMahon-Pratt et al. 1993, Infect. Immun. 61, 3351; Xu and Liew 1993, Immunology 84, 173).

An antigen of 30 kDa from amastigote forms of L. (L.) amazonensis was characterized and showed to be able to induce lymphoproliferative responses in BALB/c mice mediated by CD4⁺ Th1. The antigen presents cysteine proteinase activity and conferred partial protection against homologous infection (Beyrodt et al. 1997, Infect. Immun. 65, 2.052). In order to clone and sequence the gene encoding the p30, a fragment of 500 bp was amplified by PCR by using genomic DNA of L. (L.) amazonensis amastigotes and a pair of primers derived from evolutionary conserved active sites of Dictyostelium discoideum cysteine proteinase. After cloning and amplification the 500 bp fragment was also subcloned in pGEX expression vector in phase with glutathione S-transferase (GST) gene, resulting one fusion protein of 47 kDa. This recombinant protein has been tested in both lymphoproliferative assays of BALB/c mice and active immunization of animals followed by challenge with L. (L.) amazonensis.

The aim of our work is to compare the ability of the native and recombinant p30 to induce lymphoproliferative responses in vitro as well as to protect BALB/c mice against L. (L.) amazonensis infection.

Supported by CNPq/PADCT and FAPESP

IMMUNIZATION WITH DNA CONTAINING THE SEQUENCE CODING FOR 82 KDA PROTEIN PRESENT AT THE SURFACE OF TRYPANOSOMA CRUZI METACYCLIC TRYPOMASTIGOTES

Boscardin, S.B., Santori, F., Ramirez, M.I., Yoshida, N. & Franco da Silveira, J.

Depto de Microbiologia, Imunologia e Parasitologia, UNIFESP, Escola Paulista de Medicina, Rua Botucatu ,862, CEP: 04023-062, SP - Brasil.

The metacyclic forms of T. cruzi express a surface glycoprotein of 82 kDa (gp82) that is involved in the penetration of parasites into the host cells. A cDNA recombinant clone carrying the complete open reading frame (ORF) of gp82 gene has been cloned by Araya et al (MBP, 65:161, 1994). Immunization with recombinant gp82 protected mice against the challenge with metacyclic forms of T. cruzi. To determine whether similar protective immune response could be achieved by immunization with DNA, the ORF of gp82 gene was subcloned in the mammalian expression vector pcDNA3 under the control of cytomegalovirus promoter. This construction was called pc-gp82. Expression of gp82 by pc-gp82 was detected in transiently transfected VERO cells using specific monoclonal antibody (mAb) named 3F6.

Groups of BALB/c mice were immunized with plasmid pc-gp82 or with non recombinant plasmid pcDNA3, as a negative control. Each mouse received four doses of 100mg of DNA intramuscularly in the tibialis anterioris, at 3 week intervals. Muscles were treated or not with cardiotoxin a week before immunization. Significant differences in anti-gp82 antibody titers were detected in mice immunized with pc-gp82 when compared with controls immunized with pcDNA3. No significant differences were observed between mice treated or not with cardiotoxin. Each group of mice (n=5) was challenged with 10⁵ metacyclic forms of T. cruzi CL strain. Our results suggest that a partial protection against acute phase infection can be achieved.

In an attempt to improve the gp82 antigen presentation to the immune system, we inserted a signal sequence in phase with the ORF of gp82 gene creating a new construction named pc-gp82+PS. VERO cells were transiently transfected with this construction and the expression of gp82 was ascertained. BALB/c mice are being immunized with pc-gp82+PS, following the same protocols described above, and will be challenged two week after the last immunizing dose.

Supported by FAPESP, CNPq/PADCT, FINEP/BID.

IMMUNOGENIC PROPERTIES OF THE GENE SEGMENT ENCODING THE C-TERMINAL REGION OF THE MEROZOITE SURFACE PROTEIN 1 OF PLASMODIUM VIVAX.

Oliveira C.I.§, Soares I.*, Levitus, G.^@., Wunderlich, G§., Rodrigues M.M.* and del Portillo H.A.§

§ICB II, Universidade de São Paulo, São Paulo, 05508900, SP, Brasil. *Universidade Federal de São Paulo,04023062, São Paulo, SP, Brasil. ^@Instituto de Ingenieria Genética y Biología Molecular, Buenos Aires, Argentina.

Nucleic acid imunization has become a valuable tool in the development of vaccines against a variety of bacterial, viral and parasitic diseases, including malaria. We have previously shown that some segments of the Merozoite Surface Protein 1 gene of P.vivax (PvMSP1) are highly immunogenic after genetic immunization of BALB/c mice, while other segments are not (XII Reunião Anual da SBPz, Abstract # 51). Strikingly, the gene segment coding for the C-terminus of PvMSP1 was among the gene segments unable to elicit immune responses in the immunized mice and yet it codes for the most immunogenic portion of this molecule in natural infections (Soares et al, Inf. Immun., 65:1606-1614, 1997). Moreover, immunization of monkeys with the PvMSP1 C-terminus produced in baculo-virus protected the animals upon challenge (John Barnwell, NYU Medical Center, pers. comm.). Due to the importance of this portion of PvMSP1 in vaccine development, we are therefore trying to render this gene segment immunogenic through three different approaches: 1. The recombinant plasmid encoding the PvMSP1 C-terminus was co-injected into Balb/c mice with a recombinant plasmid encoding the murine granulocyte macrophace colony stimulating factor. 2. This gene segment was cloned into a new expression vector that provides a signal peptide sequence that should facilitate the secretion of the translated product and consequently the elicitation of humoral immune responses. 3. This gene segment was ligated to the 5'-end of the gene encoding the hepatitis B viral surface antigen, a methodology that has been shown to increase the immunogenicity of nucleic acids in other systems. Results on the humoral and cellular immune responses of mice genetically immunized with all these recombinant plasmids will be presented. Supported by FAPESP 95/1796-3.

PRE SEQUENCES OF HBV GENOME AMPLIFY HUMORAL IMMUNRESPONSES OF IN CIS CLONED GENES ADMINISTERED AS DNA VACCINES

G. Wunderlich*^,$, F.T.M. Costa^#, M.M. Rodrigues^#, I. Raw*

*Dep. of Biotecnology, Instituto Butantan, Av. Vital Brasil 1500,

São Paulo-SP 05504-900

#Escola Paulista de Medicina, UNIFESP, R. Botucatu, 862, São Paulo 04023-062

$ present address: ICB2, USP, Av. Lineu Prestes, 1374, São Paulo-SP, 05508-900

DNA vaccines are a novel tool in vaccinology and are currently being tested in several clinical trials against infectious diseases like AIDS and HBV among others. Nevertheless, the efficiency of gene transfer into the potential expressing tissue is still very low. In an attempt to enhance the efficacy of DNA vaccines, we tested the PRE-sequence of HBV, described recently as an enhancer of expression by facilitating the export of unspliced RNA transcripts into the cytosol. During experiments, Balb/C mice were vaccinated intramuscularly or intradermally with a CMV-promoter plasmid construct containing the HBV-S gene or the CS gene of Plasmodium falciparum, followed by PRE sequences or not.

After analysis of the humoral immune response by ELISA, an at least twofold increase of antibody titers against the constructs was found in mice vaccinated with constructs containing the PRE sequences, independent of the gene used for vaccination, or the site of injection of the DNA solution. Results show that the PRE sequences of HBV function as a general enhancer module, acting in the natural context of the HBV genome as well as in non-homologous systems. Moreover, the activity of this element is not limited to a certain tissue such as primate kidney or human liver cells, because similar results were found in both ways of vaccine application (intradermal or intramuscular). Currently, experiments are under way to show if the PRE-sequence is a more potent activator of expression than the Intron A sequence which drives otherwise unspliced RNAs into the splicing apparatus of the nucleus, thus facilitating export of the corresponding RNA.

This project was sponsored by CAPES DCI/1146-14/95

FLOW CYTOMETRY ANALYSIS OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH OCULAR TOXOPLASMOSIS.

Araujo L.C.¹, Kanashiro, M.M.¹, Martins-Filho, O.A. ², Gama, L.M.¹, Kipnis, T.L. ¹, Gazzinell,G.³; Artiles, N.F.¹, Correa-Oliveira, R. ³ & Bahia-Oliveira, L.M.G¹.

1-UENF, CBB, LBR, Campos dos Goytacazes, RJ; 2- Deparatamento de Bioquímica e Imunologia, ICB-UFMG, Belo Horizonte, MG;3- Centro de Pesquisas René Rachou- FIOCRUZ, Belo Horizonte, MG.

Ocular form of Toxoplasmosis in Campos dos Goytacazes (north of Rio de Janeiro State) has reached an alarming prevalence. Flow cytometry analysis of Peripheral Blood Mononuclear Cells (PBMC) from patients with ocular disease was undertaken in order to analyze the cellular populations present in the peripheral blood of infected patients with ocular toxoplasmosis and to evaluate its possible relationship with this clinical form of the disease. A comparative analysis was also performed with seropositive patients without ocular lesions. We identified two groups of patients: one presenting only one recent episode of ocular disease (group I) and a second presenting several recent episodes of ocular symptomathology (group II). We observed that patients belonging to group I had similar profile of cell surface markers as Non infected individuals (NI). Some patients belonging to the group II but none of group I had CD8 T cells expressing IL-2 receptor (CD25). Follow up of patients from the group I and group II will be of great value to clarify the significance of CD8+/CD25+ T cells on the pathogenesis of ocular Toxoplasmosis.

Support by: FENORTE, CNPq/ FINEP- 1282/94(PRONEX).

A PREVIOUS INFECTION WITH TOXOPLASMA GONDII PROTECTS BALB/C MICE AGAINST INFECTION WITH LEISHMANIA MAJOR

Helton C. Santiago*, M. A. P. Oliveira*, A. M. C. Faria, E. Bambirra*, R. T. Gazzinelli*, L. Q. Vieira*

Departamento de Bioquímica e Immunologia, ICB e Departamento de Patologia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.

Simultaneous infection with different parasites is a common occurrence in medical practice. However, few studies have been done addressing this important issue. In order to investigate the effect of a previous infection on the development of a subsequent infection with a different parasite, we chose two highly prevalent parasites: Toxoplasma gondii, the ethiological agent of toxoplasmosis, which may reach 90% prevalence in European countries, and Leishmania major, the ethiological agent of cutaneous leishmaniasis in the Old World. In mice, L. major may trigger a Th1 response in resistant mice such as C57BL/6, characterized by the production of high levels of IL-12, IFN-g and IL-2 and low levels of IL-4 and IL-5. On the other hand, susceptible mice such as BALB/c develop a Th2 response with production of IL-4, IL-5 and IL-10 and downregulation of the production of IL-12 and IFN-g. Moreover, the first few days after infection are fundamental in the choice between a protective response and a response that will lead to susceptibility. Since T. gondii will induce a type 1 response in the L. major-susceptible BALB/c mouse strain, we hypothesized that these mice, if previously infected with T. gondii, would develop benign lesions in response to L. major and eventually heal, due to the type 1 environment provided by T. gondii. Therefore, we infected BALB/c mice with T. gondii and challenged with L. major 5 days or 7 weeks later. Our results show that BALB/c mice developed small lesions that eventually healed in both cases. However, lesions were significantly smaller when mice were infected with L. major during the acute phase of toxoplasmosis (5 days) than during the chronic phase (7 weeks). In addition, mice previously infected with T. gondii presented a Th1-type response to L. major with the production of higher levels of IFN-g and IgG2a, and lower levels of IL-4 and IgG1, when compared to mice infected solely with L. major. Histopathological analysis of the site of infection with L. major showed that there was a smaller cellular infiltrate and fewer parasites in the group infected with both L. major and T. gondii if compared with infection with just L. major. These results indicate that the environment in which and infection occurs may alter significantly its outcome.

Supported by FAPEMIG, CAPES and CNPq

*Fellows of CNPq

LOW FREQUENCIES OF CELLULAR PROLIFERATIVE RESPONSES AND INTERFERON-GAMMA (IFN-G) PRODUCTION INDUCED BY PLASMODIUM VIVAX ANTIGENS IN INDIVIDUALS EXPOSED TO MALARIA IN ENDEMIC AREA IN BRAZIL

Braga, EM¹, Carvalho,LH, Fontes, CJF² & Krettli, AU¹

Centro de Pesquisas René Rachou, FIOCRUZ, Minas Gerais, ¹ Departamento de Parasitologia, Universidade Federal de Minas Gerais; ² Universidade Federal de Mato Grosso

Studies on the mechanisms of immunity to Plasmodium falciparum clinical malaria naturally acquired in hiper and holoendemics areas in Africa and Asia suggest that after life-long exposure, immunity gradually develops via both humoral and cellular immune mechanisms. In Brazil, since the transmission and morbidity are unstable, and since P. vivax is responsible for about 60% of the half million malaria cases reported annually from this country , studies are required to clarify whether resistance against the infection is being developed. Our present goal is to evaluate cellular (in vitro proliferation and g-IFN production) responses to P. vivax recombinant proteins of sporozoites (circumsporozoite protein-rCSPv) and assexual blood stage antigen (Pv200) which are potential vaccine candidates, in a malaria endemic population living in the north of Mato Grosso state for about 11 years. The PBMC's were obtained from 74 residents of an endemic malaria area in Mato Grosso State (average residence time in the area = 11 years), including: (A) convalescents who had more than 10 episodes of clinical malaria (poly-infected, n=48); (B)convalescents from a single episode of clinical malaria (primo-infected, n=8); (C) subjects with no previous malaria (n=18); and controls (D) living outside the endemic area (n=16). Their PBMC's were stimulated with rPvCS, rPv200 or the mitogen (PHA). The lymphoproliferative assay to both P. vivax antigens showed that the responses were very low in all groups (£12%). The IFN-g was measured in supernatants using a two-site ELISA assay. Values above 200pg/ml were considered positive. The induction of IFN-g production was also low. Thus, IFN-g was induced by rPvCS or rPv200 respectively, in 25% or 19%, of the poly-infected group; up to 13% of responders among groups B and C; in none of group D. In contrast, PHA induced high levels of IFN-g in most subjects tested (72%). We concluded that PBMC's from individuals exposed for many years in areas of unstable transmission of malaria in Brazil are able to proliferate and produce IFN-g but their responses to recombinant P. vivax antigens was specifically depressed.

Supported by CNPq, FAPEMIG, CAPES

IMMUNOLOGICAL STUDIES IN MICE INFECTETED WITH NEOSPORA CANINUM.

Carvalho L.J.M ¹; Gonchigoogin B.²; Igarashi I.²; Satoma H.²; Daniel-Ribeiro C.T.¹; Ferreira-da-Cruz M.F.¹; Ishii H.²; Saito A.²; Toyoda Y.²; Nagasawa H.²; Dubey J.P.³ and Suzuki N.^2,4

¹Laboratory on Malaria Research, Department of Imunology, IOC-Fiocruz-Rio de Janeiro, Brazil; ²The Research Center for Protozoa Molecular Immunology, Obihiro, University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japão, ³USDA, ⁴Kitassato University.

Neospora caninum is a recently recognized coccidian protozoan which infects dogs, cattle and other animals. Until 1988, it was misdiagnosed as Toxoplasma gondii. Since it has been only recently identified, there are not many data yet about this parasite, and host immunological responses to Neospora are still poorly understood. We studied several aspects of the immunological responses of mice during infection by N. caninum . When BALB/c mice were infected with N. caninum tachyzoites, they developed splenomegaly and high titers of serum anti- Neospora IgG. Giemsa-stained sections of spleen showed that there was strong activation in the B-cell and also in the T-cell regions, specially 1 week after infection, and despite decreasing afterwards it remained until 4 weeks after infection, when was a predominance of differentiation over proliferation. Flow cytometry studies reveled that during the infection there was a percentual increase in the splenic B-cell population and a parallel decrease in the T-cell population due to a drop in both CD4+ and CD8+ subsets. Studies on cytokine expression using PCR did not allow us to define patterns in IL-2, IL-4, IL-10, IL-12. IFN-g and TNF-a expression during infection. We were not able to detect N. caninum tachyzoites or cysts in brain, lungs and spleen up to 4 weeks of infection. Three and 4 week-infected mouse spleen cells responded to in vitro stimulation by soluble Neospora (Nc)-Antigen, which showed also mitogenic activity by stimulating proliferation of normal mouse spleen cells, although in a weaker way as compared to infected mice. Altogheter, these results showed that BALB/c mice respond strongly to infection by N. caninum and this seemed to be due mainly to reaction in B-cell compartment, caused by both specific and mitogenic stimulations.

CRYPTOSPORIDIOSIS AND ISOSPORIDIOSIS IN HIV-INFECTED PATIENTS IN THE REGION OF RIBEIRÃO PRETO, SP.

Capuano, D.M., Okino,M.H.T., Bettini,M.J.C.B., Souza,C.A.& Ferreira,C.G.

Instituto Adolfo Lutz - Lab.I de Ribeirão Preto, Rua Minas, 877 - Ribeirão Preto-SP - CEP 14085-410.

Ribeirão Preto city, that is about 329 Km north of the capital of São Paulo State, represents an important center for a region in service of infected patients with the human immunodeficiency virus (HIV). Among the enteric pathogens that attack these patients are opportunistic protozoans Cryptosporidium sp and Isospora belli. The purpose of this investigation was to establish the frequency of cryptosporidiosis and isosporidiosis in HIV positive patients in Ribeirão Preto region. From April 1990 to June 1997 in Adolfo Lutz Institute, 3372 stool samples were examined from 1862 symptomatic patients or not, with the ages between 10 months and 55, attended in Ribeirão Preto AIDS Outpatient Departments. For the detection of these pathogens was employed the formalin-ether method in stool specimens, followed by modified Ziehl-Neelsen stain. The frequency of these protozoan oocysts among all the examined stool samples were 9,2%. We observed 207 (11,1%) patients with these protozoan parasites. Cryptosporidium sp was identified in 117 (6,3%) patients and Isospora belli in 81 (4,3%) patients. Nine (0,5%) patients were infected by both parasites. Cryptosporidium sp and Isospora belli have been recently recognized as enteric pathogens. They have been associated as common causes of diarrhoea that is self-limited in patients with normal immune function but can be chronic in immunocompromised patients, accompanied by several dehydration, malabsorption and wasting. In conclusion, we hope that our study shows the importance of realization of routine examination for these protozoan oocystis in stools from HIV positives patients, no matter if they are symptomatic or not.

IMMUNE RESPONSE TO TOXOPLASMA GONDII TACHYZOITES SUBMITTED TO ⁶⁰COBALT IRRADIATION, FORMALDEHYDE AND CHEMOTHERAPY

Hiramoto, RM*, Almeida, BSV; Alves, IC 7 ANDRADE Jr., HF

Lab.Protozoologia - Instituto de Medicina Tropical de São Paulo - FMUSP and *Superv.Radiobiologia/IPENICNEN-SP Av.Dr.E.C.Aguiar, 470 - 05403-000 - São Paulo, SP, Brazil

Toxoplasnta gondii, an Apicomplexa parasitic protozoa, affects warm blood aninials and man, usuahy with few symptoms, except in antenatal infections or in immunossupressedldeficient patients. Despite those few symptoms in most infected persons, some groups at risk could be beneflt of a sterjle vaccine, especially seronegative transplant patients at risk of organ infection. Most vaccine attempts deal with attenuatcd strains, that could be not used in immunosupressed patients. Gamma radiation was frequently used as a tool in the production of immunogens, especially when viability wàthout reproduction is expected, vvith good results, mainly in schistosonúasis. Scveral reports dealing with cysts and tachyzoites irradiation had shown that the parasite could be lcilled at low doses, but no s@ was conducted to the biochemical alterations and immune response against irradiated Tgondii tachyzoites. Here, we present preliminary results of gamma radiation on the parasitos, and the antibody and delayed hipersensitivity rcsponse of ísogenic núce challenged m4th irradiated parasitos, comparing these results with formolized parasite challcnge or carly treatmcnt ofacute infection. Peritoneal washing tachyzoites from mice infected with RH strain were treated with 200 G y 6OCo radiation and used as irradiated parasites. Protozoa maintains its viability higher than 90% aftcr irradiation, in a usual TP~ Blue assay, wlthout any physiological dcath as radio induced apoptosis, as detected by TUNEL assay, vvith an electron niicroscopic usual appearance. When IO' irradiated parasitos wcre injected in mice, those are no dcaths or other disease symptoms nor tachyzoites couldbe found in peritoneal washing after three days. The induced immune response of this challenge was compared with mice injected with formolized tachyzoites or wlth mice infected with naive parasites and immediately effectively treated with sulfadoxine and pyrimetamine. We compare the antibody response, as specific anti Tgondii IgG detection in an ELISA assay using ~ine Tgondii extract as coat, in those three groups of mice, with the greater response seen in the infectcd-treated group, with similar, but not so high levels in irradiated challenged mice. Formolized parasitos failed in induction of detectable antibody. We also test the qualitv of thc antibody response bv Westem Blot with similar few bands labeled by abs from infected-treated and irradiatcd challenged nuce. We also testcd the dclayed hypersensitivity by- the paw edema assay in those three groups, using few animais. showing that significant response could be dctected in the three groups, with clear paw edema formation, presenting high levels in the infcctcdltreated and, curiously, also in the formolized challenged group. Those preliminary results shows that gamma rachation induces some modífications in the reproduction of tachyzoites of Tgondii and also affects the inunune response to the agent, as show by the diverse antibodv and delayed hypersensitivity results. Vaccine development against Tgondii must consider all of those immune aspects in order to provide a sterile and effective immunogen.

Thís work was supported by FAPESP n 96/5875-8 and 96101763-O(BSVA), and LIMHCFMUSP-49. RMIH is a fellow of CNPQ.

TRYPANOSOMA CRUZI IN PHILANDER OPOSSUM AND DIDELPHIS MARSUPIALIS (MARSUPIALIA, DIDELPHIDAE): A COMPARATIVE STUDY OF HUMORAL IMMUNE RESPONSE IN NATURAL AND EXPERIMENTAL INFECTIONS.

Legey, A.P.¹, Xavier, S.C.C.¹, Leon, L.L.²& Jansen, A.M.¹

(1) Laboratório de Biologia de Tripanosomatídeos, Departamento de Protozoologia, (2) Núcleo de Bioquímica de Tripanosomatídeos, Departamento de Imunologia. Instituto Oswaldo Cruz, Caixa Postal 926, 21045-900, RJ, Brasil.

Knowing that Philander opossum and Didelphis marsupialis (Marsupialia, Didelphidae) maintain the parasitism by Trypanosoma cruzi without apparent disease or any important tissular lesion , we decided to follow up the humoral immune response of naturally and experimentally infected didelphids and also evaluated their humoral immune response to different immunization routes (intradermic, subcutaneous and intraperitoneal) with T. cruzi antigens. It was observed that both didelphids showed high serological titers to the different immunization routes. Serological titers of naturally infected P. opossum displayed a significant individual variation, while those of D. marsupialis remained stable during all the following up. The serological titers of the experimentally infected animals varied according to the inoculated strain. P. opossum sera recognized more antigens than D. marsupialis. Moreover, recognition pattern did not show any change during the whole experiment. A peptide of 66 kDa was the most prominent antigen recognized on the soluble and enriched membrane fractions, by both, naturally and experimentally infected didelphids. Another peptide (90 kDa) was strongly recognized on the epimastigotes soluble and enriched membrane fractions, by experimentally infected P. opossum and D. marsupialis. Our data demostrate that: (i) the didelphid marsupials are able to built up an efficient humoral immune response as compared to placentals mammals, (ii) in spite of both didelphids have presented high imunoglobulins titers to T. cruzi antigens, P. opossum recognized intensivelly more peptides than D. marsupialis, (iii) the differences in the serological titers in natural and experimental infections may be explained by the interaction of P. opossum with different types of subpopulations of T. cruzi, since P. opossum are "biologic filters" less restrict than D. marsupialis, (iv) probably the 66 kDa peptide must be a transialidase enzyme, however we need some additional data to confirm this statement , (v) the early recognition of all antigens could be significant in the maintenance of this well balanced host-parasite interaction.

Furthermore, our results allow us to say that the didelphids P. opossum and D. marsupialis must have been infected by T. cruzi after their divergence, since these phylogenetically close related species selected distinct strategies to successfully handle the parasitism by T. cruzi.

Supported by FIOCRUZ/ FAPERJ/CNPq/PAPES

EFFECTS OF OVARIECTOMY AND ORCHIECTOMY ON THE HUMORAL RESPONSE OF CALOMYS CALLOSUS INOCULATED WITH THE Y STRAIN OF T. CRUZI.

Prado Jr., JC^1,2, Levy, A.M.A³, Leal, M. P.² & Kloetzel, J.K,^2,4..1-Dept. Parasitologia, Faculdade de Ciências Farmacêuticas USP Ribeirão Preto, Av. do Café s/n - Ribeirão Preto, 14049.903 Brasil. 2-Instituto de Medicina Tropical S.Paulo 3-Instituto Dante Pazzanese de Cardiologia 4-Dept.Parasitologia, Instituto de Ciências Biomédicas USP

The wild rodent Calomys callosus, a natural reservoir of T.cruzi, is being used as a resistant model of Chagas'disease. Y strain infected animals overcome parasitemia, with no mortality. We have also shown that female C.callosus are more resistant to the infection than males, and that castration inverts the patterns of resistance of both sexes. Here we study the effects of ovariectomy and orchiectomy on the humoral response of C.callosus. Groups of male and female C callosus were divided into O = operated, Sham=S (simulated operation) and C =C (controls). Approximately 30 days after surgery, all animals were inoculated i.p. with 4x10³ blood trypomastigotes of the Y strain. On days 9, 12, 30and 45after infection, blood was collected from 4 or 5 animals of each group and samples were individually tested by complement mediated lysis. Lysis was assessed by counting motile parasites at time 0 and 60 min. after adding complement to a suspension of bloodstream trypomastigotes and sera. A higher than 30% lysis rate was considered positive. At the peak of parasitemia (7^th or 9^th day) similar lysis rates (30%-40%) were observed in animals of all groups. On the 30^th day, when parasitemia is sub-patent, lysis of C and S females was negative (9-18%), while that of the O group was positive (35%). On this day high lysis rates (70%) were obtained for C and S males, although those from the O group were also positive, but to a significantly lesser degree (44% lysis). On the 45^th day lysis rates of all groups of both sexes were negative. Lytic antibodies are known to be stimulated only by live trypomastigotes. We therefore conclude that the lower rates of lysis on the 30th day after infection, of the more susceptible groups of animals, indicates a longer persistence circulating parasites, thus stimulating higher of antibody levels.

Financed by CNPq, FAPESP and LIM 49 HC.

T CELL RECOGNITION OF RECOMBINANT B13 PROTEIN FROM T. CRUZI: HLA RESTRICTION AND EPITOPE MAPPING.

¹Abel LCJ, ¹Goldberg AC, ¹Fae K, Ianni B, ¹Mady C, ²Hammer J, ²Sinigaglia F, ²Gallazzi F, ²Raddrizanni L, ²Bono E, ¹Kalil J and ¹Cunha-Neto E.- ¹Instituto do Coração, HC-Faculdade de Medicina USP, São Paulo, Brazil and ²Roche Milano Ricerche, Milano, Italy

Evidence from the literature suggests the participation of molecular mimicry in pathogenesis of Chronic Chagas' disease Cardiomyopathy (CCC). Our group identified T cell clones from peripheral blood and heart infiltrate crossreactively recognizing cardiac myosin and the immunodominant tandemly repeated recombinant protein B13 from T. cruzi. We studied the HLA-DR and HLA-DQ profile of Chagasic patients (asymptomatic ASY and CCC) and normal individuals (N) showing PBMC proliferative responses to B13 protein. Furthermore, B13-derived synthetic peptides were subject to HLA-binding assays to the HLA-DR alleles corresponding to: DR1, DR2,DR51, DR3 (17), DR4, DR5 (11), DR7 and DR8: and HLA-DQA1*0501/DQB1*0301. Results showed that B13-derived peptides bound detectably to DR1, DR51 and HLA DQA1*0501/DQB1*0301 molecules. Analysing the HLA-DR/DQ profile of responder individuals, we found that 25/26 responder individuals carry at least one of such B13-binding HLA-class II alleles. When ranked according to HLA alleles, responder individuals were 17/27 (63%) among HLA- DQA1*0501 carriers, 8/15 (53%) among DR51 carriers and, 6/16 (38%) among DR1 carriers. This association between HLA profile and T cell response to B13 was conserved in the three clinical groups tested. It is of note that three HLA alleles were equally distributed among clinical groups; ca. 80% of individuals in the CCC, ASY or N population are carriers of at least one of the alleles. Employing overlaping synthetic peptides covering the B13 tandemly repeated sequences (incluing the degenerate sequences) it was found that the 10-mer FGQAAAGDKP was recognized as the minimal epitope by a B13-specific T cell line, presented by HLA-DQA*0501/DQB1*0301. These results may indicate that T cell recognition of B13 alone may be a necessary but not sufficient "checkpoint" for the development of CCC. Furthermore, mapping of the MHC/T cell receptor contacts on the B13 T cell epitope reported here may lead to identification of the B13-crossreactive T cell epitope in cardiac myosin.

Supported by FAPESP,CNPq, Roche, HHMI..

LACK OF ASSOCIATION BETWEEN HLA CLASS II ALLELES AND CHAGAS' DISEASE CARDIOMYOPATHY

Faé, K.C., Cunha-Neto, E., Drigo, S., Chiarella,J.M., Ianni, B., Mady, C., Kalil, J., Goldberg, A.C. Lab. Imunologia de Transplantes, Instituto do Coração, Universidade de São Paulo, São Paulo, SP.

It is believed that autoimune mechanisms may participate in the pathogenesis of Chronic cardiomyopathy (CCC). T. cruzi antigens and cardiac tissue proteins have been shown to crossreact both at the antibody and T cell levels. Only 30% of the patients develop cardiomyopathy, while the others remain asymptomatic. Together with familial studies that indicate a genetic component in the differential susceptibility to CCC, data suggest that immunogenetic factors may influence CCC pathogenesis. Considering the central role of the MHC (Major Histocompatibility Complex) molecules in the immune response, we typed HLA class II antigens in patients with different clinical forms of the disease. As CCC and asymptomatic seropositive patients (ASY) are from the same endemic regions, the former have been used as a T. cruzi-infected control group to evaluate HLA profiles. We analysed 183 patients (121 CCC and 62 ASY) by PCR-SSP and PCR-SSO techniques for HLA-DR and HLA-DQ. The results indicate that HLA profiles in both CCC and ASY groups are similar. A trend towards the accumulation of HLA-DR4 antigen was observed in the severe CCC group (EF <0,4) compared to patients with the mild CCC group (EF>0,4). HLA-DR4 allele typing did not show significant differences between severe and mild CCC groups, ruling out the possibility of a CCC-related preferential antigen presentation by a given DR4 allele. We conclude that class II HLA molecules do not define susceptibility to the development of Chagas'disease cadiomyopathy. However, the functional implication of this finding is that there may be several different HLA alleles capable of presenting antigen associated to the immunopathology in CCC.

Supported by FAPESP, HHMI.

ANALYSIS OF ANTIBODY REPERTOIRE IN CHRONIC CHAGAS' DISEASE

Bessa, MC, Dos Reis, GA, Nobrega, AF. Instituto de Microbiologia, Depto de Imunologia, UFRJ, Rio de Janeiro, RJ, Brasil; Instituto de Biofísica , UFRJ, Rio de Janeiro, RJ, Brasil.

Serum IgM and IgG antibodies from BALB/c mice chronically infected with Trypanosoma cruzi, were analysed by Western blotting for reactivity on extracts of homologous tissues (liver, brain, heart, kidney) and on extracts of epimastigote and metacyclic tripomastigote parasites. The reactivity profiles of infected mice were compared to age-matched control animals. The available repertoire of splenic B lymphocytes of the same groups of animals was analysed after in vitro stimulation of B cells with LPS, by testing IgM reactivity onto the same antigenic extracts. Classification of reactivity patterns were made by multivariate statistical analysis, revealing repertoire deviations characteristic of the infection. This technique will be applied to study control of antibody repertoire selection by CD4 T cells in the course of T. cruzi infection.

This work was supported by PADCT, CNPq, FINEP and CEC.

POLYCLONAL ACTIVATION INDUCED BY PLASMODIUM CHABAUDI CHABAUDI INFECTION: ITS EFFECTS ON THE EFFECTOR AND MEMORY SPLEEN COMPARTMENTS.

L.R. Sardinha, R.A. Cavinato, M.R. D'Império Lima and J.M. Álvarez. Depto de Imunologia, ICB-USP, São Paulo, Brasil

In the acute phase of infections by a diverse array of parasites, the immune system responds with B and T cell polyclonal activation (PA), with no gain in parasite-specific frequencies. We have investigated if effector and memory lymphocytes are included in the populations polyclonally expanded by P. c. chabaudi infection. Moreover, we investigated if plasmodium induced PA, which shows a Th1 profile, changes the pattern of an ongoing Th2 response. BALB/c mice immunized with OVA in Al(OH)₃ were injected with 10⁶ infected erythrocytes, at the peak of OVA effector response (7 days after immunization), or when the OVA-specific B cell effector response had declined (memory stage; 80 days after immunization). A week after parasite challenge, the numbers of total and OVA-specific Ig-producing spleen cells of the different isotypes were determined by ELISASPOT. Effector B cell responses were expanded by PA without modifying the Ig-isotype pattern. While immunized controls (Im) showed 24600 IgG1 OVA-spots/spleen, in immunized-plasmodium-infected mice (Im-Pc) OVA-spots raised to 83520. PA induced a minor gain in IgM OVA-spots (from 706 in Im to 3990 in Im-Pc) and in IgG2a OVA-spots (from 0 in Im to 675 in Im-Pc). PA did not affect memory antibody responses since P. c. chabaudi infection of 80-days-immunized mice did not alter OVA-specific IgG1 response (from 2134 in Im to 1383 in Im-Pc), in spite that these mice contained memory cells which could be recalled by an OVA challenge (from 2134 in Im to 38573 in Im-OVA). IgG3 and IgG2b OVA-spots were not detected. In 80-days-immunized mice, however, OVA-specific T cells were activated by PA. Cytokine analysis of OVA-stimulated cells from these mice showed a striking increase of IL-4 levels (from 0,01 ng/ml/5x10⁶cells in Im to 0,15 ng/ml/5x10⁶cells in Im-Pc). These IL-4 levels might even be higher considering the increases in cellularity induced by infection. No IL-4 production was detected in OVA-stimulated cultures of unimmunized infected controls. IFNg levels of immunized mice did not exhibit major changes after PA, whereas IL-10 levels were reduced (from 0,468 ng/ml/5x10⁶cells in Im to 0,163 ng/ml/5x10⁶cells in Im-Pc). Results are discussed in the contexts of the physiopathology of polyclonal activation, and of non-specific maintenance/expansion of effector and memory cells. Supported by FAPESP.

ABSORTION OF HUMAN SERUM ANTIBODIES ANTI-TOXOPLASMA GONDII USING OOCCYSTS AND OOCYSTS/SPOROZOITES OF CRYPTOSPORIDIUM PARVUM.

Mercado, R.

Departamento de Parasitología. Facultad de Medicina. Universidad de Chile Casilla 9183 Santiago Chile.

Cryptosporidosis has been recently recognized as a human begin infection. As toxoplasmosis, is considered a cosmopolitan parasitosis. Campbell and Current (1983) first reported the demostration of serum antibodies to Cryptosporidium sp in normal and inmunodeficient humans with cryptosporidiosis. As well as, very little cross-reactivity with other coccidia like Toxoplasma, Sarcocystis and Isospora sp. ocurred in the immunofluorecent procedure. Mercado, Castro & Sanhueza (1992), described than ten million of tachyzoites of T. gondii were suffficient to absorb the antibodies from human sera presenting titer 1:16 to 1:256 by using the Sabin and felman or Dye Test(DT). This test is considered the standart exam for the diagnosis of toxoplaasmosis.

To evaluate croos-reactivity of the human T.gondii antibodies againts oocysts and oocyts/sporozoites of C.parvum absortion experiments were donne. Serum samples from two patients with DT titer of 1:16 and 1:64 were tested. In two of four test tubes. 0.2 ml of serum sample of each patients were disposed . Two samples were absorbed with: 10⁶ oocysts and with 10⁶ oocysts sporozoites of C.Parvum . As controls one sample was absorbed with 10⁶ tachyzoites of the RH strain of T.gondii, and the other with none antigen. After suspention and incubation at 37 C for 60 minutes. the parasites were pelleted by centrifugation, removed and the sera were tested by using DT. Results were as follows:

10⁶ whole Parasites Human Serum DT 1:16 Human Serum DT 1:64 Without C.parvum 1:16 1:64 With oocyts of C.parvum 1:16 1:16 With oocysts/sporozoites C.parvum 1:64 1:16 With tachyzoites of T.gondii Negative Negative

Campbell & current reported a decrease in titer from 1:2560 to 1:1280 of C.parvum antibodies serum sample absorbed with zoites of T.gondii whole antigen. The titer of C.parvum antibodies remain the same.

The presentes results suggest that DT human antibodies were not absorbed with oocyts and oocyts sporozoites of C.parvum.

Suported by funds of the DP/FM/U.de Chile.

SPECIFIC AND HETEROLOGOUS HUMORAL IMMUNE RESPONSE AFTER BALB/C INFECTION WITH TRYPANOSOMA CRUZI STRAINS OF DIFFERENT LEVELS VIRULENCE.

Silva Filho, H.H.; Carobrez, S.G.; Oliveira, D.A.S.*; Steindel, M.; Mineo, J.R.* Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, SC, Brasil. *Departamento de Patologia, Universidade Federal de Uberlândia, MG, Brasil.

The majority of the immunological studies regarding T. cruzi infection have employed strains with high virulence. Considering the lack of studies using strains with low and median virulence, the present work objective was to compare the humoral immune response among T. cruzi strains with different levels of virulence. Moreover, we evaluated the possibility that the different immune response against the parasite would modify the humoral immune response against sheep red blood cells (SRBC).To perform theses experiments, we employed 6 groups of BALB\c females, 10 - 12 week-old, which were inoculated intraperitoneally with T. cruzi strains. Two group with 100 blood forms parasites of the Y strain (high virulence), two group with 5 x 10³ blood forms parasites of the SC38 strain (median virulence), two group with 2 x 10⁷ culture forms parasites of the PF strain (non virulent) were set. Seven days later, three groups of mice, which were previously inoculated with each strain of T. cruzi, were also inoculated with 2,5 x 10⁸ SRBC to evaluated the heterologous antibodies production. We also set two control groups. One was inoculates with physiological solution and another with SRBC. The sera samples were collected at 7, 15, 30 45, 60, 90 days. The levels of heterologous antibodies against SRBC was measure by hemaglutiation. IgG class antibodies against T. cruzi (ELISA method), specific antibodies against T. cruzi from isotypes IgG1, IgG2a and IgE (ELISA method) were determined also in each serum samples. The results showed a significant reduction in the heterologous antibodies production, which varies in according to the degree of virulence of the parasites and may be related with the virulence of each strain. The non virulent strain, PF, produces IgG1, even though in lower level when compared with other two strains. The IgE production was detected in serum samples from animals infected with the Y and SC38 strains but without significant difference.The infection with median virulence strain SC38 elicited the higher production of IgG2a antibodies when compared to the other strains. These data may indicate that one mechanism is involved in the T. cruzi control and in the heterologus humoral immune response against SRBC. It may involue the modulation of the Th1 and Th2 response by the parasite therefore. Consequently, this modulation may infer that the SRBC response is regulated by the parasite immune response. Supported by CNPq

IgG ISOTYPES IN EXPERIMENTAL TRYPANOSOMA CRUZI INFECTION

Silva-Gonçalves AJ, Sampaio ALF & Pirmez, C.

Dept. Biochemistry & Molecular Biology , Instituto Oswaldo Cruz & Lab. Applied Pharmacology, Farmanguinhos, FIOCRUZ, Rio de Janeiro, Brasil.

The genetic background may influence the course of an infection, either through a particular antigen presentation or the particular cytokine profile produced during an immune response. It has been previously suggested that type 2 cells may have a key role in the immunopathological processes occurring in Trypanosoma cruzi infection (Res Immunol 142:137, 1991). Type 2 response is usually accompanied by a high antibody response, with a predominance of some Ig isotypes such as IgE and/or IgG1. We thus investigated the reactivity of anti-T. cruzi IgG isotypes in experimental Trypanosoma cruzi infection using three different mouse haplotypes.

Mice of the H2^b (C57BL/6), H2^d (BALB/c) and H2^k (CBA) were experimentally infected with 50 trypomastigotes of T. cruzi, Colombian strain. Sera were collected at days 21, 30, 40, 50 and 300 days after infection. Pooled sera from normal and chronically infected (more than 7 months) BALB/c mice were used as controls. IgG isotypes were evaluated by ELISA using T. cruzi epimastigote soluble antigen and rat monoclonal antibodies specific for each mouse IgG isotype (Isotyping kit, Pharmigen).

Increasing levels of total IgG were observed in all strains studied, but particularly in BALB/c mice. This augment was mainly due to IgG1 and IgA isotypes, which were significantly higher in this strain in comparison to the other ones. The peak of IgA isotype was found 21 days after infection, decreasing to levels equivalent to those found in non-infected normal mice. In contrast, IgG1 increased continuously, reaching maximum levels during the chronic phase. No significant differences were detected in IgG2a levels, which were increased in all strains, during all the examined period. Levels of this isotype however, represent half of the amount found for IgG1 isotype.

BALB/c mice, in comparison to the two other strains, develop a more severe disease as depicted by the progressive inflammation of heart tissue and higher parasitemia and mortality during the acute phase. Putting together, we may speculate that the high susceptibility of BALB/c mice could be a consequence of a predominant type 2 response.

Supported by PAPES/FIOCRUZ.

IS PARASITE PERSISTENCE RESPONSIBLE FOR THE IMMUNOLOGICAL MEMORY IN HUMAN LEISHMANIASIS?

Pirmez C, Oliveira-Neto MP, Andrade TCB, Oliveira MP & Conceição-Silva F.

Hospital Evandro Chagas, Depts. Biochemistry & Molecular Biology and Protozoology, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil.

In ten years of surveillance in an endemic area of Leishmania (V.) braziliensis transmission in Rio de Janeiro, only 5% of the infected individuals had the appearance of new cutaneous lesions after clinical healing (unpublished observation). Although reinfection can not be ruled out since these patients live in an endemic area where transmission occurs continuously, we have the following indication that parasites may persist within the host for a period that may be life-long: parasite DNA can be detected by PCR a) in 80% of scars from patients treated with Glucantime (Mem Inst Oswaldo Cruz 91:185, 1996) or b) in the peripheral blood of approximately 30% of patients with active cutaneous (LCL) or mucosal (MCL) disease, patients clinically healed (HL) after therapy with Glucantime, or in individuals who had no past history of leishmaniasis, but are Montenegro skin test positive and live in endemic areas (MTN) (Camera et al, this forum). In addition, patients who have concomitant HIV infection and leishmaniasis often relapse, despite the apparent clinical healing after specific therapy (Mattos et al, this forum).

We have re-examined 21 individuals ten years after the clinical cure, still living in the endemic area. All of them were clinically healed, and had no mucosal lesions. Montenegro skin test was significantly greater (p<0,05) in 21/21 tested individuals, the reaction being at least two-fold the initial diameter. In addition, peripheral blood mononuclear cells isolated from a total of 36 HL individuals after a period of two to ten years after clinical cure by therapy with Glucantime, 29 (80%) presented a strong positive response to Leishmania antigens in vitro. Furthermore, the in vitro response was also strong in 7/12 (58%) MTN individuals.

If protective response is dependent on parasite persistence, this phenomenon should be considered in vaccination strategies. On the other hand, even if that is the case, the opposite effect in candidates for secondary mucosal lesions produced by L. (V.) braziliensis may also occur. Our experience in endemic areas of Rio de Janeiro State, however, suggests that parasite-host equilibrium seem to be achieved in the majority of the population at risk.

Supported by FAPERJ.

A MONOCLONAL ANTIBODY RAISED AGAINST PLASMODIUM CHABAUDI CHABAUDI SOLUBLE ANTIGENS PRESENT IN THE SERUM OF INFECTED MICE RECOGNIZES A 250 KDA SCHIZONT GLYCOPROTEIN THAT IS SECRETED DURING SCHIZOGONY

Furtado, G.C.; Moura, I.C.^*; Pudles, J.^*; Alvarez, J.M.; D'Império Lima, M.R. Dept. Imunologia and * Dept. Parasitologia, Universidade de São Paulo, São Paulo SP.

Soluble malaria antigens are released from erythrocytes infected with Plasmodium at the time of schizont rupture and also during merozoite re-invasion. It has been suggested that these antigens could be associated to many pathological manifestations observed in the malaria disease once they can stimulate macrophages to overinduce the production of TNF, IL-1 and IL-6. In order to characterize parasite soluble antigens present in the acute serum of mice infected with P. c. chabaudi, we immunized the animals with acute malaria serum and produced a panel of monoclonal antibodies. Thirty seven out of 38 parasite-specific clones recognized a band of 250 kDa in the purified parasite fraction obtained from enriched schizont cultures. The 1C29F9 MAb was used to characterize this 250 kDA molecule that was neither expressed on the infected erythrocyte surface nor on ring and early trophozoite stages. Flow cytometry analysis showed that the MAb 1C29F9 labeled the parasites when schizogony takes place. The biochemical characterization of this molecule revealed that the 1C29F9 MAb immunoprecipitated a high molecular weight protein of 250 kDa that is secreted and probably cleaved into smaller polypeptides of 220, 180, 148, 110 and 61 kDa in the supernatant culture. The secretion of this molecule is brefeldin A sensitive which indicates that the transport of the derived 250 kDa fragments to the supernatant culture is dependent on the classical protein transport system. The 250 kDa molecule is a glycoprotein as demonstrated by its binding capacity to Concanavalin A, and also, by in vitro incubation of infected cells with D-[U-¹⁴C]mannose, followed by immunoprecipitation of the molecule with the 1C29F9 MAb, from the parasite extracts. Additionally, we observed that animals treated with 1C29F9 MAb delayed the rinse of parasitaemia and enhanced the animal survival, even though it did not protect the animals from the infection. Finally, schizont infected cells pre-treated with the MAb 1C29F9 prior to addition to normal erythrocytes were substantially blocked in the capacity to re-invade new cells in vitro. These results indicate a role of a soluble molecule of 250 kDa in the infection process of this experimental malaria disease.

Supported by CNPq/FAPESP.

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST MSP-1 OF P. YOELLI

Lilian Spencer and Anthony A Holder

Division of Parasitology, National Institute for Medical Research, The Ridgway, Mill Hill, London NW7 IAA.

Monoclonal antibodies (Mabs) have been useful tools in the study of maian'a antigens, because they can inhibit parasite invasion of host cells and define inlportant epitopes . One of the most important surface antigens 'm the blood stage ls tbe merozoite surface prote'm-l (MSP-I), a protein

found 'm all species.

T'he C-temu'nal 19 kDa fragment Plasmodium yoelii MSP-1 ls cysteine-n'ch, contain'mg two epiderinal growth factor-like domains. ln our group we have expressei 'm bacteria this sequence fused to glutathione S-transferase. ln previous work thls recomb'mant protem was highly effective m protecting nu'ce against challenge 'mfection; protection was dependent upon the formation of confortnational epitopes maintained by disulphide bonds. To defíne the critical epitopes that are involved in the inhibition of invaslon into the red blood cell, Mabs have been produced.

Six Mabs assessed in passive transfer experiments, and 3 of them were pratectives (lOOO/o) to parasites 'mfection of P. yoelii YM strain (lethal strain). Odier 2 Mabs were partially pratectives and one was non-protective, but it can recognise adier fragrnent of MSP-1 (33 kDa).

T'hls ls the first time has been described Mabs against other fragments of MSP-1 in this rodent model. ln addition one of pratective Mabs recognlsed a 42 kDa fragment and this suggest that 19 kDa fraginent is not unique fragment important in the antigenicity of MSP-I.Now a panel of Mabs aga'mst MSP-1 of P.yoelii exlst and they could be useful in the studies of proteolytic processmg.

SPECIFIC MONOCLONAL ANTIBODY (SST-1) TO LEISHMANIA (VIANNIA) BRAZILIENSIS PROMASTIGOTE GLYCOLIPIDS

Silveira , TGV, Takahashi, HK & Straus, AH.

Departamento de Bioquimica, Universidade Federal de São Paulo/EPM, Caixa Postal 20.372, São Paulo, 04023-900, SP, Brasil

Monoclonal antibodies (MoAbs) were produced by immunization of BALB/c mouse with membranes of Leishmania (Viannia) braziliensis promastigotes using acid-treated Salmonella minnesotae as adjuvant. Relevant MoAbs were screened by solid-phase radioimmunoassay (RIA) using 96-well plates coated with L. (V.) braziliensis promastigotes. A clone secreting an IgG3 antibody specific for glycolipid fraction of L. (V.) braziliensis promastigotes was isolated and termed SST-1. By solid-phase RIA, MoAb SST-1 did not show any reactivity with other parasites such as: Leishmania (Leishmania) amazonensis (promastigotes), Leishmania major (promastigotes) and Trypanosoma cruzi (epimastigotes), and this MoAb did not cross-react with glycolipids isolated from promastigote forms of L. major, and from amastigote and promastigote forms of L. (L.) amazonensis.

Glycolipids from promastigote forms of L. (V.) braziliensis were purified by a combination of chromatography on Octyl-Sepharose, Silica-Gel 60 and C-18 columns, and by HPLC using Iatrobeads columns. By HPTLC immunostaining it was observed that SST-1 recognizes two glycolipid components distinct of the glycolipids recognized by MoAbs ST-3 and ST-5. Mild treatment of L. (V.) braziliensis glycolipids with sodium periodate (20 mM of sodium periodate in 0.1 M of sodium acetate buffer pH 4.5) followed by reduction with borohydride abolished the reactivity with MoAb SST-1, indicating that it recognizes a carbohydrate epitope.

By Western Blot of total extract of L. (V.) braziliensis promastigotes only a low molecular weight component, with the same migration of the glycolipid fraction on SDS-PAGE, was reactive with MoAb SST-1, suggesting that the main antigen recognized by MoAb SST-1 is a glycolipid.

Supported by: CAPES, CNPq, FAPESP, PRONEX

REACTIVITY OF ANTI-TERMINAL GALACTOFURANOSE MONOCLONAL ANTIBODY (MEST-1) WITH TRYPANOSOMATIDS. ROLE OF LEISHMANIA MAJOR GIPL-1 IN THE MACROPHAGE INVASION

Suzuki, E, Toledo, MS, Takahashi, HK & Straus, AH.

Departmento de Bioquímica, Universidade Federal de São Paulo/ EPM, Caixa Postal 20.372, São Paulo, 04023-900 SP, Brasil

Recently, it was produced in our laboratory a monoclonal antibody (MoAb) MEST-1 which recognizes terminal residue of b1-3/6 galactofuranose (Suzuki et al., 1997, Glycobiology, 7: 463). The expression of antigens with terminal residues of galactofuranose recognized by MoAb MEST-1 was analyzed in different Trypanosomatids such as: amastigotes and promastigotes of Leishmania (Leishmania) amazonensis, promastigotes of Leishmania (Viannia) braziliensis, promastigotes of Leishmania major, and epimastigotes of Trypanosoma cruzi (CL and Y strains). By indirect immunofluorescence, labeling was observed only in L. major and T. cruzi. Glycolipids were extracted from these Trypanosomatids with a mixture of isopropyl alcohol/hexane/water (55:20:25; v/v/v), and identified by high performance thin layer chromatography (HPTLC). By solid-phase radioimmunoassay (RIA), HPTLC immunostaining and Western blot, it was verified that MEST-1 reacted only with L. major and T. cruzi glycolipids. Immunochemical analysis identified GIPL-1 and LPPG as the antigens recognized by MEST-1 in L. major and T. cruzi, respectively. It was also verified by solid-phase RIA that MoAb MEST-1 presents high affinity to GIPL-1 (detect as low as 1 ng/well) and low affinity to LPPG (200 ng/well). By Western blot of total extracts of L. major and T. cruzi no protein/glycoprotein antigens reactive with MoAb MEST-1 were detected, confirming that GIPL-1 and LPPG are the major antigens recognized by MEST-1. Involvement of L. major GIPL-1 in the macrophage invasion was studied by pre-incubating promastigotes with Fab fragments of MEST-1, or with Fab fragments of an irrelevant MoAb. Under these condition, MEST-1 was able to inhibit 80% of macrophage invasion by L. major promastigotes. This is the first report associating GIPL-1 with binding and/or infectivity of host cells by parasites.

Supported by: FAPESP, CNPq, PRONEX

CHARACTERIZATION OF STAGE-SPECIFIC GLYCOLIPID ANTIGENS OF LEISHMANIA (VIANNIA) BRAZILIENSIS. INHIBITION OF INFECTIVITY BY ANTI-GLYCOLIPID MONOCLONAL ANTIBODIES

Silveira, TGV, Straus, AH & Takahashi, HK.

Department of Biochemistry, Universidade Federal de São Paulo/EPM, Caixa Postal 20.372, 04023-900-São Paulo, SP, Brazil

Glycolipid antigens of Leishmania (Viannia) braziliensis, were isolated and their reactivity with monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 analyzed. By HPTLC, it was verified that the pattern of total glycolipid as well as the glycolipid reactivity profile with the three MoAbs are distinct for L. (V.) braziliensis (Leishmania braziliensis complex) and Leishmania (Leishmania) amazonensis (Leishmania mexicana complex). In amastigote forms of L. (L.) amazonensis ST-3, ST-4 and ST-5 recognize the glycan sequence: Galb1-3Gala1-4Galb1-4Glc. Glycolipids antigens containing the above sugar sequence were not detected in amastigote forms of L. (V.) braziliensis. However, in promastigote forms of L. (V.) braziliensis it was detected an unique glycolipid antigen reactive with ST-3 and ST-5. Sugar analysis of the L. (V.) braziliensis reactive glycolipid by acid hydrolysis followed by high performance thin layer chromatography (HPTLC) showed that it is constituted by glucose, galactose, and hexosamine. We are currently sequencing the carbohydrate moiety of this glycolipid antigen.

By indirect immunofluorescence it was shown that ST-3 and ST-5 are reactive with promastigote forms of L. (V.) braziliensis, whereas no fluorescence was observed with amastigote forms. The promastigotes fluorescence was greatly reduced after treatment of the parasites with organic solvent (chloroform/methanol 2:1; v/v) indicating that the main antigen recognized by these MoAbs is a glycolipid. To clarify the biological function of this glycolipid, we carried out in vitro assays to assess the invasive capacity of promastigotes of L. (V.) braziliensis into macrophage monolayers. An inhibition of 65% of the macrophage invasion was observed when the parasites were pre-incubated with Fab fragments of MoAbs ST-3 and ST-5. The results of the inhibition assays clearly indicate that this newly described glycolipid antigen is involved in macrophage binding and/or infection.

Supported by: CAPES, CNPq, FAPESP, PRONEX

ANTIBODY RECOGNITION OF THE IMMUNODOMINANT EPITOPE IN T. CRUZI B13 PROTEIN IS DEPENDENT ON AMINO ACID SIDE CHAIN DISPOSITION

Iwai, L.K.^{1, 2}, Duranti M.A.², Kalil J.², Juliano M.A.¹, Cunha-Neto E.², Juliano L.¹, ¹Departamento de Biofísica - EPM_UNIFESP, ²Lab. Imunologia de Transplantes -Instituto do Coração - INCOR-HC-FMUSP

Protein antigens of several pathogenic protozoa frequently display immunodominant tandemly repeated sequences. Evidence indicates that the immunodominance of such tandem repeats can protect the parasite by diverting the immune response away from neutralizing antibodies directed against functional domains, the so-called "smoke screen" hypothesis. Little is known, yet, about the mechanisms underlying the general immunodominance of repeated sequences, apart from their multi-valency that can make them activate B cells directly and behave as T-independent antigens. On the other hand, it is accepted that epitope conformation can be relevant for antibody binding of peptide antigens. We have been studying the conformational features of the immunodominant tandemly repeated epitope (LFGQAAAGDKPS)n present in the recombinant T. cruzi antigen B13. It is known that the B13-derived peptide S4 (FGQAAAGDK-NH₂) contains the immunodominant epitope and assumes a helical conformation. Further, the antigenicity of short AAAGDK-containing synthetic peptides is critically dependent on the ability of the peptide to assume the secondary structure. Topological related analogues of the parent peptide can be used as probes in structure-antigenicity studies. In order to further assess the role of side chain disposition on antibody recognition of the helical peptide S4, we studied the effect of reversal of backbone polarity on antibody recognition of S4 peptide. Peptides [FGQAAAGDK-NH₂] (S4) and its retro analogue [KDGAAAQGF-NH₂] (retro-S4) were used in competitive ELISA with B13 as the solid phase antigen to test antibody binding in sera from 12 Chagas' disease patients. Interestingly enough, the retro-S4 analogue was not recognized by sera from 12 chagasic patients. Results are reported as the average (min-max) of percent inhibition in B13 competitive ELISA on Chagasic sera.

Peptide concentration 100 mM 500 mM Avg. % inhib S4
Avg. % inhib retro-S4 100 (96-100)
4 (0-15) 100 (99-100)
5 (0-20)

These results demonstrate that the antibody recognition of the S4 epitope is not palindromic; thus, the disposition of the peptide side chains might be essential for antibody recognition of the B13 immunodominant epitope along with the secondary structure of the peptide as recently described. Further studies of the antigenicity of additional peptidomimetics of the same natural sequence are in progress.

Supported by FAPESP, HHMI

CONFORMATIONAL PREFERENCES ASSOCIATED WITH THE IMMUNODOMINANCE OF TANDEMLY REPEATED EPITOPE OF B13 ANTIGEN OF TRYPANOSOMA CRUZI.

^1,4Duranti, M; ²Franzoni, L; ²Sartor, G; ²Benedetti, A; ²Spisni, A; ¹Iwai, L; ³Gruber, A; ⁴Zingales, B; ⁵Guzman, F; ³Diez, M.; ¹Kalil, J and ¹Cunha-Neto, E.

¹Lab. of Transplantation Immunology, Heart Institute, University of São Paulo; ²Institute of Biological Chemistry, Univ. of Parma, Italy, ³Faculty of Veterinary Medicine and Zootechny and ⁴Institute of Chemistry, University of São Paulo, Brazil, ⁵Institute of Immunology, National University of Colombia, Bogotá, Colombia

Little is known about the reasons underlying the immunodominance of tandemly repeat regions observed in several antigens of pathogenic protozoa. The recombinant protein B13 of Trypanosoma cruzi is an immunodominant antigen made of 12 amino acids tandem repeats (Gruber & Zingales, Exp. Parasitol. 76, 1, 1993). Previous data on epitope mapping with overlapping 9-mer synthetic peptides derived from the B13 sequence showed that peptide S4 (FGQAAAGDK) contained the immunodominant epitope, in a small number of human chagasic sera (Cunha-Neto et al. PNAS, 92, 3541, 1995). In order to analyze structure-activity correlations, we studied the relative antigenic activity of peptides pB13 (GDKPSLFGQAAAGDKPSLF), S4/S5 (FGQAAAGDKPS), S4, S5 (QAAAGDKPS) and hexa pB13 (AAAGDK) by competitive ELISA with recombinant B13 protein and synthetic peptides in 46 sera from Chagas'disease patients. Solution conformation of peptides pB13, S4 and S5 was also studied by analysis of CD and 2D ¹H-NMR TOCSY, DQF-COSY and NOESY spectra in PBS and 80%TFE-20%PBS. Results of competitive ELISA show that in 87% (40/46) of sera tested, the immunodominant epitope of B13 was contained in the 19-mer pB13 peptide. Among those sera, the median percent inhibition of competitive B13 ELISA was: 91% for pB13 and S4/S5, 86% for S4, 68% for S5 and 22% for hexa pB13 peptides. Although all peptides contain the AAAGDK epitope "core", their distinct antigenicity suggests that possessing this sequence may be necessary but not sufficient for optimal antigen recognition. The significant difference in antigenic activity exhibited by S4 and S5, both 9 residues long, suggests that flanking residues could somehow influence the antigenicity and recognition of the epitope core. NMR results show a population of folded, helical conformers on the AAAGD region with a gradual decrease in TFE-induced a-helical content in the peptides pB13, S4 and S5. One major conclusion from this study is the parallelism between the antigenicity and the ability to assume helical structure along the hexapeptide "core" region in each peptide: the repetitive helical character of the epitope may be involved in the immunodominance of B13 protein. Supported by FAPESP, CNPq, MURST-CNR.

TWO SECUENCES PEPTIDES FROM T. CRUZI ANTIGENTS WITH ANTAGONIC CHARACTERISTICS

¹Briceño, L ;¹Marquez, J ; ²Noya, O &¹Mosca, W.

¹ Instituto de Biomedicina, Facultad de Medicina, UCV.

² Instituto de Medicina Experimental, Facultad de Medicina, UCV.

We have been compared the immune response of infected asymptomatic patients, with those who develop Chagasic cardiomyopathy. In previous studies we have demostrated by T cell blot with different subcellular fractions that only a fraction of all antigens assayed did not induce suppression and give differences in the immune response between the two groups of patients. Currently, we are purifying and evaluating these antigens in a mice model. In this study we present our resullts with one of them. A band of 48 Kda, from the cytosol soluble epimastigote´ fraction (Cs) was sequenced, originating two sequences. From these sequences the polymeric peptides 150 and 152 were synthesized and evaluated in protection trials with non isogenic mice (NMRI). The results show that with a lethal 80 dose, 60% of all the mice immunized with the 152 peptide survived and did not develop parasitaemia, while with the 150 peptide all the mice developed high parasitaemias and died, rendering animals more susceptible to acute infection. Considering the pathogenic properties of the 150 peptide, we decided to confirm the results by challenge with a lower lethal dose (20) and in the resistant mouse line C57Bl/6. The results corroborated earlier observations. 100% of the mice died and the time of mortality was significantly earlier than in the control group. In order to obtain more information about the protein domain distributions or shared epitopes in proteins from the Cs fraction, we produced antibodies directed against these peptides. With an immunoblot test, the policlonal sera were assayed, in denaturation and reduced conditions. The serum directed against peptide 152 recognized four bands (71, 60, 48 and 36 Kda), while the anti 150 serum, recognized two bands in the same position as the 152 antiserum (71 and 36 Kda). Regarding parasite recognition in an immunofluorescence test, we found that the anti-152 serum give cytoplasmatic labeling in the epimastigote stage, while in the trypomastigote stage the labeling was in the parasite membrane, suggesting that the proteins who contain the epitopes have a stage-specifc regulation. Our results show that in the same band we find out two motifs with antagonic characteristics: one with pathogenic properties and the other inducing a a good protection index in nonisogenic mice, without parasitaemia.

Financial support: CONICIT proyecto BTS 51

THE OLIGOPEPTIDASE B AND THE CATHEPSIN B-LIKE PROTEASE OF TRYPANOSOMA CRUZI ARE ANTIGENIC IN HUMAN AND RABBIT INFECTION

Fernandes, L.C.^a, Bastos, I.M.D.^a, Lauria-Pires, L.^a, Grellier, P.^b, Schrével, J.^b, Teixeira, A.R.L.^a, and Santana, J.M.^a

^aLab. Multidisciplinar de Pesq. em Doença de Chagas, Dep. de Biologia Celular e de Patologia, Universidade de Brasília. 70919-970, Brasília-DF, Brasil. ^b Museúm d'Histoire Naturelle, 61 rue Buffon, 75231, Paris.

The Trypanosoma cruzi alkaline proteinase (oligopeptidase B) is expressed in all of its forms, and displays narrow proteolytic activity (Santana et al., 1992, Biochem. Bioph. Res. Comm. 187: 1466-1473). This enzyme is involved in the generation of a Ca⁺⁺-signaling factor important in host cell invasion by the parasite (Burleigh et al., 1997, J. Cell Biol. 136: 609-620). On the other hand, the 30 kDa cysteine protease was shown to be a cathepsin B-like enzyme with a broad substrate specificity at acidic pH.

To study humoral immune response against these proteases in humans and rabbits infected with T. cruzi, we purified both enzymes to homogeneity. The purified proteases (2 mg/ml) were used to perform conventional ELISA with sera from 13 rabbits in the acute and chronic phase of the infection, 21 patients with chronic Chagas disease (CCD), from 10 patients with visceral leishmaniasis (VC), and from 10 patients with cutaneous leishmaniasis (CL). 10 normal sera were used as control. Four and three out of the 21 sera from CCD did not recognize the cathepsin B-like protease and the oligopeptidase B, respectively. Both enzymes were recognized by antibodies from 100 % of the sera from rabbits in the chronic infection. In the acute phase, 62 and 85 % of these sera reacted with oligopeptidase and cathepsin B-like, respectively. Although the sera strongly reacted with both antigens, the antibody titers for cathepsin B-like protease were higher than those observed for the oligopeptidase B. Different level of cross reactions were observed with sera from VC and CL. Host immune response to enzymes important for the life cycle of T. cruzi could be an efficient mechanism to prevent cell invasion by the parasite and inespecific tissue damage. However, neither the sera or the purified IgGs prevented cathepsin B-like enzymatic activity in vitro.

Supported by CEE, CNPq and FAPDF

COMPLEX GLYCOLIPIDS FROM TRYPANOSOMA CRUZI DM 28C STRAIN ARE RECOGNIZED BY RABBIT HYPERIMMUNE SERUM

Villas Bôas, MHS & Barreto-Bergter, E.

Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio deJaneiro, RJ, BR - E mail: inmigbci@ã@microbio.uf@.br

Chaga's discase is a common cause of congestive heart failure and sudden death. After the acute phase, most patients progress to thc chronic phase iiianifcsted by cardiovascular, digestivo and autononúc nervous system disordcrs, particularly in licart and eosopliagus (I).

Glycosplúngolipids, as components of tlie cell surface, are bclieved to be involved in the cell-cell interaction, differcntiation, imtiiunogcnícity and oncogenesis. Trypanosoma cruzi Dm 28c epimastigote cells were extractcd with chlorofoniiL/niethanol 2:1 and I:2 (v/v). The extracts were pooled and concentrated to dryness under reduced pressure. The crude lípid extract was fractionated on silíca gcl 60 column chroinatography and complex glycolipids were cluted with chloroform/inethanol I: I (v/v). These compounds were separeted into bands on HMC (high-perforinance thin-layer chroinatography) and their migration properties conipared to standards of neutral glycosphingolipids. Two main bands migrated closely to authentic ceramide trihcxosíde and globoside standards. This fraction was testcd by Elisa (Enzylne-Linked lmmunoadsorvent assay) and showcd to be highly rcactivity when compared with others fractions (ceramide monohexosídes). The present study shows that glycolipid fractions isolated from T. cruzi may be important antigens in eliciting the host's inunune rcsponse.

(I) losa, D., Massarí, D.C. & Dorscy, F.C. (l991) Am. Heart J., 122, 775-785

Supported by: CNPQ, FINEP, CEPG/UFRJ and PRONEX.

COMPARISON OF BIOLOGICAL AND ANTIGENIC CHARACTERISTICS OF TRYPANOSOMA CRUZI CLONE CL BRENER AND CL STRAIN

Katia de Almeida,¹ Norival Kesper Jr,¹ Bianca Zingales² and Eufrosina S. Umezawa¹

1- Instituto de Medicina Tropical de São Paulo - FMUSP, Av. Dr. Enéas de Carvalho Aguiar 470, CEP 5403-000; 2- Instituto de Química - USP

CL Brener is a clone derived from the CL strain and used as the reference organism in the Trypanosoma cruzi Genome Project. The biological characteristics and molecular typing of clone CL Brener have been defined (Zingales et al., Acta Tropica, 1997).We have further compared some biological parameters and the antigenic composition of CL Brener and the parental CL strain. It has been observed that CL Brener trypomastigotes are less infective to LLC-MK₂ cultured mammalian cells, both at 33^o and 37^oC, when compared to CL parasites. In addition, it was verified that the differentiation of epimastigotes to metacyclic trypomastigotes in axenic medium is drastically reduced after serial sub-culturing of CL Brener, contrary to what is observed for the CL strain. On the other hand, the antigenic profile of CL Brener and CL is very similar. This was analysed by immunoblotting of total extracts of either trypomastigote or epimastigote forms, as well as with antigens secreted/excreted by trypomastigote forms (TESA) of the two T. cruzi stocks. Sera from experimentally infected rabbits at the acute and chronic phases, as well as sera of rabbits immunized with T. cruzi extracts were employed in these analyses. Polyclonal monospecific sera of rabbits immunized with several T. cruzi recombinant proteins (B13, JL8, H49, SAPA/TS and C11) also resulted in similar reactivity patterns with parasite extracts from both CL Brener and CL strain. Immunoblotting patterns of TESA from CL strain and CL Brener clone probed with rabbit acute phase sera revealed the presence of several members of the Transialidase/Neuraminidase (TS) family. On the other hand, when the chronic phase sera were used only a major 150-160 kDa antigen from CL strain and CL Brener clone was identified. This band corresponds to a non-polymorphic, highly immunogenic T. cruzi antigen, already described by us in other studies performed with different T. cruzi strains (Umezawa et al, J.Clin.Microbiol.1976).

Financial support: FAPESP, LIM-49/FMUSP

U.S. BLOOD DONORS AT LOW RISK FOR TRYPANOSOMA CRUZI INFECTION: PREVALENCE OF ANTIBODIES AND POSSIBLE FAMILIAL ASSOCIATED INFECTIONS.

Leiby, D.A.¹; Fucci, M.-C.²; & Stumpf, R.J.³

¹Transmissible Diseases Department, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA; ²Southwest Region, American Red Cross, 10151 East 11th Street, Tulsa, OK 74128, USA; ³Abbott Laboratories, Abbott Park, IL 60064, USA.

Recent investigations have demonstrated that the seroprevalence of antibodies to Trypanosoma cruzi in high risk blood donors from Los Angeles and Miami is approximately 1 in 8,800. Relative to other infectious agents transmitted by blood this rate is high. Therefore, expanded investigations of T. cruzi transmission by blood transfusion were initiated to better understand the extent of this problem in the U.S. While extensive data exists for blood donors in high risk populations, little information is available regarding the prevalence of T. cruzi among donors in low risk populations. Thus, blood donors in the Southwest Region of the Red Cross, which is comprised of three areas ranging from low to moderate risk, were tested for T. cruzi antibodies. Over the course of 10 months, all allogeneic blood donors located in portions of Texas and Oklahoma were tested by enzyme immunoassay (EIA; Chagas Antibody EIA Generation 2.0; Abbott Laboratories), and if repeatedly reactive, confirmed by radioimmunoprecipitation (RIPA). Previous donations by infected donors were traced to identify potentially exposed recipients for testing. A total of 100,089 blood donors were tested by EIA, 150 (0.15%) of these were repeatedly reactive, and ultimately 3 were confirmed as positive by RIPA. Overall this suggests a seropositivity rate of 1 in 33,000 blood donors. However, all 3 positive donors were from a single collection area near Waco, Texas that collects approximately 23% of the blood for the Southwest Region. Thus, in the area around Waco, approximately 1 in 7,700 donors were positive for T. cruzi antibodies. One positive donor was born in an endemic area of Mexico, but the other 2 donors were born in the U.S. and reported no apparent risk factors for infection. One of these latter donors described a family history that included relatives with enlarged hearts, arrhythmia's, and sudden onset of death, symptoms suggestive of possible congenital transmission. Studies are underway to determine the source of infection in the 2 donors without risk factors. Specifically, we are investigating congenital and vector-borne transmission. Investigations of previous donations by infected donors identified and tested one recipient who was negative for T. cruzi antibodies. In conclusion, blood donors seropositive for T. cruzi are present in low risk populations, but at lower rates. If screening for T. cruzi is implemented in U.S. blood banks at some date in the future, universal testing may be necessary .

Supported by American Red Cross Biomedical Services.

CONFIRMATORY TESTING FOR TRYPANOSOMA CRUZI ANTIBODIES: VALIDATION OF RADIOIMMUNEPRECIPITATION ( RIPA ) TESTING.

D. A. Leiby; S. Wendel; D.T. Takaoka; R. Fachini; L.C. Oliveira; L.P. Lima & M.A. Tibbals; - Hospital Sírio Libanês, São Paulo, Brazil and American Red Cross, Rockville, MD, USA.

Background: Several published and ongoing studies investigating the seroprevalence of Trypanosoma cruzi among blood donors in the US have used radioimmuneprecipitation (RIPA) as a confirmatory test. The RIPA, however, remains controversial. To asses the reliability of RIPA, we compared test results for RIPA with several alternative test procedures using a panel of sera from Brazilian blood donors with positive or inconclusive results for T. cruzi antibodies.

Methods: A total of 220 sera from blood donors with positive or inconclusive test results for T. cruzi antibodies were tested and compared by indirect immunofluorescence (IFA), 2 types of indirect hemagglutination (IHA - HEMAGEN and BIOLAB), 4 different ELISAs (ABBOTT, EMBRABIO, ORGANON and GULL), and RIPA.

Results: Data were initially grouped for comparative purposes based on observed IFA titers. All controls (n = 19) were negative on all tests. At 1:20 (n = 9), 3 sera were positive by the third and fourth ELISA (neg, on all other tests), and one sample was positive only by the third ELISA, while one sample was positive only by RIPA, suggestive of an indeterminate. For sera at 1:40 (n = 35), 32/35 (91%) were RIPA positive, conflicting results were observed for IHAs (97% vs. 69%), and ELISA results were similar to RIPA. At higher titers (1:80, n = 56 & 1:160, n = 101) all sera were positive by RIPA and the first IHA, 95% positive by second IHA, and one sample was missed by ELISA (negative on the first and second ELISA).

Conclusions: A serological test for T. cruzi that can be designated as the "gold standard" remains elusive. However, a comparison of RIPA with other test methods currently in use suggests that RIPA is valid for use as confirmatory test, particularly in a research setting.

DETECTION OF POTENTIALLY DIAGNOSTIC ANTIGENS FOR CHAGAS DISEASE IN THE TRYPANOSOMA CRUZI INSOLUBLE FRACTION

Pereira, V.R.A.¹, Gomes, F.C.², Furtado, V.C.² , Abath, F.G.C.² & Gomes, Y.M.²

1-Departamento de Bofísica e Radiobiologia, UFPE, Brazil. 2-Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Cidade Universitária, 50670-420, Recife-PE, Brazil.

A safe diagnosis to confirm infection with T. cruzi is still a crucial problem, specially because of cross-reactivity with other parasitic organisms. In previous studies carried out with soluble antigens of T. cruzi from three different strains (Y, WSL and Colombiana), a polypeptide was described of 26 kDa which was recognized by patients with the cardiac form of the disease, which seemed to be an indicator of this clinical form (Mem. Inst. Oswaldo Cruz, Suppl. 1996, 256: 391). As an extension of our previous studies, sera of patients with different clinical forms (cardiac form = 12 and asymptomatic form = 13) of the disease were analysed by immunobloting, using epimastigotes insoluble T. cruzi antigens. Although reactivity was variable among individual patient sera, some antigens (58 and 46 kDa) were almost always detected independently of the strain used. When the same analysis was undertaken in patients harboring other parasitic diseases (Leishmaniasis, Toxoplasmosis, Amebiasis and others) the above mentioned antigens were not recognized. Thus the detection of the 58 and 46 kDa insoluble antigens may be indicative of T. cruzi infection. Further studies are in progress to caracterize these antigens.

Supported by: CAPES, CNPq, FACEPE and FIOCRUZ.

CHAGAS' DISEASE - PROFILE OF PROTEIN ANTIGEN RECOGNITION BY IMMUNOBLOTTING

Moriya, N.¹*; Alves Jr., A.M.¹*; Rodrigues, A.M.¹; Oliveira, E.C.²; Luquetti, A. O.² & Stefani, M.M.A.¹

1. Departamento de Microbiologia, Imunologia, Parasitologia e Patologia IPTESP/Universidade Federal de Goiás 2. Laboratório de Pesquisa em Doença de Chagas HC/Universidade Federal de Goiás

Immunoblotting has been demonstrated to be a useful technic for diagnosis and follow up assessment. This study was conducted in order to define the protein antigen recognition profile of patients with Chagas' disease by immunoblotting. Sera from 47 well studied patients in the chronic phase of Chagas' disease with 3 serological tests positive for Trypanosoma cruzi infection were used in this study. These patients presented different clinical forms (25 megacolon/megaesophagus, 13 cardiopathy and 9 indeterminate ). Protein antigens from T. cruzi epimastigotes, grown in LIT medium, prepared in the presence of protease inhibitors were separated in 8% SDS-PAGE gel and electroblotted overnight onto nitrocelulose membrane. Free binding sites were blocked and NC strips incubated individually with sera. Antigen recognition by antibodies was demonstrated by the use of anti-human IgG conjugated to alkaline phosphatase and NBT/BCIP. Proteins with estimated molecular weights from 14 to 150 kD were recognized by the majority of sera. Two groups of proteins were mostly evident. One group, presented high molecular weight proteins varying from 80 to 150 kD, in which a doublet of 110-120 kD proteins was immunodominant. The other group, represented by proteins ranging from 14 to 50 kD, showed the predominant recognition of proteins in the scope of 40-50 kD. This profile of recognition was observed by the sera of all chagasic patients regardless of the clinical form. This methodological approach pointing out protein antigens mainly recognized by sera of patients with different clinical forms, can be helpful in the indication of candidates for new diagnostic methods.

Supported by FUNAPE/UFG

*Supported by CNPq/PIBIC

SERODIAGNOSIS OF TRYPANOSOMA CRUZI INFECTION USING PARTICLE GEL IMMUNOASSAY - ID -PaGIA.

Rabello A¹, Luquetti A O², Furtado E M³, Gadelha F⁴, Melo L⁵, Schwind P⁶. ¹Centro de Pesquisas René Rachou - FIOCRUZ, Caixa Postal 1743, Belo Horizonte, M.G., Brasil. ²Universidade de Goiás, ³Hemocentro de Pernambuco, ⁴Fundação Ezequiel Dias, ⁵DiaMed Brasil, ⁶DiaMed AG-Switzerland.

The ID-Chagas antibody test is a Particle Gel Immuno Assay (PaGIA). Red coloured polymer particles are sensitised with three different synthetic peptides representing antigenic sequences of T.cruzi: Ag2, TcD and TcE (Peralta et al., J.Clin.Microbiol.32, 971-4,1994). When these particles are mixed with serum containing specific antibodies, particles agglutinate. The reaction mixture is centrifuged through a gel filtration matrix allowing free non-agglutinated particles to deposit on the bottom of the microtube while agglutinated particles remain trapped on the top or distributed within the gel. The result can be read visually. In order to investigate the ability of the ID-PaGIA to discriminate negative and positive sera, 198 samples, 117 positive and 81 negative, collected in four different Institutions in Brasil were tested by each of the participants of the same Institutions. All sera were previously defined as positive or negative according to results obtained with three conventional serological tests (IFI, IHA and ELISA). Results of ID-PaGIA Chagas were considered positive or negative by visual interpretation agreement of three technicians not aware of conventional serology classification. Sensitivity rates varied from 95.7% to 97.4% with mean sensitivity of 96.8% and specificity rates varied from 93.8% to 98.8% with mean specificity of 94.6%. The overall Kappa test was 0.94. The assay presents as advantages the simplicity of operation and the reaction time of 20 minutes. In this study, the ID-PaGIA showed to be highly sensitive and specific. Extended field studies are under way.

EVALUATION OF CHAGAS INNOLIA ASSAY AS A CONFIRMATORY TEST FOR CHAGAS DISEASE.

Sabino, EC¹, Salles N¹, Stoops E², Santos ML¹, Saez-Alquezar, A¹, Zrein, M².

1. Fundação Pró-Sangue/Hemocentro de São Paulo, 2. Innogenetics, Gent/Belgium

Screening of blood donors for Chagas disease using currently available serologic tests is complicated by the lack of adequate sensitivity, discordant results between tests and the absence of a gold standard. The development of a confirmatory test is a major challenge in the diagnostic of Chagas disease. A INNOLIA assay was developed using 6 peptides derived from most representative T cruzi immunodominant proteins and one recombinant protein. This assay was evaluated with reactive blood donors from whom epidemiological data was available. Samples were tested with IFI, ELISA and IHA and classified as reactive in 0, 1, 2 or 3 tests. A sample received a score F when the donor refered someone in their family with Chagas disease, and S when the donor had always lived in an urban area and did not recall any contact with the Chagas bug. The results are shown in the table

INNOLIA Chagas Results

Score Posn
(%) Indn
( %) Negn
(%) Total 3F 64 (97) 2 (3) 0 (0) 66 1F 3 (15.7) 1(5.3) 15 (79) 19 1S 1(2) 1 (2) 48 (96) 50 0S 0 (0) 0 (0) 46 (100) 46 L.donovani 0 0 4 4

Conclusion: INNOLIA Chagas MAYBE a suitable assay for the confirmation of Chagas disease.

A RECOMBINANT ANTIGEN AND PEPTIDE LINE IMMUNOASSAY (LIA) AS AN ALTERNATIVE DIAGNOSIC TEST FOR CHAGAS' DISEASE

da Costa GCV⁽¹⁾, Teixeira MGM⁽¹⁾, Borges-Pereira J⁽²⁾, de Castro JAF⁽³⁾, Coura JR⁽²⁾ , Vanderborght BOM^(4,5), Stroops E ⁽⁴⁾, Zrein M⁽⁴⁾ , Peralta JM⁽¹⁾ & Oelemann WMR⁽¹⁾,

(1) Instituto de Microbiologia, UFRJ, Rio de Janeiro, (2) Departamento de Medicina Tropical, IOC-FIOCRUZ, Rio de Janeiro and (3) Departamento de Medicina tropical , UFPI, Teresina, (4) Innogenetics, Gent, Belgium, (5) HUCFF, UFRJ, Rio de Janeiro

Chagas´ disease is endemic throughout Latin America and represents a major cause of morbidity and death in the affected countries. In Brazil, successful vector control programmes abolished almost completely naturally occuring transmission of Trypanosoma cruzi, but transmission by transfusion of blood from infected donors accounts for an estimated number of 20,000 new cases per year. In the urban centers infected blood donors usually immigrated from endemic rural areas where a great variety of circulating T. cruzi strains and different forms of manifestation of the disease are found. Therefore, an efficient donor screening is very important in order to discard contaminated blood while not negatively affecting the country's blood supply. In blood bank screening serological tests are at present the only feasible means. In this study we evaluated the diagnostic performances of a commercial line immunoassay (LIA). This assay consists of seven T. cruzi antigens which have been coated as discrete lines to a nylon membrane with plastic backing. In addition, the strips contain control lines for sera with strong, moderate and weak (cut-off) reactivity. Positive or negative results are determined visually by comparing the antigen lines with the controls.

Serum panels were obtained in four different Brazilian areas where Chagas' disease is either endemic or sporadic. Their serological status was defined using concordant results of IIF and an in-house ELISA employing the cytosolic fraction of strain Y epimastigotes as antigen.

Seventy-eight sera were obtained in the Amazon region (3,8% positive, 96,2% negative) where Chagas disease is sporadic; 261 sera are from Minas Gerais (69% positive, 31% negative) where the cardiac and digestive forms of the disease are frequent; and 246 sera were obtained in Piauí (82,1% positive, 17,9% negative) and 440 sera in Paraíba (30,7% positive, 69,3% negative), where the indeterminate form of the disease is common.

As a whole, we employed 520 (50,7%) positive and 505 (49,3%) negative sera in the evaluation of the prototype version of the INNO-LIA CHAGAS Ab test (Innogenetics Inc., Gent, Belgium). Our results show that the LIA presents a relative sensitivity of 99,8%; a relative specificity of 97,4%; and a precision of 98,5%.

DETECTION OF ANTIBODIES ANTI-LEISHMANIA AND ANTI-T.GONDII USING DOT-DYE-IMMUNOASSAY

Mouta-Confort, E. ¹ ;Nascimento, R. G. ¹ ;Alves, A. S. ² ; Marzochi, M. C. A¹ ;Amendoeira, M. R. R. ²

¹Lab. Imunodiagnóstico Departamento de Ciências Biológicas - ENSP - FIOCRUZ - Cx. Postal 926-CEP-21.041-210 ² Departamento de Protozoologia - IOC-FIOCRUZ - Cx. Postal 926-CEP 21045-900

Actually the test ELISA and IFI used, for the sera diagnosis for leishmaniasis need highly coasted equipments which limit its application in the field conditions. The use of the textile dyes in immunoassays (dot-Dye-immunoassay; dot-DIA), was recently described by Snowden & Hommel, (1991) being also used for the diagnosis of Schistosomiasis mansoni. (Rabelo et al. 1992). This test has been demonstrated to be a simple method, rapid and presents a high sensibility and specificity. In this work we are trying to standardize the dot-DIA for the anti-Leishmania antibodies detection in dogs serum provided by the endemic areas of LVA and LTA and the anti-T.gondii antibodies detection in human serum. In this assay, the positive reactions can be easily seen by the color development in the antigen application site in the nitrocellulose membrane. Have been used two antigens: Leishmania major-like extract frequently used in ELISA for the leishmaniasis diagnosis and T-gondii extract dotted in the nitrocellulose membrane in concentrations of 0,3 and 100 mg of protein/ml. Different dyes concentration of anti-dog immunoglobulin, and human anti-IgG, between 12 and 100 mg/ml were used for both preparation. In the assays for the research of anti-Leishmania antibodies analysis, the serum were diluted from 1:50 to 1:1600. In the assays for the research of antibodies anti-T.gondii the dilution used varies between 1:16 to 1:256. At this stage we used 6 dogs serum provided by the endemic areas of LTA in the state of Rio de Janeiro with positive reactions in IFI and ELISA, and 6 negative reaction. We also used 6 human positive reaction and 3 negative serum reactions in the methods. At the present moment, the preliminary results show that in all antigen and conjugate concentrations of dot-DIA presented a 100% concordance with ELISA and IFI in the minor dilutions of serum used. However, the false positive results were observed in these dilutions. These unespecific reactions were eliminated in antigens concentrations of 12 to 50 mg/ml. The number of the serum must be increased for a better definition according to the best concentration of reagents and for calculation of various predictive values.

Support by CNPq and FIOCRUZ

ELISA WITH PLASMODIUM VIVAX-LIKE/P.SIMIOVALE CS REPEATS IN HUMAN SERA AND IN ANOPHELINES FROM THE STATE OF ACRE, BRAZIL.

Marrelli, M.T.^1,2, Branquinho, M.S.³, Hoffmann, E. H.E.¹, Benevento, C.M.³, Natal, D.⁴, Taipe-Lagos, C.B.⁴ & Kloetzel, J.K.^1,2 1-Inst. de Med. Tropical de S.Paulo, Av.Eneas de Carvalho Aguiar, 470, S.Paulo, Brasil, 05403-000, 2-Depto. de Parasitologia ICB-USP, 3- SUCEN-S.Paulo, 4-Fac. de Saúde Pública USP.

Polymorphism of the Circumsporozoite (CS) protein of P.vivax has been described by several authors. Besides the original tandemly repeated amino-acid sequence ("classic" P.vivax, Type I), the variant VK247 (Type II) and the one corresponding to human P.vivax-like are now known. The latter, morphologically similar to P.vivax, has a repetitive sequence identical to that of P.simiovale. A previous study from our group in the State of Acre, Brazil, 3056 anophelines captured in 1991-1992 tested by two-site ELISA, found 1.3% positivity for P.vivax VK247 and 2.3% for "classic" P.vivax in Anopheles oswaldoi (n=2610), 0.8% and 0.3% in An.deaneorum (n=362), besides positivity for P.falciparum (Branquinho et al., 1993). Sporozoites were also found in salivary glands of 1/34 An.oswaldoi in the same region, in 1995 (Branquinho et al., 1996). In the present study we evaluated anti-P.vivax-like antibodies in human sera from the same region, as well as anopheline infection. Sera were collected from 120 adults of both sexes. Samples were tested by ELISA against the synthetic peptide (APGANQEGGAA)₃. Extracts of 1375 anophelines, which had been kept at -70^oC, from the 91-92 capture, were also processed by capture-ELISA, with monoclonal antibody Pam 172, directed against the same repetitive region of the P.vivax-like CS protein. 10.7% (13/120) of the sera tested positive in ELISA. 1207 of these anophelines were An.oswaldoi with 12 positives (1.0%), while 168 were An.deaneorum, with 2 positives (1.2%). The correlation in the same geographical region between human and anopheline serology for the repetitive CS sequence of human P.vivax-like/P.simiovale is a strong indication that sporozoites of the variant migrate to the salivary glands of these vectors, and can be inoculated into men. This is an additional indication that An.oswaldoi may be a malaria vector in that region and that the parasite is circulating in the area.

Financed by Fundação Nacional de Saúde, LIM49-HC, Superintendência de Controle de Endemias (SUCEN).

SEROLOGICAL DIAGNOSIS OF CUTANEOUS LEISHMANIASIS BY THE ELISA TECHNIQUE: DEFINITION OF PARAMETERS FOR COMPARING EXPERIMENTAL INFECTIONS IN THE CEBUS APELLA (PRIMATES: CEBIDAE) MODEL WITH NATURAL INFECTIONS IN MAN.

Souza, R. A¹; Ramos, P.K.¹; Silveira, F.T.¹; Garcez, L.M.¹; Brigido, M.C.²; Muniz, J.P.C.² & Shaw, J.J.^{1 & 2}

¹ Programa de leishmaniose, Instituto Evandro Chagas/FNS, Av. Almirante Barroso, 492 - 66090-000 Belém, Brazil - email <[e-mail]>; ² Centro - Nacional de Primatas/FNS; ³ Depto de Parasitologia, Instituto de Ciências Biomédicas, USP.

One of the axioms for using a non-human primate model to study American Cutaneous Leishamaniasis (ACL) is that their immune responses need to be simialr to those of man. The objective of the present study was to define diagnostic paramaters of ACL using the ELISA technique for future comparison with those obtained from experimental infections in Cebus apella. The plasmas of 27 patients diagnosed parasitologically as having cutaneous caused Leishmania (Leishmania) amazonensis were examined by the ELISA technique. Of these individuals 19 were cases in which the lesions were limited and 8 were cases of anergid diffuse cutaneous leishmaniasis (ADCL). Besides these parasitologically proven cases the serum of 53 normal control individuals with no signs of disease were also tested. ELISA antigens were prepared from cultures of L. (L.) amazonensis and L. (Viannia) braziliensis by consecutively freezing (-182ºC) and thawing (37ºC) washed cultures 10 times. The optimal antigen dilutions were determined by testing serial dilutions of known serologically positive sera against serial antigen dilutions. A human anti-IgG/peroxidase conjugate was used with an OPD substrate and reactions were read at 492nm. Antibody titres obtained with the different antigens were compared statistically by the analysis of variance. The ELISA titers of the ADCL patients were significantly higher with the homologous antigen and were also significantly higher than those of patients with normal ACL. The sensitivity of the test varied in function of the antigen and was 100% with the L. (L.) amazonensis antigen and 47.4% with L. (V.) braziliensis antigen for patients with normal ACL. The difference between the titers obtained with the two antigens was highly significant (p=0.002684). For normal sera the specificity was 67.9% for the L. (L.) amazonensis antigen and 86.8% for the L. (V.) braziliensis antigen. When considering the low specificity for the normal sera, particularly with the L. (L.) amazonensis antigen, it has to be remembered that we could not rule out the possibility of undetected infections. These results show that the ELISA is a senstive test for uncomplicated ACL caused by L. (L.) amazonensis, especially with the homologous antigen but that the specificity of test for human serum needs to be re-evaluated using normal sera from a non-endemic region.

Financial support: Instituto Evandro Chagas/FNS, Centro Nacional de Primatas/FNS, CNPq., Programa PCMAM/FNS

COMPARATIVE SEROLOGIC SURVEY OF SERA AND BLOODSPOT ELUATES SAMPLES IN THE DIAGNOSIS OF CANINE AMERICAN TEGUMENTARY LEISHMANIASIS

Mendes da Cruz, D.A., Duarte, R. & Marzochi, M.C.A.

Laboratório de Imunodiagnóstico, ENSP - FIOCRUZ, Caixa Postal 926, cep. 21041-210, RJ, Brazil

The relation between canine and human leishmaniasis has been shown by the presence of infected dogs in endemic areas, making evident its importance to the infection epidemiology and explaining the utilization of canine material in epidemiologic studies. In the serodiagnosis of leishmaniasis, ELISA (Enzyme-linked immunosorbent assay) and IFT (Indirect immunofluorescent test) have presented satisfactory results, generally using sera samples in quantitative tests. However, this kind of sample presents difficulties to collection, transportation and storage. In order to try to solve these problems, blood samples collected on filter paper for qualitative tests or screenings in epidemiologic studies are currently being used. For elution, filter paper discs with dried blood samples are cut and put in tubes with phosphate-buffered saline (PBS). In this study we compared the results of canine sera and bloodspot eluates tested by ELISA-IgG and IFT-IgG for American tegumentary leishmaniasis (ATL), aiming at a better utilization of this kind of samples on laboratory routine. Both samples were collected from 72 dogs of São Lourencinho, at Vale do Ribeira, São Paulo State, recognized as endemic area of ATL and analyzed by IFT-IgG. Only sera samples were analyzed by ELISA-IgG. The results obtained with sera were 6 (8.3%) positive by IFT-IgG and 11 (15.3%) were positive by ELISA-IgG. The IFT-IgG performed on bloodspots eluates had 4 (5.5%) positive. The concordance (Kappa coefficient - k) IFT/ELISA performed on sera was considered regular, and the concordance IFT-sera/IFT-eluates were considered good. Although IFT-sera is considered the reference method for ATL serodiagnosis, ELISA-sera has shown higher positivity indices, being indicated for screenings studies, and IFT-eluates in this study showed less sensitivity than the other methods utilized.

CANINE LEISHMANIASIS: VALUATION AND CONFRONTATION OF THE IMMUNOENZIMATIC TECHNIQUES FOR THE DETECTION OF IMMUNOGLOBULIN G.

Laurentino-Silva, V.; ⁽¹⁾ Freire, R.B. ⁽²⁾ & Marzochi, M.C.A. ⁽¹⁾

⁽¹⁾ Laboratório de Imunodiagnóstico - DCB-ENSP-FIOCRUZ

⁽²⁾ Laboratório de Imunologia Animal - UFRRJ

The purpose of this study was to provide a brief overview and emphasize the coolness of immunoenzimatic assays ELISA and Dot - ELISA on the canine leishmaniasis diagnosis. Three different species of the genus Leishmania (L. brasiliensis, L. chagasi and L. major-like) were cultured during 7 days in Schineider's liquid medium originating 12 different antigenic preparations which were evaluated, according to their relative reactivity. The immunoenzimatic assays were performed using 29 test sera obtained from dogs with visceral leishmaniasis (VL), 30 test sera from dogs having the tegumentar form of the disease (TL) and 30 samples of serum obtained from uninfected dogs (healthy). The infected animals were from endemic areas, whereas the uninfected ones were from non endemic regions of Rio de Janeiro State, Brazil. The potential of immunoassays, ELISA and Dot - ELISA, for the diagnostic of canine VL has proven to be extremely similar, once there was a concordant result in almost every antigenic preparation assayed, either by spectrophotometer or by visual observation. The sensitivity values, related to the visual ELISA test, ranged from 82.8 % to 89.7% when L. chagasi was the source of antigens. Using the same method to make the diagnostic of canine TL, the sensitivity values were lower than 50% when the antigenic preparations were from L. chagasi or L. major-like. Using antigenic preparations obtained from L. brasiliensis the sensitivity raised to 73.3 %. One of such antigens, named Lb SPR gave rise to a 90 % of sensitivity, showing its further useful application as a diagnostic antigen. Although inaccurate results were mostly achieved when results were negative, mainly in the TL - Dot - ELISA , wherein the sensitivity varied from 10.0 % to 66.7 %, the VL - Dot - ELISA test, because of its high fix and variable qualities, defeated the conventional ELISA. The results achieved using canine leishmaniasis - ELISA, when evaluated as an assembly, comparatively to the reference assay (indirect - immunofluorescence-assay, IFA), should be considered as regularly concordant. In spite of that, an excellent concordance was achieved when Dot - ELISA tests, using integral promastigotes as antigen, were carried out. The further utilization of enzymatic immunoassays for the diagnostic of canine leishmaniasis was discussed, and practical limitations, concerned mainly to the TL immunoassays, led to considerations about the relationship between the levels of circulating antibodies and their detection in such form of disease.

DETECTION BY IMMUNOBLOT ANALYSIS OF POTENTIALLY DIAGNOSTIC LEISHMANIA BRAZILIENSIS ANTIGENS FOR HUMAN CUTANEOUS LEISHMANIASIS

BRITO, M.E.F.*; MENDONÇA, M.G**.; GOMES, Y. M*.; & ABATH, F.G.C*

*Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-FIOCRUZ, Cidade Universitária , 50670-420, Recife-PE, Brazil. ** Departamento de Dermatologia, Universidade Federal de Pernambuco-UFPE, Recife-PE, Brazil.

American cutaneous leishmaniasis (ACL) is endemic in some regions of Brazil. Routine diagnosis is generally based on clinical findings together with the confirmation of the parasite by examination of smears, and culture or biopsy of the lesions . In the present study we evaluated Western blot analysis as a tool for diagnosis of cutaneous leishmaniasis.

We tested 50 sera of patients diagnosed clinically with cutaneous leishmaniasis, 20 sera of individuals with other parasitic diseases, 10 sera of normal individuals from endemic areas, and 10 sera from normal individuals from non-endemic areas. Soluble and non-soluble antigens (l5mg/well) obtained from L. braziliensis were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transfered to nitrocelullose. IgG antibody binding was then determined for individual sera. Analysis of the soluble and non-soluble antigenic pattern allowed the identification of a diagnostic profile showing two doublets with 30 and 27 kDa in the soluble fraction and bands of 60, 43, 40, 19 and 16 kDa in the non-soluble fractions. Although there was some cross-reaction with patients diagnosed as Kala-azar and Chagas' disease, the antigenic pattern allowed differentiation in most cases. Our suggest results that immunoblotting may be a useful approach for diagnosis of human cutaneous leishmaniasis.

Supported by: FIOCRUZ.

THE FML-ELISA VERSUS IF IN THE ANALYSIS OF SERA FROM DOGS OF A KALA-AZAR ENDEMIC AREA (SÃO GONÇALO DO AMARANTE, RN)

da Silva , VO¹, de Azevedo Pereira, R¹, Borja-Cabrera GP¹, Palatnik,M² & Palatnik de Sousa, CB¹.

Instituto de Microbiologia "Prof. Paulo de Góes"¹, Fac. Medicina-HUCFF², Universidade Federal do Rio de Janeiro, Caixa Postal 68040, Rio de Janeiro, 21941-590, RJ, Brasil.

The FML-ELISA assay (100% sensitivity, 96% specificity in diagnosis and prognosis of human Kala-azar) showed 100% sensitivity, 100% specificity and 100% correlation with IF and in the diagnosis of canine overt Kala-azar. The analysis of the anti-Leishmania donovani antibody prevalence in dogs from São Gonçalo do Amarante (RN), disclosed 83/350 seropositive animals. This 23.7% of seroreactivity might explain the high endemicity of this district that account for 6% of the annual cases of human Kala-azar. In December 1996, a first screening was developed in the area Q41-58. Among 138 dogs tested, 28 were identified as seroreactive in the FML-ELISA assay. Only 6/28 were also positive, and 3/28 considered at the threshold level by the IF assay regularly performed by FNS-RN. Two consecutive screening of sera from 160 dogs were performed in the Q20-40 area. In October 1996 41/160 sera (25.6%) were reactive in the FML-ELISA and 0/160 (0%) in the IF analysis. The second screening (March 1997) disclosed that 10/41 FML-reactive dogs and 10//119 non-reactive died during this period. 3 of them showed typical Kala-azar and were removed by the FNS while another one died in the area showing all characteristic symptoms. The number of total obits and of obits due to Kala-azar was significantly associated to the seroreactivity in the FML-ELISA assay (c ²= 7.126p<0.01 and c ²= 8.241 p<0.005, respectively). Furthermore 24 dogs considered non-reactive in 1996 developed anti-FML antibodies during this interval while only 8 seropositive individuals loosed reactivity. Our results prove the higher sensitivity and the predictive value of the FML-ELISA assay when compared to the IF assay performed by FNS on the diagnosis of canine Kala-azar.

Support: PNUD-FNS, PRONEX, CNPQ, RHAE-CNPQ, FINEP, CAPES, FUJB-CEPG-UFRJ.

THE MICRO-ELISA TECHNIQUE IN THE SERODIAGNOSIS OF CUTANEOUS LEISHMANIASIS IN DIDELPHIS MARSUPIALIS (MARSUPIALIA: DIDELPHIDAE)

Souza-Leão^# , S; Franco, AMR* & Silva, MH**

#Departamento de Protozoologia, *Departamento de Imunologia, Instituto Oswaldo Cruz, FIOCRUZ, CP. 926, RJ & **Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21941, RJ, Brasil.

Didelphismarsupialis is considered as the main reservoir host of Cutaneous Leishmaniasis in the Amazon region, Brazil. From this metatherian mammal, Arias et al. (1981) isolated Leishmania guyanensis and L. amazonensis. Parasitological methods are not always succesful in detecting leishmanial infections, mainly in sylvatic animals. The parasites could not be easily isolated from living or necropsied infected opossums with L. amazonensis through culture or hamster inoculation of skin, blood, spleen, liver and bone-marrow (Franco, A M.R. et al. 1986. Franco, A M.R. et al. 1990). The enzyme-linked immunosorbent assay (ELISA) could be an important investigative tool to use as diagnosis of natural parasitic infection of wild mammals in epidemiological trials. In this present study, micro-ELISA has been applied to detect specific IgM and IgG levels of antibodies in the sera of opossums D. marsupialis. It was used different groups of animals. Sera of normal, immunized and experimentally inoculated and/or infected opossums with L. amazonensis (MHOM/BR/77/LTB0016) were tested. To evaluate antibody responses, IgM and IgG preparations were purified from the sera of D. marsupialis (Souza-Leão, S.M. et al. 1987). These two immunoglobulin preparations were used to generate specific goat antibodies to apply in ELISA-based serology. Different levels of Igs were found and the cut-off with normal sera of opossums was determined. Results have been compared with the related data obtained in indirect fluorescent antibody tests (IFAT). Our data demonstrate that ELISA assay is available and could be applied for field surveys of New World leishmaniasis in opossums. Experiments using the dot- ELISA on nitrocellulose membrane are in progress.

PREVALENCE OF MONTENEGRO SKIN TEST POSITIVITY TO AMERICAN CUTANEOUS LEISHMANIASIS IN HIGH ENDEMIC REGION OF PERNAMBUCO, BRAZIL.

Sinval P. Brandão-Filho, Maria E. Felinto de Brito, Clediane A. Pereira Martins* & Iara B. Sommer*.

Instituto Aggeu Magalhães/FIOCRUZ. Av. Moraes Rego, S/N, Recife-PE, Brasil, CEP 50670-420. *FACEPE scholarship.

An epidemiological study has been developed since 1991 in three highly endemic localities for American Cutaneous Leishmaniasis (ACL) in the 'Zona da Mata' of Pernambuco. Here we presents the results of the study to compare the prevalence of ACL in two different periods (1991 and 1996) by Montenegro Skin Test (MST). It was used the antigen (leishmanin) producted according Mayrink et al. (Mem Inst. Oswaldo Cruz, 88 (Suppl.): 226, 1993). In the first survey (1991) the test was applied in 412 individuals habitants of Refrigerio, Tranquilidade and Raiz de Dentro. The MST positivity in this year was 31%. The frequency distribution of the posivites by social and profissional activities consisted of 50.35% of farmer workers, 19.60% of house people (predominantly wifes), 15.24% of students and 14.81% of childrens under 5 years old. During the survey conducted in 1996 in 458 individuals residents in the same localities, it was noticed an increase of 12.5% (43.56%) in the MST positivity. The distribution of the frequency was similar to the first survey (49.06%, 21.90%, 14.86% and 8.56%, respectively). These data shows that the same pattern of transmission may be involved which corresponds the maintenance in these areas of the same social economic activitiy and ecological characteristics.

Supported by FACEPE and CPqAM/FIOCRUZ.

CHARACTERIZATION OF T CELL RESPONSES IN SUBJECTS WITH SUB-CLINICAL L.BRAZILIENSIS INFECTION

Follador, I, Bacellar, O, Araújo, C, Almeida, R, & Carvalho, EM.

Serviço de Imunologia, Hospital Universitário Prof. Edgard Santos, Universidade Federal da Bahia, Rua João das Botas s/n, Canela, Salvador, 40110-160, Bahia, Brasil

L.braziliensis infection is associated with cutaneous or mucocutaneous leishmaniasis. In a cohort study of 555 subjects living in an area of L.braziliensis transmission, it was documented that 39 individuals converted to positive a skin test with leishmania antigen in a two-years follow-up. These subjects had neither previous history of leishmaniasis or clinical evidence of active disease during the follow-up. Lymphocyte proliferative response measured by ³[H]-Thymidine uptake and cytokine production (IFN-g and IL-5) were determined after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with leishmania antigen in 22 of these individuals and these data were compared with those observed in patients with classical cutaneous leishmaniasis (CCL). In vitro lymphocyte proliferative response to leishmanial Ag was observed in 14 (64%) of the 22 subjects with sub-clinical infection versus in 95% of CCL patients. The mean + SD of IFN-g production in subjects with sub-clinical infection (185+406pg/ml) did not differ from the mean of IL-5 (124+145pg/ml). IFN-g and IL-5 production were observed in 59% and 91% of the supernatants of lymphocyte cultures of these individuals with sub-clinical infection respectivelly. These data show that there is a Th0 activivation in patients with sub-clinical L.braziliensis infection and that only a few of these subjects are high IFN-g responders. Since these subjects with sub-clinical infection are putative resistant to leishmania these data indicate that protective immune response in humans are associated with Th0 activation.

Supported by NIH Grant 30639, PRONEX-FINEP and CNPq.

TNF-a IN CUTANEOUS AND MUCOSAL LEISHMANIASIS

Bacellar, O, Lessa, H, Barral, A, Machado, PL, D'Oliveira Jr, A, Almeida, RP, Cruz, A & Carvalho, EM.

Serviço de Imunologia, Hospital Universitário Prof. Edgard Santos, Universidade Federal da Bahia, Rua João das Botas s/n, Canela, Salvador, 40110-160, Bahia, Brasil

Control of leishmania infection is associated with T cell response and IFN-g is the cytokine that mediate leishmania killing. Although IFN-g is produced in the course of L.braziliensis infection, progression of the infection and tissue damage associated with an inflamatory response are observed. TNF-a have been detected in the serum and expression of mRNA for TNF-a is observed in tissue of patients with mucosal leishmaniasis. In the present study TNF-a and IFN-g levels were measured in the supernatants of lymphocyte cultures after 48 hours of in vitro stimulation with leishmania antigen before and after therapy of cutaneous (N=10) and mucosal (N=10) leishmaniasis. Additionally in situ TNF-a was measured in mucosal tissue by immunochemistry. Both cytokines were produced in all patients studied. IFN-g and TNF-a levels were 1794+1258 pg/ml and 299+189 pg/ml in CL and 1923+893 pg/ml and 474+224 pg/ml in mucosal leishmaniasis respectivelly. Additionally, TNF-a was documented in tissue by immunochemistry. Two months after therapy IFN-g levels were reduced by 39% and TNF-a by 61% in patients with mucosal disease. Finally in 6 patients with mucosal disease that were refractory to antimonial therapy, cure of the lesions was observed when antimonial was associated with pentoxifyline, an inhibitor of TNF-a production. Putting together, these data argue in favor that although TNF-a and IFN-g are important for parasite killing, these cytokine may be involved in tissue damage in cutaneous and mucosal leishmaniasis.

Supported by NIH Grant 30639, PRONEX-FINEP and CNPq.

SEROCONVERTION IN SCHOLAR CHILDREN POSITIVE FOR T. CRUZI TREATED WITH BENZNIDAZOLE: EPIDEMIOLOGICAL ANALYSIS

Revollo, S.¹, Postigo J. R.¹, Soto M. L.¹, Alcazar J. L.², Flores, M.³, Yaksic, N. ⁴, Terrazas G.¹ and Illanes, M.¹

1 Facultad de Ciencias Farmacéuticas y Bioquímicas, Instituto de Servicios de Laboratorio de Diagnóstico e Investigación en Salud (SELADIS), UMSA, Av. Saavedra, Nº 2224, La Paz, Bolivia.

2 Agroquímico - ORSTOM, Universidad Mayor de San Simón, Cochabamba, Bolivia.

3 ORSTOM - Instituto Boliviano de Biología de Altura (IBBA), CP9214, La Paz, Bolivia.

4 Instituto Boliviano de Biología de Altura (IBBA), Facultad de Medicina, UMSA, c/Claudio Sanjines s/n, La Paz, Bolivia.

The Chagas disease, has been recognized as a major problem of public health these last years. In Bolivia, the infantile population presents a high infection risk by Trypanosoma cruzi in endemic zones. In this study, 1.479 serums originating from children of 4 to 10 years of a peri-urban zone at Cochabamba were analyzed. Two serologics tests [IFI] and[ ELISA] were used for antibodies detection anti Trypanosoma cruzi. Thirty five children were treated with Benznidazole and evaluated according clinical and epidemiological schedules.

There were serological tests accomplished before and post treatment for detection of antibodies anti IgG for T. cruzi through ELISA, IFI and ELISA-RA (recombinants antigens A13 and H49) techniques. Also, a control of reinfection was accomplished, smudging all the houses and their environment of the treated children.

A 5,6% seropositivity was found in the group studied. This population is composed two groups of children originating from two different social strata (Group 1 and Group 2). More seropositive children in Group 2 was found (7,2%) than in the Group 1 (4,6%). This situation is significantly related with the infection risk by Trypanosoma cruzi, since Group 2 is composed lower economic resource children than the Group 1. The children of group 1 were submitted to a specific treatment with Benzonidazole at a dosis of 8 mg/kilogram/weight during 60 continuous days, controlled by medical staff.In the results obtained by classic methods (ELISA and IFI) it was observed a seroconversión in a 26% of the total of children treated after 6 months the treatment ended. Not occurring the same with the ELISA-RA.On the other hand, the statistical analysis performed among the variable clinics, hematologic and antropometrics in relation to the infection by Trypanosoma cruzi, do not demonstrate a net dependency among these variables.This infantile population could be said probably cured. However, other studies will be realized using more specific antigens.

Supported by UNDP/World Bank/WHO/TDR (Ref. ID-940856) .

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Publication Dates

• Publication in this collection
  30 Nov 2000
• Date of issue
  Nov 1997