S1-4 – Morbidity and Regional Variation in Chagas Disease in Brazil (*)

Despite the fact that Leishmania parasites are transmitted exclusively by the bite of an infected sand fly, there are few reports concerning the relationship of this factor on Leishmania infection. Two models were developed, one using needle injection and the second based on natural transmission by the vector. During the natural transmission of Leishmania, sand flies inoculate low numbers of metacyclic promastigotes, along with saliva, into the skin of the mammalian host. It has previously been revealed by experimental mouse models of cutaneous Leishmaniasis that infection in a subcutaneous site is enhanced following co-inoculation of parasites with sand fly saliva. Extending these observations to a dermal model of disease, we take into account the fact that most individuals who live in endemic areas are exposed to saliva from uninfected flies before the bite that transmits infection. We developed a model of cutaneous Leishmaniasis with L. major in BALB/c and C57Bl/6 (B/ 6) mice that seeks to mimic these natural conditions of infection: low number of metacyclic promastigotes (1000), co-inoculation with salivary gland sonicate (SGS, 0.2 gland pair equivalents) from a natural vector, Phlebotomus papatasi, and intradermal inoculation into the ear dermis of naive mice or of mice pre-exposed to sand fly saliva. Our studies reveal a dramatic exacerbating effect of saliva on lesion development in the dermal site, and a complete abrogation of this effect in mice pre-sensitized to sand fly saliva. In both BALB/c and B/6 mice, the dermal lesions in saliva naïve mice appeared earlier, progressed to a larger size, were more destructive, and contained greater numbers of parasites following infection in the presence of SGS. Furthermore, the dermal lesions in normally resistant B/6 mice failed to heal for up to 7 months after infection in the presence of saliva. Flow cytometric analysis permitted detection of cytokines produced by epidermal cells at the single cell level and disease exacerbation was found to be associated with the ability of saliva to elicit an early (6 hr.) increase in the frequency of cells producing Type 2 cytokines in the epidermis. The early Type 2 responses elicited by SGS in the epidermis were not observed in mice previously injected with SGS. These mice made anti-saliva antibodies that were able to neutralize both the ability of saliva to enhance infection and to elicit IL-4 and IL-5 responses in the epidermis. These results are the first to suggest that in individuals at risk of vector borne infections, the exposure history to vector saliva might influence the outcome of exposure to transmitted parasites. The second approach was to define a reproducible murine ear model based on the transmission of Leishmania major by bite was established. The model was used to investigate the effect of host pre-sensitization to sand fly saliva via exposure to uninfected sand fly bites on the course of infection but also to analyze the initial events occurring in the site during natural transmission. This parameter is of epidemiological importance since the natural rate of infection in sand fly populations is low, and inhabitants of endemic regions are repeatedly subjected to the bite of uninfected flies before coming into contact with the parasite throughout the follow up period (P < 0.01). Moreover, 92% (11/12) of ears in naive mice developed lesions compared to 67% (8/12) in ears of sensitized mice, and the number of lesions per ear was also higher in the naïve mice. The above data clearly demonstrates an increased resistance to Leishmania as a result of host pre-exposure to uninfected sand fly bites. These results are the first to suggest that in individuals at risk of vector borne infections, their exposure history to vector saliva might influence the outcome of exposure to transmitted parasites. S5-2 – NATURAL BARRIERS TO LEISHMANIA INFECTIONS IN THE SANDFLY VECTOR Pimenta, P. F. P. , Secundino, N.F.C. and Nieves, E. Laboratory of Medical Entomology, Centro de Pesquisas Rene Rachou CPqRR, Fundação Oswaldo CruzFIOCRUZ. Av. Augusto de Lima 1715, Belo Horizonte-MG Brazil. Fax: (31) 2953115 e-mail: [email protected] The completion of the life cycle of the Leishmania inside its sandfly vector is the main factor for successful of transmission to the vertebrate host. The parasite needs to deal with several challenges in the environment after beong ingested by the insect. A very fine balance exists between the sandfly and the developing pathogen and there are many physical and chemical natural barriers that may interrupt the parasite’s life cycle. These barriers may lead to death of the parasite, a shortened life span or allow insufficient time for the parasite to develop and mature in order to be passed on to the vertebrate host. The ability of a Leishmania sp overcome these barriers, associated with susceptibility of certain sandfly species to infection could be determinants for vectorial capacity. The parasite needs to able to: (a) resist enzymatic activities present in the insect midgut; (b) escape from the peritrophic matrix that surrounds the bloodmeal; (c) attach to the epithelial cells; (d) develop a complete life cycle ending with differentia33 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 tion to metacyclics, the infective form that is introduced into the vertebrate hosts by the bite of the sandfly.The first natural barrier that any microorganism including Leishmania needs to deal with is to resist or avoid the enzymatic activities in the insect midgut. The level of proteases in the gut is usually small but can rise 20-fold immediately after ingestion of bloodmeal. Adler et al (Proc. Royal Soc. London, B15:491, 1938) were the first to notice that nonsusceptible species of Leishmania were killed inside the bloodmeal during the digestion in the vector species. Experiments done with different natural pairs of species of Leishmania and their sandfly vectors from New and World have demonstrated that during blood digestion the numbers of parasites inside the gut decrease from two to eight-fold, mainly due the trypsin activity (Borosvsky & Schlein Med. Vet. Entomology 1:235, 1987 ; Pimenta et. al. Parasitology 115:359, 1997; Nieves et. al. 1999 J. Med. Entomology in press). The susceptibility of the parasite to the digestive enzymes appears to be stage specific with most mortality occurring in the intermediate forms. The completely transformed promastigotes are resistant due to the acquisition of a specific lipophosphoglycan coat on the cell surface (Schlein et al. Trans. Royal Soc. of Trop. Med. Hyg. 84: 353, 1990; Pimenta et al. 1991). The defective LPG Leishmania mutants are killed 48 h after ingestion by the vector enzymes, but in parasites with the ability to express the molecule this condition is inverted and they able to develop infection. Schlein and collaborators (Exp. Parasitology 62:376, 1986) also demonstrated that L. major is able to block a huge amount of digestive enzyme production in its natural sandfly vector, P. papatasi, this was not seen for L. donovani which is not naturally transmitted by this insect. Again the LPG molecules appears to be involved in modulating the activity of proteolytic enzymes, and therefore might delay digestion fo the bloodmeal and promote parasite survival. In conclusion, several parasites are killed inside the bloodmeal due the natural physiological conditions of sandfly digestion and this factor is relating to the non-susceptible Leishmania species. The surviving parasites need to pass another barrier involving their escape from the peritrophic matrix. In blood-feeding Diptera including sandflies, the peritrophic matrix is formed immediately after the ingestion of blood. It consists of a network of chitin in a matrix composed of proteins and proteoglycans. The major roles ascribed to this structure include preventing damage or clogging of microvilli by luminal contents, compartmentalization of digestive events by acting as a permeability barrier for digestive enzyme and protection against microbes by acting as a physical barrier to their development. Loss of Leishmania infection in unnatural sandfly vectors was found to be due to the failure of the parasite to escaper from the peritrophic matrix prior to bloodmeal elimination (Feng Peking Nat. Hist. Bull. 19;327, 1951; Walters et al. Am. J. Trop. Med. Hyg. 46:211, 1992), an event which is thought normally to be facilitated by the action of a parasitederived chitinase (Schlein et al. Proc. Royal Soc. of London, B245:121, 1991). In studies designed to define more conclusively the role of the peritrophic matrix in controlling the remarkable specificity of some Leishmania-sandfly interactions using sandflies fed on bloodmeals containing exogenous chitinase, a novel role for protecting Leishmania from the proteolytic activities of the blood digestion was explored by Pimenta et al. (Parasitology 115:359, 1997). The author provided direct evidence that the peritrophic matrix, while clearly acting as a potential barrier to the development of infection, also promotes early parasite survival by moderating the exposure of parasites to the hydrolytic activities of the bloodfed midgut, prior to their differentiation into protease-resistant forms. As the peritrophic matrix breaks down, the parasites need to attached to the gut epithelium to remain in the gut. In nonpermissive Leishmania species the promastigote are not destroyed by the digestive enzyme or expelled with the digested bloodmeal. Within an appropriate vector, the attachment of promastigotes to the microvillar lining of the abdominal and thoracic midgut is thought to be critical. As with other trypanosomatids, this attachment to the wall of the alimentary tract allows the parasite to remain in position during the bloodmeal excretion. It has been shown that LPG is directly involved in mediating attachment of procyclic promastigotes to the midgut (Pimenta et al. Science 256:1812, 1992; Pimenta et al. PNAS 91:9115, 1994) and these experiments suggest that gut-associated lectins or lectin-like molecules, which have been described for sandflies (Wallbanks et al. Trop. Med. And Parastol. 37:409-13; 1986) serve as parasite attachment sites. After the attachment the parasite needs to multiply and differentiates into the infective stage, the metacyclics being the only form that is able to survive in the vertebrate host. Since the work of Adler and Ber (Indian J. of Med. Research 29:803, 1941) it is believed that one particular form of promastigote exist in the sandfly adapted for life in the vertebrate. However only in the 1980’s, Sacks and Perkins (Am. J. Trop. Med. Hyg. 34:456, 1985) recovered promastigotes from infected midgut on days 4-7 after infection and demonstrated that they became progressively more virulent for vertebrates. This process known as metacyclogenesis is common to all other trypanosomatids, and is essential for vector competence. In all the Leishmania species, metacyclogenesis means that the parasites become preadapted to survival within the vertebrate host while within the vector. Differentiation into metacyclic is the final challenge for the Leishmania parasite inside the sandfly vector allowing it to complete its life cycle. Financial support: FIOCRUZ, WHO, CNPq and PRONEX. 34 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S5-3 – THE STUDY OF MOSQUITO EMBRYOGENESIS IS ONE OF THE REQUIREMENTS TOWARDS THE DEVELOPMENT OF A MALARIA VECTOR CONTROL PROGRAM Denise Valle (1,2), Abreu-Manso, P. P. (1,2,3), Cardoso, S. G. C. (1,2), Jesus-Martins, A. Jr. (1,2), Pereira-daCunha, M. (2), Monnerat, A. T. (1), Soares, M. J. (4) and Lima, J. B. P. (1,2) (1) Depto. Entomologia, FIOCRUZ, (2) Instituto de Biologia do Exército, (3) Depto. Patologia, FIOCRUZ, (4) Depto. Ultraestrutura e Biologia Celular, FIOCRUZ Nowadays an alternative strategy aiming at the control of malaria vectors is in development by different laboratories throughout the world. This novel strategy, based on the construction and field release of genetically modified Anopheles mosquitoes, is intended to block malaria transmission in a short period of time. To accomplish this, different research lines are being carried on, almost exclusively with Anopheles gambiae, the main african malaria vector: isolation of (a) gene(s) able to render the mosquito refractory to Plasmodium infection, characterization of gut-specific promoters, identification of mosquito transforming systems, genome mapping and isolation of mutants. Until recently neotropical malaria vectors could not take part in this process, because free-mating colonies of these mosquitoes were not available. Since the establishment of stable (more than 60 generations) Anopheles (Nyssorhynchus) albitarsis and Anopheles (Nyssorhynchus) aquasalis free-mating colonies (J. B. P. Lima), this bottleneck is overcome. We are presently taking advantage of these colonies to unravel some relevant aspects of the subgenus Nyssorhynchus biology aiming at the establishment of this novel malaria vectors’ control program at Brazil. One of our research lines is related to Anopheles embryogenesis. We consider that the analysis of the embryogenesis process is an additional requirement towards the establishment of this kind of vector control. It will be necessary to define time and place of the exogenous DNA injection in order to attain high transformation efficiencies. Detailed knowledge of the whole mosquito embryogenesis will also help in the characterization of mutants and transgenic lines to be generated and, consequently, in the feasibility of application of this strategy in the field. And, it is expected that data acquired with neotropical Anopheles can be applied to An. gambiae, helping transformation experiments. The external morphological characterization of An. albitarsis embryogenesis is being carried on, by laser scanning microscopy. An adequate protocol is under development in order to perform a detailed analysis of mosquito internal structures morphogenesis. Egg permeabilization assays (based on the use of sclerotization/melanization inhibitors) are also in progress and will hopefully contribute not only to the set up of the molecular basis of embryonic development but also to the weakening of eggshell layers necessary to the exogenous DNA injection. Supported by: UNDP / WORLD BANK / WHO, PAPES II Program (Fundação Oswaldo Cruz), USAMRU-B. S5-4 – OOGENESIS IN RHODNIUS PROLIXUS : THE ROLE OF FOLLICULAR EPITHELIUM CELLS HATISABURO MASUDA*, ANA C.A. MELO**, MARIO A.C. SILVA-NETO*, DENISE M. BOUTS*, GABRIELA.O.PAIVA-SILVA*, ANA P. SALERNO*, MARIA J. PORTO*, ELEONORA KUTENBACH*, JOSÉ H. M. NASCIMENTOO E MASAKO MASUDAO * Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, UFRJ. Rio de Janeiro, Brasil. **Departamento de Patologia, Universidade Federal do Pará, UFPa. Pará, Brasil O Laboratório de Eletrofisiologia Cardíaca, Instituto de Biofísica Carlos Chagas Filho, UFRJ Rio de Janeiro, Brasil. The process of egg production, as in many other insects, depends on the concerted metabolic effort of different organs and tissues. In Rhodnius prolixus, the blood taken from a vertebrate, is digested and the building blocks of macromolecules, absorbed by posterior midgut and transported to several tissues where they are used to sinthesize different molecules. Part of it are transformed in proteins, lipids and sugars or lipoglycoproteins, nucleic acids etc, necessary for the embryo growth. All these molecules must be transported in the hemolymph to the ovary where they are taken up by the growing oocyte by receptor mediated endocytosis. Among proteins, vitellogenin and Rhodnius Heme-binding protein (RHBP) synthesized by fat body, and Calcium binding protein (RCBP) are taken up by the oocyte. The several classes of lipids are transported by and delivered at the oocyte by a lipoprotein named lipophorin. Besides these molecules synthesized by the fat body, here we present evidences for the participation of follicle epithelial-cells as an important source of a variety of molecules such as enzymes, vitellogenin that accumulates in the oocyte and also proteins presenting multiple functions. A protein synthesized by the follicular epithelial cells, besides binding vitellogenin also protects the egg against fungi invasion. The function of this protein and the role of follicle epithelial cells on the oogenesis will be discussed. Supported by: PADCT, PRONEX and CNPq 35 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S6-1 – CYTOKINES, CHEMOKINES, AND APOPTOSIS IN THE REGULATION OF IMMUNE RESPONSE TO TRYPANOSOMA CRUZI João S. Silva*, Gislaine A. Martins*, Fabiana S. Machado*, Júlio C. S. Aliberti*, Janeusa T. Souto*, Fernando Q. Cunha°, Mauro M. Teixeira# and Ricardo T. Gazzinelli#. *Department of Immunology and Pharmacology°, School of Medicine of Ribeirão Preto-USP, Ribeirão Preto – SP, #Dept of Biochemistry and Immunology, ICB/UFMG, Belo Horizonte MG, Brazil. The resistance of mice to infection with T. cruzi has been associated with the production of the pro-inflammatory cytokine, IL-12, which triggers the production of IFN-γ by NK and T cells. The IFN-γ produced, in turn, activates macrophages to release nitric oxide and kill the obligate intracellular amastigote forms of the parasite. TNF-α, another cytokine associated with macrophage activation provides a second signal to induce microbicidal activity in IFN-γ activated macrophages by stimulating NO production. Since T. cruzi-infected macrophages produce TNF-α, this cytokine appears to exert its trypanocidal activity in an autocrine fashion. The control of NOS induction is mediated by TGF-β and IL-10, both produced during the acute phase of infection. Despite its importance as a microbicidal agent, NO is involved in the establishment and maintenance of lymphocyte unresponsiveness in mice infected with several parasites. In addition, NO induces apoptotic cell death in many different cells. In this regard, we have previously shown that splenocytes from T. cruzi-infected mice exhibit high levels of apoptosis, which seems to be mediated by nitric oxide (NO). In the present work we investigate the role of IFN-γ in modulating NO production, apoptosis induction and host protection during T. cruzi infection in mice. We found that the IFN-γ-/mice were highly susceptible to T. cruzi infection, exhibited significant inhibition of NO production and apoptosis levels in splenocytes, but normal lymphoproliferative response compared to the infected WT mice. Furthermore, we found that T. cruzi leads to an enhancement of Fas and Fas-L expression which is modulate by IFN-γ, since the IFN-γ -/infected mice showed significant lower levels of Fas and Fas-L expression. The addition of recombinant IFN-γ to splenocytes from infected IFN-γ -/mice increased apoptosis levels, Fas expression and NO production. In presence of IFN-γ and absence of NO (by addition of LNMMA) the apoptosis levels were significantly reduced, but still higher than those found in splenocytes from normal mice. In addition, Fas expression was maintained, despite the low levels of NO production, suggesting that the high levels of NO do not modulate Fas expression. Moreover, in vivo, Fas expression and NO production appear to be two distinct and independent phenomena, since the treatment of infected WT mice with the iNOS inhibitor, aminoguanidine, led to decreased NO and apoptosis levels but not Fas expression. Taken together, these results indicate that besides being of crucial importance in mediating resistance to T. cruzi infection in mice, IFN-γ controls the immune response, through apoptosis modulation by mediating Fas and Fas-L expression and NO production. Regarding to the resistance to the infection, we have investigated the role the other parasite induced-molecules, PAF and chemokines. We found that, PAF induced NO secretion by T. cruzi-infected macrophages and the secreted NO inhibited intracellular parasite growth. The addition of a PAF antagonist, WEB 2170, inhibited both NO biosynthesis and trypanocidal activity. The iNOS/L-arginine pathway mediated trypanocidal activity, since it was inhibited by treatment with L-NMMA, an L-arginine analog. PAF-mediated NO production in infected macrophages appears to be dependent on TNF-α production since the addition of a neutralizing anti-TNF-α mAb inhibited NO synthesis. In order to test the role of PAF in mediating resistance or susceptibility to T. cruzi infection, infected mice were treated with WEB 2170, a PAF-antagonist. These animals had increased parasitemia and earlier mortality as compared to vehicle-treated mice. Altogether our results suggest that PAF belong to a group of mediators that coordinate the mechanisms of resistance to infections with intracellular parasites. Regarding to β-chemokines, we have previously demonstrated the expression of the MIP-1α, MIP-1β, RANTES and JE/MCP-1 mRNAs in heart of T. cruzi-infected mice. We next investigated whether T. cruzi trypomastigotes triggered β-chemokine mRNA expression and protein production and whether these chemokines were involved in the regulation of NO production. We found that in vivo infection with T. cruzi led to MIP-1α, RANTES and JE/ MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in β-chemokine MIP-1α, MIP-1β, RANTES and JE mRNA expression by macrophages. The expression of the β-chemokines MIP-1α, MIP-1β, RANTES and JE proteins by murine macrophages cultured with trypomastigote forms of T. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by β-chemokines. Hence, treatment with anti-βchemokine specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of exogenous β-chemokines MIP-1α, MIP-1β, RANTES and JE/MCP-1 induced an increased T. cruzi uptake, leading to an enhanced NO production and control of parasite replication in a dosedependent manner. L-NMMA, a specific inhibitor of the L-arginine: NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with b-chemokines. Among the βchemokines tested, JE was more potent to inhibit parasite growth, although it was much less efficient than IFN-γ. Nevertheless, JE potentates parasite killing by macrophage incubated with low doses of IFN-γ. Together, these results suggest that in addition to their chemotactic activity, murine β-chemokines may also contribute to enhance parasite uptake and promote control of parasite replication in macrophages and may play a role in resistance to T. cruzi infection. 36 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 We next addressed the possible role of chemokines in the pathogenesis of myocarditis that occurs in T. cruziinfected mice. Then, we asked about the mediators that trigger leukocyte migration to the heart as well as the source of these possible mediators. We have previously shown mRNA expression for chemokines in total heart cells of mice infected with T. cruzi, which was correlated with the type and intensity of the inflammatory infiltrate. In this work, we investigated 1) nitric oxide synthase (NOS) induction; 2) nitric oxide (NO) synthesis; 3) trypanocidal activity; and 4) chemokine and cytokine mRNA expression in isolated cardiac myocytes infected with T. cruzi. We found that T. cruzi trypomastigotes induced TNFα, IL-1 a and inducible NOS (iNOS) mRNA expression as well as NO synthesis in murine embryonic cardiac myocytes. The addition of IL-1a, IFN-γ, or TNF-α to cultured cardiac myocytes resulted in significant NO production, although the cells exhibited only low trypanocidal activity. However, simultaneous addition of IL-1 a, IFN-γ and TNFα to cultures resulted in high levels of NO, activation of iNOS and a marked trypanocidal activity. The iNOS/L-arginine pathway mediated this latter activity since it was inhibited by treatment with L-NMMA. In addition, myocardial cells infected with T. cruzi expressed mRNA for the chemokines KC, JE, Crg-2, RANTES and Mig. These results indicate that iNOS activation and pro-inflammatory cytokines and chemokines produced by cardiac myocytes are likely to participate control the parasite growth, cell influx and in the pathogenesis of chagasic cardiomyopathy verified T. cruzi infected mice. Supported by FAPESP and CNPq. S6-2 – ANALYSIS OF ANTIGENS REACTIVE T-CELLS DERIVED FROM LESIONS OF AMERICAN TEGUMENTARY LEISHMANIASIS PATIENTS Da-Cruz, A.M., Oliveira-Neto, M.P., Bertho, A.L. & Coutinho, S.G. Laboratório de Imunidade Celular e Humoral, Instituto Oswaldo Cruz-FIOCRUZ, Av. Brasil 4365, CEP 21045900, Rio de Janeiro, Brasil. E.mail: [email protected] The higher frequency of Leishmania antigens reactive T-cells in lesions suggests that crucial immunological events are occurring at the site of the lesions. Immunopathological studies have contributed for characterization of the in situ inflammatory infiltrate, however little is known about the antigen reactivity of the T-cells present in the lesions. Our objective was to analyze the in vitro responsiveness of those infiltrating T-cells against different parasite antigens. Mononuclear cells obtained from lesions (LEC) or peripheral blood (PBMC) from 40 cutaneous leishmaniasis patients (CL), four mucosal patients (ML) and four patients with disseminated lesions (DissL) were phenotypicaly characterized for B, T, CD4+, CD8+, γδ, macrophages, IL-2 receptor (IL-2R) and IFN-γR by flow cytometry. Results showed that the percentages of T-cell subpopulations (mean±s.e) were similarly distributed in the inflammatory infiltrates from CL, ML and DissL patients. Lesions older than three months showed higher percentages of CD4+ and CD8+ T cells. The higher percentages of IL-2R (87.2±5.2%) and IFN-γR (81.3±6.6%) observed in ML as compared to CL and DissL, may be associated with the severity of mucosal lesions. Assays of lymphoproliferative responses (LPR) of LEC induced in vitro by L. braziliensis (Ag-Lb) antigens as well as by concanavalin A (Con-A), T. gondii (Ag-Tg) and T. cruzi (Ag-Tc) antigens were performed. Positive LPR to Ag-Lb (stimulation indies – SI ≥ 2.5 over the background cultures) were seen in 13 out of 24 CL patients (∆cpm x 103=4.9±1.8). LEC from 10 out of 16 CL patients seroreactive to Toxoplasma proliferate in response to Ag-Tg (∆cpm x 10-3=8.3±5). The LPR to T. cruzi was positive in only one out of 18 examined CL patients (SI=3.4). The supernatants of those cultures were harvest for type 1 (IFN-γ) and type 2 (IL-4, IL-5) cytokine quantification. IFN-γ production was detected in LEC cultures stimulated with Con-A, Ag-Lb and Ag-Tg. The percentages of lesions with activated LEC T-cells (IFN-γ production and/or T-cell proliferation) after in vitro stimulation with Con-A, Ag-Lb or Ag-Tg were respectively 100%, 85.7% and 84.6%. The LEC from seven patients with no serological evidence of past T. gondii infection did not proliferate or produce IFN-γ after in vitro stimulation with Ag-Tg. Nor IL-4 neither IL-5 were detected in the supernatants from Lb-stimulated cultures. PBMC and LEC cultures stimulated with AgLb were able to produce MIP-1 a e RANTES. Leishmania-reactive T-cells obtained after four days in cultures were also separated using a Percoll gradient for CD4+ and CD8+ phenotypic analysis. The percentages of CD4+ and CD8+ Lb-reactive T-cells were variable: three patients had CD4+ > CD8+ and three had CD8+ > CD4+. The present results show that T-cells from leishmaniasis lesions are able to proliferate and produce IFN-γ not only in response to leishmanial antigens but also after stimulation with other not related parasite antigens (Ec: Tg). Thus, T cell populations reactive to other antigens can also migrate to the site of the leishmanial lesions. Their role for healing or aggravation of the lesions is not yet well determined. Supports: CNPq and Economic European Community (EEC). 37 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S6-3 – CELLULAR INTERACTIONS IN THE IMMUNOPATHOLOGY OF MALARIA Carlos Eduardo Tosta Laboratório de Imunologia Celular, Departamento de Patologia, Faculdade de Ciências da Saúde, Universidade de Brasília, 70.910-900 Brasília, DF [email protected] The main pathological manifestations of malaria result from the ability of plasmodia to grow within erythrocytes, and particularly, from the interplay of the parasite and its antigens with the immune system. The consequences of these interactions are anemia, pulmonary edema, renal dysfunction and neurological abnormalities, which cause over one million deaths a year. Malaria-associated anemia is due to multiple and complex factors, including the mechanical destruction of erythrocytes by the growing parasite, alterations of erythropoiesis, spleen sequestration of erythrocytes, and blood loss as a consequence of the coagulopathy. The expression of parasite antigens or neo-antigens in the membrane of erythrocytes elicits different mechanisms of anemia related to the action of antibodies (including auto-antibodies), complement and phagocytosis by macrophages. The peculiar open circulation of blood in the spleen adds an extra hazard to erythrocyte survival in malaria. To return to the venous sinuses erythrocytes have to squeeze themselves through the narrow interendothelial gaps. Parasitized erythrocytes make a slower cross, which causes splenic stasis, and may be deparasitized during this event. Released parasites are phagocytosed by red-pulp macrophages, while deparasitized erythrocytes (espherocytes or ropalocytes) return to the circulation as cells with a shortened mean life. Two kinds of cellular interactions play a crucial role in the immunopathology of brain, lung and kidney involvement in malaria: parasitized erythrocytes with endothelial cells, and macrophages with these cells. Binding of parasitized erythrocytes (e.g. by Plasmodium falciparum) to endothelial cells (and to unparasitized erythrocytes) involves different adhesion molecules and receptors, and represents a critical step for the life cycle of the parasite. It is possible that the particular microenvironment generated by this sequestration facilitates the local activation of monocytes and lymphocytes, and the subsequent release of inflammatory cytokines (e.g. TNF-α, IL-6), which may activate endothelial cells, and eventually damage them. The ability of injected recombinant TNF-α to cause several pathological features of severe malaria, together with the finding that the plasma levels of this cytokine are usually elevated in infected individuals were considered as possible evidence of the responsibility of this cytokine in the pathogenesis of malaria. However, the attempts to interfere with the pathogenesis of malaria by inhibiting TNF-α synthesis with drugs (e.g. pentoxifylline), or its action with monoclonal antibodies have achieved frustrating results. Since it is recognized that hyperactivation of the immune system is associated with the pathological manifestations of severe malaria, it can be anticipated that a better understanding of the interactions of macrophages and lymphocytes, and of these cells with endothelial cells and parasitized erythrocytes, will open new avenues for the adoption of novel methods for reducing the morbimortality of malaria. S6-4 – ROLE OF NF-KB IN THE REGULATION OF INNATE AND ADAPTIVE IMMUNITY TO TOXOPLASMA GONDII Jorge Caamano*, Jim Alexander+, Kendra spiers*, Christopher Hunter* *University of Pennsylvania, Philadelphia; +Strathclyde University, Glasgow T. gonddi is an opportunistic pathogen in patients with AIDS as well as patients with other defects in T cell medated immunity and remains an important cause of congenital disease. The NF-KB transcription factors are an ancient family associated with the regulation of innate immunity to infection. These transcription factors can regulate the production of factors (IL-1, IL-2, TNF-α, IL-2, IFN-γ, iNOS) and the responses of immune cells to stimuli (IL-1, TNF-α, IL-12, CD28) involved in resistance to T. gondii. In addition, the activation of NF-KB in response to cytokines enhances replication of HIV1 and it has been proposed that during the immune response to opportunistic infections there is activation of NF-KB and increased replication of HIV which hastens the course of disease. However, there are few studies which have examined the role of NF-KB in resistance to the opportunistic infection taht affect patientes with AIDS. Our in vitro and in vivo studies have shown that there are high levels of NF-KB activation following infection with T. gondii. The functional significance of this observation was shown by our studies with mice deficient in different NF-KB family members. We found that the NF-KB family member RelB is required for the ability of T cells to produce IFN-γ and activation of NK cells after infection with T. gondii whereas NF-KB2 appears to be involved in the maintenance of the immune response to this parasite. We hypothesize that the activation of NF-KB represent the very earliest elements of the innate response to T. gondii. These studies address fundamental questions about the role of NF-KB in the regulation of the innate immune response and may help to understand the molecular and cellular basis of these factors in resistance to the opportunistic infection that affect patients with AIDS. 38 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S7-1 – FUNCTIONAL ANALYSIS OF THE GPI:PROTEIN TRANSAMIDASE OF LEISHMANIA MEXICANA James D. Hilleya, Jody L. Zawadzkib, Malcolm J. McConvilleb, Graham H. Coombsc and Jeremy C. Mottrama,* a Wellcome Centre for Molecular Parasitology, University of Glasgow, The Anderson College, 56 Dumbarton Rd, Glasgow G11 6NU, Scotland, UK b Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Royal Parade, Victoria 3052, Australia c Division of Infection & Immunity, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, UK Glycosylphosphatidylinositol (GPI) anchors are used as a means of tethering surface proteins to the plasma membrane in all eukaryotes, but are particularly abundant among parasitic protozoa such as Leishmania. For example, the major surface protein of Leishmania is a 63 kDa metalloproteinase (gp63) that is attached to the surface membrane with this type of lipid anchor. The anchors are added to precursor proteins within the endoplasmic reticulum by a process that involves a proteolytic cleavage followed by a transamidation event. This is carried out by a multi-component complex called the GPI:protein transamidase. We have cloned from Leishmania mexicana a single copy gene, GPI8, which encodes the catalytic subunit of the transamidase complex. GPI8 is a member of the C13 family of cysteine peptidases. We created GPI8 null mutants (DGPI8) through targeted gene replacement. Surface-located gp63 is absent from DGPI8 mutants; episomal-expression of GPI8 restores gp63 to the cell surface. DGPI8 mutants are viable as promastigotes in culture and are capable of differentiating into amastigote-like forms in vitro. In addition DGPI8 mutants can invade and proliferate in macrophages in vitro, which shows that GPI-anchored surface proteins are not essential for entry into and survival of L. mexicana within host cells. DGPI8 mutants do not express detectable levels of any GPI-anchored proteins and accumulate a putative protein-anchor precursor and an inositol-acylated form of this precursor. Pulse chase labelling suggests that this acylated compound is not an intermediate in protein-anchor biosynthesis but rather a metabolic end-product. The cellular levels of two classes of non-protein-linked GPIs, a lipophosphoglycan (LPG) and a family of free GPI glycolipids (GIPLs), are not altered in the DGPI8 mutant. S7-2 – THE MYSTERIOUS GPI-PLC WHICH RELEASES THE VSG OF AFRICAN TRYPANOSOMES Cardoso de Almeida, M.L.*, Geuskens, M. & Pays, E. Molecular Parasitology Laboratory, Free University of Brussels, rue des Chevaux, 67, B-1640Rhode-St.Genèse, Belgium *on leave from the Dept. Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, UNIFESP, São Paulo, SP, Brazil African trypanosomes are coated by 10 million copies of a single variant specific glycoprotein (VSG) which are held in the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. A GPI-specific phospholipase C (GPI-PLC) triggers fast VSG release upon cell lysis but in vivo it is safely controlled and topologically concealed from its substrate by being intracellular (Bülow et al., 1989, J. Cell Sci. 93, 233-240). One enigmatic aspect of GPIPLC action therefore consists of how it could gain access to the VSG in the exoplasmic leaflet of the membrane. The data to be presented disclosed an unexpected possible solution for this puzzle: upon cell rupture the VSG invades the cytoplasmic face of the plasma membrane which thus, becomes double coated. This unusual VSG arrangement was stable in ruptured plasma membrane from GPI-PLC null mutant trypanosomes but was transiently seen preceding VSG release by GPI-PLC in wild type (WT) trypanosomes. The formation of double coat membrane (DCM) was independent of the presence or activation of GPI-PLC, occurred both at 4°C and 30°C and was unaffected by the classical inhibitor of VSG release, p-choromercuryphenylsulfonic acid (PCM). DCMs conserved the same membrane coat thickness and association with subpellicular microtubules as in intact cells, but were prone to form vesicles following gradual detachment of the latter. Our data also demonstrated that: (i) GPI-PLC expressed by one trypanosome only targetted its own plasma membrane, being unable to release VSG of another parasite; (ii) DCMs concomitantly formed from trypanosomes expressing different VSGs did not intermix, an indication that DCM might be refractory to membrane fusion; (iii) in vivo both membrane form and GPI-PLC-cleaved molecules were detected in the lumen of the flagellar pocket. Financial support: CNPq (Brazil), Francqui Foundation (Belgium) 39 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S7-3 – CALCIUM MOBILIZATION BY NONESTERIFIED FATTY ACIDS IN TRYPANOSOMATIDS Rosana Catisti1,2, Sergio Uyemura3, Roberto Docampo2 and Anibal E. Vercesi1,2 1Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, S.P., 13083-970, Brazil, 2Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A., and 3Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, S. P., 14049, Brazil. A recent report (Eintracht, J., Maathai, R., Mellors, A. and Ruben, L., 1998, Biochem. J. 336, 659-666) proposed, based on the stimulation of calcium entry by both the phospholipase A2 activator melittin and unsaturated fatty acids, and its inhibition by phospholipase A2 inhibitors, that calcium entry in Trypanosoma brucei bloodstream trypomastigotes is regulated via a signaling pathway involving phospholipase A2-mediated generation of arachidonic acid and stimulation of a plasma membrane-located calcium channel. Here we show that Ca2+ influx in Trypanosoma brucei procyclic trypomastigotes, Leishmania donovani promastigotes and Trypanosoma cruzi amastigotes is also stimulated in a dose-dependent manner (50-400 nM) by the amphiphilic peptide melittin. This effect is also blocked by the phospholipase A2 inhibitor 3-(4-octadecyl)-benzoylacrylic acid (OBAA). The unsaturated fatty acids arachidonic, linolenic, and linoleic acid, in the range of 10-75 mmM, also induced Ca2+ entry by a mechanism sensitive to LaCl3. However, stimulation of Ca 2+ influx by myristic acid as well as Ca2+ and H+ fluxes across the membranes of mitochondria and acidocalcisomes by these unsaturated and saturated fatty acids, in the same concentration range that stimulates Ca2+ entry into the cells, and the protective effect of LaCl3 on the decrease of the mitochondrial membrane potential caused by arachidonic acid, raise some doubts about the mechanism of such stimulation of Ca2+ transport. Although the present data do not rule out the possibility of a signaling pathway involving Ca2+ entry via phospholipase A2-mediated generation of free fatty acids, other possibilities for the stimulation of calcium transport through the plasma membrane and intracellular compartments such as transmembrane flip-flop of the fatty acid-cation complex or cation leakage due to fatty acid-induced bilayer packing defects might also be considered. S7-4 – ETHER LIPIDS X CERAMIDES IN GPI ANCHORS OF TRYPANOSOMATIDS: FIRST ROUND Heise, N.1, Lima, A.C.P.1, Hart, D.T.2, Mendonça-Previato, L.1 and Previato, J.O.1 1Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21944-970, Rio de Janeiro-RJ, Brasil; 2Infection and Immunity Research Group, King’s College London, London W8 7AH, UK One of the common features of all trypanosomatids is the presence of high amounts of glycoconjugates linked to the surface via glycosylphosphatidylinositol anchors. The lipid moieties of these anchors may include lyso-acyland di-acyl-glycerolipids, ether lipids in the form of lyso-alkyland/or alkyl-acyl-glycerolipids, sphingolipids or both ether lipids and sphingolipids. Despite the extensive compositional analyses done so far with several species and the studies of fatty acid remodelling and lipid exchange in Trypanosoma brucei, little is known about the mechanisms of ether-lipid and sphingolipid biosynthesis. In a first round, we will consider the potencial use of these biosynthetic pathways as suitable targets for chemotherapeutic intervention in human-pathogenic species. The three initial steps of ether-lipid biosynthesis are associated with glycosomes of T. brucei and Leishmania species and, as described for mammalian cells, the synthesis follows the dihydroxyacetonephosphate pathway. So far, a direct connection of ether-lipid synthesis and GPI anchor formation has not been demonstrated in Leishmania. However, ether-lipid analogues were shown to be extremely toxic against Leishmania and have been selected by the WHO for clinical trials against leishmaniasis. Toxicity, but at a lower level, has also been observed against Trypanosoma spp.The exact reason for this toxicity in not known but it has been suggested that the mode of action of these analogues would be the result of a perturbation of GPI-anchor biosynthesis, phospholipid biosynthesis and/or disturbance of signal transduction by inhibition of protein kinase C. Because of these observations, the glycosomal ether-lipid biosynthetic pathway may serve as a suitable drug target. The initial steps of sphingolipid biosynthesis in fungi and animal cells are conserved. However, the description of selective inhibitors suggested functional differences between the enzymes present in the two groups. Therefore, those enzymes became excelent targets for the action of new and potent anti-fungal chemotherapeutic agents. In addition, fungi do not form sphingomyelin like mammalian cells. Instead, fungi carry out the transfer of a phosphatidylinositol moiety to the hydroxyl group of carbon 1 of ceramide for the production of inositolphosphoceramide. Because of the similarity in the production of several components containing inositolphosphoceramides and of the chemical characteristics described to date, the sphingolipid biosynthetic pathway may also be explored as a putative chemotherapeutic target against T. cruzi. Supported by CNPq, FUJB, FAPERJ, Finep, HHMI, Pronex and CEPG-UFRJ 40 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S8-1 – THE INTRACELLULAR TRASPORT OF PROTEINS IN PLASMODIUM FALCIPARUM Berry, L., Couffin, S. and Mattei, D. Unité de Biologie des Interaction Hôte-Parasite, CNRS URA 1960, Institut Pasteur, France During the intraerythrocytic cycle, Plasmodium falciparum synthesis several proteins that are exported beyond its membrane. These secreted antigens represent an important virulent factor by modifying the morphological and antigenic properties of the red blood cell. The mechanisms and pathways involved in proteins trafficking are poorly understood. In order to better understand the mechanism implicated in protein secretion by the parasite, we analysed the transport of the histidinerich protein I (PfHRPI), the exported protein 1 (exp-1) and the 41-2 antigen. The exp1 and 41-2 antigens are associated to membranous structures in the red blood cell cytoplasm, whereas PfHRPI is located at the erythrocyte’s membrane. Synchronised parasite cultures were treated with Brefeldin A (BFA), processed for immunofluorescence and visualised on a confocal laser scanning microscope. Our results show that the transport of 41-2 and exp-1 is blocked by BFA while that of PfHRPI is insensitive to the drug. This data suggests that PfHRPI is secreted through an alternate pathway, different from the ER-Golgi apparatus. We further analysed the secretion of these antigens by in vitro translation/translocation studies. The genes coding for the three antigens were cloned into the pBluescript vector, transcribed and translated with a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomal membranes. As expected, exp-1 is translocated and PfHRPI is not translocated into the microsomes. The 41-2 antigen does not cross the microsomal membranes in contradiction with the results obtained in vitro with the BFA-treated parasites. The 41-2 translocation might occur post-translationally in the heterologous system. Our results suggest that Plasmodium possesses two secretion pathways: the classical pathway represented by the ER-Golgi apparatus and an alternate, BFA-insensitive, not characterised pathway. This later may represent and important target for the development of new drugs against the parasite. S8-2 – THE ENDOCYTIC PATHWAY IN TRYPANOSOMA CRUZI EPIMASTIGOTES Isabel Porto Carreiro, Kildare Miranda, Marcia Attias, Wanderley de Souza and Narcisa Cunha e Silva Laboratório de Ultraestrutura Celular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, 21941-900, Rio de Janeiro, Brasil. (E-mail: [email protected]) It has been known for many years that the trypanosomatids require exogenous essential growth factors such as heme and cholesterol in order to divide. Additional data showed that they are able to take in exogenous macromolecules such as ferritin, peroxidase, albumin, etc and that two structural components are involved in starting this process: the flagellar pocket and the cytostome. Although the flagellar pocket plays a fundamental role in the endocytic process occurring in several trypanosomatids, the cytostome is the main structure involved in such process in epimastigote forms of T. cruzi. One-minute endocytosis of gold labeled tracers revealed very few cells with a small number of particles inside the flagellar pocket, whereas more than 98% of the cells analized presented the markers entering the cytostome. Freeze-fracture studies showed that it appears as a specialized, smooth region of the plasma membrane very poor in intramembranous particles, but limited by a row of closely associated particles. Using the fracture-flip technique it has been shown that the actual surface of the cytostome is rugous containing a material rich in carbohydrates which can be revealed by ruthenium red and several lectins. After two, five and fifteen-minute endocytosis, gold particles were seen inside vesicles and tubules, prior to fusing with reservosomes. Three-dimensional reconstruction of these tubules and vesicles showed they are interconnected, forming an intrincated and branched network, distributed from the perinuclear region to the posterior end of the cell. Whole unfixed parasites that had uptaken gold-protein conjugates for fifteen minutes were washed and dried onto electron microscope grids. Observation with energy loss filtering TEM revealed long gold-filled tubules at the cell posterior end. The acidic nature of the early endosomal network was verified using acridine orange. Based on pH and protein uptake kinetics we propose that the vesicle-tubular network is the early endosome of Trypanosoma cruzi epimastigotes. PRONEX, FINEP, CNPq, and FAPERJ have supported the work carried out in the author’s laboratory. 41 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S8-3 – ACIDOCALCISOME: A NOVEL CALCIUM STORAGE COMPARTMENT IN PROTOZOAN PARASITES Silvia N.J. Moreno and Roberto Docampo. Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802-6178, USA. In all eukaryotic cells, acidification is driven by ATPases of the vacuolar type. Additionally, some cell types have H+-pumps which are driven by pyrophosphate. Vacuolar H+-pyrophosphatases (V-H+-PPases) had, until recently, been found mainly in vacuoles of plants, ranging from the unicellular alga Acetabularia to higher plants (1). The known range of organisms possessing V-H+-PPases was recently greatly expanded by our discovery of this activity in Trypanosoma cruzi (2). Our results showed that much of the activity was associated with a vesicle rich in calcium, phosphorus, and magnesium, which we had previously identified as the acidocalcisome (3). This organelle was first described in T. brucei (4). We initially defined the acidocalcisome in permeabilized T. brucei functionally, as an organelle that was acidic and that imported Ca2+ by the action of a vanadate-sensitive Ca2+-ATPase. Subsequently, acidocalcisomes were detected in other trypanosomatids, i.e., T. cruzi (5), and Leishmania amazonensis (6), and in the apicomplexan parasite Toxoplasma gondii (7). In recent work, we found that both bloodstream and procyclic trypomastigotes of T. brucei (8), as well as L. donovani promastigotes (8), T. gondii tachyzoites (9) and Plasmodium berghei trophozoites (10) possess a V-H+-PPase with features in common with the T. cruzi and plant activities, and used this activity as a marker for the purification of acidocalcisomes. The purified organelles from T. brucei were shown to possess Na+/H+ exchange activity and to generate a pyrophosphate-dependent membrane potential. In permeabilized procyclics it was confirmed that Na+ could diminish proton gradients established via H+-ATPase activity. Na+ had the same effect on pyrophosphate-generated proton gradients, if ADP was present. Together, these data suggest co-localization of H+-ATPase and H+-PPase activities in T. brucei and provide evidence that the isolated acidocalcisome is the same organelle as that identified initially on a functional basis. 1. Rea, P.A. et al. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:157-80; 2. Scott, D.A. et al. (1998) J. Biol. Chem. 273:22151-8; 3. Scott, D.A. et al. (1997) J. Biol. Chem. 272:28020-9; 4. Vercesi, A.E. et al. (1994) Biochem. J. 304:227-33; 5. Docampo, R. et al. (1995) Biochem. J., 310:1005-12; 6. Lu, H.-G. et al. (1998) Mol. Cell. Biol 18:2309-23; 7. Moreno, S. N. J. et al. (1996) Biochem. J. 313:655-9; 8. Rodrigues, C.O. et al. (1999) Biochem. J. 340, 759-66, and submitted; 9. Moreno, S.N.J. et al. (1999) submitted. 10. Marchesini, N. et al. (1999) submitted. S8-4 – CA2+ SIGNALING IN PARAMECIUM DURING SYNCHRONOUS TRICHOCYST EXOCYTOSIS H. Plattner, M. Hardt, N. Klauke Faculty of Biology, University of Konstanz, P.O.Box 5560, D-78434 Konstanz, Germany In the ciliated protozoan, Paramecium tetraurelia, polyamines like aminoethyldextran (AED) can stimulate synchronous exocytosis of dense core secretory vesicles (trichocysts) within 80 ms. This depends on an increase of cortical free Ca2+ concentration, [Ca2+], which is the result of a massive increase of total Ca concentration [Ca]. Both these components have been analyzed, (i) [Ca2+] by injected fluorochromes, eventually using fast confocal laser scanning microscopy (CLSM) from 33 ms (image build-up time) on, and (ii) by quenched-flow combined with freeze-substitution and energy-dispersive x-ray microanalysis (EDX) attached to a scanning transmission EM (STEM), for analysis from 30 ms (dead time) on. In both cases, analyses were run with or without extracellular Ca2+, Cae. This time frame corresponds to minimal Ca2+-activated current signals registered in whole cell patch recordings. In fluorochrome analyses, we see local [Ca2+] increase already at 30 ms AED stimulation, paralleled by exocytosis. [Ca2+] signals then sweep towards the interior of the cell. Cortical [Ca2+] increases from resting values of ~65 nM to maximally 800 nM, while injections of Ca2+-buffers of different Kd and t-values indicate requirement of local [Ca2+] of ~5 μM to induce exocytotic membrane fusion. Without Cae (generated by brief complexation), AED also causes cortical [Ca2+] increase, but with lower amplitude. For extrusion of trichocyst contents, Cae is definitely required. To determine [Ca] transients, samples were mixed with AED +/Cae (normally 500 μM, eventually substituted for by Sr2+) for different sub-second time intervals and rapidly cryofixed by spraying into liquid propane, followed by freeze-substitution with fluoride (to retain Ca or Sr). 500 nm thick sections were analyzed by EDX. Element-specific CaKa or SrKa signals were quantified after calibration. Under resting conditions we find Ca signals selectively in alveolar sacs. Without Cae, Ca 2+-stores are depleted by ~50 % already within 80 ms AED stimulation. With Cae added, store depletion is immediately superimposed by Ca 2+ (or Sr2+) influx into the stores, also within 80 ms. This may optimize signal response by site-directed Ca2+ flux during stimulation. 42 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 Summary. Balance calculations from results obtained by the widely different approaches indicate that the celloperates with an excess by orders of magnitude of Ca. The anatomical arrangement of subcellular and molecularcomponents guarantees maximal effect on the respective targets. ReferencesPlattner, H., C. Lumpert, G. Knoll, R. Kissmehl, B. Höhne, M. Momayezi & R. Glas-Albrecht 1991 Eur. J. CellBiol. 55:3-16.Knoll, G., C. Braun & H. Plattner 1991 J. Cell Biol. 113:1295-1304.Knoll, G., A. Grässle, C. Braun, W. Probst, B. Höhne-Zell & H. Plattner 1993 Cell Calcium 14:173-183.Erxleben, C. & H. Plattner 1994 J. Cell Biol. 127:935-945.Länge, S., N. Klauke & H. Plattner 1995 Cell Calcium 17:335-344.Erxleben, C., N. Klauke, M. Flötenmeyer, M.P. Blanchard, C. Braun & H. Plattner 1997 J. Cell Biol. 136:597-607.Klauke, N. & H. Plattner 1997 J. Cell Sci. 110:975-983.Plattner, H., C. Braun & J. Hentschel 1997 J. Membr. Biol. 158:197-208 Hardt, M. & H. Plattner submitted. S9-1 – UPTAKE OF APOPTOTIC T CELLS MEDIATED BY VITRONECTIN-RECEPTOR DRIVES THEGROWTH OF TRYPANOSOMA CRUZI IN MACROPHAGES Freire de Lima, C. G1.; Nascimento, D. O1.; Soares, M. B. P2.; de Mello, F. G1.; Bozza, P. T3.; Castro-Faria-Neto,H. C3.; DosReis, G. A1. & Lopes, M. F1. 1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Riode Janeiro, 21944-970, Rio de Janeiro, RJ, Brasil. 2Instituito Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil.3Centro de Pesquisa Gonçalo Moniz, FIOCRUZ, Salvador, Ba, Brasil. Trypanosoma cruzi is the causative agent of Chagas’ disease, a debilitating heart disease, which affects millionsof people (1). In experimental Chagas’ disease, cytokines regulate the ability of infected macrophages to kill theparasite and play a decisive role in the host response to infection (2). Activation induced cell death (AICD) has beendescribed in CD4+ T cells during the acute phase of experimental Chagas’ disease (3, 4). The onset of AICD inCD4+ T cells increased T. cruzi growth in cocultured macrophages (5), and blockade of Fas-FasL interaction inhib-ited both cell death and parasite replication (5). These results prompted us to investigate the direct effects of apoptoticT cells on macrophage capability of dealing with T. cruzi.Apoptotic, but not necrotic cells exacerbated T. cruzi growth in macrophages either infected in vitro, or derivedfrom infected mice. In agreement with in vitro results, in vivo injection of apoptotic, but not necrotic T cells in T.cruzi infected mice increased parasitemia. In order to investigate if apoptotic bodies interfere within macrophagetrypanocidal activity, in vitro experiments were conducted with macrophages activated by INF-γ and LPS. ApoptoticT cells increased T. cruzi replication even in macrophages activated by those potent NO synthase inducers. More-over, NO production induced by INF-γ/LPS was significantly reduced by apoptotic T cells.Experiments with RGDS and RGDE peptides identified an important role for an integrin in the uptake of apoptoticT cells by infected macrophages. Moreover, RGDS, but not RGES, inhibited parasite growth induced by apoptoticT cells. We then, tested the role played by the Vitronectin Receptor (VnR or αv/β3 integrin). Fab fragments from ananti-αv mAb inhibited both apoptotic cell binding and apoptotic cell-driven T. cruzi growth in macrophages. Inaddition, both intactanti-αv andanti-β3 mAbs were able to induce T. cruzi replication in infected macrophages, inthe absence of apoptotic cells.PGE2 and TGF-β have been reported to inhibit pro-inflammatory cytokine production by human macrophages treated with apoptotic cells (7). We found that prostaglandin antagonists blocked the pro-T.cruzi activity of apoptoticcells in vitro and almost completely eliminated parasitemia after in vivo injection in infected mice. Apoptotic, butnot necrotic T cells, induced intense PGE-2 and TGF-β production by both uninfected and infected macrophages.High levels of PGE-2 and TGF-β were also detected following engagement of VnR byanti-αv mAb. Neutralizationof TGF-β blocked T.cruzi growth in a dose-dependent manner. It has been demonstrated that TGF-β deviates argi-nine metabolism from NO to both urea and ornithine production, by inducing arginase activity (8). Ornithine is a necessary precursor for putrescine synthesis by ornithine decarboxylase (ODC). ODC is expressed by the host, butnot by T.cruzi. We found that apoptotic T cells induced high levels of ODC activity and putrescine production inmacrophage cultures. To investigate the involvement of host-derived putrescine in parasite replication, a competitive ODC inhibitor, a-methylornithine (MO), was added to cultures, and suppressed the apoptotic cell effects on T.cruzi growth.These results suggest that ongoing T cell apoptosis and phagocytosis of apoptotic bodies by macrophages can be one of the mechanisms involved in parasite persistence in Chagas’ disease. In addition, widely employed Prostag-landin antagonists could be helpful to control T. cruzi burden and to reduce parasite-dependent consequences onchronic Chagas’ disease. 43Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S9-2 – TRANS-SIALIDASE FROM TRYPANOSOMA CRUZI IMMUNOSTIMULATES HOST T-LYMPHO-CYTES; INVOLVEMENT OF THE MAJOR LYMPHOCYTE MUCIN CD43 Todeschini, A.R.,*Nunes, M.P.,†Lopes M.,‡Pires, R.S.,*Previato, J.O.,*Mendonça-Previato, L.*& DosReis, G. A.‡*Departamento de Microbiologia Geral, Instituto de Microbiologia and ‡Instituto de Biofísica Carlos Chagas Filho,CCS, Cidade Universitária, Universidade Federal do Rio de Janeiro, 21944-970, Rio de Janeiro, † Instituto OswaldoCruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. The surface of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas’ disease (AmericanTrypanosomiasis) in man, displays a unique biological system comprised both by an enzyme known as Trans-sialidase (TS) and by sialic acid acceptor molecules known as sialoglycoproteins or mucins like molecules (1-3).This functional complex is thought to play an important role in host-parasite interaction (4). Recent studies demon-strated that, besides a role in mammalian cell invasion, the soluble form of TS also functions as a virulence determi-nant molecule (5), and therefore, could have relevant biological effects on the host immune system. The studies byChuenkova and Pereira (5) showed that in vivo injection of minute amounts of purified TS increased subsequentparasitemia and mortality in T. cruzi-infected mice. The mechanisms responsible for these effects were not deter-mined but, since TS injection into immunodeficient SCID mice did not affect parasitemia or mortality, it wassuggested that TS acts on host lymphocytes of the acquired immune system (5). T lymphocytes bearing conven-tional αβ TCRs are required for control of parasitemia and mortality in murine infection by T. cruzi (6). In order tobetter understand molecular mechanisms of Chagas’disease, it is important to investigate the effects of TS on hostT-lymphocyte functioning, and to correlate these effects with immunopathological changes induced by the parasite.In the present study, we show that native and recombinant TS exert multiple stimulatory effects on host CD4+ Tlymphocytes. TS activates CD4+ T cells in vivo and in vitro. An in vivo injection of TS increased CD44 expressionon CD4+ T cells from draining lymph node. In vitro studies showed that, in the presence of a costimulus, TSinduced mitogenic responses and synergized with TCR stimulation for activating CD4+ T cells from normal BALB/c mice. By immunoblotting, TS induced ERK-1/ERK-2 nuclear translocation, and synergized with TCR signals toenhance MAP kinase translocation. TS induced maximal TNF-α secretion in the absence of any TCR stimulationand increased IL-2 secretion and by TCR-activated CD4+ T cells. Exposure of CD4+ T cells from T. cruzi-infectedmice to TS also induced co-mitogenic response, and completely blocked activation-induced cell death (AICD)triggered by TCR ligation. Following infection with T. cruzi, surface expression of CD43 was upregulated in CD8+T cells and in a subset of CD4+ T cells. Pre-treatment of T cells with TS prevented the binding of anti-CD43 mAb,but not the binding of mAbs to CD4, CD8 or to CD45, another sialylated protein. CD43 ligation blocked TCR-induced AICD, while CD45 ligation also enhanced lymphocyte killing. AICD was induced in CD4+ T cells fromeither wild-type (WT) or CD43 knock out (KO) mice infected with T. cruzi; TS failed to rescue CD43KO, but notWT T cells from cell death. TS action leads to CD43 shedding from the T-cell surface, presumably inducing themto a hyper-responsiveness state. These results indicate that TS exerts multiple stimulatory effects on host CD4+ Tcells, and that at least some of the effects could be mediated through CD43 binding. Our results strongly suggest thatTS could be one molecule involved in host polyclonal lymphocyte activation and in immunopathology in the Chagas’disease. Finantial support: PADCT-CNPq, FAPERJ, CEPG/UFRJ, FINEP, PRONEX-MCT and HHMI. 1. Previato, J.O., Andrade, A.F., Pessolani, M.C. and Mendonca-Previato, L. (1985) Mol. Biochem. Parasitol. 16:85-96.2. Schenkman, S., Ferguson, M.A.J., Heisen, N.; Cardoso de Almeida, M.L.; Mortara, R.A. and Yoshida, N. (1993)Mol. Biochem. Parasitol. 59: 293-304.3. Previato, J.O.; Jones, C., Goncalves, L.P.B., Wait, R., Travassos, L.R. and Mendonca-Previato, L. (1994) Biochem.J., 301: 151-159. 4. Burleigh,B.A. and Andrews,N.W. (1995) Annu. Rev. Microbiol., 49: 175-200.5. Chuenkova, M. and Pereira, M.E.A. (1995). J.Exp.Med., 181: 1693-1703.6. DosReis, G.A. (1997) Parasitol. Today, 13: 335-342. S9-3 – ANTI-LAMININ ANTIBODY BLOCKS HEART GRAFT REJECTION TRIGGERED BY T CELLSFROM TRYPANOSOMA CRUZI-INFECTED MICE: RELATIONSHIP WITH LAMININ-BINDINGCYTOKINES S.D.S. Barbosa; I. Riederer; K.R.F. Lima-Quaresma &. W. Savino Laboratory on Thymus Research, Department of Immunology; Oswaldo Cruz Institute, Oswaldo Cruz Foundation Brazil. It is well established that extracellular matrix proteins, such as laminin for example, act on the site specific positioning of lymphocytes. In this context, the main goal of the present work was to evaluate the simultaneousinvolvement of laminin and its receptor VLA6, in the myocardium T cell autoreactivity generated during experi44Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 mental Trypanosoma cruzi infection. For that we used the mouse model of syngeneic heart transplantation, and localinjection of CD4+ T lymphocytes derived from chronically chagasic donors. We observed that CD4+ T cells frommice chronically infected with Colombian strain of T. cruzi triggered graft rejection, and that treatment of the cellswith anti-laminin receptor antibody prior to injection prevented graft rejection. These findings were confirmedwhen an anti-laminin antibody was injected adjacent to the grafts.In a second vein, we found an enhancement of VLA6 density on the CD4+ T cells to be injected, and a progres-sive increase in local laminin deposition, which was paralleled to the appearance of the cellular infiltrate.Moreover, we observed that the pattern of laminin was also different, exhibiting a thinner laminin-containingnetwork when rejection was prevented by anti-laminin antibody treatment. In this respect, it is noteworthy that thesame treatment was able to modulate the deposition of laminin-binding pro-inflammatory cytokines IFN-γ andTNF-α, which is largely present along with graft rejection (including within the grafts), and that become restrictedlylocated in the periphery of the heart, except for some rare cytokine-positive cells within the transplanted organ.From a conceptual point of view, our results correspond to in vivo evidence demonstrating the concept ofecotaxis, which takes into account the migrating cells as well as the microenvironment where the cell migrates. Financial support: FIOCRUZ; CNPq; CNPq-PADCT; CNPq-PRONEX. S9-4 – TRYPANOSOMA CRUZI-INDUCED HIPER-RESPONSIVENESS IN MICE: POSSIBLE CONTROLMECHANISMS Martins, G. A.1, Nascimento, M. C.1, Machado, F. S.1, Tanowitz, H. B. 2, and Silva, J. S.1.1Dept. of Immunology, FMRP, USP, Ribeirão Preto, SP, Brazil. 2Dept. of Pathology, Albert Einstein College ofMedicine of Yeshiva University, New York, EUA. Infection of hosts with Trypanosoma cruzi leads to many immunological disturbances, including a progressiveinflamatory destruction of nervous and/or muscle cells, which can lead to the fatal compromising of either the heartand the digestive system. This immunological attack to the host heart tissue has been ascribed to hiper reactivitymechanisms probably triggered by the chronic persistence of the parasites (1), suggesting that a defective control ofimmune response could be implied in autoreactive response developed after T. cruzi infection.The induction of apoptosis has been implied as one of the most important mechanisms to control the immuneresponse (2). In fact, we and others have previously shown that lymphocytes from mice acutely infected with T.cruzi present enhanced spontaneous (3) and activation-induced apoptosis (AIA) (4) in the acute phase of infectionwhen compared to non-infected mice. While AIA was ascribed to the Fas-FasL system (5), spontaneous apoptosiswas reported to be due to nitric oxide (NO) which is largely produced in mice acutely infected with T. cruzi (3).The role of NO in modulating apoptosis was demonstrated by treating T. cruzi infected mice with iNOS inhibi-tors (3) or by infecting iNOS knockout mice (Martins et al, manuscript in preparation). Both, inhibition and geneticdepletion of iNOS led to a significant decrease of apoptosis levels during the infection with T. cruzi. However, evenin absence of the iNOS enzyme, infection with T. cruzi still increased apoptosis levels, which is certainly due to thehigh expression of Fas and Fas-L found after infection. Interestingly, this increase in Fas and FasL expressionobserved in the acute phase of infection was demonstrated to be modulated by IFN-γ but independent of NO (6).The expression of Fas and Fas-L in cells from mice chronically infected with T. cruzi is significantly smaller than the expression of these molecules in cells from mice in the acute phase of infection. However, significantlyenhanced Fas-L expression is easily induced in splenic cells from chronically infected mice after 96 hours culturewith IL-2, live trypomastigotes or with parasite antigens. Interestingly, this enhancement in Fas-L expression wasIFN-γ dependent, since the addition of neutralizing antibody anti-IFN-γ to the cultures blocked the increase in Fas-L expression. Moreover, when stimulated with IL-2, live parasite or parasite antigens, spleen cells from chronicallyinfected but not from normal mice were able to exhibit cytotoxic activity against T. cruzi-infected macrophages in vitro. This cytotoxic activity is mediated mainly by CD8+ T lymphocytes, since the depletion of CD8+, but notCD4+ T cells led to a significant reduction in cytotoxic activity. Addition of mAb anti-Fas-L to these culturesinhibited the cytotoxic activity in more than 60%, indicating that the mechanism underlying the CD8+ T cell-mediated citotoxicity is Fas-L-dependent. These results suggest that a IFN-γ -dependent and Fas-Fas-L-mediatedmechanism of cytotoxicicy could be implied in limiting the parasite replication during the chronic phase of experi-mental T. cruzi infection. Limiting parasite replication could be crucial to avoid hyper-responsiveness and the tissue damage reported to occur in T. cruzi-infected hosts (1).In an effort to better understand the importance of the Fas-Fas-L pathway in immune response to T. cruzi weevaluated the course of infection in mice deficient in Fas expression (C57Bl/6-lpr/lpr). The results demonstrated that these mice are highly susceptible to the infection with the Y strain of T. cruzi. In addition, the lpr infected miceshowed decreased NO production, and reduced levels of apoptosis in vivo and in vitro if compared to the WT controlinfected mice. The reduced NO production and increased parasitemia observed in the lpr mice could be a conse45Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 quence of the decrease production of IFN-γ and enhanced production of the cytokines IL-10 and IL-4 in response toantigen stimulation in vitro. These data are in striking accordance with a recent report showing similar results in theFas-L deficient (gld) mice (5) infected with T. cruzi. Together, these results suggest that Fas-FasL-induced apoptosismight be important to control the hiper-responsiveness and favor an efficient immune response against T. cruzi andmaybe to promote the establishment of a chronic disease. References1. R. L. Tarleton and L. Zhang, 1999. Paras. Today, 15: 94-99.2. Van Parijs, L. and Abbas, A. 1998. Science 280: 243-248.3..Martins, G. A., M. A. G. Cardoso, J. C. S. Aliberti, J. S. Silva. 1998. Immunol. Letters 63:113-120.4. Lopes, M. F., F. Veiga, A. R. Santos, M. E. Fonseca, G. A. DosReis. 1995 J. Immunol. 154, 744-752.5. Lopes, M. F., M. P. Nunes, A. Henriques-Pons, N. Giese, H. C. Morse III, W. F. Davidson, T. C. Araújo-Jorge, G.A. DosReis. 1999. Eur. J. Immunol. 29: 81-89.6. Martins, G. A., L. Q. Vieira, F. Q. Cunha, and J. S. Silva 1999. Infec. Immunity, in press. Supported by grants # 96/04304-7 and 96/4118-9 from FAPESP S10-1 – TELOMERASE AND TELOMERE ASSOCIATED PROTEINS IN KINETOPLASTID PARASITICPROTOZOA Cano, M. I. N. 1, Blake, J. 2, Dungan, J. 2, Agabian, N. 2, Blackburn, E. H. 31.Núcleo de Pesquisa e Pós-Graduação, Lab. Biologia Molecular de Microorganismos, UNIBAN, 02071-013 SãoPaulo-SP, Brazil, [email protected]; 2. Stomatology and Pharmaceutical Chemistry Dept., Univ. California-San Francisco; 3. Microbiology and Immunology Dept., University of California-San Francisco, 531 ParanassusAve, 94143-0422 CA, USA. Telomeres are specialized protein:DNA complexes that form the physical ends of eukaryotic chromosomes. Inalmost all organisms the telomeric DNA is formed mainly by sequences repeated in tandem such as in human andsome Kinetoplastida protozoa whose telomeres consist of 5’-TTAGGG-3’ repeats (Moyzis, 1988; Fu e Barker,1998; Chiurrillo et al., 1999). The proteins associated to the telomeric DNA are found binding specifically to boththe double-stranded repeat and to the single-strand G-rich overhang. In some protozoa, like T. brucei telomereenvironment, although yet obscure, is the place of important events such as antigenic variation and recombination.We have first identified telomerase activity in extracts from insect form cells of three evolutionarily divergeKinetoplastida species: Trypanosoma brucei, Leishmania major and Leishmania tarentolae. Telomerase activitywas detected using two modifications of the TRAP assay described by Kim et al. (1994). The activity in T. bruceiextracts was sufficiently robust to enable its detection by primer-extension or the conventional telomerase assay. Itwas demonstrated that enzyme processivity is low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA (TER) of T. brucei. (Cano et al., 1999). Preliminary results using antisensemodified oligonucleotides (2’-O-methyl-RNA) to the hypotethical TER template sequence inhibited T. bruceitelomerase activity. In the future, this may provide the means to target parasite telomeres for the development of anti-parasitic drugs.Knowing that telomerase and single-strand telomere-binding proteins share the same substrate specificity, theyboth bind to the G-rich telomeric DNA overhang, we decide to use telomerase positive extracts of T. brucei to isolate possible components of the telomerase holoenzyme or factors that could interact or specifically regulate T.brucei telomere length. We were able to identify three distinct protein:DNA complexes by gel mobility shift assay(GMSA) and UV cross-linking. One complex, called C3, exhibits high affinity to the G-strand telomere sequence and shows similar features to other single-stranded telomere ending-binding proteins previously described. It is alsohighly possible that complex C3 is tightly associated to an RNA component as the complex is completely eliminatedby RNase or chemical nuclease treatment. The protein components of complex C3 were isolated from a 2D gel, eluted and their peptide sequences are under way. * Funded by Pew For Latin American Fellows, FAPESP, NIH. 46Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S10-2 – IN VITRO TRANSPOSITION AND LEISHMANIA GENOME ANALYSIS Luiz R. O. Tosi and Stephen M. Beverley*Departamento de Bioquímica, Faculdade de Medicina de Ribeirão Preto – USP, Ribeirão Preto, SP*Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO Genome analysis is undergoing a shift from mapping and sequencing efforts to approaches marked by a func-tional perspective. This transition is characterized by the design and development of genome-wide experimentalstrategies to evaluate gene function using the information and reagents generated by structural genomics. Thepurpose of functional genomics is to produce a background and starting point for further genetic and/or biochemicalanalysis.The Leishmania Genome Project has generated extensive structural information. A complete cosmid-basedphysical map of the genome is available and sequencing of the smallest chromosome has been completed (http://mercury.ebi.ac.uk/parasites/leish.html).We have developed a convenient in vitro transposition system that constitutes a useful tool for genomic studies.The transposable element mariner is a member of the mariner/Tc1 superfamily of transposons, which are found ina wide range of species. While many mariner elements are defective, the Mos1 element from Drosophila mauritianaencodes an active transposase. We have purified the Mos1 transposase and shown its ability to mediate transpositionin vitro.Sequence analyses of insertions into a variety of targets showed that mariner inserts randomly into target TAs.Mariner transposes with high efficiency (up to 10-3) even into large targets, such as cosmids. Also, cis requirementsfor transposition are restricted to short stretches of sequence within the transposon structure. These parameterspermitted the construction of large insertional libraries and the design of modified mariner elements. We have usedmariner insertion libraries to rapidly sequence the H region of L. major following a primer-island sequencingapproach and also to map genes in Leishmania spp. by insertional mutagenesis.Modified mariner elements containing reporter genes were constructed and will allow the generation of transla-tional fusions when insertional libraries are introduced into the parasite. This strategy will identify expressed se-quences and easily locate their products within the Leishmania cell. Furthermore in vitro insertions of appropriatetransposons into Leishmania genes will generate ready-to-use reagents for systematic knockout approaches. Supported by FAPESP and PEW Latin American Fellows Program. S10-3 – TELOMERES OF PLASMODIUM FALCIPARUM: NOT JUST THE END Figueiredo, L., Pirrit, L., Bakhsis, N., Bottius, E. and Artur ScherfCNRSURA 1960-Biology of parasite-host interaction, Institut Pasteur, Paris, France Telomeres play an essential role in a number of biological processes in eukaryotic cells. They ensure completereplication of chromossomes and are necessary for protection against and fusion. Chromossome ends of the proto-zoan malaria parasite Plasmodium falciparum are composed of degenerate G-rich repeats, in which GGGTT (T/C)A is the most frequent and consist of distinct structural regions: the telomere and the polymorphic subtelomericregion. We observed a dimorphic chromatin structure in P. falciparum telomeres: a non-nucleosomal structure at thechromosome end and nucleosomal organisation further upstream. Enzymes involved in telomere replication andchromosome length maintenance are of fundamental importance for organisms such as Plasmodium with a highlyproliferative life cycle. One of these enzymes is the specialised reverse transcriptase, telomerase, which has recently been identified in P. falciparum cell extracts. We developed an assay which allowed us to study the de novo synthe-sis of thenhighly cariable telomere repeats added to the 3’ end of DNA oligonucleotide primers by plasmodialtelomerase activity in P.falciparum bloodstage parasite cell extracts. In addition to elongating pre-existing telomere sequences, P. falciparum telomerase can also add telomere repeats onto non-telomeric 3’ ends. The efficient of non-telomeric primer elongation was dependent on the presence of a G-rich cassete upstream of the 3’ terminus.Olidonucleotide primers derived from natural P. falsiparum chromosome breakpoints are efficiently used as telomerase substrates. These results imply that P. falciparum telomerase contributes to chromosome maintenance and to denovo telomere formation on broken chromosomes. Reverse transcriptase inhibitors such as dideoxyguanosine triph-osphate (ddGTP) efficiently inhibit P. falciparum telomerase activity in vitro. These data point to malaria telomerase as a new target for the development of drugs that could induce parasite cell senescence.Genes coding for virulence factors are localised in the subtelomeric regions of P. falciparum chromossomes, insites which are known to be highly recombinogenic. In several organisms, telomere-associated regions function as kind of adaptive domain, mediating rapid evolution of DNA seuquences located in these regions. A recent study inour laboratory detected frequent ectopic recombinant between two members of the var gene family located ondifferent chromosome ends. Ectopic recombination appears to be an important mechanism involved in the generation of diversity of the va repertoire. 47Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S11-1 – RESEARCH TRENDS WITH ANAEROBIC AND AEROTOLERANT PROTOZOA—TRICHOMO-NAS VAGINALIS J. F. Alderete, Ph.D. Department of Microbiology, University of Texas Health Science Center, San Antonio, TX, USA Trichomonas vaginalis is an ancient microaerophilic protist responsible for trichomonosis (vaginitis), the num-ber one, non-viral sexually transmitted disease (STD). The parasite survives in the constantly changing and adverseenvironment of the female urogenital tract. Research trends for this, and other, protozoa should focus on continuingto understand basic and fundamental aspects of the biology of the parasite itself and of the complex host-parasiteinterrelationship. Areas of emphasis include the following: Metabolism: Continued characterization of the uniqueaspects of the metabolism of parasites will yield insights into new targets and therapies. Novel pathways for proto-zoa may be discovered through the use of continuous flow cultivation under in vivo-like conditions (pH, nutrientlimiting or excess, generation times, etc.) as compared to general batch cultures. Drug resistance: Research into themechanisms of resistance concomitant with studies on metabolism is prerequisite for development of new drugs.This area of research, however, will yield information on the existence of general multidrug resistance genes versusunique pathways for resistance, such as gene amplification or inactivation of drugs resulting from direct modifica-tion. Microbicides: In addition to pharmacologic drug targeting resulting from studies on metabolism, the effect ofknown microbicidal molecules (e.g., bile, defensins, etc.) must be examined. Further, it may be possible to generatemonoclonal antibodies that are cidal in a complement-independent fashion, with such reagents useful during mu-cosal infections. Pathogenesis: Understanding the mechanisms of pathogenesis is fundamental to vaccine develop-ment. These studies will identify virulence factors with specific functional properties, such as parasite moleculesmediating tissue tropism and/or cytoadherence, neutralizing immune effector molecules, and acquiring nutrients.The heterogeneity among infecting subpopulations must be delineated to understand the antigenic and functionalcomplexity(ies) among parasites. Epidemiology/surveillance: There is a need to carefully establish relationshipsbetween disease outcomes and symptomatology with the infecting isolate types. Similar correlates with specificvirulence properties and molecules is warranted. For example, isolate types may be defined on the basis of thepresence or absence of a double-stranded (ds) RNA virus and satellite dsRNAs within protozoa. Genomics: Inves-tigators in the future will have access to entire genomic sequences for protozoa. This post-genomics era will havesequence databases from which to make comparative analyses with other known pathogens, providing informationon novel pathways of metabolism, strategies for immune evasion, molecules for tissue parasitism, and unique nutri-tional requirements. A holistic approach: A knowledge base that will lead to infection and disease interventionstrategies will require a holistic approach to the basic biological research on pathogenic protozoa. S11-2 – THE REVERSIBLE INTERNALIZATION OF THE FLAGELLA IN TRITRICHOMONAS FOETUS-THE PSEUDOCYST FORMATION Marlene Benchimol1, Wanderley de Souza2 and Bruce Granger3 .1Universidade Santa Úrsula. Rua Jornalista Orlando Dantas, 59. Rio de Janeiro.2 Instituto de Biofísica CarlosChagas Filho. Universidade Federal do Rio de Janeiro.3 Montana University, USA. When stimulated the eukaryotic cell has the ability to either change its surface topography, forming projectionsor depressions, or internalize significant portions of the plasma membrane through endocytic processes. Studies carried with some protozoa, such as Tritrichomonas muris and Trichomitus batrachorum, have shown that undercertain conditions trophozoite forms transform into rounded motionless forms known as pseudocyts. Early morpho-logical investigations showed that a true cyst wall was not observed in such forms, however, the undulating membrane and the flagella appeared to be internalized. In the present study using the cattle pathogenic protozoanTritrichomonas foetus we established conditions to induce a reversible internalization of the flagella and analyzedthis process using scanning and transmission electron microscopy, videomicroscopy, freeze-fracture, cytochemistry and several drugs such as colchicine, cytochalasine, nocodazol and starvation. In order to induce transformation twoprocedures were used: (1) The parasites were cooled from 37oC to 0oC and maintained at this temperature for 60min, and then rewarmed to 16, 18, 23, 30 or 37oC. Samples were analyzed by light microscopy after 3, 20, 120 and240 min; (2) The cells were maintained under starvation conditions in PBS for 3 hs and then examined. About 300cells were examined and the percentage of cells showing the (a) 3 anterior and 1 recurrent flagela, (b) only the 3anterior flagella, (c) two anterior and 1 recurrent flagella, (d) only two anterior flagella, (e) 1 anterior and 1 recurrent 48Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 flagella, (f) only 1 anterior flagellum, and (g) no exposed flagellum. Observation by light microscopy of T. foetussubmitted to variation in temperature or starvation conditions showed that the cells become rounded and the flagellagradually disappeared. This process was further analyzed in experimentes in which the cells were submitted tovariation in temperature. Incubation of the protozoa at 8 or 12oC led to a significant decrease in the percentage ofcells showing the three anterior flagella and the tip of the recurrent flagelum, predominating cells showing noflagella or only one anterior flagellum. This effect was dependent on the incubation time, reaching a plateau after120 minutes. No free flagella were observed in almost all cells maintained at 0oC for 90 minutes. When the cellswere first incubated for 60 minutes at 0oC and then at 16, 18, 23, 30 or 37oC the flagella gradually reappeared. Thisrecovery process was dependent on both the temperature and the incubation time, being faster and reaching higherrecovery indexes when incubation was carried out at 37oC for 120 minutes. Recovery did not occur at temperatureslower that 16oC. Reappearance of the flagellum depends on the presence of Ca2+ and Mg2+ in the incubationmedium. Supported by: CNPq, PRONEX, FAPERJ, USU. S11-4 – INTEGRINS IN TRICHOMONAS VAGINALIS Silva Filho, F. C.UFRJ Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde – Bloco G, 21949-900, Rio deJaneiro, Brasil. E-mail: [email protected] Trichomonas vaginalis is a well known parasitic protozoan which is usually found in urogenital cavities ofhumans. Most of women harbouring trophozoytes of T.vaginalis present the sexually transmitted disease (STD)known as trichomoniasis or trichomonosis. Despite of such disease to be one of the five widespread STD, mainlypresent among young people, just now the pathogenic machanisms of T. vaginalis starting to be elucidated.Trichomonas vaginalis is able to recognize each one of RGD and YIGSR-containing glycoproteins. The T.vaginaliscell surface is a mosaic of well defined molecules including adhesins, proteases, lectins, and alpha 3 beta 1 integrinsat least. The activities of each one of the components belonging the surface molecular repertoire of T.vaginalis isrevelead itself during the responses of the parasite to the various types of extracellular stimulli. These stimulliinclude host cells, soluble factors, baterial flora of the vagina, etc. Thus, it is reasonable to consider that the parasiteis able to define strategies by which it integrates diverse signals in the decision-making process. Further studiesfoccusing the functionality of each one of these surface molecules during the host cell-parasite interaction process isobviously of relevance.The extracellular matrix glycoprotein Laminin is recognized by T.vaginalis, and one of the consequences ofsuch recognizing is a detectable activity of an unusual activity of a matrix metalloproteinase (MMP) in the parasite.Such proteinase of M(r) 92.000 could also be detected in the parasite when it was pre-treated with nanomolaramounts of 12-O-tetradecanoylphorbol-13-acetate (TPA). The activity of such proteinase was inhibited when para-sites were previously treated with TIMP-1 which is in turn, a well known inhibitor of MMP-9. Since MMP-9 is amember of the MMP family of endopeptidases with critical role in hydrolysis of ECM we did decide to investigate the sensitivity of such MMP activity to endoglycosidase H (endo H).Parasites were consecutively treated or not with each one of TPA and Laminin-1, and Endo H. Following thesetreatements, they had their surfaces biotinylated. Surface biotinylation and immunoprecipitation with anti-MMP-9 antibodies revealed the presence of enzyme activity only on the cell surface of Laminin-1 or TPA-treated parasites.These results clearly pointed out that the intracellular signalling starting by the binding of Laminin-1 to alpha 3beta integrins in T.vaginalis may result in the activation of a MMP-9 proteinase at the cell surface of the parasite. Such results may help to explain how T.vaginalis degrades host tissues. Supported by CNPq, FAPERJ, and FUJB-UFRJ 49Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S12-1 – DISTINCT IMMUNE RESPONSES DETERMINE THE DEVELOPMENT OF DIGESTIVE ANDCARDIAC CLINICAL FORMS OF CHAGAS DISEASE Correa-Oliveira, R.1, Gomes, J. A. S.1,2, Lemos, E. M.1,2, Cardoso, G. M.1, Reis, D. D. A.3, Adad, S. J.4, Crema,E.4, Martins-Filho, O. A.1, Rocha , M. O. C.5, Gazzinelli, G.1,Taboada, D. C.1, and Bahia-Oliveira, L.M.G6.1-Centro de Pesquisas Rene Rachou Fiocruz; 2-Departamento de Bioquimica e Imunologia, ICB-UFMG; 3-Departamento de Morfologia, ICB-UFMG; 4-Faculdade de Medicina do Triangulo Mineiro;5Faculdade de Medicina/Hospital das Clinicas, UFMG;6Laboratorio de Biologia do Reconhecer, Universidade Estadual Norte Fluinense-UENF, Campos do Goytacazes, RJ. The involvement of cell-mediated immunity is undoubtedly of major importance in the pathology of Chagasdisease. Studies on the characterization of the inflammatory infiltrates in the chronic cardiac form have demon-strated that they are composed predominantly of small lymphocytes, macrophages, plasma cells and segmentedleukocytes. Immunohistochemical studies demonstrated that the cells present in the tissues are mainly CD8+ manygranzyme A+ and a few macrophages/TNF?. Analysis of the role of the various cytokines by our group has demon-strated that secretion of IFN? can be correlated with the severe cardiac form of Chagas disease. The opposite wasobserved for in vitro secretion of IL-10. These results suggest a role for IFN? on the development of the severepathology in Chagas disease and of IL-10 in controlling morbidity. Intracytoplasmic staining for cytokines demon-strated that IL-10 is produced mainly by macrophages/monocytes (CD14+high) whereas IFN? Is produced byCD3+ cells, both ?? and ?? In cardiac patients the majority of the CD3+ cells were IFN?+ in contrast with indeter-minate patients. In gastrointestinal clinical form of the disease a significant decrease in the absolute number ofCD3+ T cells as well as in CD19+ B lymphocytes were detected. The most striking observation was an inversion ofthe CD4/CD8 ratio, contrasting with results from cardiac chagasic patients where the ratio of these cells is normal.A decrease of the percentage of CD4+CD28+ cells and an increase in the expression of HLA-DR on CD4+ andCD8+ cells suggest that their function may be altered by the lack of CD28 expression. Immunohistochemicalanalysis of esophagus and colon tissues demonstrated a predominance of CD3+ T lymphocytes, CD68+ and highernumber of CD4+ cells when compared to the CD8+ population. An interesting observation was the presence of cellsexpressing TIA-1+. Finally, we also observed that all chagasic patients with mega presented denervation associatedwith inflammation, whereas patients without mega can present or not denervation, but no inflammation is observedin the latter group, suggesting a role for the inflammatory process on the development of this pathology. Financial Support: NIH AI26505, FIOCRUZ-PAPES, CNPq and CAPES S12-2 – FUNCTIONNALLY ACTIVE MUSCARINIC ANTIBODIES IN THE CHRONIC PHASE OFCHAGAS DISEASE Campos de Carvalho, A.C.; Quintero C.C.; Costa, P.; Rose, J.L.; Almeida, N.; Cabarcas, R.; Garcia, S.; Pedroza, R.& Masuda M.O.Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Auto-antibodies to adrenergic and muscarinic receptors have been found in IgG fractions from blood samples ofchronic chagasic patients. Binding of the auto-antibodies to the receptors has been demonstrated and functionalalterations in second messengers levels and beat rate of cultured cardiac cells have been measured. We demon-strated that IgGs from a group of chronic chagasic patients with complex cardiac arrhythmias are functionallycompetent in inducing reduction in beat rate and atrial-ventricular conduction block in isolated whole rabbit heartsand that these IgGs exert a muscarinic like effect that can be blocked by atropine. Additionally, these IgGs reducewhole cell and single channel L-type calcium currents in isolated myocytes from rabbit hearts studied by the wholecell patch-clamp technique, and bind allosterically to atrial mucarinic receptors in porcine hearts. Levin and co-workers have found that many of the antibodies present in cardiomyophatic chronic chagasic patients’ sera aredirected against T. cruzi P ribosomal antigens. In addition, they have demonstrated a cross-reaction between T. cruziP0 ribosomal antigens and the adrenergic and muscarinic receptors in the heart. The targeted epitope in the cardiacmembranes seems to be a stretch of negatively charged amino-acids located in the second extracellular loop of b1-adrenergic and M2-muscarinic receptors. Following this lead, we found that the functionally active antibodiescharacterized in our previous studies were directed to intracellular antigens of T. cruzi but not of Leishmania. Wealso found that these antibodies recognize the second extra-cellular loop of the M2 muscarinic receptor and that across-reactive mechanism between this region of the receptor and the P family of ribosomal proteins of T. cruzi isinvolved in the immune recognition, since peptides derived from the ribosomal proteins of T. cruzi are able to blockthe antibody activation of the M2 muscarinic receptor. This cross-reactive mechanism seems to be rather selective,since a single amino-acid substitution in one of the T.cruzi peptides tested, a 13-mer derived from the P2b family,resulted in either partial or total loss of the blocking peptide activity. Interestingly, the single amino-acid substitu-tions tested correspond to peptides derived from the human and Leishmania P ribosomal proteins, respectively. Financial support: PRONEX, FAPERJ, CNPq, FUJB. 50Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S12-3 – GLYCOINOSITOLPHOSPHOLIPIDS ISOLATED FROM TRYPANOSOMA CRUZI STRAINS ANDFROM NON-PATHOGENIC TRYPANOSOMATIDS: UPTAKE BY HUMAN ANTIGEN-PRESENTINGCELLS Ribeiro-Gomes, F.L.*, Conceição, S.B.*, Barcelos, M.W.*., Mendonça-Previato, L.#, Previato, J.O.#, DosReisG.A.+ and Arnholdt, A.C.V.**Lab. Biologia do Reconhecer, CBB/UENF, Av. Alberto Lamego, 2000, Campos dos Goytacazes, RJ, CEP 28015-620; # Depto de Microbiologia Geral, Inst. de Microbiologia e + Programa de Imunologia, IBCCFo., UFRJ. Glycoinositolphospholipids that are not linked to either protein or polisaccharide are the major cell surfaceglycolipids in all trypanosomatids investigated to date. In previous studies, the molecular structure of GIPLs fromdifferent strains of Trypanosoma cruzi was characterized, and classified into two series based on the substituent(ethanolamine phoshate or beta-galactofuranose) on the third mannose residue (Man3) distal to inositol (Carreira etal., 1996. Glycoconjugate J 13: 955-966). Here we investigate the endocytic pathways of GIPLs from G, Y, CL,Tulahuen, and Colombiana T. cruzi strains, plus L. samuelli and P. serpens GIPLs, by human monocytes, immaturedendritic cells (iDC) and macrophages. Molecules were directly coupled to FITC and the internalization analyzedby flow cytometry after 1h of incubation at 4 and 37°C. Monocytes efficiently internalized GIPL from G>Y>CL,while iDC internalized all three glycoconjugates with similar efficiency. Macrophages clearly showed an increaseduptake of Y over G and CL, and even more over Tulahuen GIPL. Interestingly, only a small percentage of mono-cytes and iDC was capable of internalizing GIPLs from Tulahuen and L. samuelli. We performed blockage assayswith the CHO portion of G strain, and competition assays with the unlabeled molecule. Our results strongly suggestthat endocytic cells express receptors that regognize the CHO portion of GIPLs. We are currently investigating therole of pattern recognition receptors (PRRs) and the class Ib antigen presenting molecule CD1 on the binding anduptake of GIPL by these cells. Supported by FENORTE, CNPq and PRONEX S12-4 – IMMUNOLOGICAL RECOGNITION IN CHAGAS ́DISEASE KalilJ.1,2, Abel L.C.J.1, Iwai L.K. 1,3, Juliano L.3, Rizzo L.V.2,4, Teixeira, H.5, Goldberg A.C.1, Mady C.1, IanniB.1, Hammer J.6, Sinigaglia F.6, Cunha-Neto E.1,21Instituto do Coração, FMUSP; 2Disciplina de Alergia e Imunopatologia, FMUSP, São Paulo; 3Dep. Biofísica-EPM/UNIFESP; 4Dep de Imunologia, ICB/USP; ICB/UFJF, 5Juiz de Fora,MG; 6Roche Milano Richerche, Milano,Italy Susceptibility factors or pathogenesis “checkpoints” that lead 30% of T. cruziinfected patients to developChagas ́disease cardiomyopathy (CCC) after Trypanosoma cruzi infection, while the remaining 70% fail to do soare also largely unknown. Recently, our group identified T cell clones obtained from the heart lesion of a CCCpatient crossreactively recognizing cardiac myosin, the major heart protein, and the tandemly repeated, recombi-nant antigen B13 from T. cruzi, which bears sequence variants; such heart-infiltrating T cells display an inflamma-tory,T1-type cytokine profile. Furthermore, in vitro sensitization with B13 protein primes cardiac myosin-recogniz-ing T cell clones, supporting the concept of T cell molecular mimicry between cardiac myosin and T. cruzi B13 protein. It is known that the fine recognition of epitopes, as well as the cytokine profile, can determine whether animmune response will be protective or pathogenic. We studied the recognition of B13 protein and its epitopes, inPBMC from CCC or asymptomatic “indeterminate” patients (ASY) and normal individuals (N). B13 protein was recognized by PBMC from CCC, ASY or even N individuals bearing the following HLA class II genes: HLA-DQA1*0501/DQB1*0301 (DQ7, the most frequent HLA allele), DQA1*0501/DQB1*0201 (DQ2), DR1 and DR2,in line with HLA-peptide binding assays. Contact residues with HLA-DQ7 were the underlined central glycine and alanine residues in peptide KPPPFGQAAAGDKPP. Ten 15-mer synthetic peptides containing all sequence variantsin B13 protein were tested in proliferation assays among HLA-DQ7+ patients, and were differentially recognized inthe CCC and ASY groups. In addition, ELISPOT assays on PBMC indicated that the frequency of cells producingIFN-γ after PHA stimulus was significantly higher in PBMC from CCC than ASY or N. Among CCC patients, thedistinct recognition repertoire of B13-specific T cells, as well as the increased frequency of IFN-γ producing cells,could play a role in the pathogenesis of the disease, perhaps by influencing the generation of pathogenic, myosin cross-reactive T cells. Supported by HHMI,FAPESP, and CNPq 51Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S13-1 – BISPHOSPHONATE DERIVATIVES AS CHEMOTHERAPEUTIC AGENTS AGAINST TRYPA-NOSOMA CRUZI Julio A.Urbina1,2, Benjamin Moreno2, Eric Oldfield2, Cristina Sanoja4, Gilberto Payares4, Silvia N.J. Moreno3,Brian N. Bailey3, Wen Yan3, David A. Scott3, and Roberto Docampo3Laboartorio de Química Biológica, Centro de Bioquímica y Biofísica, Instituto Venezolano de InvestigacionesCientificas, Caracas, Venezuela, 2Departments of Chemistry and Biophysics and 3Laboratory of MolecularParasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA and4Departamento de Parasitología, Instituto de Zoologia Tropical, Universidad Central de Venezuela, Caracas, Venezuela We have recently reported, using high field 31P nuclear magnetic resonance spectroscopy (NMR) combinedwith specific enzymatic and chromatographic assays, that inorganic pyrophosphate(P2O74-, PPi) is the major highenergy phosphate compound in the parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei, Leishmaniaamazonensis, and Toxoplasma gondii, being 5-15 times more abundant than ATP (18). PPi is present in all sub-cellular fractions, but is mostly concentrated in acidocalcisomes, specialized acidic vacuoles which constitute thelargest calcium reservoir in Trypanosomatid and Apicomplexan parasites (6,8,9,15,20). New in vivo experiments inT.cruzi epimastigotes, using both 31P and 13C NMR, have demonstrated that glucose consumption is associatedwith large changes in the levels of PPi and nucleotides, indicating an active role of the former in the energy metabo-lism of this parasite. These facts are consistent with the recent discovery of several PPi-dependent enzymes inT.cruzi , including a glycosomal pyruvate, phosphate dikinase (2) and proton-pumping pyrophosphatases located inboth acidocalcisomal and plasma membranes (ref.14 and Docampo et al., unpublished observations). Bisphosphonates,metabolically stable PPi analogs which have been used in humans to treat bone resorption disorders (7,10,11), blockthe proliferation of T.cruzi amastigotes inside cultured myoblasts with IC50 of 65 μM for both pamidronate (Aredia®,Novartis) and alendronate (Fosamax®, Merck). Initial studies in a murine model of acute Chagas disease haveshown that pamidronate at 10 mg/Kg.d given intravenously for 7 days can completely suppress the proliferation ofT.cruzi in vivo while the drug pressure is maintained. Further studies with different treatment protocols and achronic model of the disease are currently in progress. The mechanism of action of bisphosphonates against T.cruzicould include selective inhibition of PPi-dependent parasite enzymes such those indicated above and/or blockade ofisoprenoid biosynthesis, as demonstrated for osteoclasts and Dictyostelium discoideum (1,3,4,10,12,13,19); in thelatter case, synergic effects should be observed when used in combination with sterol biosynthesis inhibitors (5,16,17).Taken together, the results indicate that bisphosphonates may constitute a new chemotherapeutic approach for thespecific treatment of Chagas disease. References:1. Amin, D., et al. 1992. J.Lipid.Res. 33:1657-1663.2. Bringaud, F., et al. 1998. Proc.Natl.Acad.Sci.USA 95:7963-7968.3. Ciosek, C.P., et al. 1993. J.Biol.Chem. 268:24832-24837.4. Cromartie, T.H., et al.1999. Pest.Biochem.Physiol. 63:114-126.5. Docampo, R. et al. 1997. Parasitol.Today 13:129-130.6. Docampo, R., et al. 1995. Biochem.J. 310:1005-1012.7. Licata, A.A. 1997. Am.J.Med.Sci. 313:17-22.8. Lu, H.-G., et al. 1997. J.Biol.Chem. 272:9464-9473.9. Moreno, S.N.J. et al. 1996. Biochem.J. 313:655-659.10. Rodan, G.A. 1998. Ann.Rev.Pharmacol.Toxicol. 38:375-388.11. Rogers, M.J., et al 1997. Overview of Bisphosphonates. Cancer 80:1652-1660.12. Rogers, M.J., et al. 1995. Mol.Pharmacol. 47:398-402. 13. Rogers, M.J., et al. 1997. Pharm.Res. 14:625-630.14. Scott, D.A., et al. 1998. J.Biol.Chem. 273:22151-22158.15. Scott, D.A., S.N.J. Moreno and R. Docampo. 1995.Ca2+ storage in Trypanosoma brucei: the influence ofcytoplasmatic pH and importance of vacuolar acidity. Biochem.J. 310:789-794.16. Urbina, J.A. 1997.Lipid biosynthesis pathways as chemotherapeutic targets in kinetoplastid parasites. Parasitol.117:S91-S99. 17. Urbina, J.A. 1999. J.Mol.Med. 77:332-338. 18. Urbina, J.A., et al. 1998. Mem.Inst.Oswaldo Cruz 93, Sup.II:72. 19. van Beek, E., et al. 1999. J.Bone Mineral Res. 14:722-729. 20. Vercesi, A.E., et al. 1994. Biochem.J.304:227-233. 52Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S13-2 – THE COOPERATIVE EFFECT OF THE HOST IMMUNE SYSTEM ON THE EFFICACY OFBENZNIDAZOLE THERAPY OF EXPERIMENTAL CHAGAS’ DISEASE Murta S.M.F.1,3, Ropert C.2, Alves R.O.3, Gazzinelli R.T.1,2 & Romanha A.J.31Departamento de Bioquímica e Imunologia, ICB-UFMG, 30270-010 BH, MG, Brazil 2Laboratório de Doença deChagas, 3Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou, FIOCRUZ, 30190-002 BH, MG, [email protected] The interaction of host immune system and chemotherapy against parasites has been previously reported in thetreatment of African Trypanosomiasis, Leishmaniasis (Berger & Fairlamb, 1992), Schistosomiasis (Brindley &Sher, 1987, Fallon et al., 1992) and Malaria (Target, 1992). Different authors observed that the efficacy of drugtreatment appears to be lower in immunosuppressed animals, demonstrating that the immune system of infectedhost, can play an important role in successful chemotherapy. Toledo et al. (1991) observed that cyclophosphamide-induced immunosupression decreases the percentage of cure in mice inoculated with different T. cruzi strains andsubmitted to specific treatment with benznidazole (BZ). Michailowsky et al. (1998) also demonstrated that neutral-ization of endogenous IFNγ or IL-12 in vivo, reduces the efficacy of BZ treatment during the acute phase of experi-mental Chagas’ disease. In fact, it is well known that the treatment with nitroheterocyclic derivatives is moreeffective during the acute phase of Chagas’ disease, when a strong activation of the cellular compartment of theimmune system and high levels of IFNγ, IL-12 and other pro-inflammatory cytokines are observed (Brener &Gazzinelli 1997).To further investigate the cooperative effect of BZ treatment and macrophage activation during the experimentaltherapy of acute phase of Chagas’ disease, we used a previously selected BZ resistant population of T. cruzi (Murta& Romanha 1998) and its parental Y strain (susceptible). Mice infected with either parasites were treated at the peakof parasitemia with a high dose of BZ (500 mg/Kg of body weight). Trypomastigotes isolated from untreatedinfected mice, as well as, three hours after treatment with BZ were incubated with inflammatory macrophages andused to study phagocytosis, parasite destruction, cytokine release and reactive nitrogen intermediates (RNI) synthe-sis. According to previous study using the Y strain of T. cruzi (Lages-Silva et al. 1990), our results demonstrate thatphagocytosis and destruction of the drug-susceptible parasites were significantly enhanced by drug treatment. Theseenhancements were accompanied by an increase in cytokines (IL-12 and TNFα) and RNI release by murine inflam-matory macrophages primed with IFNγ. In contrast, BZ treatment of mice infected with drug-resistant T. cruzipopulation showed no effect whatsoever. The synthesis of IFNγ and RNI by splenocytes of mice infected with eithersusceptible and drug-resistant parasite populations, before and after treatment with BZ were also studied. Only thesplenocytes from mice infected with the drug-susceptible parasites treated with BZ produced high levels of IFNγand RNI. Our findings indicate that BZ acts on the drug-susceptible T. cruzi parasites by enhancing the phagocytosisand the production of cytokines and RNI, thus, favoring the destruction of the intracellular parasites by the cellularcompartment of the immune system.Comparative studies of drug susceptibility on T. cruzi, demonstrate no correlation between parasite drug suscep-tibility in vitro and in vivo (Scoth & Mathews 1987, Neal & Van Bueren 1988, Ribeiro-Rodrigues et al. 1995). Thesestudies suggest the involvement of the immune system in the efficacy of treatment of T. cruzi infections. Takentogether, our results suggest the existence of a cooperation between BZ treatment and the immune response againstT. cruzi, which ensure the efficacy of drug therapy. Interestingly, this cooperative effect is observed on the suscep-tible but not on the drug-resistant T. cruzi population and may explain, in part, a mechanism by which T. cruziparasites may resist to chemotherapy in vivo. Supported by CNPq, PRONEX No 2704, PAPES/FIOCRUZ and FAPEMIG. ReferencesBerger B.J. & Fairlamb A.H. (1992). Parasitol. 105, 871-878. Brener Z. & Gazzinelli R.T. (1997). Int. Arch. Allergy Immunol. 114, 103-110.Brindley P.J. & Sher A. (1987). J. Immunol. 139, 215-220.Fallon P.G., Cooper R.O., Probert A.J. & Doenhoff M.J. (1992). Parasitol. 105, 41-48. Lages-Silva E., Filardi L. & Brener Z. (1990). Mem. Inst. Oswaldo Cruz 85, 401-405.Michailowsky V., Murta S.M.F., Carvalho-Oliveira L., Pereira M.E.S., Ferreira L.R., Brener Z., Romanha A.J. &Gazzinelli R.T. (1998). Antimicrob. Agents Chemother. 42(10), 2549-2556. Murta, S.M.F. & Romanha A.J. (1998). Parasitol. 116, 165-171.Neal R.A. & Van Bueren J. (1988). Trans. R. Soc. Trop. Med. Hyg. 82, 709-714.Ribeiro-Rodrigues R., Dos Santos W., Oliveira A.B., Snieckus V., Zani C. & Romanha A.J. (1995). Bioorg. Med. Chem. Letters 5, 1509-1512.Scoth V.R. & Mathews T.R. (1987). Am. J. Trop. Med. Hyg. 37, 308-313.Target G.A.T. (1992). Parasitol. 105:61-70. Toledo M.J.O., Machado G.B.N., Pereira M.E.S. & Brener Z. (1991). Mem. Inst. Oswaldo Cruz 86, 237. 53Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 S13-3 – GPI-BIOSYNTHESIS: FROM BIOLOGY TO THE DEVELOPMENT OF STRATEGIES FOR THEIDENTIFICATION OF THERAPEUTIC TARGET MOLECULES IN TRYPANOSMA CRUZI Nisha Garg and Rick L. Tarleton.Department of Cellular Biology, University of Georgia, Athens, GA 30602 Parasitic protozoans utilize glycosylphosphatidylinositol (GPI) molecules as the major mode of surface anchor-ing of proteins. We have previously addressed the critical requirement of GPIs in the development, infectivity andvirulence of T. cruzi and Leishmania by heterologous expression of T. brucei GPI specific phospholipase C encod-ing cDNA. The expression of GPI-PLC in T. cruzi and Leishmania resulted in an apparent deficiency of GPIs, areduction in the surface expression of GPI-anchored proteins and the inability to maintain replication as amastigoteforms. These results have provided the impetus for the identification, cloning and characterization of the genes thatare essential for amastigote replication and are the targets for designing inhibitors of chemotherapeutic importance.We have used reverse genetic approaches that are based upon the presence of significant homology for the geneof interest among different organisms for the identification of a GPI8 homologue of T. cruzi. In addition, we arepursuing direct genetic approaches for the isolation of GPI-biosynthesis genes that do not share significant homol-ogy with yeast or human GPI-biosynthesis genes. We have devised a robust mutagenesis/screening approach for theselection of mutants that were capable of in vitro growth as epimastigotes and differentiation into infective metacyclics,but are unable to sustain replication as intracellular amastigotes the phenotype seen in GPI-PLC transfectants. Aproportion of these “replication incompetent” mutants exhibit incomplete GPI-biosynthesis and the accumulationof various GPI-intermediates. The inability to make complete GPIs was associated with a reduction in the surfaceexpression of GPI-anchored proteins GP50/55 and 1G7 in epimastigotes. These results suggest that our selectionstrategy has allowed us to identify T. cruzi “replication incompetent” mutants that are defective in the GPI-biosyn-thesis gene(s) function. Future studies will focus on the identification and functional characterization of the genesinvolved in GPI-biosynthesis in these parasitic protozoans. S13-4 – EFFECT OF CHEMOTHERAPY WITH BENZNIDAZOLE ON THE IMMUNE RESPONSE ANDHEART INFLAMMATORY LESIONS IN MICE INFECTED BYTRYPANOSOMA CRUZI Olivieri, B.P., De Souza, A.P., Cotta-De-Almeida, V., De Castro, S.L. & Araújo-Jorge, T.C.Laboratório de Biologia Celular, DUBC, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, RJ. Chagas’disease is an important public health problem in Latin America and the main cause of myocardium dis-eases. In Brazil, only one drug with trypanocidal effect is available for clinical use: N-benzyl-2-nitroimidazole-aceta-mide (benznidazole, Bz). Bz shows many side effects and has restricted efficacy over different parasite strains. In thisstudy, we analysed clinical and immunological parameters in the lymphoid organs, as well as cardiac involvement ofmice infected by the Y strain (Bz-sensitive) of Trypanosoma cruzi and submitted to chemotherapy with Bz. Parasiteload abortion (100mg/kg of Bz per os 0-8 days post infection dpi) or reduction schemes (0.25mg/mL of Bz in thedrinking water 7-21 dpi) were compared. We did not observe any noticeable effect of Bz in non-infected animals. Theclassically described characteristics of experimental T. cruzi were found: typical parasitaemia and survival curves,strong cachexia, reversible strong splenomegaly and lymphadenopathy, lymphocyte expansion of B and T populations(patent in the 9th dpi, and reversed until 14th dpi), important percentage of cells expressing activation markers such asCD69 and CD25, thymic atrophy and lymphocyte apoptosis. Since myocarditis is a prominent feature of both acute and chronic stages, we made histopathological study of heart inflammation, in paralell to measures in changes ofcreatine kinase (CK-MB) levels, a marker of heart lesion very often used in humans. About 38% of the infectedanimals showed high CK-MB levels at the 21st dpi, that positively correlated to the intensity of the inflammatoryprocess, measured by the number of inflammatory infiltrates quantified on heart tissue sections.The main differences found in infected Swiss mice treated with Bz compared to non-treated ones were: a) partial(reductive scheme) or total (abortive scheme) reduction of parasitaemia and mortality; b) reversal of cachexia; c) efficacy of abortive treatment in the reversion of all immunological parameters analysed, including the thymicatrophy, as well as the decrease of cardiac inflammation and CK-MB leakage during the 2nd and 3rd weeks. How-ever, two unexpected results were consistently found: a) exacerbation of splenomegaly and lymphadenopathy inmice submitted to reductive treatment, with an increase in the number of T CD4+ cells, parallel to partial reversal ofthymic atrophy; b) increase in the frequency of mice submitted to abortive scheme with high CK-MB levels duringthe 4th dpi. With the abortive scheme, despite the treatment starting at the moment of infection, alterations in thehomeostasis of the host immune system were yet observed, such as mild splenomegaly and lymphadenopathy in the9th dpi, indicating cellular expansion, which were reverted in the 14th dpi. The abortive treatment prevented theappearance of acute phase lesions until the 3rd week post-infection, but at the 4th and 8th week 42% and 50% of miceshowed high CK-MB levels, respectively. The reductive scheme with 0.25 mg/mL led to a potentialization of the 54Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. II, Nov. 1999 splenomegaly, specially in the 14th dpi, with a decrease of the B cells, and an increase of the CD4+ cells number,suggesting that only this latter population was proliferating in vivo. We also observed partial reversal in the expres-sion of activation markers in the spleen and lymphnodes, indicating that the cells remained partially activated, butdid not follow the activation pathway up to apoptosis induction. We concluded that the treatment with Bz of T. cruzi-infected animals should not be considered simply as a way of reducing parasitaemia and mortality. As Bz treatmentcauses alterations in the immune system and cardiac enzyme leakage that do not show correlation with the parasiticload, it is necessary a long-term experimental study about the repercussion of these alterations not only on the laterstages of the disease, but also in auto-immune reactions and resistance to re-infections. If similar results could beobtained in acute human cases of T. cruzi infection, then treatment in endemic areas with ongoing active parasitetransmission should be re-evaluated. Financial Support: CNPq, CAPES, PAPES/FIOCRUZ & IOC/FIOCRUZ.