Round Tables

• RT01 -

  ^{CELLULAR BIOLOGY}

• RT02 -

  ^BIOCHEMISTRY

• RT03 -

  ^MALARIA

• RT04 -

  ^{MOLECULAR BIOLOGY I}

• RT05 -

  ^CHEMOTHERAPY

• RT06 -

  ^PROTOZOOLOGY

• RT07 -

  ^{IMMUNOLOGY I}

• RT08 -

  ^BIOCHEMISTRY

• RT09 -

  ^{IMMUNOLOGY II}

• RT10 -

  ^{MOLECULAR ENTOMOLOGY AND VECTORS}

• RT11 -

  ^{NON-PATHOGENIC PROTOZOA}

• RT12 -

  ^{IMMUNOLOGY III}

• RT13 -

  ^{MOLECULAR BIOLOGY II}

• RT14 -

  ^EPIDEMIOLOGY

RT01 - ^{CELLULAR BIOLOGY}

A NOVEL ORGANELLE DISTINCT FROM LYSOSOMES IN TRYPANOSOMATIDS

Roberto do Campo, University of Illinois, IL, USA

Abstract not received.

THE HYDROGENOSOME

Marlene Benchimol

Universidade Santa Úrsula, R. Jornalista Orlando Dantas, 59/^9o andar. Botafogo-CEP 22231-010. Rio de Janeiro, Brazil

During the evolutionary process eukaryotic microorganisms appeared presenting special cytoplasmic organelles. One example is the hydrogenosome initially found in protozoa of the Trichomonadida order and which contains enzymes that participate in the metabolism of pyruvate formed during glycolysis and was the site of formation of molecular hydrogen and ATP. Later on this organelle was described in some fungi, in a number of anaerobic rumen ciliates and very likely in certain free-living ciliates. The hydrogenosomes were described as spherical or slightly elongated granules with 0.5-2.0 mm diameter, closely associated to cytoskeletal structures such as the axostyle and costa. We have shown that two closely apposed unit membranes envelop the hydrogenosome. A flattened, membrane-bounded vesicle was found at the periphery of the hydrogenosome. We have shown that the electron-dense amorphous core described in this organelle is in fact an artifact.

Analysis of thin serial sections used to 3-D reconstruction and freeze-fracture replica show that the hydrogenosome resembles a sphere, but presents a protrusion towards the cytoplasm. The vesicle varies in size from organelle to organelle and represents about 8.5% of the volume of it. Based on the fact that the vesicle (a) presents a distinct morphological appearance from the hydrogenosome matrix, (b) was the main site of Ca⁺⁺-accumulation, (c) presents phosphatase activity and (d) its membrane presents N-acetyl-glucosamine-containing glycoconjugates, as revealed by incubation of cryosections in the presence of gold-labeled WGA, we conclude that it represents a specialized sub-compartment of the hydrogenosome.

The morphogenesis of hydrogenosomes in several trichomonad species Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence shows that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming elongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.

BCECF (2^', 7'-bis- (2-carboxyethyl) _5(and-6)-carboxyfluorescein is a fluorochrome widely used for the measurement of intracellular pH. It is usually loaded into cells as the membrane-permeant acetoxymethyl (AM) ester, and is hydrolysed by cytosolic esterases to give free BCECF, which, being charged, is trapped in the cytosol. The dye has been much used for the measurement of cytosolic pH in mammalian cells and also trypanosomatid protozoa, although in plants and fungi sequestration into the vacuole occurs, and there are reports indicating localization of the dye in endosomes and mitochondria in mammalian cells. When we loaded Trichomonas vaginalis with BCECF/AM, it was immediately apparent by microscopic examination that the dye was present not only in the cytosol, but was also accumulating in, or binding to, hydrogenosomes.

The structure of hydrogenosomes of the anaerobic fungus Neocallimastix frontalis was analyzed by using routine preparation for transmission electron microscopy, freeze-fracture and immunocytochemistry. They appeared as round or elongated structures, always enveloped by two distinct, but tightly apposed membranes. Images of organelle division were very similar to those observed in trichomonad protozoa. These observations suggest that hydrogenosomes are homologous organelles in unrelated species weakening the hypothesis of a polyphyletic origin and support the evidence that fungal hydrogenosomes are probably derived from an endosymbiont relationship.

Supported by: FENORTE, FINEP, CNPq, AUSU.

NOVEL REGULATED SECRETORY VESICLES IN GIARDIA LAMBLIA, A PRIMITIVE PARASITE

Frances Gillin, University of California, San Diego, CA, USA

Abstract not received.

REVISITING TRYPANOSOMA CRUZI WITHIN TISSUES FROM CHAGASIC PATIENTS

Mortara, R.A & Silva, S.

Disciplina de Parasitologia, Escola Paulista de Medicina - UNIFESP, R. Botucatu, 862, 6º andar 04023-062, São Paulo, SP, Brasil

Supported by FAPESP, CNPq and PADCT-CNPq

Since the discovery of Chagas' disease some 80 years ago, a lot has been learned about the causing parasite, how it interacts with host cells in vitro and in vivo. During the course of the disease, the parasite survives within the mammalian host escaping the immune response mainly by its sheltered intracellular location. It is within host cells, heart fibers, nervous cells and a variety of other cell types, that amastigote forms slowly and continuously perpetuate the infection. Back in the early 1900's when Carlos Chagas first identified patients harboring T. cruzi bloodstream trypomastigotes (3,4), and Vianna (8) and Torres (6) reported the presence of amastigotes within brain mononuclear cells and heart muscle fibers, several investigators described in detail the role of the parasite in the development of the disease, mainly using immunohistological and electron-microscopic tools (1).

The advent of confocal microscopy in the late 80' enabled a great quantitative and qualitative jump in the observation of biological structures that was previously not possible (5).

We have, with this study, began the analysis of the distribution of parasite and host cell components in tissues from chagasic patients using confocal fluorescence microscopy. Our long-term goal is to evaluate the intimate relationships that develop between the two parts, leading to altered distribution of important components of either part. Another aspect that we will be examining relates to the mechanisms underlying reactivation of the disease following immune depressed states.

In the first series of experiments, we have examined the distribution of T. cruzi amastigotes in tissues from patients in both chronic and acute phases of the disease. We used a series of monoclonal antibodies that react with: i) amastigote surface components (Mab 1D9, 2B7, 3B9, 4B9); ii) intracellular trypomastigotes (Mab 3B2) and Mab 4B5 that localizes to the amastigote membrane in dividing forms and concentrates near the flagellar pocket region in elongated amastigotes (2). By using a combination of Mab and DNA dyes, we were able to access the physiological state of the parasites within host cells.

We have been able to positively identify, in most specimens, amastigotes by their appearance under Nomarski differential interference contrast (DIC). In these samples, reaction with our Mabs or with a polyclonal anti- T. cruzi amastigote rabbit antiserum (7)were consistently positive.

In these preliminary studies we have found:

Chronic forms: Nests were usually scarce and well encircled by a relatively thick membrane. Reaction with Mab 1D9 was positive and some variations were observed with the other Mabs.

Congenital and acute forms. We found that in patients that were developing acute forms of the disease, Mab 1D9 reacted with all samples examined, and amastigote nests were abundant and present in different tissues (lung, brains, kidney, heart). Moreover, we could detect reactivity with Mab 3B2 and 4B5, indicative of active proliferation and differentiation of the parasites.

Immunodepressed patients. We have examined skin biopsies that appeared in two cases of Chagas' disease (re)activation following transplants. In the case of heart transplant, we detected positive reaction with all Mabs, and found positive reaction for 3B2 very near the epidermal layer, possibly within Langerhans' cells. In another case of T. cruzi infection following liver and kidney double transplant, we also identified T. cruzi amastigotes in skin lesions. As previously reported by several groups, AIDS patients bearing Chagas' disease can also develop severe forms of encephalitis and brain tumors. We have found extensive parasitism is brain biopsies and necropsies of such patients, and also positive reaction with all our Mabs. Amastigotes are usually found within glial-mononuclear cells.

Acknowledgments: we are grateful to our colleagues that provided specimens for this study: M. Lourdes Higuchi, Edison Reis Lopes, Ademir Rocha, Francy R. S. Patrício, Márcia M. Souza, Dirceu Almeida, Alberto Alain Gabbai, Rodrigo Correa Oliveira.

References

1. Andrade, Z. A. 1991. Pathogenesis of Chagas' disease. Res. Immunol. 142:126-129.

2. Barros, H. C., N. V. Verbisck, S. Silva, M. F. Araguth, and R. A. Mortara. 1997. Distribution of epitopes of Trypanosoma cruzi amastigotes during the intracellular life cycle within mammalian cells. J. Euk. Microbiol. 46:203-233.

3. Chagas, C. 1909. Nova tripanozomíase humana. Estudos sobre a morfologia e o ciclo evolutivo do Schizotrypanum n.gen, n. sp., agente etiológico de nova entidade mórbida do homem. Mem. Inst. Oswaldo Cruz (Rio de Janeiro) 1:159-218.

4. Chagas, C. 1911. Nova entidade morbida do homem. Resumo geral de estudos etiologicos e clínicos. Mem. Inst. Oswaldo Cruz (Rio de Janeiro) 3:219-275.

5. Lichtman, J. W. 1994. Confocal Microscopy. Sci. Am. 271:30-35.

6. Torres, M. 1917. Estudo do miocardio na molestia de Chagas (fórma aguda). I- Alteraçoes da fibra muscular cardíaca. Mem. Inst. Oswaldo Cruz (Rio de Janeiro) 9:114-139.

7. Verbisck, N. V. 1997. Estudos imunoquímicos e moleculares de antígenos de superfície de formas amastigotas do Trypanosoma cruzi. Tese de Mestrado, EPM-UNIFESP.

8. Vianna, G. 1911. Contribuição para o estudo da anatomia patolojica da "molestia de Carlos Chagas" (Esquizotripanose humana ou tireoidite parazitária). Mem. Inst. Oswaldo Cruz (Rio de Janeiro) 3:276-294.

RT02 - ^BIOCHEMISTRY

CYSTEINE PROTEINASES OF LEISHMANIA: STRUCTURE, FUNCTION AND POSSIBILITIES OF EXPLOITATION

Graham H. Coombs, University of Glasgow, Glasgow, UK

Abstract not received.

CYSTEINE PROTEASE INHIBITORS: INSIGHTS ON THEIR MECHANISM OF ACTION IN TRYPANOSOMA CRUZI AND LEISHMANIA MAJOR.

Juan C. Engel, Paul Selzer, Patricia S. Doyle, and James H. McKerrow

Department of Pathology, University of California, San Francisco, and Department of Anatomic Pathology, VA Medical Center, 4150 Clement St., San Francisco, CA 94121, USA

Cruzain (a.k.a. cruzipain, gp57/54), the major cysteine protease of T. cruzi, is a potential chemotherapeutic target. Cysteine protease inhibitors (CPI), such as fluoromethyl ketone (FMK) or diazomethane derivatized dipeptides, can block the parasite life cycle (Ashall et al., 1990 Biochem. Biophys. Res. Comm. 170, 923; Souto-Padrón et al., 1990 J. Cell Sci. 96, 485; Bonaldo et al., 1991 Exp. Parasitol. 73, 44; Meirelles et al., 1992 Mol. Biochem. Parasitol. 52, 175; Cazzulo et al., 1994 Fems Microb. letters. 124, 81). In previous reports, we showed the in vitro effect of two FMK-derivatized peptides, Z-Phe-Ala-FMK and Z-Phe-Arg-FMK, on several developmental stages of T. cruzi (Harth et al., 1993 Mol. Bioch. Parasi. 58, 17). These compounds have little effect and short half-lives in vivo probably due to peptide cleavage. To minimize degradation, a new generation of inhibitors was synthesized with substituted non-natural aminoacid (pseudo-peptides) chains. The thiol reactive FMK was replaced by vinylsulfonyl benzene (VSPh) which reacts specifically with the cysteine protease thiol group (Brömme et al. 1996 Biochem. J. 315, 85) Mu-F-hF-VSPh cured T. cruzi-infected cells without any toxicity for mammalian cells. In vivo, Mu-F-hF-VSPh reduced parasitemia and protected mice against a lethal dose of T. cruzi trypomastigotes (McGrath et al., 1995 J. Mol. Biol. 246, 251). Assays with purified cathepsin B and L, and with cell extracts showed that Mu-F-hF-VSPh also inhibits mammalian cysteine proteases. We are currently investigating the different susceptibilities of T. cruzi and mammalian cells to this CPI.

Radiolabeled or biotinylated CPI bound exclusively to cruzain in living parasites confirming that cruzain is accessible to, and targeted by the inhibitors. Ultrastructural changes were observed when antiproliferative concentrations of CPI were added to epimastigotes and T. cruzi-infected cells. Several T. cruzi organelles showed significant morphological and Ultrastructural alterations after 36 h of CPI-treatment. Mitochondria were considerably swollen, and the endoplasmic reticulum was dilated. Severe alterations in the Golgi apparatus were evident including peripheral dilations of cisternae, where cruzain accumulated. A concomitant reduction of cruzain transported to reservosomes/lysosomes in epimastigotes and to the surface of amastigotes occurred. Prolonged treatment (>96 h) increased the size and number of membranes accumulated throughout the parasite cytoplasm. Similar results were obtained with another reversible hydrazide derivatized, a non-peptide selected by molecular modeling (Ring et al., 1993 Proc. Natl. Ac. Sci. 90, 3583).

Both CPI-treated, and -resistant T. cruzi epimastigotes secreted species of higher M_r than native cruzain into their supernatants. These results suggest that CPI induce alterations in the ER-Golgi-Endosomal/Lysosomal pathway which prevent the normal autocatalytic processing of cruzain. Abnormally processed cruzain molecules would then be actively secreted instead of being routed to epimastigote reservosomes.

In Leishmania major, CPI did not induce alterations in neither mitochondria, endoplasmic reticulum, nor Golgi apparatus. Ultrastructural changes included abnormally dilated lysosomes containing dense bodies and mulivesicular bodies, and also enlarged flagellar pocket filled with cysteine protease-containing membranes/vesicles. These results are consistent with a CPI-induced alteration of the Leishmania secretory pathway at a post-Golgi complex level. The development of an extended cytostome in close association with the Golgi complex and the flagellar pocket in CPI-resistant promastigotes also suggests an enhanced exocytosis.

Thus, CPI interfere with the normal processing and/or trafficking of cysteine proteases through the secretory pathway in T. cruzi and Leishmania, and the organisms seem to respond by enhancing secretion of incorrectly processed cysteine proteases. In contrast, CPI-treated mammalian cells do not exhibit any evident alteration in secretory mechanisms. The differences in susceptibility to CPI between parasites and mammalian cells may be further exploited to develop specific inhibitors with increased parasitocidal activity.

This work was supported by the American Heart Association, National Institutes of Health, and Burroughs Wellcome Cysteine protease inhibitors were provided by Prototek Inc., Arris Pharmaceuticals, and Dr. Fred Cohen, UCSF.

MOLECULAR CHARACTERIZATION OF CHAGASIN, A TRYPANOSOMA CRUZI INHIBITOR OF PAPAIN-LIKE CYSTEINE PROTEINASES.

Ana Carolina Monteiro^a Magnus Abrahamso^b, Marcos Andre Vannier^c, Ana Paula Lima^a and Julio Scharfstein^a .

^a Laboratório de Imunologia Molecular, IBCCFUF.R.J; ^bDepartment of Clinical Chemistry, University of Lund,Sweden; ^claboratório de Ultraestrutura Celular Hertha Meyer, IBCCF, UFRJ.

Introduction. ln spite of the identification of papain-like cysteine proteinases (CP) as key enzymes of protozoa and helminth parasites, the biochemical mechanisms that regulate their enzymatic activities inside the cell are not known. Mffibition of catabolic functions mediated by these lysosomal enzymes may spare the parasite's own polypeptide reserves at times of nutritional stress. At least for some helminths, there is evidence that CP regulation is important to molting ll]. Uncontrolled proteolysis by endogenous proteases may be a potential threat for the integrity of the eukaryotic cell. ln the case of lysosomal proteinases, stressful conditions may allow leakage of their contents into the cytosol or deliver them into undesirable compartments of the secretory system. The regulatory constraints acting upon lysosomal proteinases may be detennined by a "built-in" mechanism of inactivation. For example, it is known that mammalian cathepsin L rapidly unfolds at neutral pH, due to intramolecular confonnational changes. Other lysosomal CP's (for example, cathepsin S) are more robust and may thus require the intervention of other endogenous molecules to be effectively controlled and degraded. This might well be the case of some lysosomal cysteine proteinases from úypanosomatids, such as the major cysteine proteinase from T cruzi (cruzipain). Because of its broad pH activity profile and high stability, cruzipain inactivation may depend on the activity of other endogenous factors. Recent studies [2] have indicated that the developmental regulation of cruzipain expression occurs predominantly at the translational and/or post-translational level. In view of the broad disúibution of cystatin-like inhibitors in nature, the search for related molecules in úypanosomatid species [3] seemed worth undertaking in the T cruzi species.

Biochemical properties of cystatins. Most of the available inforrnation on CP-regulation by protein derives from studies of the cystatin superfamily [41. Widely distributed in mammalian body fluids and tissues, and also present in plants and lower organisms, the cystatins are representei by 3 major families. Members of family I and 2 are single-chained proteins of relatively low molecular mass, which bind target proteinases at a 1: I ratio. The family I proteins (also known as cystatins A and B or stefins) proteins have a Mr of 11-12 kDa, lack intrachain disulfide bridges and carbohydrate, and are thought to accurnulate in the cytosol of mammalian cells. Members of family 2 (ccystatins C, D, S, SN, SA and E) are of 13-14 kDa, display two intra-chain di-sulfide bridges and are found in secretions. The family 3 cystatins are related to kininogen, a high- Mr plasma glycoprotein that contains 3 cystatin domains (only two of which are functional). These tight-binding proteins inhibit papain-like enzymes by a competitive mechanism. The X-ray crystallographic analysis of chicken cystatin and a cystatin B-complex formed with papain revealed that cystatins are elongated proteins containing a protuberant wedge-shape structure that fits perfectly in the active site cleft. Formed by three cystatinsegtnents, the binding site portion that interacts with the subsite specificity pockets of the target protease is located at the N-terlninal side of the Gly-1 l residue (in the case of family 2 cystatins).

Molecular characterization of the endogenous inhibitor of cysteinyl proteases. ln view of the high content of cruzipain(s) in epimastigotescell lysates, we initially failed detect a CP inhibitory activity in cell extracts. Assuming that the CP inhibitors would most likely bind to CP targets, we then chose to work with a mutant cell line (R-Dm28) that expresses lower contents of cruzipain (refer to Yong. et ai., this volume). Extracts from these R-Dm28 were readily depleted of residual CP activity by gel filtration, allowing for detection of minute quantities of an inhibitory activity. Tentatively identified as an 11 kDa protein, this protein formed tight reversible complexes with purified cruzipain, but its presumed relationship to cystatins could not be proved. In order to characterize the primary structure of the cruzipain-inhibitor, a lgtl1 cDNA expression library from Dm28c epimastigotes was screened by ligand-binding assays, using capam as a probe. Out of 10 clones obtained, 8 showed closely-related sequences. lnterestingly, the CDNA sequence did not show any similarity with known cystatins, or with any other sequence available in data banks. A 65Obp CDNA was expressei in E. coli as a fusion protein using the pHD3l3 system, previously used for mammalian cystatins. The recombinant fusion protein, heretofore designated as r-chagasin, displayed potent CP inhibitory activity. N-terminal microsequencing of r-chagasin revealed the expected amino acid sequence. A polyclonal antiserum raised against r-chagasin identified a 11 kDa inhibitory protein in lysates from both wild type DM28c and R-Dm28 epimastigotes. The natural Dm28 protein homologue was purified and subjected to N-tenninus microsequencing. Confirining our predictions, its sequence was closely-related to that of r-chagasin. We then sought to compare the Ki values of the natural and recombinant chagasin proteins. This was performed using (i) natural cruzipain, isolated from Dni28 [5] (ii) r-cruzain, the engineered (E. coli) protease whose C-term portion was deleted (gift from J.H. McKerrow, UCSF) [6] (iii) r-cruzipain 2, a functionally distinct isoform [7] expressei in S. cerevisae, likewise expressei without the C-term domain. The Ki values (refer to Monteiro et al., this volume) obtained for n/r-chagasins were very similar (in the picomolar range). lnterestingly, chagasin was more efficacious against r-cruzain than r-cruzipain 2, further suggesting that these isofonns differ considerably in their active-site structure [8]. Similarly to members of the cystatin superfamily, chagasins display broad target specificities: the T cruzi protein inhibited CP's from mammals (eg. cathepsin B), vegetal organisms (eg., papain) and other pathogenic parasites (eg., falcipain, one of the trophozoite proteases from P. falciparum). Northem blot analysis indicated that chagasin trancripts are of approximately 800 nt and are expressei by all developmental forns of T cruzi. lmmunolocalization studies performed with afflnity purified antibodies to r-chagasin identified these molecules in the flagellar pocket and flagellum, and also in secretory vesicles. lnterestngly, that pattern of surface expression of chagasin is not the same in the various developmental forras, the immunoreactivity being markedly intensified on amastigotes. lnterestingly, the levei of chagasin expression during development appears to be inversely corrrelated with cruzipain (the inhibitor being expressei in higher levels in trypomastigotes, while cruzipain contents are higher in replicating fortns). Albeit circumstantial, these data suggest that chagasin may play a role in the post-translational control of cruzipain expression. lt will be interesting to know if the rates of catabolic degradation of the lysosomal cruzipain(s) are at least in part controlled by chagasin binding and enzyme inactivation.

References

1. Lustigrnan et ai. (l992). J Biol. Chem. 267:17339

2.Tomas, AM and Kelly, J. (l996) Mol. Biochem. Parasitol. 76:9]

3. lrvine et ai. (i 992). FEMS Microbiol. Lett. 96:6 7

4. Abrahamson, M. (I 993) Ciência& Cultura 45(5):299

5. Eakin et ai. (i 992). J Biol. Chem. 267:741 1.

6. Lima et ai. (I 992). Mol. Biochem. Parasitol. 56:338

7 Lima et ai. (I 994). Mol. Bioch. Parasitol. 67:333

8. Dei Nery et ai.. (I 997) J Biol. Chem (in press).

STUDY ON THE EFFECT OF THE HERBICIDE TRIFLURALIN IN TRYPANOSOMA CRUZI

Ortigão, M., Sampaio, M.C., Amorim, I. and Traub-Csekö, Y.M.

Depto. de Bioquímica e Biologia Molecular, Fiocruz, Ave. Brasil 4365, Rio de Janeiro, Brasil

Chagas’Disease is an important illness afflicting a large number of people in Brazil.Up to now there are only two drugs available which are effective against T. cruzi, and there is a strong need for the development of new therapies for the treatment of such disease. Trifluralin, which has been used as a herbicide in the USA since the 1960’s has been shown to kill pathogenic trypanosomatids in vitro through the binding to b tubulin, destabilizing them. Such an effect however is not seen in mammalian cells. We are currently testing Trifluralin against T. cruzi, and our preliminary results show an effect of the drug in the range of 50 mM. Apparently there is a relationship between susceptibility to Trifluralin and the b tubulin sequence of a given organism. b tubulin proteins are well conserved among a large number of organisms varying from lower eukaryotes to humans. Nevertheless important differences are found within the aminoacid sequences of different organisms. These differences may occur even among organisms of the same family. Our group has previously cloned and sequenced the tubulin genes of T. cruzi. We intend to, by studies of sequence comparison among mammals, plants and trypanosomatids, be able to determine which changes in the aminoacid sequence are crucial for Trifluralin resistance or susceptibility. We hope that answers regarding these and other questions may provide us with information which can de used to develop new strategies in the fight against this parasite. “Cellular targeting of cysteine proteinases in Leishmania pifanoi”.

RT03 - ^MALARIA

ANTIGENIC VARIATION IN P. FALCIPARUM: ECTOPIC RECOMBINATION INVOLVING SUBTELOMERIC VAR GENES

Bottius, E., Hernandez-Rivas, R., 1Pouvelle, B., 1Gysin J., 2Wellems, T.E. and Scherf, A.

Institut Pasteur, Paris; 1UFR de Meédecine, Universiteé d'Aix-Marseille II,France; 2NIH, Bethesda, USA

Human erythrocytes infected by Plasmodium falciparum parasites acquire adhesive properties that are an important virulence factor in the pathogenesis of malaria. These adhesive modifications cause erythrocytes infected by mature bloodstage parasites to attach to postcapillary venular endothelium and thereby sequester from the circulation, enabling them to avoid destruction by the spleen. The sequestration of infected erythrocytes within critical organs is thought to correlate with the development of severe malaria syndromes, including the feared cerebral form. Adherence of the sequestered cells is mediated by a family of 200,000-350,000 Mr molecules (Plasmodium falciparum erythrocyte membrane protein 1, PfEMP1) that are highly polymorphic, antigenically variant, and expressed on the erythrocyte surface at electron-dense protrusions termed knobs. These variant surface molecules are encoded by a large and extremely diverse family of genes (var). Individual parasites have been found to have unique complements of 50-150 var genes in their chromosomes (accounting for 2-6% of the nuclear DNA), indicative of tremendous repertoires of variant types. Transcripts from var genes have been mapped to central as well as subtelomeric regions approximately 20 to 40 kb from the telomere repeats. DNA hybridization analysis revealed that var genes are not commonly shared by genetically different parasite strains. In some cases, comparisons of individual isolates have shown identical or very closely related single-copy var genes in the subtelomeric regions of different chromosomes, suggesting that the var genes undergo transposition events and are therefore mobile. Saimiri monkey erythrocytes infected by P. falciparum in vivo and human erythrocytes infected in vitro have been shown to spontaneously switch their antigenic forms and adherence types. Evidence indicates that these changes are accompanied by switches in the expression of individual var genes, indicating the presence of important mechanisms governing selective transcription and exclusive expression from the var repertoire.

We analysed the significance of the observed var mobility with regard to the mechanism(s) that control switches of var gene expression and the diversity of the var gene repertoire in genetically different parasite strains. Parasite subpopulations with different cytoadherent phenotypes were selected from a cloned laboratory parasite line. PFG and fine mapping analysis of those parasites did not reveal any repositioning of the activated var genes. In switch events, in situ activation, possibly involving trans-activating factors, may play an important role in var gene regulation. However, after meiosis, we observe high frequencies of DNA rearrangements involving var genes. In nearly half of the progeny clones from two crosses of P. falciparum, recombination events between var genes on different chromosome ends lead to the transposition of var sequence from one chromosome to the other, or to the presence of a duplicated copy of the var sequence on a different chromosome (ectopic recombination). Our finding suggests that ectopic recombination plays a crucial role in the generation of var gene repertoire diversity.

GENOME ORGANIZATION IN PLASMODIUM VIVAX MALARIA

Anamaria A. Camargo¹, Carmen Fernández-Becerra¹, Katja Fischer², Michael Lanzer² and Hernando A. del Portillo¹

¹Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Lineu Prestes 1374, São Paulo, SP 05508-900, Brasil. ²Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany

Studies on the genome organization of parasitic protozoa is one of the fastest evolving fields in molecular parasitology. Unfortunately, only one study has been reported on the genome organization of Plasmodium vivax, the most widely distributed human malaria and the most prevalent species in Brazil. In it, the presence of two DNA components with different G+C contents (30% and 18%) as well as other minor isochores as determined by buoyant density CsCl gradients, clearly indicated that the genome organization of P. vivax is remarkably different from that of the other major human malaria, P. falciparum (McCutchan et al. 1984. Science, 225:808). To further studies on the genome organization of P. vivax, we have constructed a genomic library in Yeast Artificial Chromosomes (YACs). As P. vivax can not be maintained continuously under laboratory conditions, the P. vivax DNA necessary for the library construction was isolated from a single human patient presenting himself to a local hospital in the Brazilian Amazon with vivax malaria. Thus, this YAC library is the first of its kind to be generated from patient-derived material. The YAC library consists of 560 clones with an average insert size of 180 kb. Of nine published P. vivax genes, eight were found to be present in the library. In addition, twelve P. vivax telomeric YAC clones were identified. Results on different projects initiated with this library will be presented. Supported by FAPESP, CNPq and INCO-DC Programmes."

CHONDROITIN SULFATE AND CYTOADHRENCE OF PLASMODIUM FALCIPARUM - INFECTED ERYTHROCYTES

J. Gysin, Institut Pasteur de Lyon, Lyon, France

Abstract not received.

THE MOLECULAR PATHIGENESIS OF SEVERE MALARIA

Mats Wahlgren

Microbiology & Tumor Biology Center, Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden

Adhesion of Plasmodium falciparum infected erythrocytes in the vascular capillaries plays an important role for parasite survival but also contributes to the pathogenesis of complicated malaria: excessive binding may cause obstruction of the blood flow. The virulence of the parasite is associated with the capacity of the infected erythrocyte to adhere to endothelial cells and to erythrocytes (rosetting). Cerebral malaria is the most malignant state of the infection due to massive sequestration of infected and uninfected erythrocytes in the brain micro-vasculature. Adherence of the sequestered cells is mediated by a family of 200,000-350,000 Mr molecules (Plasmodium falciparum erythrocyte membrane protein 1, PfEMP1) that are highly polymorphic, antigenically variant, and expressed on the erythrocyte surface at electron-dense protrusions termed knobs. PfEMP1 has features of an adhesive molecule and has been associated with the cytoadherent properties of the infected red cell. E.g. the expression of PfEMP1 or its gene var has been shown to correlate with the capacity of the pRBC for binding to host receptors, including CD36 and ICAM-1. Recently we have shown that the DBL-1 domain of rosetting PfEMP1 expressed in bacteria adhere directly to erythrocytes and disrupt pre-formed rosettes. A second group of low molecular weight polypeptides, sc. rosettins, are also expressed at the Prbc surface. The rosettins are available for iodination and their solubility properties suggest them to be associated with the erythrocyte cytoskeleton. The importance of the rosettins in rosetting however remains unknown although the expression of the rosettins has also been shown to correlate with the adhesive capacity of the parasitized erythrocyte.

Taken together it can be said that the sequestration of infected and uninfected RBC in the microvasculature needs to be further explored, at all biological and scientific levels, in order to be able to develop a deeper understanding of the pathogenic mechanisms, adjunct therapy and vaccines that prevent the development of the severe forms of the disease.

RT04 - ^{MOLECULAR BIOLOGY I}

BIOCHEMICAL AND GENETIC VALIDATION OF TRYPANOTHIONE REDUCTASE AS A CHEMOTHERAPEUTIC TARGET

Jorge Tovar & Alan H. Fairlamb

Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom

Supported by the Wellcome Trust.

Trypanothione reductase (TR) is the flavoprotein oxidoreductase responsible for the enzymatic regeneration of the thiol pool in trypanosomatid protozoa. It helps maintain a reduced intracellular environment by keeping the majority of the unique peptide-polyamine conjugate, trypanothione (N₁,N₈-bis(glutathionyl)spermidine) in its reduced form (T(SH)₂). Trypanothione is the main low molecular mass thiol in these organisms and the enzymatic and non-enzymatic regeneration of other low molecular mass thiols such as glutathione, glutathionyl-spermidine and ovothiol is mostly dependent on it. Through a number of redox and disulphide exchange reactions that result in the oxidation of trypanothione (T(S)₂), trypanosomatid parasites protect themselves against the toxicity of heavy metals and xenobiotics and from the damage caused by free radicals generated during aerobic metabolism and, most importantly, during the host defence response. T(S)₂ is then regenerated into T(SH)₂ by TR in an NADPH-dependent reaction. As the trypanothione system is absent in host cells and its central enzyme appears to fulfil a relevant physiological role, TR has been proposed as a potential target for the chemotherapy of trypanosomatid infections [1]. The enormous costs involved in the development of new drugs against specific cellular targets demands the thorough validation of potential candidates by biochemical and genetic means. We have gathered evidence that TR is indeed essential for parasite survival using reverse genetics methodologies.

A trans-dominant mutational strategy was initially used to downregulate TR activity levels in Leishmania donovani, the causative agent of visceral leishmaniasis. TR is a homodimeric enzyme containing two active sites. Each associated monomer contributes at least one essential residue to the formation of each active site [2]. By site-directed mutagenesis we created a trans-dominant mutant version of the Trypanosoma cruzi TR that, upon dimerisation with wild type monomers, forms inactive heterodimers. Extrachromosomal, heterologous expression of such mutant enzyme in L. donovani resulted in a dramatic decrease in endogenous TR activity levels. Cells depleted of up to 85% of TR activity were significantly impaired in their ability to regenerate low molecular mass thiols (the basis of a reducing intracellular environment) following diamide oxidation. During growth in culture these cells were nonetheless capable of maintaining a reduced intracellular environment despite their highly decreased levels of TR activity. Furthermore, under these conditions, TR was found not to be rate-limiting in the metabolism of hydrogen peroxide in vitro. However, in a murine model of Leishmania infection, cells expressing the trans-dominant mutant TR displayed a markedly decreased ability to survive inside cytokine-activated macrophages, highlighting the importance of TR for parasite intracellular survival.

Gene targeting is a powerful genetic tool for the study of gene function and regulation. In protozoan parasites such as Leishmania, the plasticity of the genome allows for a positive identification of essential gene products through the generation of extra allelic copies of presumably vital targeted genes [3]. We used precise, targeted gene replacement to remove all allelic copies of the TR-encoding gene (tryA) in virulent L. donovani LV9. Not unexpectedly, and analogous to complementary gene disruption studies [4], the attempted replacement of the last tryA allele invariably led to the generation of an extra allelic copy of the gene resulting in partial trisomi. Karyotypic and Southern blot analyses of genomic DNA revealed the precise nature and fidelity of all replacement events; FACS scan analyses confirmed that no changes in general ploidy occurred during the transfection and selection of recombinant clones. Replacement of all chromosomal copies of the L. donovani tryA gene locus was only possible in cells expressing TR from an episome, providing unequivocal evidence that TR and its encoding gene are essential cellular components in L. donovani. In a murine model of Leishmania infection, partial replacement mutants were also drastically affected in their ability to survive inside bone-marrow, cytokine-activated macrophages. Since no compensatory mechanism for the partial loss of TR activity was observed in any of our mutant lines nor was it possible to obtain viable Leishmania completely devoid of TR catalytic activity, we conclude that TR fulfils an essential, non-redundant cellular role. It should now be possible to rationally design specific inhibitors of this enzyme to obtain novel anti-leishmanial agents for chemotherapeutic use.

References

1. Fairlamb AH, Cerami A. 1992. Metabolism and functions of trypanothione in the kinetoplastida. Ann Rev Microbiol 46:695-729.

2. Borges A, Cunningham ML, Tovar J, Fairlamb AH. 1995. Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase. Eur J Biochem 228:745-752.

3. Cruz AK, Titus R, Beverley SM. 1993. Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc Natl Acad Sci USA 90:1599-1603.

4. Dumas C, Ouellette M, Tovar J, Cunningham ML, Fairlamb AH, Tamar S, Olivier M, Papadopoulou B. 1997. Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages. EMBO J 16:2590-2598.

STUDIES OF SUBCELLULAR LOCALIZATION OF RAB7 IN TRYPANOSOMA CRUZI

Araripe, J.R .*; Leal, S.T.*; Cunha e Silva, N.L.**; de Souza, W.** & Rondinelli, E*

* Lab. Metabolismo Macromolecular Firmino Torres de Castro, ** Laboratório de Ultraestrutura Celular Hertha Meyer - Instituto de Biofísica Carlos Chagas Filho, UFRJ, 21949-900, Rio de Janeiro, RJ.

The small monomeric GTPases of the rab subfamily are key elements of the machinery that controls membrane traffic in eukaryotic cells. Approximately 30 different rab genes have been identified in a variety of mammalian species and localized to many different intracellular organelles on both the endocytic and exocytic pathways. This suggest that each step of membrane traffic might involve a different rab protein acting as a catalyst, accelerating the fusion reaction between donor and acceptor vesicles promoted after v/t-SNARE complex assembly. The Rab7 protein is associated with late endosomes in mammalian cells and several results suggest that this protein is involved in the anchoring step of these vesicles during endocytosis. The rab7 gene of Trypanosoma cruzi (tcrab7)has already been sequenced and characterized in our laboratory. We obtained transformed cell lines stably expressing Rab7 protein (pTAG) and a similar protein but without the last three C-terminal residues (DCXC). This Rab7 mutant fails to associate with membranes. We used a polyclonal antibody raised against a synthetic peptide comprising amino acid residues localized in the C-terminal region of Rab7 in order to localize the protein by immunofluorescence microscopy. This antibody showed that Rab7 is located at the anterior region of the cell, near the kinetoplast and the bottom of the flagelar pocket; this structure is duplicated in dividing cells. The intensity of staining was the same for all cells lines studied, including wild type CL Brener.These results are in agreement with those described for Trypanosoma brucei (Field,H. & Field, M.C., 1997, J. Biol. Chem., 272:10498). We are currently studying the distribution of Rab7 by immunoelectronmicroscopy. We also obtained a single transformant clone that is partially knocked out for the rab7 gene (Araripe et al, acompanying abstract). Its morphological characterization showed alterations at the anterior region of the parasite when observed at electron microscopy. Further characterization of this transformant is in progress.

Supported by CNPq, FAPERJ, CAPES and FINEP

GENETIC ANALYSIS OF THE THROMBOSPONDIN-RELATED ANONYMOUS PROTEIN (TRAP); A MALARIAL SPOROZOITE PROTEIN

Ali Sultan, Vandana Thathy, Ute Frevert, Kathryn Robson, Andrea Crisanti, Ruth S. Nussenzweig, Victor Nussenzwieg and Robert Menard.

Division of Immunology, Department of Pathology New York Univesity Medical Center and Department of Medical and Molecular Parasitology New York University USA, MRC Molecular Haematology Unit, Institute of Molecular Medicine, Oxford UK, Department of Biology, Imperial College of Science, Technology and Medicine, London, UK

Malaria parasites undergo sporogonic cycle in Anopheles mosquito vector. Sporozoites, the infective form to the vertebrate host, develop inside mosquito oocyts before they migrate and eventually invade the salivary glands. The thrombospondin-related anonymous protein; originally was identified as blood stage antigen but later was detected and studied as sporozoite protein, TRAP,

along the circumsporozoite protein, are thought to be involved in sporozoite attachment and invasion of the host hepatocytes.

We undertook a genetic approach to study the role of TRAP protein in the life cycle of malaria parasites, using the newly developed transformation technology in the rodent malaria parasite; Plasmodium berghei. By gene targeting we have generated TRAP (-) P. berghei parasite lines. Analysis of TRAP (-) phenotype demonstrated that TRAP is critical for infection of mosquito salivary gland and host hepatocytes. In addition TRAP is essential for gliding motility of sporozoite in vitro.

OVEREXPRESSION OF A STAGE-REGULATED GENE IN LEISHMANIA

Silvia R. B. Uliana¹ and Deborah F. Smith^{2 (1)} Dept. Parasitologia, Instituto de Ciências Biomédicas, USP, São Paulo, Brasil. ⁽²⁾ Dept. Biochemistry, Imperial College of Science, Technology and Medicine, London, UK

The meta 1 gene, initially characterised in L. major, is expressed exclusively during the promastigote stage of the parasite life cycle. The gene is conserved between different species of Leishmania and encodes an 11.5 kDa protein with increased levels of expression in stationary phase promastigotes (Nourbakhsh et al., Mol. Biochem. Parasitol. 76:201, 1996).

The L. amazonensis meta 1 homologue gene has also been characterised and shows the same regulated pattern of expression previously observed in L. major. The deduced aminoacid sequences of the meta 1 protein are highly conserved in different species of Leishmania but do not show significant homologies to any sequence in database banks. The peptide sequence does not show any potential trans-membrane domain or motifs for addition of GPI anchors.

Immunogold electron microscopy shows that the protein is present in the cytoplasm or inside vacuoli, preferentially in the region around the flagellar pocket. The expression pattern and the localisation data could suggest a role for this protein in the control of cellular homeostasis, specifically related to the changes in the extracellular environment, ocurring during the metacyclic stage.

Experiments using gene targetting were performed to investigate the function of this conserved protein in L. major and L. amazonensis. Targetted replacement of two alleles did not result in the knock out of the meta 1 gene, since genomic rearrangements led to the presence of a third wild type allele. Refractoriness to gene inactivation has been previously described in Leishmania (Cruz, A.K. et al., Proc. Natl. Acad. Sci USA 90, 1599-1603, 1993; Dumas,C. et al., EMBO J. 16:2590-2598, 1997) and is currently attributed to an essential function for a gene.

As an alternative approach to address the question of function for the meta 1 protein, a vector was designed for overexpression in Leishmania (Uliana,S.R.B.; Floeter-Winter,L.M.; Smith,D.F.; unpublished). The plasmid was built as to have the L. amazonensis ribosomal promoter (Uliana et al., Mol. Biochem. Parasitol. 76:245-255, 1996) driving the transcription of the L. major meta 1 and hygromycin phosphotransferase genes. Transfected promastigotes showed a 10 to 20-fold increase in the expression of meta 1 protein compared to wild type metacyclic promastigotes.

Growth curves of overexpressor lines did not show any significant difference and there were no morphological changes in promastigotes kept in liquid culture. Preliminary results indicate that BALB-c mice infected with meta 1 overexpressor L. amazonensis develop lesions of increased size in comparison to animals infected with the wild type strain, suggesting that the overexpression of the meta 1 gene might render the parasite more virulent.

Supported by FAPESP and Wellcome Trust."

RT05 - ^CHEMOTHERAPY

STEROL BIOSYNTHESIS INHIBITORS: A RATIONAL STRATEGY TOWARDS CHEMOTHERAPY OF CHAGAS DISEASE

Julio A. Urbina. Laboratorio de Química Biológica, Centro de Bioquímica y Biofísica,

Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela. e-mail: [e-mail]

The rational development of chemotherapeutic agents for the treatment of Chagas disease relies in the exploitation of specific metabolic differences between the causative agent, Trypanosoma (Schizotrypanum) cruzi, and its vertebrate hosts. Lipid biosynthesis pathways have been generally considered "soft" chemotherapeutic targets [1], based on the idea that these cellular components and/or the metabolic pathways that produce them are not sufficiently specific among the different phylogenetic groups to allow selective targeting by chemical intervention. Contrary to this idea, the drugs most widely used in the treatment of fungal diseases are compounds which interfere with sterol synthesis or function [2,3] and several specific phospholipid biosynthesis inhibitors have found applications as agrochemicals [4]. Work from our laboratory and other groups have shown that T.cruzi, as many fungi and yeasts, requires specific endogenous sterols to maintain cell viability and proliferation [5-17]. However, sterol biosynthesis inhibitors which have been used successfully for the treatment of human fungal infections [2,3] are unable to eradicate T.cruzi from infected humans or experimental animals [18-20]. Recent studies in our group [1,21-23] have shown that the bis-triazole derivative D0870, the R(+) enantiomer of the experimental compound ICI 195,739 [13,14,24-26], was 30-50 times more potent than nifurtimox or ketoconazole in prolonging the survival of mice infected with a lethal dose of T.cruzi and was able to protect 85-100% of animals form death when given at _ 15 mg/Kg.d on alternate days for a total of 28 doses. Furthermore, using six different criteria, including parasitological, serological and recently developed polymerase chain-reaction (PCR)-based test [27-29], it was found that >60% of infected animals attained parasitological cure while no cures were observed with currently available drugs such as nifurtimox and ketoconazole given daily for a total of 43 doses [21]. In a model of chronic (long-term) Chagas disease D0870 at 15-20 mg/Kg.d given on alternate days as indicated above provided 90-100% protection from death with 80-90% parasitological cures, while conventional drugs had no significant effects on survival or number of cures when compared with controls [21]. This was the first report of parasitological cure of experimental chronic Chagas disease. The anti-T.cruzi activity of D0870 was highly stereospecific as the S(-) enantiomer was 30-100 times less active ([21] and Liendo et al. submitted), indicating a very selective interaction between the drug at its biological target, the parasite's sterol C-14 demethylase. Moreover, studies carried out with nine T.cruzi strains with very different susceptibilities to nifurtimox and benznidazole, showed that D0870 was able to produce radical parasitological cure even in several of the highly-drug resistant strains [30] and its activity increased when the same total dose was spread in time by dosing every other day (Molina et al., unpublished). These results showed that D0870 has an exceptional in vivo anti-T.cruzi activity, not previously observed with any other compound. However, a detailed analysis of the in vitro antiproliferative and biochemical effects of this compound showed that, although it was very active (minimal inhibitory concentration against amastigotes in cultured Vero cells of 10 nM, ref. [21]), it was not significantly superior to other azoles such as ketoconazole or itraconazole, which have no curative effects in vivo; this led to the conclusion that the special in vivo antiparasitic activity of the bis-triazole is a composite result of a potent and selective intrinsic anti-T.cruzi activity and particular pharmacokinetic properties (serum half time in mice is 50 h [31] and in human HIV patients ca. 72 h [32]). Work is underway towards clinical trials.

In a continuation of our studies we have investigated SCH 56592, a new triazole currently in development as a systemic antifungal agent [33-35], and found it to be 30-100 times more potent in vitro than ketoconazole or D0870 as antiproliferative agent and sterol biosynthesis inhibitor against epimastigotes and amastigotes of T.cruzi [1]. In the murine model of acute Chagas disease described above this compound given daily at _10 mg/Kg.d for a total of 43 doses allowed 85-100% survival and produced 90-100% cures of the surviving animals [1]. As D0870, SCH 56,592 was found to be highly active in protecting experimental animals form death and inducing parasitological cure in a series of T.cruzi strains, including strains partially and totally resistant to benznidazole (Molina et al., unpublished). It should be noted that this compound also has a relatively long half-time (ca. 24 h) in a series of animal models [36,37], a fact which supports the notion that sustained serum and tissue levels of the drug could be a critical factor for the successful outcome in this type of treatment.

Taken together, the results suggest that some recently developed azole derivatives, with improved biochemical and pharmacokinetic properties, could be useful in the treatment of human Chagas disease.

Work at the author's laboratory was supported by the UNDP/World Bank/World Health Organization Programme for Research and Training in Tropical Diseases (grant 930161), the National Research Council of Venezuela (CONICIT, Grant RP-IV-110034) and the Venezuelan Institute for Scientific Research (IVIC).

References

[1] Urbina, J.A. (1997)Parasitol. 117, In press.

[2] Lyr, H.(1995) Modern Selective Fungicides, Gustav Fisher Verlag, Jena.

[3] Yamaguchi, H., Kobayashi, G.S., and Takahashi, H.(1992) Recent Progress in Antifungal Chemotherapy, Marcel Dekker, New York.

[4] Robson, G.D., Wiebe, M., Kuhn, P.J., and Trinci, A.P.J. (1990) in: Biochemistry of Cell Walls and Membranes in Fungi (Kuhn, P.J., Trinci, A.P.J., Jung, M.J., Goosey, M.W., and Copping, L.G., Eds.) pp.245-281, Springer-Verlag, Berlin .

[5] McCabe, R.E., Remington, J.S., and Araujo, F.G. (1984)J. Infect. Dis. 150,594-601.

[6] McCabe, R.E., Remington, J.S., and Araujo, F.G. (1986)Am. J. Trop. Med. Hyg. 35,280-284.

[7] McCabe, R.E., Remington, J.S., and Araujo, F.J. (1987)Trans. Roy. Soc. Trop. Med. Hyg. 81,613-615.

[8] Raether, W. and Seidenath, H. (1984)Z. Parasitenkunde 70,135-138.

[9] Beach, D.H., Goad, L.J., and Holz, Jr.,G.G. (1986)Biochem. Biophys. Res. Comm. 135,851-856.

[10] Goad, L.J., Berens, R.L., Marr, J.J., Beach, D.H., and Holz, Jr.,G.G. (1989)Mol. Biochem. Parasitol. 32,179-190.

[11] Larralde, G., Vivas, J., and Urbina, J.A. (1988)Acta Cient. Venez. 39,140-146.

[12] Urbina, J.A., Lazardi, K., Aguirre, T., Piras, M.M., and Piras, R. (1988)Antimicrob. Agents Chemother. 32,1237-1242.

[13] Urbina, J.A., Lazardi, K., Aguirre, T., Piras, M.M., and Piras, R. (1991)Antimicrob. Agents Chemother. 35,730-735.

[14] Lazardi, K., Urbina, J.A., and DeSouza, W. (1991)Antimicrob. Agents Chemother. 35,736-740.

[15] Urbina, J.A., Lazardi, K., Marchan, E., Visbal, G., Aguirre, T., Piras, M.M., Piras, R., Maldonado, R.A., Payares, G., and DeSouza, W. (1993)Antimicrob. Agents Chemother. 37,580-591.

[16] Urbina, J.A., Vivas, J., Visbal, G., and Contreras, L.M. (1995)Mol. Biochem. Parasitol. 79,199-210.

[17] Urbina, J.A., Vivas, J., Lazardi, K., Molina, J., Payares, G., Piras, M.M., and Piras, R. (1996)Chemotherapy 42,294-307.

[18] McCabe, R.E. (1988)J. Infect. Dis. 158,1408-1409.

[19] Moreira, A.A.B., DeSouza, H.B.W.T., Amato Neto, V., Matsubara, L., Pinto, P.L.S., Tolezano, J.E., Nunes, E.V., and Okumura, M. (1992)Rev. Inst. Med. Trop. Sao Paulo 34,177-180.

[20] Brener, Z., Cançado, J.R., Galvão, L.M., da Luz, Z.M.P., Filardi, L.d.S., Pereira, M.E.S., Santos, L.M.T., and Cançado, C.B. (1993)Mem. Inst. Oswaldo Cruz 88,149-153.

[21] Urbina, J.A., Payares, G., Molina, J., Sanoja, C., Liendo, A., Lazardi, K., Piras, M.M., Piras, R., Perez, N., Wincker, P., and Ryley, J.F. (1996)Science 273,969-971.

[22] Quintas, L.E.M., de Castro, S.L., Urbina, J.A., Borba-Santos, J.A., Pinto, C.N., Siqueira-Batista, N., and Miranda Filho, N. (1996) in: Molestia de Chagas (Siqueira-Batista, R., Correa, A.D., and Higgins, D.W., Eds.) pp.125-170, Editora Cultura Medica, Rio de Janeiro.

[23] Croft, S.L., Urbina, J.A., and Brun, R. (1997) in: Trypanomiasis and Leishmaniasis (Hide, G., Mottram, J.C., Coombs, G.H., and Holmes, P.H., Eds.) pp.245-257, CAB International, London.

[24] Boyle, F.T., Gilman, D.J., Gravestock, M.B., and Wardleworth, J.M. (1988) in: Antifungal drugs (St.Georgiev, V., Ed.)pp.86-100, Annals of the New York Academy of Sciences, New York.

[25] Ryley, J.F., McGregor, S., and Wilson, R.G. (1988) in: Antifungal drugs (St.Georgiev, V., Ed.)pp.310-328, Annals of the New York Academy of Sciences, New York.

[26] Maldonado, R.A., Molina, J., Payares, G., and Urbina, J.A. (1993)Antimicrob. Agents Chemother. 37,1353-1359.

[27] Wincker, P., Britto, C., Borges-Pereira, J., Cardoso, M.A., Oelemann, W., and Morel, C.M. (1994)Am. J. Trop. Med. Hyg. 51,771-777.

[28] Britto, C., Cardoso, M.A., Wincker, P., and Morel, C.M. (1993)Mem. Inst. Oswaldo Cruz 88,171-172.

[29] Britto, C., Cardoso, M.A., Monteiro Vanni, C.M., Hasslocher-Moreno, A., Xavier, S.S., Oelemann, W., Santoro, A., Pirmez, C., Morel, C.M., and Wincker, P. (1995)Parasitol. 110,241-247.

[30] Molina, J., Araújo, M.S.S., Pereira, M.E.S., Urbina, J.A., and Brener, Z. (1996)Mem. Inst. Oswaldo Cruz 91, Sup. 1,515.

[31] Clemons, K.V., Hanson, L.H., and Stevens, D.A. (1993)Antimicrob. Agents Chemother. 37,1177-1179.

[32] De Wit, S., O'Doherty, E., Smith, R.P., Yates, R., and Clumeck, N. (1995)ICAAC Abstracts 35,F97.

[33] Sugar, A.M. and Liu, X.-P. (1996)Antimicrob. Agents Chemother. 40,1314-1316.

[34] Perfect, J.R., Cox, G.M., Dodge, R.K., and Schell, W.A. (1996)Antimicrob. Agents Chemother. 40,1910-1913.

[35] Pfaller, M.A., Messer, S., and Jones, R.N. (1997)Antimicrob. Agents Chemother. 41,233-235.

[36] Girijavallabhan, V.M., Saksena, A.K, Lovey, R.G., Bennet, F., Pike, R.E., Wang, H., Pinto, P., Liu, Y.T., Patel, N., and Ganguly, A.K. (1995)ICAAC Abstracts 35,F61.

[37] Nomeir, A., Kumari, P., Hilbert, M.J., Loebenberg, D., Cacciapuoti, A., Menzel Jr., J., Moss Jr., E., Hare, R., Miller, G.H., Cayen, M.N., and Lin, C.C. (1995)ICAAC Abstracts 35,F68.

BENZNIDAZOLE IN TREATMENT OF CHILDREN WITH EARLY TRYPANOSOMA CRUZI INFECTION

Ana Lucia S Sgambatti de Andrade

Benznidazole (BZ) a nitroimidazole derivative has been licensed in several South American countries as a trypanosomicidal drug for the treatment of acute and congenital Trypanosoma cruzi infection.The efficacy of the treatment is based on the clearance of parasitemia and negative seroconversion of T.cruzi antibodies. We conducted a Phase III randomized double-blind placebo-controlled field trial, from 1991 to 1995 in na endemic area in Central Brazil (Andrade et al. 1992, Andrade et al. 1996) to evaluate the safety and efficacy of a regular 60-day course of BZ in the treatment of early chronic phase of infection (first years of infection). 129 asymptomatic children aged 7-12 years old with 3 positive conventional serological tests to T.cruzi were randomly assigned to receive either BZ (n=64) or a placebo preparation (n=65), at 7.5mg/kg/day x 60 days. We also performed a highly sensitive and specific chemiluminescent ELISA (Almeida et al. 1995). The primary end-point of the trial was negative seroconversion of specific antibodies by the end of a 3-year follow-up period. The secondary end-point was the reduction of antibodies titres evaluated through repeated serological tests. Full compliance with treatment and follow-up was achieved by 87% of participants. Minor side effects requiring no specific medication were recorded in a small proportion of individuals. Treatment was terminated in only one child due to allergic manifestation. A time trend analysis indicated that T.cruzi antibodies levels measured by indirect immunofluorescence (IIF), hemagglutination and ELISA decreased consistently after treatment in the BZ group. The final IIF geometric mean titres was 5 fold lower in the drug group than in the placebo, 195.8 (95%CI 147—256) versus 1068 (95%CI 809-1408) (p<0.01). A chemiluminescent ELISA with purified trypomastigote glycoconjugate, positive in all subjects at the beginning of the trial, was found to become negative in 63.8% of those individuals in the BZ group. The efficacy of BZ treatment was estimated to be 55.8% (95%CI 40.8-67.0). Although morbidity and mortality evaluations were outside of the trial na amazing result was the detection of 5 incident cases of myocardiopathy, 4 of them in the placebo group, at the 3-year-follow-up, indicating early development of cardiac lesions. A prolonged cohort study from this trial participants (treat and untreated children) would enable to assess the preventive effect of BZ in the progression from infection to disease, mild to severe forms and consequently mortality.

References

1. Andrade ALSS, Zicker F, Luquetti AO, Oliveira RM, Silva SA, Souza JMP, Martelli CMT 1992. Surveillance of Trypanosoma cruzi transmission by serological screening of schoolchildren. Bulletin of the World Health Organization 70(5): 625-629.

2. Andrade ALSS, Zicker F, Oliveira RM, Silva SA, Luquetti AO, Travassos LR, Almeida IC, Andrade SS,Andrade JG, Martelli CMT 1996. Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection. Lancet 348: 1407-1412.

3. Almeida IC, Salles NA, Santos MLP et al 1995. Serum diagnosis of American trypanosomiasis in blood banks: a highly sensitive and specific carbohydrate-rich trypomastigote antigen and why there are so many inconclusive results. Mem Inst Oswaldo Cruz 90: 72-74.

CORRELATION OF MOLECULAR MARKERS AND TRYPANOSOMA CRUZI STRAINS NATURALlY RESISTANT AND NON-RESISTANT TO NITROHETEROCYCLIC DERIVATIVES

Murta, S.M.F. ^a,c, Gazzinelli R.T. ^b,c, Brener Z. ^b & Romanha A.J.^a

^a Laboratório de Parasitologia Celular e Molecular ^b Laboratório de Doença de Chagas, Centro de Pesquisas "René Rachou" - FIOCRUZ, Caixa Postal 1743, CEP 30190-002, B.H., MG-Brazil,^c Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, UFMG, B.H., MG, Brazil- Email: [e-mail]

Differences in the susceptibility of Trypanosoma cruzi strains to drugs [1-3] may explain in part, differences in drug efficiency in treatment of vertebrate host infected with T. cruzi. Several authors have demonstrated that geographical differences might interfere in the efficiency of therapeutic regimens. Patients from Chile and Argentina and Southern Brazil (State of Rio Grande do Sul), treated with nifurtimox showed more than 80% of cure. In contrast, only 40% of cases treated with the same drug in the states of São Paulo, Minas Gerais, Bahia and Goiás-Brazil, were cured [4]. Although association of drug susceptibility with geographic areas has been reported, no parasite genetic correlation with drug susceptibility has been established yet.

Enzyme electrophoresis studies have demonstrated distinct T. cruzi populations (zymodemes) circulating in the domestic and sylvatic transmission cycles, providing therefore a good epidemiological marker for Chagas'disease [5]. More recently, kDNA (schizodemes)[6], DNA fingerprinting [7] and randomly amplified polymorphic DNA (RAPD) [8,9] have been used in studies of genetic diversity of trypanosomes and defined highly variable groups of T. cruzi. In addition, 2 independent nuclear markers, ribosomal RNA and mini-exon genes, allowed the division of T. cruzi strains into two major phylogenetic lineages [10].

In the present study, twenty seven Trypanosoma cruzi strains naturally resistant and non-resistant to nitroheterocyclic derivatives, benznidazol and nifurtimox, were analyzed for different molecular markers as follow: 1) isoenzymatic profiles of 6 different enzymes ASAT, ALAT, GPI, PGM, ME and G6PD; 2) genetic variability by Randomly Amplified Polymorphic DNA (RAPD) profiles using 2 different primers; 3) a polymorphic segment of T. cruzi Tcpgp gene that codifies for a membrane phosphoglycoprotein, associated with drug resistance in Leishmania, Plasmodium and Entamoeba; 4) a polymorphic segment of T. cruzi gene, that codifies the hypoxantine-guanine phosphoribosyl transferase (HGPRT) enzyme [11], a rational target for Chagas'disease therapy; 5) a T. cruzi DNA that codifies for ribosomal RNA and 6) a T. cruzi DNA that codifies for mini-exon gene. Of the 6 molecular markers, RAPD and isoenzyme profiles divided the T. cruzi strains into 3 groups, whereas the other 4 markers allow us to divide the T. cruzi strains in 2 groups. In RAPD groups I and II or isoenzyme zymodemes Z1 and Z2, respectively, were indistinctly found naturally resistant and non-resistant strains. In contrast, in RAPD group III or the zymodeme B, the heterozygote profile, were only found drug non-resistant strains. All strains from the latter group, except one (MR), were polymorphic for HGPRT and Tcpgp genes, confirming their heterozygote genotype. No correlation was observed between drug susceptibility and dimorphism of ribosomal RNA and mini-exon genes.

In the genetic variability study, the strains were clustered according to the isoenzymatic and RAPD profiles rather than drug susceptibility. In order to analyze if drug susceptibility is more related to geographic origin than T. cruzi zymodeme, 18 T. cruzi strains Z1, Z2 and ZB from two different regions of Brazil were studied. The in vivo susceptibility of T. cruzi strains to benznidazole was evaluated by rapid treatment. Ten T. cruzi strains from zymodeme B, eight from Rio Grande do Sul and two from Minas Gerais, were sensitive to benznidazole. Whereas the other eight strains Z1 and Z2, were of medium resistance and sensitive to benznidazole independently of the State origin. Altogether, the relationship between drug susceptibility and zymodeme profile of 45 T. cruzi strains, 27 reported in [3] and 18 by us, showed that all 19 T. cruzi strains zymodeme B from different geographical origins (3 from Minas Gerais, 1 from Goiás, and 12 from Rio Grande do Sul Brazil and 3 from Argentina) were highly sensitive to treatment. The high prevalence of heterozygous T. cruzi strains, in South Brazil [12], Argentina [13] and Chile [14] coincides with the areas where the chemotherapy for human Chagas'disease has been reported as more effective. Further suggesting that heterozygous T. cruzi strains are associated with drug-sensitive phenotype.

Supported by CNPq, PAPES/ FIOCRUZ and PRONEX

References

1) Haberkorn, A. and Gonnert, R. (1972). Arzneimittel-Forschung 22, 1570-1580.

2) Andrade, S., Magalhães, J.B. and Pontes, A. L. (1985). Bull. World Heath Organiz. 63, 721-726

3) Filardi, L. S. and Brener, Z. (1987). Trans R Soc Trop Med Hyg 81, 755-759.

4) Rassi A. and Luquetti A. O. (1992). In: Chagas disease (American Trypanosomiasis): its impact on transfusion and clinical medicine (ed. Wendel S., Brener Z., Camargo M.E. and Rassi A.), pp. 237-247. ISBT Brazil"92 São Paulo, Brazil.

5) Miles MA, Souza AA, Povoa MM, Shaw JJ, Lainson R, Toyé PJ (1978). Nature 272: 819-821.

6) Morel CM, Chiari E, Camargo EP, Mattei DM, Romanha AJ, Simpson L (1980). Proc Natl Acad Sci USA 77: 6810-6814.

7) Macedo AM, Martins MS, Chiari E, Pena SDJ (1992). Mol Biochem Parasitol 55: 147-154.

8) Steindel M, Dias Neto E, Menezes CLP, Romanha JA, Simpson AJG (1993). Mol Biochem Parasitol 60: 71-80.

9) Tibayrenc M, Neubauer K, Barnabé C, Guerrini F, Skarecky D, Ayala F (1993). Proc Natl Acad Sci USA 90: 1335-1339.

10) Souto R., Fernandes O., Macedo A., Campbell D. and Zingales B (1996). Mol. Biochem. Parasitol. 83, 141-152.

11) Allen T. and Ullman B. (1994). Mol. Biochem. Parasitol. 65, 233-245.

12) Fernandes C.D et al. (1997). Mem. Inst. Oswaldo Cruz 92, 343-351.

13) Montamat E, D'Oro G., Gallerano R., Sosa R. and Blanco A. (1996). Am. J. Trop. Med. Hyg. 55, 625-628.

14) Apt W., Aguilera X., Arribada A., Gomez L., Miles M. and Widmer G. (1987). Am. J. Trop. Med. Hyg. 37, 302-307.

MOLECULAR RECOGNITION OF B-DNA MINOR-GROOVE BINDERS. DRUG DESIGN OF ANTILEISHMANIASIS COMPOUNDS

Montanari, C. A.

Núcleo de Estudos em Química Medicinal-NEQUIM, Departamento de Química/ICEx/UFMG, Campus da Pampulha, 31270-901 - Belo Horizonte - MG - Brazil email: [e-mail]

The aromatic bis(amidine) compound pentamidine is currently in widespread clinical use for the treatment of PCP in AIDS patients. Its analogues are effective against a number of microbial infections including Trypanosoma rhodesience, Giardia lamblia, Cryptosporidium parvum, and also against leishmaniasis. Pentamidine is still being used against antimony-resistant leishmaniasis. Pentaminide analogues have DNA binding affinity, as estimated by stabilisation of the DNA helix to coil thermal denaturation transition (T_m). Footprinting studies have also shown that the molecule binds to AT-rich regions of duplex DNA. Thus, the mode of action for these dicationic molecules has been linked to their selective binding to the minor-groove of DNA at AT rich sites and their ability to selectively interfere with the normal functioning of the parasite topoisomerases. The X-ray crystal structure of pentamidine bound to the DNA sequence d(CGCGAATTCGCG)₂ has been determined and shows binding within the AT-rich region of the DNA minor groove. The interest in the rational drug design and synthesis of sequence selective or specific DNA binding agents have been continuously sought. There is a great goal in designing novel ligands for biologically important receptors based primarily on knowledge of the three-dimensional structure of the receptor. This structure-based ligand design has the potential to greatly increase the number of lead compounds discovered for the development of new therapeutic agents. This paradigm is cyclical in nature, and the information on the receptor is used to design putative ligands which are synthesised and then evaluated for binding to the targeted receptor. By the comparison of predicted versus observed binding properties, rationally selected ligand modifications can then be employed in subsequent designing cycles to improve both the design methodology and the binding affinity and selectivity of its resulting ligands.

This study examines the combined approach to structure-based-3D QSAR ligand design in deriving new drug molecules that would be better binders than pentamidine itself, mainly those with constrained structures.

How molecules interact with the minor groove of the nucleic acids has intrigued chemists and biologists. They can be summarised as follows: (i) genome regulatory proteins show affinity for non-specific sites; (ii) this non-specific binding is largely 'electrostatic' in nature; (iii) specific binds involves the interaction of a matrix of DNA hydrogen bond donors and acceptors, located in the groove; (iv) close van der Waals contact. Small molecules primarily bind in the narrow minor groove of B-DNA whereas larger molecules such as proteins utilise the wide major groove. The extensively studied antitumor antibiotics netropsin and distamycin bind to the minor groove of DNA at AT sequences. These molecules can be regarded as being assembled with cationic ends responsible for electrostatics and NH groups resulting in hydrogen bonding that would be needed for binding though not being essential. The electrostatic attraction of doubly charged ends reads the AT sequence which is the deepest negative potential wells in the minor groove of DNA. Nevertheless, we have shown that for a set a 37 bisamidines active against Leishmania mexicana amazonensis the QSAR model does include the minimal electrostatic potential as being important for binding to AT sequence of B-DNA. The replacement of the pyrrole rings with imidazole moieties alters the recognition from AT to GC base pairs. By examining the molecular models of a number of aryldiamidines it seems that they fall into a particular spatial classes.

The QSAR obtained using the similarity index generated for bisamidine moieties takes into account differences in conformation of the reference structure in the receptor site - the pharmacophoric conformation. The biological data set of 37 bisamidine analogues against Leishmania mexicana amazonensis are uncovered by the following structural variations: the amidine group is ortho or para, the chain length is (CH₂)_n where n = 2-6, the bioisosteric substitution of -O- by -NH-, inclusion of ortho-substituents and substitution of amidines by dihydroimidazoles. The most active compound has a -NH- bioisostere with six methylene groups and the least active has a nitro group ortho to the ether moiety and two methylene groups.

Supported by CNPq, FAPEMIG and FINEP.

RT06 - ^PROTOZOOLOGY

MICROSPORIDIA AND HUMAN MICROSPORIDIOSIS

Hércules Moura & Alexandre J. DaSilva

Departamento de Parasitologia, FCM, UERJ, Rio de Janeiro, & 2Centers for Disease Control and Prevention, Atlanta, Ga., USA

Microsporidia is the term used to refer to emerging unicellular eukaryotic parasites that are obligatorily intracellular and belong to the order Microsporida of the phylum Microspora. Mcrosporidia infect practically all groups of animais, including humans and other protozoans. More than 100 genera with over 1,000 species of núcrosporidia have been described. To date, seven species, classified to six genera (Enc~tozoon, Enterocytozoon, Pleistophora, Trachipleistophora , Nosema and Vittaforma) have been identified as human pathogens (l6). Nficrosporidia are increasingly recognized as important opportunistic pathogens predominantly, but not exclusively, in persons with AIDS and cause gastrointestinal (GI) as well as disseminated microsporidiosis (DM). GI disease accounts for 30% of diarrhea in patients with AIDS.

Of the several microsporidia that infect humans only Enterocytozoon bieneusi is known to cause strictly GI disease, whereas Enc~tozoon intestinalis (formerly Septata intestinalis) causes a GI as well as a DM disease involving the lungs, kidneys, and eyes (13). The other two species in the Encephalitozoon genus, Encephalitozoon cuniculi and Enc~tozoon hellem have been reported as agents of disseminated infections involving the urinary tract, respiratory tract, the eyes and the central nervous system, whereas Vittaforma and Nosema spp. are believed to be agents of keratitis and thus confined to the eye (12, 14). The genus Trachipleistophora has been identified as agents of myositis in AIDS patients. ln addition, with improved diagnostics and wider interests, there are increasing reports of microsporidial infections in immunocompetent persons (16).

Human microsporidiosis constitute a group of rapidly emerging parasitic diseases for which very little is known regarding risks for infection or modes of transmission. There are evidences for person-to-person transmission of spores either by fecal-oral contaifflnation or via aerosolized respiratory secretions, besides the possibility of transn-ússion by sexual means or transplacental as occur among many mammalian hosts including rabbits, dogs and squirrel monkeys. The possible role of an animal reservoir was not yet detemúned for the núcrosporidia species which infect humans (16).

Nficrosporidial spores are characteristic refringent ovoid structures with 1.0x1.5 mm for E bieneusi and l.0x2.5 mm for Encephalitozoon. They present a unique coiled polar filament through which infective sporoplasm is injected into the host cell. Electron n-úcroscopic analysis revealed a thin extemal membrane, the exospore, and a thick internal membrane, the endospore. No mithocondria or Golgi membranes have been detected within the microsporidial spores.

Diagnosis of human núcrosporidiosis is dependent upon the identification of spores ín clinical samples, e.g. stools, urine, conjunctival smears, and nasopharingeal fluids. The detection of spores in biologic samples, however, is a laborious, challenging, and time-consuming task-, these tiny organisms can easily be niissed, even by trained núcroscopists (8). During the past few years procedures for concentration of spores in stools and their visualization by staining have been descríbed (11). Among the procedures reported to detect niicrosporidial spores in clinical samples were Giemsa, toluidine blue, Gram, periodic acid Schiff stains, chromotrope 2R and modifications thereof. Chemofluorescent agents and polyclonal and monocional antibodies have also been used for spore detection. The modified trichrome stain of Weber et al. (15) provided a breakthrough for the differential staining of spores which made it easier for the detection of rácrosporidial spores in stools and body fluids, thus beconúng the standard staining technique for diagnosis. Grarn-chromotrope staining of rácrosporidial spores is being used in several laboratories with good results, as it is a rapid diagnostic procedure which combines the properties of the Gram staining with those of Weber's chromotrope (8, 10).

Moiecular diagnosis is very useful for the diagnosis of microsporidial infections. DNA amplification by using polimerase chain reaction (PCR) has been widely used for detection and identification of núcrosporidia at species level in clinical samples, including stools. PCR using primers designed on the basis of the small subunit RRNA has proved to be very reliable for this purpose (4, 5, 6). Xficrosporidia species identification can be performed using electron n-úcroscopy (EM), but for those species not distinguishable by morphology (e.g. E. cuniculi and E. hellem) the use of non-morphologic analysis such as protein profiles and DNA probes is recomendei.

The first report of human microsporidiosis in Brazil occurred in 1993 when microsporidial spores were found in stools of two persons with chronic diarrhea in Rio de Janeiro (1,2,7). These parasites have been detected later in people from several states in Brazil (Ceará, Pemambuco, Rio de Janeiro and São Paulo). As is observed in different parts of the world, GI is the most common clinical presentation of n-úcrosporidiosis in MV positive persons but in one occasion microsporidial spores have been found in stools of a non-FHV child with posttransplant immunosuppression with chronic diarrhea (9). There are two case reports of ocular microsporidiosis from São Paulo and Rio de Janeiro (1,3). ln Rio de Janeiro niicrosporidial spores have been detected in clinical samples such as stools, urine, bile, duodenal lavage and tissue, bronco-alveolar nasal sinus lavages and corneal scrapings from several different patients. Mcrosporidia strains have been isolated from the urine of 4 Brazilian patients and are currently being studied. E. bieneusi spores detected in the bile of a Brazilian patient with intestinal microsporidiosis had been used to design specific PCR diagnostic primers. Mcrosporidiosis seems to be a burgeoning problem in Brazil and further studies are warranted.

References

1- Brasil, P.- Lima, D.B. & Moura, H. 1997. Nficrosporidiose humana na síndrome de imunodeficiência adquirida. Rev.Ass.MédBrasil. 43(3):254-64.

2- Brasil, P.; Sodré, F.C.; Cuzzi-Maya, T.; Gutierrez, M.C.G.F.S., Mattos, H. & Moura, H. 1997. Intestinal microsporidiosis in MV-positive patients with chronic unexplained diarrhea in Rio de Janeiro, Brazil: diagnosis, clinical presentation and follow-up. Rev.Inst.Med. Trop.Sío Paulo 38(2):97-102.

3- Benchimol, E.; Moura, H.; Sued, M.; Gonçalves Neto, C.R.P.; Coelho, J.M.C.O. & Serapião, M.J. 1993. Retrospective identification of n-úcrosporidial keratoconjunctivitis in a patient with AIDS in Rio de Janeiro, Brazil (abstract). Folia Parasitologica (,Praha) 40:250.

4- da Silva AJ, Schwartz DA, Visvesvara GS, Moura H, Slemenda SB, Pieniazek NJ. 1996. Sensitive PCR diagnosis of infections by EnteroWozoon bieneusi (Mcrosporidia) using primers based on the region coding for small-subunit RRNA. i CliíL MicrobioL 34:986987. S- da Silva AJ, Slemenda SB, Visvesvara GS, Schwartz DA, Wilcox CM, Wallace S, Pieniazek NJ. 1997a. Detection of Septata intestinalis (Nficrosporidia) Cali et al. 1993 Using polymerase chain reaction primers targeting the sanúl subunit ribosomal RNA coding region. Molecular Diagnosis 2: 47-5 1.

6- da Silva A J, Bornay-Llinares FJ, dei Aguila de Ia Puente C, Moura R Peralta JM, Sobottka I, Schwartz DA, Visvesvara GS, Slemenda SB, Pieniazek NJ. Diagnosis of Enterocytozoon bieneusi (Nficrosporidia) infections by PCR in Stool Samples Using Primers Based on the Region Coding for Small Subunit Ribosomal RNA. Arch Pathol Lab Med. 1997, in press.

7- Moura, H.; Cardoso, R. R.; Veloso, V. G.; Cavalcante, S. C. & Visvesvara, G. S. Nficrosporidia e diarréia crônica em AIDS: identificação de Enterocytozoon em 2 pacientes no Rio de Janeiro. XXIX Congresso da Sociedade Brasileira de Medicina Tropical. Fortaleza, Ceará: SBMT-Resumos, março, 1993. Page 180.

8- Moura, H.; DaSilva, J.L.; Sodré, F.C.; Brasil, P.; Wallmo, K,; Wahlquist, S.; Wallace, S.;Croppo, G.P. & Visvesvara, G. S. 1996. Gram-chromotrope: a new technique that enhances detection of microsporidial spores in clinical samples. J. Eukar. MicrobioL 43:94S-95S.

9- Moura, H.; lfirschfeld, M.P.; Brasil, P.; Sodré, F.C. & Palhares, M.C.A. 1993. Nficrosporidiosis among AIDS patients in Brazil: prelinúnary results (abstract). Folia Parasitologica (,Praha) 40:25 1.

10- Moura, H.; Schwartz, D.A.- Bornay-Linnares, F.; Sodré, F.C.; Wallace, S. & Visvesvara, G. S. A new and improved "quick-hot Gram-chromotrope" technique that differentially stains núcrosporidia spores in clinical samples including paraffin-embedded tissue sections. Arch Pathol Lab Med. 1997, in press.

11- Sodré, F.C.; Borges, A.L.V.; Brasil, P.; Cunha, R.C.C. & Moura, H. 1995. Descrição de um método modificado para a concentração de esporos nas fezes para o diagnóstico das microsporidioses intestinais. JBras.PatoL 31(4):126-133.

12- Visvesvara GS, Leitch GJ, DaSilva AJ, Croppo GP, Moura R Wallace S, Schwartz DA, Moss DM, Bryan RT, Pieniazek NJ, 1994. Polyclonal and monoclonal antíbody and polymerase chain reaction amplified small subunit ribosomal RNA identification of the núcrosporidian, Encephalitozoon hellem, from a case of disseminated infection in a patient with AIDS. J. Clin.Microbio. Lab. (11):2760-2768.

13- Visvesvara GS, Leitch GJ, DaSilva AJ, Croppo GP, Pieniazek NJ, Leitch G, Ferguson D, Moura H, Wallace S, Slemenda SB, Tyrrel I, Moore DF, Meador J. 1995. In vitro culture and serologic, and molecular identification of Septata intestinalis isolated from the urine of a patient with AIDS. J.Clin.MicrobioL 13(4):930-936.

14- Visvesvara, G. S.; Leitch, G. J.; Moura, H.; Wallace, S. Weber, R. & Bryan, R. T 199 1. Culture, electron núcroscopy, and immunoblot studies on a microsporidian parasite isolated from the urine of a patient with AID S. J. ProtozooL 38(6):105 S- I I I S.

15- Weber R, Bryan RT, Owen RL, Wilcox CM, Gorelkin L, Visvesvara GS. 1992. Improved light-núcroscopical detection of microsporidia spores in stool and duodenal aspirares. N. EngLJ.Med. 326:161-166.

16- Weber R, Bryan RT , Schwartz DA, Owen RL. Human niicrosporidian infections. 1994. Clin MicrobioL Rev.7:426-461.

CYCLOSPORA CAYETANENSIS: MOLECULAR DIAGNOSLS BY PCR AND TAXONORM'C ASPECTS BASED ON SSU-RRNA CODING REGLON

^l Alexandre J. da Silva & ²Hércules Moura

'Centers for Disease Control and Prevention, Atlanta, Ga., USA - & ²Departamento de Parasitologia, FCM, UERJ, Rio de Janeiro

Cyclospora cayetanensis is an emerging and opportunistic coccidian previously referred to as either "cyanobacterium-like body" or big Cryptosporidium. This parasite had not been fully classified until 1993 when Ortega et al. (I), on the basis of morphologic aspects and sporulation characteristics included it in the genus Cyclospora. Later on, Ortega et al. 1994 (2), created a new taxon, Cyclospora cayetanensis, for this parasite, based on studies of a Peruvian isolate. Very little is known regarding epidemiology, pathogenic aspects and possible existente of reservoirs for this coccidian. ln 1996, different outbreaks of diarrhea due to Cyclospora infection took place in the US and Canada. These were linked to the consumption of raspberries imported from Guatemala (3). At least one isolated case of human cyclosporiasis was reported in Brazil, Rio de Janeiro in MVinfected patient, which had no intestinal symptom (4). Reports in the literature describing this parasite in nonhuman hosts are scarce but there are ate least two where Cyclospora oocysts were identified in stools of primates as baboons and chimpanzees (5, 6). There is also an indication that Cyclospora may be found in birds in Brazil, as it was reported in swans of the lbirapuera Park in Sao Paulo (Dr. Mariza Hirrshfeld, personal communication).

Routine diagnosis of infections due to Cyclospora is based on microscopic observation of oocysts in stool smears stained by the modified Kinynoun's acid-fast stain (7). Recently, a modification of the safranin procedere by Baxby et al. (8) has been applied successfully to stain Cyclospora oocysts (9). The uniform staining obtained by this procedere facilitares the identification of the parasite in the smears and may consequently increase the sensitivity of the detection.

Application of PCR for the detectou of Cyclospora in stools is linúted, and to date, only one of such an application was reported in the literature (I O). ln this study, only 62% of the positive samples analyzed by n-úcroscopy could be confirmei by using this approach. Nevertheless, no false-positive result was generated by PCR amplification. Sensitivity of the PCR could be circumstantially related to few points. First, the optimum extraction of the parasite DNA from stool samples is crucial to generate high sensitivity. lf extraction is not efficiently performed, samples may either carry PCR inhibitors or have low arnount of the template (I I). To date, the methods describing DNA extraction from parasites in stools, are not suitable enough to provide I OO% of confidence regarding yield of DNA free of PCR inhibitors.

Another point to be considered, is the possible existence of more than one species of Cyclospora that may be associated to the cause of human disease. Different species may show variation in different regions of SSU-RRNA coding regions. ln this way, the set of primers that were used for PCR detection may fail in amplifying the template, if genetic variability among species is observed at the level of the target sequence.

Phylogenetic analysis of Cyclospora cayetanensis based on the SSU-RRNA coding region was performed by different authors and results have shown that this parasite is closely related to genus Eimeria (l2, 13). These results are important since analogies can be depicted in order to develop culture systems and to establish animal models that may apply to the isolation of Cyclospora. ln addition, such investigations will contribute to clarify aspects of the life cycle of this parasite, as well as be of invaluable importance for epidemiological studies. Finally, these future efforts may reveal sources of infection and potential reservoirs, which is an important task towards the creation of prophylaxis measures to control the transnússion of cyclosporiasis among risk population.

References

1-Ortega YR, Sterling CR, Gilman RH, Cama VA, Diaz F. Cyclospora species- a new protozoan pathogen of humans. N Engl J Med 1993;328:1308-12.

2-Ortega YR, Gilman M Sterling CR. A new coccidian parasite (Apicomplexa:Eimeriidae) from humans. J Parasitol 1994;80:625-9.

3-Herwaldt BL, Ackers ML, and the Cyclospora working group. An outbreak in 1996 of cyclosporiasis associated with imported raspberries. N Engl J Med 1997;336:1548-1556.

4-Schubach TM, Neves ES, Leite AC, Araujo AQC, Moura H. Cyclospora cayetanensis in a assymptomatic patient infected with HIV and HTLV- 1. Trans R Soc Trop Med and Hyg1997;91:175.

5-Ashford RW, Warhust DC, Reid GDF. Human infection with cyanobacterium-like bodies. Lancet 1993;341:1034.

6- Smith HV, Paton CA, Girdwood RWA, Mtambo MMA. Cyclospora in non-human primates in Gombe, Tanzania. The Veterinary Record 1996;138:528.

7-Ash LR, Orihel TC. Collection and preservation of faeces, p. 5-53. ln Parasites: a guide to laboratory procedures and identification, I" ed. American Society of Clinical Pathologists Press, Chicago 111.

8-Baxby D, Blundell N and Hart CA. The development and perfortnance of a simple, sensitiva method for the detection of Cryptosporidium oocysts in faeces. J. Hyg. Camb.

9-Visvevara GS, Moura H, Kovacs-Nace E, Wallace S, Eberhard ML. Unifonn staining of Cyclospora oocysts in fecal smears by a modified safranin technique with núcrowave heating. J Clin Xficrobiology 1997-730-733.

10-Pieniazek NJ, Slemenda SB, da Silva AJ, Alfano EM & Arrowod MJ. PCR confinnation of infection with Cyclospora cayetanensis. Emerging Infectious Disease 1996;2:357-359.

11 - da Silva AJ, Bomay-Llinares FJ, del Aguila de Ia Puente C, Schwartz DA, Visvesvara GS, Slemenda SB & Pieniazek NJ. Diagnosis of Enterocytozoon bieneusi (Mcrosporidia) infections by PCR in unfixed and fixed stool samples using primers based on the region coding for small subunit ribosomal RNA. Acrh Patol Lab Med 1997; in press.

12-Relman DA, Schdmidt TM, Gajadhar A, Sogin M, Cross J, Yoder K, Omtipa S, Echeverria P. Molecular phylogentic analysis of Cyclospora, the human intestinal pathogen suggests that it is closely related to Eimeria species. J Infect Dis 1996;173:440445.

13-Pieniazek NJ, Herwaldt BL. Reevaluating the molecular taxonomy: is humanassociated Cyclospora a mammalian Eimeria species? Em Infect Dis 1997; 3:1-3.

HUMAN INFECTION BY CRYPTOSPORIDIUM PARVUM

Pedro Paulo Chieffi - Instituto de Medicina Tropical de São Paulo (LIM 06)

Protozoans belonging to the genus Cryptosporidium are parasites that grow and reproduce within epithelial cells of the digestive and respiratory tracts of several vertebrate species (4). The first publlshed descripúon of Cryptosporidium infecfion occurred in 1907, when Tyzzer found this protozoan in the gastric glands of mice maintained in laboratory conditions. Only in 1976 the occurrence of hwnan infection by Cryptosporidium was reported for the first time (i i).

However, firom the beginning of the eighties on, infection by Cryptosporidium parvum lias been recognized as a frequent cause of enten'c alterations, and less commonly extra-intestinal dlsease in immunosuppressed patients, mainly those who have AIDS, with several grades of severity, depending on the level of their immune system ínvolvement (l3). Cryptosporidiosls lias been observed as well in non-immunosuppressed individuais, with a self-limited clinlcal evolution (l2).

After the development of coprological diagnostic methods with good sensitivity for the diagnosis of Cryptosporidium infecfions (I,8) it was realized that cryptosporidiosis should be a very firequent infection in children with diarrhoea (2) and, sometimes, there are even non-diarrhoeal patients shedding Cryptosporidium oocysts in the stool.

The main sources of human cryptosporidian infection are the consumpfion of food and water contanu'nated by Cryptosporidium parvum oocysts, as well as the contact with either human beings or animais infected by this coccidian.

As Cryptosporidium oocysts are reslstant to drugs commonly used ln water disinfecfion, the conventional chlorination of water supplles is not a safe method to avold human infection by thls coccldlan. ln some occaslons the contammation of water supply systems by Cryptosporidium pane~ oocysts had been resulted in epldemic episodes of cryptosporidiosls. The most important waterbome outbreak of human cryptospon'diosis occurred, in 1993, in Milwaukee (USA), affecting over 400.000 persons (g).

One of the most common modes of Cryptosporidium parvum transmission envolves person-to-person contact. So, this kind of transmission ls the principal responsible for the occurrence of children's cryptosporidiosis in day-care centers, for infection ainong sexual partners and in intranosocomlal transmission (6).

Cryptosporidiosis shows worldwide distribution and has been diagnosed at higher prevalence rates in developing countries (2). ln Brm'l it lias frequently been found in AIDS patients (3) and in other immunosuppressed 'mdividuals (5). On the other hand, it lias also been diagnosed in non-immunosuppressed diarrhoeal children (io) and, less commonly, in paúents with non-diaithoeal faeces (7).

Currently there is no effective therapy for cryptosporidial infeCfiOn. lmmunocompetent patients show a self-limited dlsease but, sometimes, they need to be submitted to rehydration at home or even hospitalizei. ln immunosuppressed patients, depending on the level of thelr lnimune system compromis'mg, cryptosporidiosls may be a life-threatening challenge. ln this lánd of patients many drugs have been tested, however, only a few ones (spiramycin or paromomyc'm) showed some results in controlling the watery diarrhea during a short period of time.

References

1.Bronsdon, M. A - Rapid dimethylsulfoxide modified acid-fast stain of Cryptosporidium oocysts in stool specimens. J. Clin. Microbiol., 19: 952-953, 1984.

2.Current, W.L. & Garcia, L.S. - Cryptosporidiosis. J. Infect. Dis., 160: 325-358, 1991.

3.Dias, R.M.D.S. et ai. - Cryptosporidiosis among patients with acquired immunodefíciency syndrome (AIDS) in the county of São Paulo, Brazil. Rev. Inst. Med. Trop. S.Paulo, 30: 310-312, 1988.

4. Fayer, R. & Ungar, B.L.P. - Cryptosporidium spp. and cryptosporidiosis. Microbiol. Rev., SO: 458-483, 1986.

5. Jabur, P. et al. - Criptosporidiose e outras enteroparasitoses em pacientes submetidos a transplante renal ou hemodiálise. J. brasil. Nefrol., 18: 239-242, 1996.

6. Juranek, D.D. - Cryptosporidiosis: sources of infection and guidelines for prevention. Clin. Infect. Dis., 2l(Suppl. I): 557-561, 1995.

7.Loureiro, E.C.B.- Linhares, A C. & Mata, L. - Acute diarrhoea associated with Cryptosporidium sp. ln Belém, Brazil. Rev. Inst. Med. trop. S.Paulo, 28: 138-140, 1986.

8.Ma, P. & Soave, R. - Three-step stool examination for cryptosporidiosis in 10 homosexual men with protated watery diarrhea. J. Infect. Dis., 147: 824-828, 1983. 9. Mac Kenzie, W.R. et al. - A massive outbreak in Nfilwaukee of Cryptosporidium infection transnútted through the public water supply. N. Engl. J. Med., 331: 161-167,1994.

10.Mangini, A C.S. et al. - Parasitismo por Cryptosporidium sp. em crianças com diarréia aguda. Rev. Inst. Med. trop. S.Paulo, 34: 341-345, 1992.

11. Nime, F.A et al. - Acute enterocolitis in a human being infected the protozoan Cryptosporidium. Gastroenterology, 70: 592-598, 1976.

12.O'Donoghue, P.J. - Cryptosporidium and cryptosporidiosis in man and animais. lnt. J.Parasit., 25: 139-195, 1995.

13.Ungar, B.L.P. Cryptosporidiosis in humans (Homo sapiens). ln: Dubey, J.P.; Speer, C.A & Fayer, R. (eds.) Cryptosporidiosis of man and animais. Boston, CRC Press, p. 59-82, 1990.

TOXOPLASMOSIS – RECENT APPROACH

Maria Regina Reis Amendoeira

Departamento de Protozoologia-Instituto Oswaldo Cruz- FIOCRUZ , Av. Brasil, 4365 Manguinhos, Rio de Janeiro, Brasil.s

Toxoplasrnosis is a zoonosis of worldwide distribution caused by Toxoplasma gondii , an obligate intracellular coccidian that has an extremely wide host range and that can survive in all nucleated cells of almost all warm-blooded animal species, including humans. The parasite was discovered in the wild rodent Ctenodactylzís gondii (Nicolle and Manceaux, 1908) and in Brazil in a laboratory rabbit (Splendore, 1908) .

Until 1970 , when its life cycle was elucidated (Frenkel et al.,Science,l67:893-896,1970, Hutchison et al., Trans.R.Soc.Trop.Med.Hyg.,65: 380399,1971), the mode of transmission of Tgondii remained unclear. Since that time diverse transmission mechanisms have been discovered (Amendoeira, An. Acad. Nac. Med.;155(4): 224-225, 1995) . ln humans, for example , the protozoa is generally acquired by ingestion of raw or undercooked meat contaminated by parasite cysts (pork, lamband, beef) , raw vegetables or water contarninated by oocysts from infected cat feces (the definitive hosts) , and by transplacental passage of parasite from mothers primarily infected during pregnancy (congenital toxoplasmosis). On the other hand, the possibilities for inter-human direct transmission from tachyzoites have been put forward. Chathie, (Latncet, 266: 813-814,1954) found the parasite in the saliva during the acute phase of infections, sometimes without any clinical signs (Amendoeira & Coutinho, J Infect. Dis.,145:187, 1982).

Congenital toxoplasmosis may cause fetal death or result in a grave disease in the infant causing cerebral damage and retinochoroiditis (Desmonts & Couvreur, Bull. N. Y Acad. Med, 50: 146-59, 1974). ln France, where Toxoplasma serologic screening during pregnancy is mandated by law, the risk of infection is 6 - 10 per I 000 pregnancies (Pinon et al., Rev. Int. Pediatr., 157: II-16, 1986), on the hand in the Unites States it ranges from I per I 000 to I per 8000 live births (Wong & Remington, Clin. Infect. Dis., 18: 853-862,1994). For other countries , like Brazil, in the absence of systematic serologic screening, there are insufficient data to allow determination of the incidence of Toxoplasma infection during pregnancy. The tiniing in the pregnancy period in which the woman has contact with the parasite is very important for the pathogenicity of infection.

Infection of immunocompetent persons is usually asymptomatic, although in some cases the acute infection is accompanied by cervical lymphadenopathy with moderate fever (37,0 - 37,5'C). During the chronic phase of toxoplasmosis, Toxoplasma tissue cysts are well controlled by the host immune system which is continually stimulated by parasite antigens. This leads to the acquisition of definitive protective immunity against reinfection, however, there ls evidence of possible reinfection with L gondii in children (Coutinho et al, J Infect. Dis., 146 (I)-. 1982). But when the immune system is deeply affected, reactivation of quiescent encysted parasites occurs with intense proliferation of tachyzoites. This is observed in patients with malignant hemopathies with immunosuppressive therapy for cancer and in graft recipients ( mainly in the case of chronically infected donors).

Since the early 198Os with the outbreak of human HIV infection the cases of life-threatening toxoplasmosis have increased (Holliman, J Infect., 16: 121-128, 1988). Actually, toxoplasmosis is the most common opportunistic pathogen causing encephalitis or focal intracerebral lesions in AIDS patients (Remington, Clin. Infect. Dis 15:211-222, 1992).

Generally, the laboratory diagnosis of toxoplasmosis is based on the detection of specific immunoglobulins of the lgG and lgM classes . ln infected humans , specific IgM is usually first detected. lgG antibodies appear later and its level decreases gradually to low titer , which persists for life. However, the use of sensitive methods to detect IgM antibodies considerei to be characteristic of the acute phase of toxoplasmosis , can now be detected by assays for extended periods of time after infection ( months to years ) .

Detection of specific lgA antibodies can be evidenced in early infection and they disappear earlier than lgM antibodies. The simultaneous detection of lgm and lgA points out the acute phase of toxoplasmosis and allows a better evaluation of risk in pregnant women . The specific lgE antibodies are demonstrable in humans and other animais. Those antibodies could also be considerei good markers for acute toxoplasmosis because lgE appears in early infection and is transient ( Camargo, Am . Acad Nac Med, 155 (4) : 236-239 , 1995) The classic immunological methods - enzyme linked immunosorbent assay (ELISA), indirect immunofluorescence test and passive hemaglutination - are used for serological diagnosis but the gold standard test consists of the detection of T. gondii (of suspect materiais) in isolation by mouse inoculation (requires 6-8 weeks ) and by fibroblast cell culture (this is sensitive but very time consuming , requiring 4 weeks , and may also fail to detect some cases ) . The coagglutination test for urine samples ( Fachado et al , Mem. Inst. Oswado Cruz 87 : 3034 , 1994 ) has been used. The other direct detection procedures such as histological examination and immune histology, sometime are either insensitive or time -consuming.

ln recent years molecular biology techniques have successfully been applied to the detection of parasite DNA in clinical samples. The most used method is polymerase chain reaction ( PCR ) and more recently dot-blot hybridization (Angel et al, J.Clin.. Microbiol. 35 (3): 591-595 , 1997 ). However, the finding of Toxoplasma DNA in blood and other biological fluids does not prove parasitemia ( presenting the viable parasites ) .

Taking into account the statements above, parasite DNA detection in patients with immunodeficiency virus infections, pregnant women and mainly in HIV - infected pregnant women may be considerei as acute toxoplasmosis.

RT07 - ^{IMMUNOLOGY I}

GENERATION OF ANTIBODIES WITH FUNCTIONAL EFFECTS IN CHAGAS DISEASE

Mariano Levin, Ingebi, Buenos Aires, Argentina

Abstract not received.

AUTOIMMUNITY IN HUMAN CHAGAS' DISEASE CARDIOMYOPATHY: CYTOKINE PRODUCTION AND ANTIGEN RECOGNITION BY T CELLS

Edecio Cunha-Neto¹, Lucia C.J. Abel¹, Luiz Vicente Rizzo², Anna Carla Goldberg¹, Charles Mady¹, Barbara Ianni¹, Juergen Hammer³, Francesco Sinigaglia³, Jorge Kalil¹¹Instituto do Coração, FMUSP, São Paulo, Brazil; ²NEI/NIH, Bethesda, USA; ³Roche Milano Richerche, Milano, Italy

The pathogenesis of Chronic Chagas' Disease Cardiopathy (CCC) is still under intense debate (1,2). Susceptibility factors or pathogenesis "checkpoints" that lead 30% of infected patients to develop the CCC after Trypanosoma cruzi infection are also largely unknown. Forty years ago, it has been hypothesized that T cells infiltrating the heart in CCC could mediate a delayed hypersensitivity process directed to the heart-specific tissue components; an autoimmune response triggered by molecular mimicry with a given T. cruzi antigen bearing similarities to heart proteins (3). This hypothesis awaited experimental confirmation, i.e. the identification of relevant antigens in molecular mimicry, and crossreactive, potentially pathogenic T cells with an inflammatory cytokine profile in the heart infiltrate. Our group identified T cell clones obtained from the heart lesion of a CCC patient (4) crossreactively recognizing cardiac myosin, the major heart protein, and the tandemly repeated recombinant antigen B13 from T. cruzi (5). Heart-infiltrating T cell lines obtained from CCC patients produce IFN-g and TNF-a in the absence of IL-4 upon PHA stimulus (6), in line with the predominant detection of IFN-g and TNF-a in immunohistochemical studies of CCC heart tissue (7, 8).

We next studied antigen presentation/recognition and systemic cytokine production by peripheral blood T cells from CCC, asymptomatic Chagasic (ASY) and normal individuals. While B13 is readily recognized in primary PBMC proliferation assays, cardiac myosin failed to be recognized even by CCC patients (9). However, cardiac myosin-recognizing T cell clones could be recovered from T cell lines induced with B13 from peripheral blood, which suggests that sensitization with B13 in vivo along T. cruzi infection may prime T cell clones able to recognize cardiac myosin (10). Antigenic B13 peptides can be presented by HLA-DQA1*0501/DQB1*0301, DR1 and DR51 as asessed by HLA-binding assays. The finding that 96% of individuals showing PBMC proliferative responses to B13 carry at least one of such B13-binding HLA-class II alleles confirm their role in T cell presentation of B13 epitopes in vivo. Interestingly enough, the HLA alleles capable of presenting B13 peptides are present in ca. 80% of the general population, CCC or ASY groups.

In order to map the T cell epitope in B13 protein, a PBMC-derived B13-specific T cell line was subject to proliferation assays with overlapping synthetic peptides covering the B13 tandemly repeated sequences. The 10-mer FGQAAAGDKP peptide was identified as the minimal T cell epitope in DQA1*0501/ DQB1*0301-positive individuals. Mapping of MHC/T cell receptor contacts on the B13 T cell epitope reported here on crossreactive T cell clones may lead to the identification of the yet unknown B13-crossreactive T cell epitope in cardiac myosin, which is essential for the further investigation of the role of molecular mimicry in CCC pathogenesis.

The cytokine production pattern of PBMC from CCC and ASY patients in response to B13 antigen was qualitatively and quantitatively shifted towards IFN-g , while responders among normal individuals (possibly sensitized to a B13-crossreactive environmental antigen) had an opposite pattern, shifted towards IL-4 production.

The fact that polyclonal heart-derived T cell lines from CCC patients, and B13-specific peripheral T cells from both CCC and ASY patients, all produce IFN-g but not IL-4 suggests that chronic T. cruzi infection in humans induces a systemic shift to a T₁ cytokine profile. This is consistent with the known IL-12 inducing activity of T. cruzi glycoconjugates (11).

In summary, our results are consistent with the view that T. cruzi infection may trigger potentially pathogenic T cells, as has been postulated for other infectious agents in organ-specific autoimmune diseases (12). The provision of the initial antigenic stimulus (B13) in the presence of costimulatory signals (13) and IL-12 production, generates "experienced", HLA-restricted, B13-specific, IFN-g producing T cells potentially able to migrate to peripheral nonlymphoid tissue in a large majority of infected individuals irrespective of the clinical phase. However, the T₁-type cytokine profile and HLA-restricted presentation/recognition of T. cruzi B13 protein are non-limiting factors shared by CCC and ASY individuals. Thus, we hypothesize that the generation of a heart-damaging T cell infiltrate may only occur in a subset of T. cruzi-infected individuals whose T cell repertoire allows crossreactive recognition of cardiac myosin by B13-specific T cells. This could be the link between low-grade persistence of T. cruzi in the chronically infected host and heart tissue damage in areas devoid of parasite antigens (4, 14). The T cell repertoire "checkpoint" (15) could also be the underlying mechanism of differential susceptibility to CCC progression. This hypothesis is currently being investigated in our laboratory.

References

1.Kalil J & Cunha-Neto E. Parasitol. Today, 12(10):396-399, 1996

2.Higuchi MD et al. Am. J. Trop. Med. Hyg. 56 (5): 485-489, 1997

3.Andrade Z. Rev. Goiana Med. 4:103-119, 1958

4.Cunha-Neto E et al. J. Clin. Invest. 98(8):1709-1712, 1996

5.Gruber A & Zingales B. Exp. Parasitol. 76,1-12 (1993)

6.Cunha-Neto E et al. Mem. Inst. Oswaldo Cruz 91(Suppl. I): 244, 1996

7.Reis DD et al. Am. J. Trop. Med. Hyg. 48:637-644, 1993

8.Reis MM et al. Clin. Immunol. Immunopathol. 83 (2): 165-172, 1997

9.Albuquerque F et al. Mem. Inst. Oswaldo Cruz 90 (Suppl. I):150, 1995

10.Abel LCJ et al. Mem. Inst. Oswaldo Cruz 91(Suppl. I):243, 1996

11.Camargo MM et al. J.Immunol. 158:5890, 1997

12.Matzinger P. Annu. Rev. Immunol. 12:991-1045, 1994

13.Frosch S et al. Infect. Immun. 65:971-977, 1997

14.Higuchi ML et al. Cardiovasc. Pathol. 2:101, 1993

15.Andre I et al. Proc Natl Acad Sci USA 93(6):2260-2263, 1996

B CELL AND NATURAL KILLER CELL ACTIVATION INDUCED BY GLYCO-INOSITOLPHOSPHOLIPIDS PURIFIED FROM TRYPANOSOMA CRUZI.

Peçanha, LMT*, Previato, JO⁺,Arruda-Hinds, LB*, Bento, CAM*, Bilate, AMB*, Melo, MB*, de Brito Gitirana, L** & Mendonça-Previato, L⁺

Departamentos de Imunologia* & Microbiologia Geral⁺, Instituto de Microbiologia Prof. Paulo de Góes & Departamento de Histologia e Embriologia**, ICB, Universidade Federal do Rio de Janeiro, Caixa Postal 68040, Rio de Janeiro, 21941-970, RJ, Brasil

Infection with T. cruzi is associated with altered function of different lymphoid cells.

One important abnormality is polyclonal B cell activation that is associated to increased circulating immunoglobulin (Ig) levels. This response is detected early during infection and can be observed throughout the chronic phase (1, 2). Polyclonal B cell activation was shown to be T cell-dependent (3). Recent data indicated that activated T cells obtained from T. cruzi-infected mice directly stimulate Ig production by normal B cells (4). Moreover, there is also a transient increase in natural killer (NK) cell cytotoxic activity during infection (5), and NK cells have been described to be important for resistance to infection (6)

Glycoinositolphospholipids (GIPLs) are one of the major glycolipids of the T. cruzi surface. The T. cruzi GIPLs have an oligosaccharide moiety phosphatidilinositol-linked to a ceramide (7). Recent studies showed that T. cruzi GIPL blocks T cell activation through its ceramide moiety (8) and that this glycoconjugate also increases proliferation and spreading of a macrophage cell line (9). Studies on the biological effects of GIPL-related glycolipids purified from Leishmania suggest that expression of this group of trypanosomatid molecules is necessary for virulence (10).

During the last years, we have focused our studies on characterizing mechanisms controlling B cell activation and Ig production during T. cruzi infection. We have used two major approaches. First, we investigated to what extent T. cruzi-derived products would stimulate B cells to produce Ig. Second, we investigated the regulatory role of NK cells on Ig secretion, based on studies showing that NK cells control Ig secretion in response to polysaccharide antigens (11).

The former studies indicated that GIPL purified from T. cruzi (G strain) is a potent in vitro murine B cell activator (12). This glycoconjugate stimulated, by itself, detectable IgM production by both low- and high-density B cells and potentiated the response induced by surface Ig ligation or cytokines. The T. cruzi-derived GIPL also stimulated Ig class switch to IgG1 or IgG3. B cell stimulatory effect of the GIPL was mediated mainly by its oligosaccharide moiety (12). We further extended those studies to verify the in vivo stimulatory effect of this glycolipid. Serum IgM levels from GIPL-injected mice showed only a modest increase 7 days after injection. However, B cells obtained 3 days after GIPL injection secreted at least 100 times higher levels of IgM in response to surface Ig ligation, IL-5 or LPS. Light microscopy analysis showed that spleens obtained from GIPL-treated mice have a white pulp hypertrophy and a consequent displacement of the red pulp, which contained a markedly elevated number of giant cells.

Although T-B cell interaction is necessary for production of Ig in response to protein antigens, it was shown that NK cells can also control Ig production (11). In order to investigate B lymphocyte-NK cell interaction during infection, we analyzed Ig production by B cells from infected mice at different periods. Those B cells were cultured with a NK cell line that supports Ig production. We observed that B cells obtained ater 45 - 55 days of T. cruzi infection produced four times higher amounts of both IgM and IgG2a in the presence of the NK cell line. Increased Ig production was not seen if B cells were obtained either earlier (15 days) or later (70 days) during infection.

Those findings suggested that B cells from infected mice would activate NK cells. We tested, additionally, the possible activation of NK cells by T. cruzi-derived molecules. We approached this by investigating if T. cruzi GIPL could activate NK cells. We observed that either the whole GIPL (obtained from either G or Tulahuem strains of T. cruzi) or the purified oligosaccharide moiety could induce an increase in cytotoxic activity of human PBMC against K562 target cells (13). In addition, GIPL increased the IL-2-induced proliferative response of the NK cell line, and potentiated the NK cell-assisted Ig secretion.

Our results suggest that the T. cruzi GIPL affects different cell populations. Interestingly, the effect of this glycolipid on B and T lymphocytes and NK cells parallels the alterations in cell function observed during T. cruzi infection. Our studies on B lymphocyte-NK cell interaction also suggest that, besides T cells, NK cells may be one cell population supporting B cell activation during T. cruzi infection. Finally, our data suggest that the GIPL purified from T. cruzi has a potent in vivo stimulatory activity and that this protozoan-derived molecule could be, at least in part, responsible for immunological changes associated with infection.

References

1- D'Imperio Lima, M, Eisen, H, Minoprio, P, Joskowicz, M & Coutinho, A. 1986. J. Immunol. 137: 353.

2- Spinella, S, Liegeard, P & Hontebeyrie-Joskowicz, M. 1992. Exp. Parasitol. 74: 46.

3- Minoprio, P, Eisen, H, Joskowicz, M, Pereira, P & Coutinho, A. 1987. J. Immunol. 139: 545.

4- Freire de Lima, C, Peçanha, LMT & dos Reis, GA. 1996. Infect. Immun. 64: 2861.

5- Hatcher, F.M., Kuhn, RE, Cerrone, MC & Burton , RC. 1981. J. Immunol, 127: 1126.

6- Cardillo, F, Voltarelli, JC, Reed, SG & Silva, JS. 1996. Infect. Immun. 64: 128.

7- Carreira, J.; C. Jones; R. Wait; J. O. Previato and L. Mendonça-Previato. 1996. Glycoconjugate J. 13: 955.

8- Gomes, NA, Previato, JO, Zingales, B, Mendonça-Previato, L & dos Reis, GA. 1996. J. Immunol. 156, 628.

9- Freire de Lima, CG, Previato, JO, Mendonça-Previato, L, Peçanha, LMT & dos Reis, GA. 1996. Glycoconjugate J. 6: 727.

10- Turco, SJ & Descoteaux, A. 1992. Ann. Rev. Microbiol. 46: 65.

11- Snapper, CM, Yamaguchi, H, Moorman, MA & Mond, JJ. 1994. J. Immunol. 152: 4884.

12- Bento, CAM, Melo, MB, Previato, JO, Mendonça Previato, L & Peçanha, LMT. 1996. J. Immunol. 157: 4996.

13- Melo, MB, Arruda, LB, Cunha, JMT, Previato, JO, Mendonça-Previato, L & Peçanha, LMT. 1996. Glycoconjugate J. 6: 754.

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

IMMUNOLOGICAL IMPLICATIONS OF THE HEAT SHOCK PROTEINS

C. Alonso, A.I. Rico, L. Quijada, M. Soto and J.M. Requena.

Centro de Biologia Molecular Severo Ochoa/UAM, Canto Blanco. Madrid 28049

The Leishmania infantum HSP70 has been described as an immunodominant antigen during both human and canine leishmaniasis. In the present study, we have mapped the antigenic determinants of the HSP70 protein showing the strong antigenic properties of the protein. Some of the epitopes are highly immunogenic as an indication of its potential as tools for diagnostic purposes. We also present evidences indicating that this protein promotes an adjuvant effect in the immune response against a covalently linked protein. The maltose-binding protein (MBP) from Escherichia coli was used as the reporter protein. Plasmid constructions were made to produce the three recombinant proteins used in this study: the MBP, the L. infantum HSP70, and the MBP-HSP70 that consists of the MBP protein fused to the amino terminal of the Leishmania HSP70. We found that immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited both humoral and cellular responses against MBP one order of magnitude higher than those elicited by the immunization either by MBP alone or by a mixture of MBP and HSP70. Covalent linkage of the MBP protein to HSP70 was found essential to elicit a strong immune response against MBP. The analysis of cytokine secretion and IgG isotypes indicated that immunization with MBP-HSP70 induce preferentially a Th1-like response. Unexpectedly, the inoculation of athymic nu/nu mice with MBP-HSP70 elicited a humoral response which features that of T cell dependent response. We discuss the possible involvement of T?d lymphocytes in the observed immunostimulatory effect of the L. infantum HSP70.

On the basis of previous investigations indicating that the Mycobacterium tuberculosis hsp70 in an adyuvant-free immunization system enhances the immune response to an accompanying protein, we have analyzed whether this stimulatory effect is shared also by the L. infantum hsp70. This Leishmania protein has been described as an immunodominant antigen during both human and canine leishmaniasis. Several plasmid constructions were made to produce the three recombinant proteins used in this study. The maltose-binding protein (MBP) from Escherichia coli was used as the reporter protein. A recombinant Hsp70 protein expression vector was developed that allows the production of the maltose-binding protein (MBP) fused to the amino terminal of the Leismania Hsp70. We found that immunization of BALB/c mice with the MBP-hsp70 fusion protein elicited both humoral and cellular responses against MBP higher than those elicited by immunization either by MBP alone or by a mixture of MBP and hsp70. The analysis of the cytokine secretion in response to stimulation with MBP and of the IgG isotypes induced by immunization with MBP-hsp70 indicated that the cellular and the humoral response is preferentially of a Th1-like. Unexpectedly, the inoculation of athymic nu/nu mice with MBP-hsp70 elicited a humoral response which features that of a T cell dependent response. We discuss the possible involvement of T?d lymphocytes in the observed immunostimulatory effect of the L. infantum hsp70. Covalent linkage of the MBP protein to hsp70 is necessary to elicit the immune response.

Supported by grants I+D 0020/94, PTR94-0091 and BIO96-0405. An institutional grant from Fundación Ramón Areces is also acknowledged.

RT08 - ^BIOCHEMISTRY

PUTATIVE HOST CELL RECEPTORS FOR TC-85 GLYCOPROTEINS FROM TRYPANOSOMA CRUZI

Alves, MJM

Departamento de Bioquimica, Instituto de Quimica, USP, S. Paulo, SP, Brasil

E-mail: [e-mail]

Trypanosoma cruzi has a complex life cycle involving mammalian and insect hosts with distinct morphological and functional stages in different environments. The parasite has to adhere to and infect a host cell in order to survive. As expected, much attention has been given to this step in the search for the receptors and counter-receptors involved and, more recently, to the events within the host cell following parasite attachment. Which parasite molecules are important for the association with the host cell? Which receptors do they interact with? Are the same molecules involved when trypomastigotes change environment? Proteins from the parasite, most of them in the range of 80-100 kDa have been implicated in the invasion of the host by different researchers.

In an attempt to identify molecules involved in the invasion step some years ago our group raised a monoclonal antibody (H1A10 mAb) that is able to inhibit the invasion of tissue culture cells by 46% to 96%. The antibody recognizes a polymorphic 85 kDa glycoprotein specific for the surface of trypomastigotes (Tc-85). Tc-85 has a half-life of 3.5-4 h and is synthesized as a 95 kDa precursor. Interestinly, heterogeneity in molecular mass, isoelectric point and carbohydrate composition was identified. Tc-85 is shed into the medium within membrane vesicles bearing an acyl-inositol modified anchor, but the shedding mechanism is still unknow. Cloning and characterization of a genomic DNA fragment allow us to include

Tc-85 into the Sialidase/Trans-sialidase supergene family, with other members that have no enzymatic activity. Also, the epitope recognized by H1A10 mAb could be identified by competition assay with synthetic peptides. Sequencing of different clones showed heterogeneity between them, including the encoding region of the epitope recognized by H1A10 mAb. The data let us to propose that all members belong to a gene family, the so called Tc-85 gene family. Tc-85 glycoproteins show a large pI range but only the acid portion binds to laminin, an important component of basal membrane. This interaction is independent of the carbohydrate from laminin or from Tc-85. Anti-idiotypic antibodies (Id) raised against H1A10 mAb recognize three polypeptides in the host cell (150-130, 73 and 43 kDa) with a major 73 kDa molecule. Also, anti-Id antibodies are able to inhibit invasion of host cells by trypomastigotes and do not react with laminin, suggesting that Tc-85 interacts with different host receptors. Anti-Id antibodies react with molecules present in liver, heart, muscles or spleen, as could be expected, since T. cruzi invades different tissues in the mammalian host, as well as tissue culture cells. Cloning the putative host cell receptors is being persuade to further characterize host-parasite interactions.

Supported by : FAPESP, PADCT/CNPq.

STRUCTURE AND DIVERSITY OF A MUCIN GENES FAMIILY IN TRYPANOSOMA CRUZI

Di Noia, J.M.; Pollevick, G.D.; Sánchez, D.O.- D'orso, 1. and Frasch, A.C.C.

Instituto de Investigaciones Biotecnológicas, UNSAM, Parque Tecnológico Nfigueletes, Edif 24, INTI, C.C. 30, San Martín, Pcia. de Buenos Aires, Argentina

Fax: [phone] E-mail: jdinoia@ jnti.edu.ar

Mucin type glycoproteins are widely spread in vertebrates where they participate in important functions like cytoprotection and cell-cell interactions. They ali share some distinctive features. A core apoprotein serves as a scaffold to a high number of carbohydrate chains covalently attached to hydroxyl group of Thr and Ser residues. Carbohydrates accounts for up to the 80% of the total moiecule mass. The core proteins show a low anúno acid sinúlarity among mucins encoded by different genes but a conunon structure can be defined for most of them. They ali have a central domain composed of up to 70% by Thr and Ser that is also very rich in Pro residues. These amino acids are generally organizei in repeatead domains with a variable number of repeats among different alleles. These central domains are flanked by unique domains and sometimes near a Cys rich domain. ln Trypanosoma cruzi, the causative agent of Chagas' disease, mucin type glycoproteins have been described in three of its four developmental stages (Almeida et al., Biochem. J 304,793-802, 1994; Previato et al., Biochem. J 301, 151-159, 1994; Schenclcman et al., MoL Biochem. ParasitoL 59, 293-304, 1993). Ali of them are rich in Thr, Ser and Pro; have a high degree of O-glycosilation and are attached to the extemal membrane by glycophosphatidylinositol (GPI) anchors. ln epimastigotes and metacyclic forms these molecules were first denonúnated 35-50 kDa antigens because of their electrophoretic behaviour. Trypomastigote mucins were described as a broad band of 60-200 kDa, ali characterized by possessing the sialylated epitope Ssp3. Ali T. cruzi mucins have been shown to be the main sialic acid acceptors in the reaction catalyzed by the Trypanosome's unique enzyme Trans-sialidase. Mucins are proposed to be involved in the processes of adhesion and penetration to the mammalian host cell by the parasite (Schenkman et al., Annu. Rev. Microbiol. 48, 499-523, 1994).

The first known mucin type gene of T cruzi was cloned by immunological screening of an expression library, using a serum from an acute human infection (Reyes et al., Gene 140, 139-140, 1994). The deduced protein had four repeats with the sequence Thr(8)-Lys-Pro(2), flanked by an N-ternúnus with a putative signal sequence and a C-temúnus ending with a sequence compatible with a GPI anchor addition. Using different regions of this gene as probes, the cromosornic, genonúc and expression pattems were analyzed and eleven genes, from seven strains, were completei sequenced. These genes resulted to be part of a very complex family of mucin type genes (Di Noia ct al., J. Biol. Chem. 270, 24146-24149, 1995), ali having a small size ranging from 350 to 650 bp. The deduced proteins have a very high degree (from 70 to 100 %) of identity within the first 24-28 and the last 50 arnino acids, but the central regions widely vary in sequence and length among them. So far, they can be divided in two groups. One with a central region composed by repetitive elements arranged head-to-tail in tandem, with the consensos sequence Thr(8)-Lys-Pro(2). The number of repeats can be from one to ten in the sequences known so far. Members of the second group have no repetitive elements but their central domains are enriched in Thr, Ser and Pro residues. Identities percentages among members of the latter group are very low in the central regions. Southern blots experiments further show that the interstrain variability at the genomic levei for this fanúly is very significant. For example, while a probe of a whole repeats-containing-gene recognizes a large number of bands in ali the strains tested up to day, the repeated regions alone only recognize a few bands in a linúted number of strains. Moreover, some non repetitive central regions seem to be absent from ali strains analyzed except the one they were cloned from. Northem blots analysis of some strains of T cruzi indicate that transcripts núgrate as a broad band with a size centered in I 000 bases. The MRNA are present both in epimastigote and trypomastigote stages. Current work on mucin genes expression involves isolation of specific MRNA either by using primers from the conservei regions to make CDNA from different stages of the parasite, or by screening of CDNA libraries. Our knowledge on the intrastrain diversity of the familiy is being increased and the MRNA structure is being analyzed to detern-únate common structures and the loci from where they come from. Aligrunent of their non encoding regions point to the existence of different groups of MRNA.

Using a monoclonal antibody directed against a carbohydrate epitope (mAb I OD8 kindly provided by Dr. N.Yoshida) and a polyclonal serum raised against the recombinant protein derived from one of the cloned genes, we obtained indirect evidence connecting this gene family with the previously described 35-50 kDa antigens from metacyclic trypomastigote and epimastigote forms of the parasite (Di Noia et ai., J. Biol. Chem. 271, 32078-32083, 1996). Work done in collaboration with Dr. Previato's laboratory have shown that mucin type molecules purified from T cruzi epimastigote, are composed by several populations of molecules with different molecular masses. The bulk of these molecules are in two populations with molecular masses of 15.9 and 17.5 for the CL-Brener clone, but these values differ with the strain thus confirming the diversity of mucin molecules in this parasite. Some direct sequencing of deglycosylated mucin cores have not yielded direct coincidence with any of the genes isolated up today. This result is not surprising since our current estimations from hybridization of ordered genonúc libraries suggest the existence of more than 500 mucin genes in CL-Brener genome. Studies on the identification of genes and their corresponding proteins are further complicated by the fact that several genes are transcribed simuitaneously as determinei from MRNA analysis.

Supportcd by UNDP/World BankfWHO Special Program for Research and Training in Tropical Diseases (TDR), Department for Research Cooperation (SAREC) from the Swedish International Development Cooperation Agency. (SIDA) The research of ACCF was support in part by an lntemational Research Scholars Grant from the Howard Hughes Medical Institute.

BIOSYNTHESIS OF GLYCOPROTEINS IN PLASMODIUM FALCIPARUM AS A POTENTIAL TARGET FOR ACTION OF NEW ANTI-MALARIA DRUGS

Katzin A.M.¹, Kimura E.A.¹, Peres V.J.¹, Uhrig M.L.², Couto A.S.²

¹Departamento de Parasitologia. ICB-USP, Brasil; ²Departamento de Química Orgánica FCEyN UBA. Argentina

Despite attempts at eradication in the 1950's, malaria is still the most important parasitic disease of man. The disease affects 300-500 million people each year, and is caused by four species of Plasmodium. Plasmodium falciparum, the predominant species in tropical Africa, Eastern Asia, Australasia and the Amazon region, may cause severe morbidity and mortality (1). Resistance of P. falciparum to most available antimalarials is now widespread (2), and the development of new drugs is regarded as a priority. This task requires a thorough knowledge of the biochemistry of P. falciparum. Yet the glycobiology of the parasite, and more precisely the understanding of the role of oligosaccharides in host-parasite interaction (3), is still a neglected area in malaria biochemistry.

Although the existence of O-linked oligosaccharides residues in glycoproteins of P. falciparum have been shown, the existence of N-linked glycoproteins was still a matter of controversy and skepticism.

We recently demonstrated the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to Concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase^®, oligosaccharides were released. It was possible to identify an N-linked glycoproteins of >200 kDa in the ring stage, and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. A glycoprotein of >200 kDa from the ring forms was excised from the gel and digested with N-Glycanase. The hydrolysate was reduced with NaB³H₄ and the released radioactive oligosaccharides were analyzed by high performance anion exchange chromatography (HPAE-PAD). The sample showed a major peak corresponding to Man₃GlcNAc₂ (1,59 glucose units, GU). This structure was confirmed by sequential treatment with a-mannosidase, b-mannosidase. Two other peaks eluting approx. 3 GU and 4 GU corresponding to N-linked chains that appear later than Man₉GlcNAc₂ were also detected. A similar analysis performed on the 200 kDa glycoprotein obtained from young trophozoites demonstrated the presence of labeled oligosaccharides eluting in the same range of glucose units. The same treatment performed on glycoproteins metabolically labeled with [¹⁴C]-glucose confirmed the presence of high molecular weight oligosaccharides. Treatment of trophozoites with 12 µM tunicamycin inhibited differentiation to the schizont stage (4).

To our knowledge, this is the first report unequivocally demonstrating the presence of N-linked glycoproteins in trophozoites of P. falciparum as well as their importance in differentiation of the intraerythrocytic stages of this parasite (4).

The presence of N-linked glycoproteins in P. falciparum suggests the existence of dolichol biosynthesis, several intermediates of this pathway until farnesyl pyrophosphate were demonstrated (5, 6). On the other hand, it has been demonstrated that inhibitors of the enzyme hydroxymethylglutaryl-CoA reductase (HMG-CoA), such as compactin, inhibit in vitro development of Plasmodium falciparum (7, 8). Preliminary results obtained in our laboratory show that several glycoproteins of trophozoite stage disappear after compactin treatment of P. falciparum in culture. When the parasites were treated with N-Glycanase or compactin, bands with similar molecular weight disappeared or decreased in intensity after these treatments. These results show that inhibition of the biosynthesis of glycoproteins P. falciparum is correlated with arrested development of the parasite. We are interested in investigating if this fact is correlated with lack of production of dolichol. Preliminary results, analyzed by HPLC from samples obtained of parasites labeled with [1-¹⁴C]acetic acid show one peak corresponding to dolichol, otherwise in samples obtained from parasites treated with compactin this peak disappear.

The inhibition of biosynthesis of dolichol or in competition with similar molecules (terpenes) could be a new target for development the anti-malaria drugs.

References.

1.- W.H.O. Weekly Epidemiol. Record. (1993). 68, 245-252.

2.- Wernsdorfer, W.H. and Kouznetsov, R.L. (1980). Bull. WHO 58, 341-352.

3.- Dieckmann-Schuppert, A., Bender, S., Odenthal-Schnittler, M., Bause, E. and Schwarz, R.T. (1992). Eur. J. Biochem. 205, 815-825.

4.- Kimura, E.A.S., Couto, A.S., Peres, V.J., Casal, O.L., Katzin, A.M.(1996) J. Biol. Chem. 271:14452-14461.

5.- Elbein, A.D. (1987) Ann. Rev. Biochem. 56, 497-534.

6.- Mbaya, B.; Rigomier, D.; Edorh, G.G.; Karst, F.and Schrevel, J.(1990). Biochem. Biophys. Res. Commun. 173:849-854.

7.- Grellier, P., Valentin, A., Millerioux, V., Schrevel, J., Rigomier, D.. (1994). Antimicrob. Agents. Chemother. 38:1144-1148.

8.- Kimura E.A., Peres V.J., Katzin A.M. (1996). Mem. Inst. Oswaldo Cruz 91(Suppl 1):88

Supported by FAPESP, CNPq, Fund Antorchas, CONICET, UBA.

GLYCO(SPHINGO)LIPID ANTIGENS OF LEISHMANIA. POSSIBLE BIOLOGICAL ROLE

Helio K. Takahashi and Anita H. Straus

Department of Biochemistry, Universidade Federal de São Paulo/EPM, Rua Botucatu, 862, 04023-900 São Paulo, SP, Brasil.

Glycosphingolipid (GSL) antigens of Leishmania were first described by Straus et al. in amastigote forms of Leishmania (L.) amazonensis (1). Although presence of glycosphingolipids in Leishmania have been also described by others (2), they were considered contamination derived from the host's tissues. Data obtained in our laboratory revealed the existence of a family of neutral GSLs in L. (L.) amazonensis amastigotes harvested from infected hamsters. By a combination of high performance liquid chromatography (HPLC), preparative high performance thin layer chromatography (HPTLC) and HPTLC immunostaining we were able to isolate at least seventeen pure GSL antigens, reactive with monoclonal antibodies (MoAbs) ST-3, ST-4, and ST-5 (directed against carbohydrate moieties of L. (L.) amazonensis GSLs) (3). In order to discard the possibility that the GSLs antigens from amastigote forms of L. (L.) amazonensis resulted from adsorption or contamination with molecules from the host cells, the reactivity of other known GSLs from different sources with MoAbs ST-3, ST-4, and ST-5 was analyzed. Only amastigote-stage-specific GSL antigens were recognized by the three MoAbs. Furthermore, by indirect immunofluorescence of infected macrophages isolated from BALB/c mouse footpad lesions, using MoAb ST-3 conjugated to FITC, we detected only labeling in the intracellular parasite, and as expected, no fluorescence was observed on the surface of the infected macrophages. These results indicate that GSL antigens of amastigote forms of L. (L.) amazonensis, contrary to those GSLs described in amastigote preparations of Leishmania donovani and Leishmania mexicana are not acquired from the host (2,4) but rather synthesized by the parasite (1,5).

Of the GSLs antigens from L. (L.) amazonensis amastigotes recognized by MoAbs ST-3, ST-4, and ST-5, we isolated six glycosphingolipids termed: b1, b2, b3, b4, b5 and b6, which were purified by a combination of HPLC and preparative HPTLC in solvent chloroform/methanol/aqueous CaCl₂ 0.2%, and analyzed by ^-FAB/MS (5). The molecular mass of the pure GSL antigens b1, b2, b3, b4, b5 and b6, were determined as: 1184 atomic mass units (a.m.u.), 1346 a.m.u. , 1498 a.m.u., 1388 a.m.u., 1660 a.m.u. and 1550 a.m.u., respectively. The sequences of hexose (Hex) and N-acetyl hexoses (HexNAc) of the bands analyzed are: b1: Hex-O-Hex-O-Hex-O-Hex-O-Cer; b2: Hex-O-Hex-O-Hex-O-Hex-O-Hex-O-Cer; b3: Hex-O-HexNAc-O-Hex-O-Hex-O-Hex-O-Cer; b4: Hex-O-HexNAc-O-Hex-O-Hex-O-Hex-O-Cer; b5: Hex-O-Hex-O-HexNAc-O-Hex-O-Hex-O-Hex-O-Cer; b6: Hex-O-Hex-O-HexNAc-O-Hex-O-Hex-O-Hex-O-Cer. Their ceramide moieties were found to contain only one type of long chain base, a sphingosine d18:1, whereas their fatty acids (f.a.) composition were: b1, f.a. 16:0 b2, f.a. 16:0; b3, f.a. 24:1; b4, f.a. 16:0; b5, f.a. 24;1 and b6, f.a. 16:0 .

By ¹H NMR, ^-FAB/MS, GC/MS, and enzymatic degradation, the structure of the smallest antigen (b1) recognized by MoAbs ST-3, ST-4, and ST-5 was identified as a b-Gal-globotriaosyl Cer (III³b-GalGb₃Cer) with the sequence: Galb1-3Gala1-4Galb1-4Glcb1-Cer, and represents a novel globoseries structure absent in hamster and in other mammalian cells (1).

The high concentration of GSLs in L. (L.) amazonensis amastigotes (1x10⁶ molecules/cell), in contrast with their absence in promastigotes, may be an important correlated with amastigotes survival and proliferation in host macrophages. These studies allowed us to obtain basic biochemical and structural information on L. (L.) amazonensis GSLs. The fact that no anti-GSL antibodies were detected in sera of patients infected with L. amazonensis suggests that these glycoconjugates may be of importance in the parasite invasion or parasite escape from immunological surveillance through mimicry of host cell components. The immune inhibitory properties of GSLs seems to be modulated by their carbohydrate moieties. A significant inhibition of ConA-induced mitogenesis was observed with the different parasite GSLs tested (1-7 sugar residues). On a molar basis, the best inhibitors were those GSLs with shorter carbohydrate chain. An attractive hypothesis would be that shedding of GSLs from the parasite may act as local immunosuppressors of both humoral and cellular responses (6).

Intact MoAbs ST-3, ST-4, or ST-5 were able to inhibit 60-80% of macrophage invasion by amastigotes. About the same inhibition was obtained with Fab fragments of ST-3 and ST-5. Taken together, these results strongly suggest that the epitope recognized by these MoAbs present in L. (L.) amazonensis amastigote surface GSLs is important in mediating binding of parasites to specific receptors on macrophages (1).

The in vivo binding capacity of the anti-glycosphingolipid MoAbs, was analyzed using ¹²⁵I labeled ST-3 antibody. The radiolabeled MoAb was injected intravenously into BALB/c mice infected with Leishmania (L.) amazonensis. After 72 h, tissue distribution of ¹²⁵I-labeled ST-3 was expressed as the ratio of radioactivity in the tissue to that present in the blood [(cpm/g tissue)/(cpm/g blood)]. The highest level of radioactivity was observed in the footpad lesions. It was observed almost three times more radioactivity in the lesion when compared to blood and lungs, no significant amount of radioactivity was found in spleen, liver, heart, and kidney. These results clearly indicate that GSLs antigens are specific markers of amastigote forms of L. (L.) amazonensis, and may be useful as potential targets for therapy/diagnosis for leishmaniasis caused by L. (L.) amazonensis (5).

Although the MoAbs ST-3, ST-4, and ST-5 were shown to recognize GSL antigens specific of amastigote forms of L. (L.) amazonensis, MoAb ST-3 presented a peculiar reactivity with promastigotes of L. (L.) amazonensis, and Leishmania (Viannia) braziliensis. The antigen recognized by ST-3 in promastigotes of L. (L.) amazonensis was identified as a subpopulation of lipophosphoglycan (about 1.5%) containing terminal residues of b1-3Gal whereas in promastigotes of Leishmania (Viannia) braziliensis the major antigen recognized by MoAbs ST-3 and ST-5 was tentatively identified as a glycolipid constituted by galactose, glucose, and hexosamine. These antigens present in promastigote forms of L. (L.) amazonensis and of L. (V.) braziliensis contain the same epitope recognized by MoAb ST-3/ST-5 in amastigote L. (L.) amazonensis glycosphingolipids. The involvement of these antigens in macrophage interaction was analyzed, using Fab fragments of MoAbs ST-3 and ST-5. Macrophage invasion by promastigotes forms of L. (L.) amazonensis was inhibited about 80% by Fab fragment of MoAb ST-3. MoAbs ST-4 and ST-5, as expected did not alter the macrophage invasion by this parasite. On the other hand, macrophage infection by promastigote forms of L. (V.) braziliensis was inhibited about 60% by Fab fragments of both MoAbs ST-3 and ST-5.

Recently, two MoAbs, termed MEST-1 and SST-1, were generated in our laboratory. The MoAb SST-1 (IgG3) is specific for promastigote forms of L. (V.) braziliensis, it recognizes an unique glycolipid antigen, and no cross-reactivity was observed with glycolipids/glycosphingolipids from promastigote/amastigote forms of L. (L.) amazonensis. SST-1 is the first monoclonal antibody described to react specifically with glycolipids of L. (V.) braziliensis. At the present time the nature and the structural characteristics of the glycolipid antigen recognized by SST-1 is still unknown. Regarding MoAb MEST-1, it is specifically directed to terminal residues of b-galactofuranose, it reacts with epimastigotes of Trypanosoma cruzi and promastigotes of L. major (7). GIPL-1 was identified as the main antigen recognized by MEST-1 in L. major promastigotes. Preliminary studies showed that MEST-1 is able to inhibit 80% of macrophage invasion by promastigote forms of L. major. In T. cruzi a weak reactivity of MoAb MEST-1 was detected by solid-phase RIA or Western blot with the fraction corresponding to LPPG.

The results described here led us to elaborate a new concept in the glycobiology of Trypanosomatids such as L. (L.) amazonensis, L. (V.) braziliensis, L. major, and T. cruzi. Thus, different glycolipids may possibly be used as biosynthetic precursors by b1-3 galactopyranosyltransferase and b1-3galactofuranosyltransferase which modulate the expression of specific glycoconjugates involved in cell binding/invasion by parasites, as shown by the inhibitory properties of MoAbs ST-3 (anti-b1-3galactopyranose) and MEST-1 (anti-b1-3galactofuranose).

References

1- Straus, A.H., Levery, S.B., Jasiulionis, M.G., Salyan, M.E.K., Steele, S., Travassos, L.R., Hakomori, S. & Takahashi, H.K. J. Biol. Chem., 268: 13723-13730, 1993.

2- McConville, M.J. & Blackwell, J.M. J. Biol.Chem., 266: 15170-15179, 1991.

3- Straus, A.H., Suzuki, E., Toledo, M.S., Takizawa, C.M. & Takahashi, H.K. Braz. J. Med. Biol. Res., 28: 919-923, 1995.

4- Winter G., Fuchs, M., McConville, M.J., Stierhof, Y-D & Overath, P. J. Cell. Science, 107: 2471-2482, 1994.

5- Straus, A.H., Valero, V.B., Takizawa, C.M., Levery, S.B., Toledo, M.S., Suzuki, E., Salyan, M.E.K., Hakomori, S., Barbieri, C.L. & Takahashi, H.K. Braz. J. Med. Biol. Res., 30: 395-399, 1997.

6- Giorgio, S., Jasiulionis, M.G., Straus, A.H., Takahashi, H.K. & Barbieri, C.L. Exp. Parasitol., 75: 119-125, 1992.

7- Suzuki, E., Toledo, M.S., Takahashi, H.K. & Straus, A.H. Glycobiology, 7: 463-468, 1997.

Supported by: FAPESP, CNPq and PRONEX

RT09 - ^{IMMUNOLOGY II}

DNA VACCINATION AGAINST INFECTION WITH A HUMAN PROTOZOAN PARASITE, TRYPANOSOMA CRUZI.

Costa, F., Pereira-Chioccola, V.^*, Ribeirão, M., Arena, F., Schenkman, S. & Rodrigues, M.

Departmento de Microbiologia, Imunologia e Parasitologia, UNIFESP-EPM, Rua Botucatu 862, São Paulo, S.P., Brazil, 04023-062 , ^*Laboratório de Xenodiagnóstico, Instituto Dante Pazzanese de Cardiologia do Estado de São Paulo, Av. Dante Pazzanese 500, São Paulo, SP Brasil.

Vaccination against pathogenic micro-organisms can now be accomplished by immunization with protein free DNA vaccines (1,2). Most studies performed so far have focused on the immunization with plasmids containing viral genes. However, DNA vaccination was also effective against Plasmodium yoelii, a protozoan parasite that causes malaria in rodents (3). Based on this prospect, we addressed the question of whether DNA vaccination could be used to elicit immunity against infection by a human protozoan parasite, Trypanosoma cruzi.. We used as antigen the trans-sialidase (TS), an enzyme expressed on the surface of T. cruzi. This enzyme is required for the parasite to obtain sialic acid from host glycoconjugates. The sialic acid is transferred to mucin-like glycoproteins, that are the most abundant molecules on the surface of bloodstream trypomastigotes (4).

We generated several plasmids containing the coding region of the catalytic domain of TS (aa 1-680). The vectors used were pcDNA3 (Invitrogen), and plasmid VR1012 provided by Vical Co., San Diego, CA.. The TS gene was introduced in each vector with or without the coding region for the TS signal peptide. Upon transfection of Cos-7 cells, trans-sialidase activity in the cell supernatant was detected only when the coding sequence for the signal peptide was present. We used these plasmids to immunize A/Sn (highly susceptible to infection) or BALB/c mice (intermediate susceptible). Animals received four doses i.m. of 100 mg of plasmid DNA. We observed that all plasmids induced IgG antibody response in both mouse strains. However, the magnitude of the IgG responses and their isotypes were modulated by a combination of plasmid and mouse strain used. The presence of the coding sequence for the signal peptide was more important to elicit an immune response in BALB/c mice. In contrast, antibody response in A/Sn mice was slightly higher after immunization with plasmids that do not contain the coding sequence for the signal peptide. In BALB/c mice, the predominant sub-class of TS-specific antibodies was IgG1. In contrast, in A/Sn mice, IgG2a and IgG2b were predominant. Antibodies elicited by immunization with TS plasmids recognize the native enzyme as determined by ELISA. Most relevant was the fact that these antibodies inhibited the enzymatic activity of native TS in vitro and parasite sialylation in culture.

Next, we assessed whether immunization with TS plasmid elicited DTH type immune response in BALB/c mice. Three weeks after the last of 4 immunizations, 50 mg of recombinant TS was injected s.c. in one of the mouse hind foot pads. Recombinant TS evoked a significant DTH response in mice immunized with TS plasmid, but not in animals immunized with control plasmid.

To determine whether DNA immunization could induce protective immunity against T. cruzi infection, immunized A/Sn and BALB/C mice were challenged with bloodstream trypomastigotes of Y strain. A/Sn mice immunized with any of the TS plasmids had a decrease in parasitemia, however there was no reduction in mortality. In BALB/c mice immunized with TS plasmid, we observed a significant reduction in the peak parasitemia when compared to animals that received control plasmid (pcDNA3). Most important, these mice had a significant higher survival rate. While all animals immunized with TS plasmid survived the infection, 8 out of 10 mice injected with control plasmid died. This result was independently reproduced in other two experiments.

In summary, our results provide the first evidence that DNA immunization can elicit protection against a human protozoan parasite.

1- Ulmer, J.B., Sadoff, J.C. and Liu, M.A . Current Opinion in Immunology 1996. 8, 531.

2- Ertl, H.C. and Xianq Z.Q. Viral Immunol. 1996. 9, 1.

3- Sedegah, M., Hedstrom, R., Hobart, P., and Hoffman, S.L. Proc. Nat. Acad. Sci. USA 1994. 91, 9866.

4- Schenkman, S., ManShiow, J., Hart, G. W., and Nussenzweig, V. Cell 1991. 65,1117-1125.

PROTECTIVE IMMUNITY INDUCED BY A RECOMBINANT ADENOVIRUS EXPRESSING THE CS PROTEIN OF MALARIA PARASITES

Rodrigues E.G.^{1) 2)}, Zavala, F.¹⁾, Nussenzweig, R.S.¹⁾, and Tsuji, M.¹⁾

¹⁾Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY; and ²⁾Department of Microbiology, Immunology and Parasitology, Federal University of Sao Paulo, SP, Brasil

A replicationdefective recombinant adenovirus expressing the CS protein of P. yoelii has been engineered and its immunogenicity has been determined. Mice were immunized with this recombinant adenovirus and their antiplasmodial immune responses were characterized, as well as their protection against sporozoite challenge. We found that a single immunizing dose of this

recombinant adenovirus induces potent cellular and humoral anti plasmodial immune responses in Balb/c mice. An ELISPOT assay, based on the secretion of IFNg by primed T cells, showed that

twelve days after the immunization, approximately one of every 350 murine splenocytes responded, in vitro, to a peptide corresponding to an epitope recognized by CSspecific CD8+ T

cells. In addition, one of every 500 splenocytes recognized another peptide representing a defined CD4+ T cell epitope of this CS protein. These immunized mice also developed rather high

antisporozoite antibody titers (IFA titer of 3200). Most importantly, we observed a 93% decrease in the hepatic parasite load, measured by the level of parasite rRNA, in the liver of

adenovirus immunized, sporozoite challenged mice. Furthermore, when challenged with highly infective P. yoelii sporozoites, 40% of the immunized mice displayed complete resistance to this

infection, compared to 100% infection in the controls. Our results indicate that this recombinant adenovirus is a very efficient vector, inducing not only large numbers of CSspecific T cells and antisporozoite antibodies, but also appreciable resistance to this infection.

POTENTIAL USE OF THE A2 GENE SYSTEM TO DIFFERENTIALLY EXPRESS SUICIDE GENES IN LEISHMANIA AMASTIGOTES

Hugues Charest, Elodie Ghedin, Wei-Wei Zhang and Greg Matlashewski

National Institutes of Health, National Institute for Allergy and Infectious Diseases, Laboratory of Parasit Diseases, Immunobiology section, Bethesda, MD USA, McGill University, Macdonald Campus, Institute of Parasitology, Ste-Anne de Bellevue, Quebec, Canada

Recovery from leishmaniasis leads to solid immunity against subsequent re-infections, and vaccination using attenuated Leishmania strains is therefore a particularly interesting approach to induce long term protection. The possibility of controlling the parasite growth as its pathologic form, the amastigote, would lower the inherent risk of using live parasites, an important parameter towards developing safe recombinant vaccine strains. With this aim in view, we have explored the possibility of using the Leishmania donovani A2 regulatory sequences to differentially express toxic gene products specifically in the amastigote form of the parasite.

The A2 gene family of L. donovani is amastigote-stage specific. A2 proteins, mainly comprised of repeated sequences, are developmentally expressed in the parasite during the promastigote to the amastigote cytodifferentiation. Although not essential for the survival of the parasite in in vitro cultures, A2 genes are required for the amastigote multiplication in vivo. The A2 developmental expression involves A2 mRNAs untranslated regions (UTRs) and we have demonstrated that A2 UTRs can regulate expression of exogenous genes throughout the Leishmania life cycle. Two different suicide gene systems were analyzed in transfection experiments involving episomal and targeted constructs: a truncated version of the L. donovani 3'nucleotidase/nuclease (3'Nt/Nu), and the Herpes simplex thymidine kinase(tk)/gancyclovir system. Both suicide genes were put under control of the A2 UTRs in recombinant cells. The L. donovani 3'Nt/Nu is normally an externally oriented enzyme involved in the purine salvage pathway. We engineered a truncated version of the enzyme by deleting the signal peptide and anchoring sequences from the gene, which led to the expression of the nuclease in the cytoplasm of the cell. Although the recombinant version of the enzyme was of low activity, increased expression of the truncated product correlated with a decrease viability of amastigotes. For the tk system, the exogenous gene was targeted into the A2 gene locus by gene replacement. In recombinant parasites, the developmental expression of the tk correlated with the differential sensitivity of promastigotes and amastigotes to the drug gancyclovir. In contrast, growth of recombinant promastigotes was unaffected by low level expression of the toxic gene, in both systems studied. These findings demonstrate the feasibility of A2 gene regulatory sequences to engineer recombinant Leishmania strains which can be cultured in vitro as promastigotes but cannot replicate as amastigotes.

CROSS IMMUNITY AMONGST LEISHMANIA AND ITS IMPLICATIONS IN THE DEVELOPMENT OF VACCINES

Shaw, J.J.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Lineu Prestes 1374. 05508-900 São Paulo, Brasil

Before any vaccine is put through the various clinical trial phases it must be carefully evaluated in the laboratory (Begg & Miller, 1990. Vaccine, 8: 180189.). A critical stage in the latter is to determine the level of protection and the immunological responses associated with the candidate vaccine in an animal model . In the case of diseases caused by different serotypes or closely related species cross protection studies are extremely important. Murine leishmanial models have been useful in untangling the different immunological systems associated with resistance and disease but their correlation with infections in man is perhaps questionable. They are also not very suitable for cross immunity studies between parasites the subgenera Leishmania and Viannia. Studies in non-human primates, however, are potentially of greater relevance because of their closer genetic relationship with man (Grimaldi, 1995. Mem. Inst. Oswaldo Cruz, 90: 553556.) and their susceptibility to parasites of the subgenus Viannia..

Experimental cutaneous and visceral leishmanial infections have been studied in Old World monkeys such as the African green monkey (Cercopithicus aethiops) and rhesus monkeys (Macaca mulatta) and in the following New World monkeys - black plumed marmosets (Callithrix penicillata), owl monkeys (Aotus sp.), squirrel monkeys (Saimiri sciureus) and capuchin monkeys (Cebus apella).

Cross protection studies between Leishmania species causing dermal infections were first performed in rhesus monkeys (Lainson & Bray, 1966. Trans. Roy. Soc. Trop. Med. Hyg., 60: 526-532) and more recently humoral and cell mediated responses were investigated to culture induced infections of L. (Leishmania) amazonensis in this same species of monkey (Amaral et al., 1996. Expl Parasit., 82: 3444.). Infections of L.(L.)donovani in the squirrel monkey, Saimiri sciureus, (Dennis, et al., 1986. Exp. Parasitol., 61: 319-314; Lujan et al., 1990. J. Parasitol., 76: 594-597 ) protected against the homologous parasite but not against L.(V.) panamensis. Recently Gicheru et al., (1997. Exp. Parasitol., 85: 109-116 ) showed that experimental infections L..(L.)donovani in the green vervet monkey, Cercopithecus aethiops, protected against subsequent infections with L..(L.)major.

The Capuchin Cutaneous Leishmaniasis Monkey (CCLM) model has been developed over the past 20 years by the Evandro Chagas Institute's leishmaniasis team. Initial studies were performed with wild caught monkeys but with the establishment of a Cebus apella (Primates: Cebidae) breeding colony at the Ministry of Health's National Primate Centre in Pará, Brazil it became possible to use laboratory bred animals.

Studies of the Cebus apella model with Amazonian Leishmania species began in the mid 70's (Lainson & Shaw, 1977. J. Trop. Med. Hyg., 80: 2935.) and have continued to the present day (Silveira et al., 1989. Revta Soc. Bras. Med. Trop. 22, 125130; Silveira et al., 1990. Revta Soc. Bras. Med. Trop. 23, 512; Silveira et al., 1990. Revta Inst. Med. Trop. S. Paulo 32, 387394; Garcez et al., Mem. Inst. Oswaldo Cruz, 90 (Supl.) 180; 1997. Acta Trop. [In press]). These studies have shown that the course of the infection and the associated pathology of experimental infections is similar to that observed in man, especially for the commoner species such as L. (L.) amazonensis and L. (Viannia) braziliensis. It is also important to remember that there is no suitable rodent model for the latter species.

One of the unquestionable advantages is that besides being able to compare the immunological responses of infections and vaccines is that the CCLM model also allows cross immunity studies of a wide range of taxonomically very different parasites. It has been shown that in man L.(V.)panamensis protected against L.(L.)mexicana, but not vice versa (Lainson & Shaw, 1966. Trans. R.. Soc. Trop. Med. Hyg., 60: 533-535). Similar observations have been found in experimental infections of Cebus apella and in man L.(V.)panamensis and L.(L.)amazonensis did no protect against L.(V.) braziliensis. (Lainson & Shaw, 1977. J .trop. Med. Hyg., 80: 29-35). More recently it has been shown that infections of L.(V.) lainsoni in capuchins does not protect against L.(V.) braziliensis (Silveira et al., 1989. Revta Soc. Bras. Med. Trop., 22:125-130; Silveira et al., 1997 [unpublished observations). Cross immunity studies have so far shown that parasites of the subgenus Leishmania do not protect against parasites of the subgenus Viannia but certain species of the latter subgenus may protect against species of the former. Although cross immunity exists between different species but it may not be reciprocal. For instance L.(V.) braziliensis protected against L.(L.)amazonensis but not L.(V.) guyanensis although L.(V.) guyanensis did protect against L.(V.) braziliensis. The latter result may be related to the way in which different T cell subsets are stimulated by different parasites. Evidence for this has been shown in mice in which different regulatory control mechanism occurred during infections of L..(L.) mexicana and L..(L.)major (Alexander & Kaye, 1985. Clin. Exp. Immunol., 61: 674-682).

In the laboratory a vaccine of dead promastigotes of L(V.)braziliensis protected dogs against infections of L.(L.)chagasi (Genaro et al., 1996. Mem. Inst. Oswaldo Cruz, 91 [Suppl., I]: 166 ), however, this same antigen failed to protect dogs when used in a field trial in the interior of Minas Gerais (Mayrink, et al., 1996. Mem. Inst. Oswaldo Cruz, 91: 695). This is one of the numerous examples in which laboratory experiments have been successful but field trials have failed.

It is generally commented that it does not matter what antigen you use for detecting humoral or cell mediated responses in leishmaniasis. This is, however, untrue and we have clearly shown that in Cebus apella both serum titres and cell mediated reactions are greater when the homologous antigen is used (Garcez et al., 1994. Revta Patol.. Trop. 23(Supl. II), 335; 1996. Mem. Inst. Oswaldo Cruz, 90 (Supl.) 180; 1997. Acta Trop. [In press]). In some situations if the homologous antigen is not used antibody responses will not be detected. It is important therefore that this is kept in mind when the responses to vaccines are being accessed.

Unpublished observations using small numbers of animals have shown that a number of factors may influence the development of a successful protection in Capuchin monkeys. The site of inoculation may influence the development of patent lesions which also effects the efficiency of the subsequent protective immunity. The development of patent lesions depends upon the size of the inoculum and shifts in the peak and intensity of both cell mediated and humoral responses in relation to lesion size have also been observed (Garcez et al.,1996. Mem. Inst. Oswaldo Cruz, 90 (Supl.) 180). Such modulation of the immune response by different concentrations of parasites is possibly very important in experimental models. Recently Menon & Bretscher (1996. Eur. J. Immunol, 26: 243-249 ) protected BALB/c mice with a low-dose of live L.(L.) major promastigotes. They suggested that this surprising result may be because the Th1 and Th2 components are differentially stimulated in accordance with the state of the memory cells that are activated when challenged.

Cross immunity studies between species of Leishmania in mice have not been as extensive as those in monkey. Experimental infections of L.(L.) major in mice protected against L.(L.) mexicana (Alexander, 1988. Parasitology; 96: 297-302 ) and in man L.(L.) major also immunises against L.(L.) mexicana.

So far varying degrees of protection against subsequent clinical manifestations in Cebus apella has been associated with the healing of active lesions. Some workers are critical of the use of non-human primate models because they consider the immune status may not relate to that observed in man. This is undoubtedly a valid argument but one can also argue that if an aspirant vaccine protects capuchin monkeys then its chances of protecting man would be greater than one that did not. Finally we must remember that there are examples in which experimental trials in animals and man gave protection but field trials using the same vaccines failed. Mice models have been responsible for tremendous advances in our understanding of the genetic control of leishmanial resistance and the complex interplay between the populations of Th1 and Th2 cells associated with disease and protection. However, non-human primate models will undoubtedly play a basic role in the evaluation and development of leishmanial vaccines for man.

The degree of cross immunity between the commoner species such as L.(L.)chagasi and L.(V.) braziliensis and their geographical races needs to be investigated. It is encouraging that parasites of one subgenera protect against those of the other and it suggests that antigens exist which are capable of promoting a broad spectrum of protection. However, it is clear that vaccines will be regional particularly for cutaneous leishmaniasis.

RT10 - ^{MOLECULAR ENTOMOLOGY AND VECTORS}

THE MOSQUITO MIDGUT INVASION BY THE PLASMODIUM MALARIAL PARASITE

Paulo F. P. Pimenta(1)^*, Mohammed Shahabuddin(3) and Sheila T. Pereira(2)

(1) Centro de Pesquisas René Rachou - CPqRR, Fundação Oswaldo Cruz - FIOCRUZ, POBox 1743. Av. Augusto de Lima 1715, Belo Horizonte-MG, 30190-002, Brasil Fax: [phone] e-mail: [e-mail]

(2) Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Univesidade Estadual do Norte Fluminense, UENF, Campos dos Goytacazes, RJ, 28025-620, Brasil

(3) Laboratory of Malaria Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 USA

Malaria, a tropical disease transmitted by bites of blood feeding mosquitoes was discovered one hundred years ago by Ronald Ross in 1897. After the ingestion of infected bloodmeal by mosquito vectors, the parasites within the mosquito midgut, transform in gametes that fertilize to produce motile zygotes or ookinetes. Ookinetes penetrate the first barrier, the peritrophic matrix and cross the epithelium in direction to the haemocoel side where beneath the basal lamina become round and transform into oocyst. Mature oocysts produce sporozoites which are liberated into haemocoel. Then, the sporozoites invade the salivary glands to be injected into vertebrate hosts during the next blood feeding, completing the life cycle of malaria parasites in the mosquito vector. Thus, malarial parasite has to cross two organs during its route within the mosquitoes, the midgut and the salivary gland. Only recently, the process of sporozoite passage through the salivary gland was morphologically characterized (Pimenta et. al. 1994). Therefore, until today it is not clear how they invade the midgut. There are several speculations regarding the way by the ookinetes attach and pass throught the midgut epithelium from the luminal side to the haemocoel side. Our purpose is to study the invasion of the midgut epithelium by the parasites and know how they cross it in direction to the haemocoel. Previous studies (Meis et. al. 1987, 1989, Torrii et. al. 1992) of the ookinete invasion of mosquito midgut utilized infected blood-fed mosquitoes, which is inefficient to follow the parasite invasion, because the process is asyncronous only few parasites are observed within the epithelial cells at the time of invasion. Therefore, to examine several infected cells by the parasites, we developed an in vitro interaction method with dissected Aedes aegypti midguts and Plasmodium gallinaceum ookinetes. We centrifuged 20 hour old ookinetes onto midgut sheets prepared from blood-fed female mosquitoes one day after the bloodmeal. The parasites were allowed to interact with the midgut cells in periods varying from 0 to 30 minutes. The parasite-exposed epithelial tissues were examined for bound and invading ookinetes by distinct morphological methods. Midguts were fixed and processed for histological and ultrastructural observations. For the histological obsevation, thick epon-sections were stained with toluidine blue and observed on light microscope. Examination of these sections demonstrated several ookinetes attached over the exposed area of the midgut epithelium immediately after stopping the interaction experiment by fixation. Actually, parasites were seen over the microvilli of the epithelial cells 5 minutes after the interaction. A large number of these parasites were attached to the microvilli by the anterior side. Besides the ookinetes were attached over the microvilli of several epithelial cells, they were seen entering a few cells. Interestingly, we notice that the invaded cells were different from the majority of the collunar epithelial cells presented in the midgut epithelium. We clearly observed that the ookinete invades a sub-population of cells poorly stained with toluidine blue. Usually, the collunar epithelial cells are stained darkly with toluidine blue. We found that this sub-population of cells represents about 26% of the epithelial cells in the mosquito midgut. Most of these target cells were invaded by more than one parasite. Scanning and transmission electron microscope identified several distinct aspects of this special type of cell preferentially invaded by the parasites. There is no or few microvilli exposed on the surface of these invaded cells. They are taller than the collunar epithelial cells (20-30 micrometers) projecting their apical surface into the midgut lumen. The cytoplasm is poor in endoplasmic reticulum, rich in vacuoles, and electron lucent when compared with the other epithelial cells. These specific features show that these cells are different to and do not comprise the majority population of midgut epithelial collunar cells. The collunar cells have the surface completely covered by microvilli, and large amount of endoplasmic reticulum in the cytoplasm, aspect characteristic of cells metabolically involved in the meal digestion and secretion of digestive enzymes. Following this observation we characterized the parasite invasion and its journey through the target cell. We clearly observed that the ookinete starts to enter the apical surface of the target cell by the anterior ends, forming a tight junction with the epithelial cell membrane. We also saw that as soon the parasite entered the host cell, it become positioned inside a vacuole. This vacuole appears to be formed by the compression of the host plasma membrane that complies with the shape of the parasite. Ookinetes well located within the epithelial cell, distant from the apical surface, were free in the cytoplasm without a surrounding vacuolar membrane. As the invasive process ends with the ookinete arriving in the basal portion of the invaded cell, the parasites were seen together forcing toward the basal plasma membrane by the anterior ends. Finally, some "rounded" parasites were seen underneath the basal lamina in recent formed oocysts. This is the final step of the midgut invasive process. In conclusion, we got a very clear idea about the mode that malarial ookinetes invade the mosquito midgut cells. This paper marks an important step in the study of the malaria parasite development in the mosquito, with the finding that the ookinete invades a specific cell type in the midgut. It is also important the first description of the whole process of the midgut invasion by the ookinete. One of the important area of study in molecular entomology is to find tools capable to make malaria refractory vectors and find molecular targets for transmission-blocking vaccines. A step toward in the identification of a possible receptor is the realization that a special cell is involved in the midgut invasion by the parasite and how this invasion occurs.

Supported by PRONEX, CNPq, FAPERJ and FENORTE

"This investigation received financial support from the UDNP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR)".

TRANSGENIC MOSQUITOES AND MALARIA: INITIAL EXPERIMENTAL APPROACHES

Valle, D.(1), Monnerat, A. T.(1), Hamedi, A.(1), Almeida, R. W.(1), Lourenço de Oliveira, R.(2), Pereira Lima, J. B.(4), Soares, M. J.(3), Freitas-Sibajev, M. G. R(1) & Galler, R.(1)

Depto. de Bioquímica e Biologia Molecular (1), Depto de Entomologia (2) and Depto. de Ultra-estrutura e Biologia Celular (3), Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365, Manguinhos, Rio de Janeiro-Brasil-CEP21045-900 and U.S. Army Medical Research Unit, Instituto de Biologia do Exército, Rio de Janeiro-Brasil (4)

Supported by FIOCRUZ, CNPq and European Community

It is estimated that 35% of all the world population is exposed to malaria, one of the major known infectious diseases, responsible for 2 million deaths per year.

Molecular Biology provided an alternative strategy that can ultimately lead to an effective malaria control through the genetic manipulation of anopheline vectors. This strategy is based on the construction and field release of transgenic mosquitoes, carrying genetic information rendering them refractory to Plasmodium infection. Reduction of vectorial capacity could thus be used as an alternative to the control of mosquito vector population.

Several laboratories around the world are trying to answer different questions directly related to the establishment of refractory mosquitoes, namely:

What? (to define mechanisms that confer mosquito refractoriness to Plasmodium)

How? (to identify a transposable element that is functional in mosquitoes, able to carry out the integration of exogenous information)

Where? (to characterize promoters of genes expressed at the organ/tissue and moment where the parasite/vector interaction first takes place, that is, inside the mosquito gut after a blood meal)

Concerning this last question, functional characterization of gene promoters encoding trypsins and chymotrypsins from both Anopheles gambiae and Anopheles stephensi is being carried out. To attain this goal transgenic Drosophila melanogaster and mosquito MOS20 cells are being used as expression systems. In our laboratory one of the research lines deals with the characterization of homologous genes in neotropical malaria vectors (Anopheles darlingi and Anopheles aquasalis). Our aim is to help in the identification of the relevant cis promoter elements of these genes, their limits and organization, in order to characterize promoters that can be used in a broadly applicable strategy.

Thus, genes coding An. darlingi and An. aquasalis chymotrypsins have been isolated and sequenced. Similarly to An. gambiae, neotropical anophelines contain 2 chymotrypsin genes, each one with 2 small introns, related in size and position to those from the African vector. Each coding unit has approximately 1 kb and, at least for An. aquasalis, both chymotrypsin genes are transcribed in the same direction. Aminoacid sequence comparison between neotropical chymotrypsins shows a high level of identity (66 to 88%) and similarity (73 to 90%). When these sequences are compared to the homologous genes from An. gambiae, the values are still high: 60 to 70% of identity and 68 to 78% of similarity.

Regarding neotropical anophelines trypsins, attempts to isolate their respective genes from genomic libraries with An gambiae derived probes were not successful. Two different approaches are being evaluated: 1) the construction of cDNA libraries in an expression vector followed by immunological screening with antibodies directed against An. gambiae trypsins and 2) the RT-PCR amplification, using primers corresponding to trypsins coding regions with sequences conserved between An. gambiae and An stephensi.

Besides the three questions mentioned above, other research lines have been implemented in order to develop Plasmodium refractory mosquitoes. One of these lines is the Anopheles genomic organization study, which will enable monitoring the different transposable elements insertions and the other one is the generation of mutants to be utilized as selection markers of this novel methodology.

Concerning transgenic mosquitoes, knowledge relative to the embryonic development is also extremely important, although it has been surprisingly neglected. Transgenic mosquitoes are obtained by exogenous DNA injection into embryos and its integration in the polar cells. Hence, detailed knowledge of early embryogenesis, putting into evidence time and position of pole cells formation, is essential. Knowledge of wild mosquito embryogenesis as a whole will also be important in the characterization of mutants that are being currently isolated and of transgenic lines to be generated by the transposable element insertion or mobilization, once it is available. In this regard we have been trying to characterize the embryonary development of neotropical anophelines. This work is possible due to the recent availability of An. aquasalis and An. albitarsis autonomous colonies, but the melanized and sclerotized nature of anopheline egg chorion is proving to be a problem. Some attempts to render anopheline embryos observable inside the egg, to define the major morphogenetic movements characteristic of embryogeny as well as to permeabilize these embryos will be discussed.

MOLECULAR SYSTEMATICS OF LUTZOMYIA WHITMANI AND RELATED VECTORS OF LEISHMANIA IN BRASIL

Ready, P.D.

Molecular Systematics Laboratory, Department of Entomology, The Natural, History Museum, Cromwell Road, London SW7 5BD, United Kingdom

Before the widespread introduction of molecular and cladistic approaches (Hillis et al., 1996), G.G. Simpson defined systematics as ' ... the scientific study of the kinds and diversity of organisms and of any and all relationships among them' (Mayr, 1969). That is to say, systematics should involve the utilitarian aim of constructing practical classifications for identification and information retrieval, as well as the more theoretical study of species relationships in a broad phylogenetic sense. Systematics also overlaps population genetics, because there are several "species concepts", including not only the "biological" one that stresses reproductive isolation but also the "phylogenetic" and "typological" ones. Each concept has its limitations for classifying haematophagous insect vectors of human disease and, often, no single classification permits us to predict accurately the distribution and inheritance of traits of epidemiological importance. DNA sequence analysis is increasingly applied to insect vector systematics, and this approach has the advantage of generating large numbers of genotypic characters that usually can be unambiguously scored. DNA ("molecular") tools can certainly help us to analyse the genetic programme that underlies reproductive isolation or other barriers to gene flow among individual members of populations. However, there is no reason why the routine use of DNA methods alone should resolve conflicting classifications that arise from different species concepts. Sandfly molecular systematics exemplify some of these points. Moderately repetitive DNA sequences have been used as probes for the identification of sister species and geographical populations of sandflies that have different vectorial roles (Ready et al., 1991; Rangel et al., 1996). Mitochondrial DNA is usually maternally inherited and does not recombine, and so it is appropriate for studying the phylogeography of sandfly vectors in order to relate their geographical and ecological distribution patterns to those of Leishmania species and strains (Esseghir et al., 1997). This "landscape epidemiology" of leishmaniasis is also being studied using vertically transmitted transposon sequences of Phlebotomus species (Booth et al., 1996). Our research on the behavioural genetics of Lutzomyia whitmani is aimed at understanding the distribution and evolution of peri-domestic transmission of human cutaneous leishmaniasis in Brasil. The number of sibling species of Lu. whitmani is being assessed by analysing geographical and local variation in mitochondrial and nuclear DNA sequences. First results indicate support for three major lineages or phylogenetic species (Ready et al., 1997). One lineage was found in the Atlantic Forest zone of the North East of Brasil, including the species' type locality, and is distinct from a second lineage located in the drier interior of Brasil, stretching from the Tropic of Capricorn to just outside Amazonia. The ranges of both lineages include populations incriminated in the peri-domestic transmission of Leishmania braziliensis. A third, Amazonian, lineage is rare in peri-domestic sites and is incriminated in the transmission of Leishmania shawi, rather than Le. braziliensis. However, the evolution of peri-domestic transmission may not be easily explained, because there is strong evidence for introgression between Lu. whitmani and Lu. intermedia, another vector of Le. braziliensis classified in the subgenus Nyssomyia (Marcondes et al., 1997). Gene flow between these sandfly species could have involved the exchange of behavioural traits of epidemiological importance.

Acknowledgements: This work was supported by CAPES, EU (CEC/DGXII), FNS,

Fundacao Oswaldo Cruz and the Wellcome Trust.

References

1. Booth, D.R., Ready, P.D. & Smith, D.F. (1996) Evolution of multiple families of non-LTR retrotransposons in phlebotomine sandflies. Genetical Research, Cambs 67, 227-237.

2. Esseghir, S., Ready, P.D., Killick-Kendrick, R. & Ben-Ismail, R. (1997) Mitochondrial haplotypes and phylogeography of Phlebotomus vectors of Leishmania major. Insect Molecular Biology 6, 211-225.

3. Hillis, D.M., Moritz, C. & Mable, B.K. (1996) Molecular Systematics. 2^nd Edition. Sinauer Associates Inc., Sunderland, Mass., U.S.A.

4. Marcondes, C.B., Day, J.C. & Ready, P.D. (1997) Introgression between Lutzomyia intermedia and both Lu. neivai and Lu. whitmani, and their roles as vectors of Leishmania braziliensis. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, in press.

5. Mayr, E. (1969) Principles of Systematic Zoology. McGraw-Hill, New York, U.S.A.

6. Rangel, E.F., Lainson, R., Souza, A.A., Ready, P.D. & Azevedo, C.R. (1996) Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera: Psychodidae, Phlebotominae). Memorias Instituto Oswaldo Cruz 91, 43-50.

7. Ready, P.D., Day, J.C., de Souza, A.A., Rangel, E.F. & Davies, C.R. (1997) Mitochondrial DNA characterization of populations of Lutzomyia whitmani (Diptera: Psychodidae) incriminated in the peri-domestic and silvatic transmission of Leishmania species in Brazil. Bulletin of Entomological Research 87, 187-195.

8. Ready, P.D., Lainson, R., Shaw, J.J. & Souza, A.A. (1991) DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae). Memorias Instituto Oswaldo Cruz 86, 41-49.

BIOCHEMICAL AND MORPHOLOGICAL CHARACTERIZATION Of THE ANOPHELES DARLINGI SALIVARY GLANDS

Marinotti, O., Moreira, C. K., and Bijovsky, A.T.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900, São Paulo, Brasil

Anopheles darlingi is the main malaria vector in Brazil. This mosquito species carries both Plasmodium falciparum and Plasmodium vivax, the two clinically most important human malarias. Although just a few studies on the relationship between the malaria parasites and Anopheles darlingi have been done.

In order to study this relationship we initiated our work by characterizing morphologically and biochemically the salivary glands of these mosquitoes.

The adult female salivary glands have a medium and two lateral lobes; extra acinar ducts emerging from the anterior part of each gland link to form a common duct that ends at the exterior through the proboscis. The lobes are acinar structures organized as a simple epithelium involved by a thin basal membrane. There is no other structure involving the organ, which is immersed in the hemolymph. Fat body cells are frequently adhered to the gland.

The acinar epithelium surrounds a salivary duct with a cuticle in the proximal portions of the gland. The cellular architecture is very similar among the different portions of the gland. Secretory material appears as large masses pushing most of the cytoplasm and the nucleus to the organ periphery; thin cytoplasmic strips separate the secretory masses.

At the proximal portion of the lateral lobes the secretory masses have a fine filamentous aspect. Cell cytoplasm contains small mitochondria in between the cisterneiform endoplasmic reticulum. Distal portion cells have a reticulate endoplasmic reticulum and denser secretory masses, with a mottled pattern. Nuclei present prominent nucleolus.

The cytoplasm of the medium lobe has abundant cisterns of rough endoplasmic reticulum and numerous plate-like mitochondria. Nuclei are irregular with large nucleolus. The secretory masses are very uniform and extremely electrondense.

A biochemical analysis of the salivary glands revealed apyrase, a-glucosidase and lysozyme activities. The apyrase is accumulated mainly in the distal portions of the gland while the a-glucosidase and lysozyme are detected mostly in the proximal lateral lobes. The differential distribution of the analyzed enzymes indicate an specialization of the different salivary gland portions for sugar and blood feeding.

A cDNA encoding the lysozyme expressed specifically in the salivary glands of Anopheles darlingi, was isolated by differential screening an adult female salivary gland cDNA library with abdomen and salivary gland cDNAs. The primary sequence of the cDNA contains a deduced coding region of 429 nucleotides and 5'- and 3'-end non-transcribed regions. A signal peptide of 23 amino acids and a mature protein of 120 amino acids are evident in the conceptual translation product. The results of RT-PCR experiments indicated that in adult mosquitoes this gene is expressed specifically in the salivary glands. Lysozyme enzymatic activity was detected in the salivary glands and abdomens of adult mosquitoes, but the pH optimum differed for each tissue. and this was interpreted to indicate the presence of more than one enzyme, each being expressed in a different tissue. The salivary gland lysozyme may be involved with protection against bacterial infection in the anterior portion of the mosquito digestive tract. Several other cDNA clones expressed in the Anopheles darlingi salivary glands were selected and will be characterized

Supported by : FAPESP and WHO

RT11 - ^{NON-PATHOGENIC PROTOZOA}

TRYPANOTHIONE-MEDIATED PEROXIDE METABOLISM IN CRITHIDIA FASCICULATA

Henryk M. Kalisz, Everson Nogoceke, Marisa Montemartini, Daniel Gommel, Peter Steinert* and Leopold Flohé*

GBF - Gesellschaftfür Biotechnologische Forschung, Mascheroder Weg I, D-38124 Braunschweig, Germany and *Department of Physiological Chemistry, Technical University of Braunschweig, clo GBF, Mascheroder Weg I, D-38124 Braunschweig, Get-many

Parasitic trypanosomatids, which comprise causative agents of debilitating or life-threatening tropical diseases, such as Chagas disease, African sleeping sickness and leishmaniasis, possess a limited capacity to cope with oxidative stress. They lack both catalase and glutathione peroxidase and contain conspicuously low concentrations of glutathione, the major antioxidant sulphydryl compound in mammalian cells. Instead they form the wúque glutathione derivative, trypanothione (T(SH)2; N',N'-bis(glutathionyl)spen-nidine), which is believed to play a central role in their antioxidant defence system. T(SH)2 can be oxidized by H2O2 to the corresponding disulphide (TS2) and is regenerated at the expense of NADPH by trypanothione reductase. A T(SH)2-dependent peroxidase activity was repeatedly reported for crude extracts of the trypanosomatids. However, a pertinent enzymatic entity could never be purified, doubts about its existence were raised and the non-enzymatic oxidation of T(SH)2 by H2O2 was even concluded to fully account for their slow hydroperoxide metabolism.

We have demonstrated that hydroperoxide metabolism in the trypanosomatid Crithidia fasciculata is enzymatic in nature involving two distinct proteins which catalyse, in a concertei action, the reduction of the hydroperoxides by T(SH)2 at the expense of NADPH. One is Cfl6, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates. The second component is Cf2l, which can be classified as a member of the peroxiredoxin family of proteins. The two proteins have been purified to homogeneity and shown to be essential for the trypanothionedependent removal of hydroperoxides. Selective derivatisation of the substrate-reduced proteins implied that the flux of reduction equivalents fiows from trypanothione to Cfl 6, Cf2l and finally to the hydroperoxide. As a consequence of these findings, we have chosen to terrn Cfl 6 tryparedoxin and Cf2l tryparedoxinperoxidase.

Tryparedoxin peroxidase was demonstrated to be a moderately efflcient peroxidase with broad specificity, efflciently reducing peroxides such as H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. Kinetic analysis of tryparedoxin revealed an enzyme substitution mechanism, with the corresponding molecular event being a reversible oxidoreduction of the disulphide bridge in the thioredoxin-related motif WCPPC. The reduction of tryparedoxin by trypanothione appears to be the rate limiting step in the trypanothione-dependent hydroperoxide reduction. The functional characteristics of tryparedoxin explain the limited capacity of trypanosomatids in coping with oxidative stress and qualify the enzyme as a potential target for the design of specific trypanocidal compounds.

The genes coding for tryparedoxin and tryparedoxin peroxidase have been cloned from C. fasciculata genomic DNA and sequenced in their entirety. The gene encoding tryparedoxin peroxidase has been incorporated into an expression vector and overexpressed in E. coli cells as active enzyme.

ECOLOGICAL RULE OF PROTOZOA IN AQUATIC ENVIROMENT

Mirna Coutinho, Universidade Federal de São Carlos, São Carlos, SP, Brazil.

Abstract not received.

TRYPANOSOMATIDS FROM INSECTS AND PLANTS: ISOLATION, CULTIVATION AND NUTRITIONAL BEHAVIOR

João Evangelista Fiorini

Departamento de Biologia - Universidade de Alfenas - Rodovia MG 179 - km 0 - Alfenas - MG - Brasil - E-mail: [e-mail]

The family Trypanosomatidae of the phylum Protozoa is characterized by a single axoneme and by an organelle known as the kinetoplast, rich in deoxyribonucleic acid (McGhee & Cosgrove, 1980). Trypanosoma and Leishmania spp are well known pathogenic parasites of man and animals (Vickerman, 1976). Some species of Phytomonas have importance as plant pathogens, causing diseases in coffee, coconut, oil palms and cassava (Dollet et al., 1977; Kitajima et al., 1986; Parthasarathy et al., 1976; Stahel, 1931) and tomatoes (Jankevicius et al., 1989; Fiorini et al., 1993). Others trypanosomatids parasitizing insects (Diptera, Hemiptera), including the genera Crithidia, Herpetomonas, Leptomonas and Blastocrithidia, are very common (Wallace, 1966). Trypanosomatids of plants are probably transmitted by the bite of phytophagous hemipterans, as suggested by various experimental and epidemiological evidences (Jankevicius et al., 1988; Brazil, et al., 1990; Itow Jankevicius et al., 1993). Many of these insects are found naturally infected by trypanosomatids (Sbravate et al., 1989) in the digestive tract, haemocoel and salivary glands. These flagellates are also known to occur in laticiferous plants (Dollet, 1984; Attias & de Souza, 1986) and in edible fruits of various plant families (Conchon et al., 1989; Kastelein & Camargo, 1990; Fiorini et al., 1993) of economical importance.

Since the first artificial medium for trypanosomes (Novy et al., 1903) several trypanosomatids have been grown in defined media, starting with that of Cowperthwaite for the insect gut parasite Crithidia fasciculata (Cowperthwaite et al., 1953). Their medium served for several species of trypanosomatids.

In our laboratory we have used the Roitman's complex and defined media (Roitman et al., 1972) for isolation and cultivation of trypanosomatids since 12 years ago. All new organisms are taken from the alimentary tract or salivary glands of insects, latex and sap of plants.

Nymphs and adults of insects, are collected in the urban areas of Alfenas, MG, Brazil. Insects are killed at -20ºC for 5 minutes and washed with a solution of antibiotics. Digestive tracts and salivary glands are screened for flagellates by phase microscopy in one drop of saline plus antibiotics. Infected digestive tracts and salivary glands are transferred to tubes containing diphasic medium made of agar base and 15% rabbit, chicken or duck blood overlaid with Roitman's media containing antibiotics. Whenever molds or yeasts persist as contaminants a migration tube is employed.

Plants are also screened for the presence of Phytomonas by direct microscopic observations. A drop of latex or sap diluted 1:1 with saline is used. Infected plants are selected, carefully washed, air-dried, and taken to a laminar flow hood where the portion to be cut is brushed with iodinated alcohol and allowed to dry. The latex drops or sap are collected in the tip of a sterilized Pasteur pipette and transferred to tubes containing diphasic medium. Cultures are maintained at 28ºC and weekly transferred to new media. Smears fixed in methanol and stained with Giemsa are used for light microscopy, drawings and measurements. Cell growth is evaluated by counting the number of cells using a Neubauer chamber.

To demonstrate the presence of endosymbionts in some isolates the parasites are cultivated in Roitman's defined medium where hemin and adenine were amitted. This is also the case for the trypanosomatids C. deanei (Mundim et al., 1974), C. oncopelti (Newton, 1957), B. culicis (Chang & Trager, 1974), C. desouzai and H. roitmani (Fiorini et al., 1989) and, more recently, for an isolate of Morus (Fiorini et al., 1995).

Furthermore, we also observed the presence of the symbionts by transmission electron microscopy in collaboration with Dr. Wanderley de Souza (IBCCF-UFRJ), Dr. Maurílio José Soares (IOC-RJ) and Prof. Paulo Márcio de Faria e Silva (EFOA - Alfenas - MG). Growth experiments are carried out in the Roitman's defined medium and essentials are identified by omission of single components. Experimental media are distributed 5mL per 18x180mm metal-capped tubes and autoclaved for 15 min. at 118-121ºC. Each tube receives 1 drop of a 48h-old culture in the defined medium. Growth is measured in a hemocytometer chamber or in a spectrophotometer at 540nm. So, requirements for amino acids, vitamins, purine, and heme are investigated.

At present, isolated specimens are being studied in collaboration with Dr. Erney P. Camargo (Depto. Parasitologia - USP) for the purpose of genera identification. In addition, the samples are rotulated and cryopreserved by Dra. Maria Auxiliadora de Souza (IOC-Rio de Janeiro - RJ).

Supported by CNPq, FAPEMIG, UNIFENAS, PRONEX.

References

1.Attias, M. & de Souza, W. J. Protozool., 33(1):84-87, 1986.

2.Brazil, R.P. et al., Mem. Inst. Oswaldo Cruz, 85(2):239-240, 1990.

3.Chang, K.P. & Trager, W. Science, 183:531-532, 1974.

4.Conchon, I. et al. J. Protozool., 36(4):412-414, 1989.

5.Cowperthwaite, J. Ann. NY Acad. Sci., 56:972-981, 1953.

6.Dollet, M. et al. Annu. Rev. Phytopathol., 22:115-132, 1984.

7.Dollet, M. et al. C.R. Acad. Sci. Paris, 284:643-645, 1977.

8.Fiorini, J.E. et al. Cytobios, 75:163-170, 1993.

9.Fiorini, J.E. et al. Mem. Inst. Oswaldo Cruz, 84(1):69-74, 1989.

10.Fiorini, J.E. et al. Mem. Inst. Oswaldo Cruz, 90(Suppl.):247, 1995.

11.Itow-jankevicius, S. J.Euk. Microbiol., 40(5):576-581, 1993.

12.Jankevicius, J.V. et al. J. Protozool., 36(3):265-271, 1989.

13.Jankevicius, J.V. et al. Mem. Inst. Oswaldo Cruz, 83(Suppl.):601-610, 1988.

14.Kastelen, P. & Camargo, E.P. Mem. Inst. Oswaldo Cruz, 85(4):413-417, 1990.

15.Kitajima, E.W. et al. Phytopathology, 76:638-642, 1986.

16.Mcghee, R.B. & Cosgrove, W.B. Microbiol. Rev. 44(1):140-173, 1980.

17.Mundim, M.H. et al. J. Protozool., 21:518-521, 1974.

18.Newton, B.A. J. Gen. Microbiol., 17:708-717, 1957.

19.Parthasarathy, M.V. et al. Science, 192:1346-1348, 1976.

20.Roitman, I. et al. J. Protozool., 19(2):346-349, 1972.

21.Sbravate, C. et al. J. Protozool., 36:543-547, 1989.

22.Stahel, G. Phytopathol. Z., 4:65-82, 1931

23.Vickerman, K. In: Biology of the Kinetoplastida. Lumsden W.H.R. and Evans D. A. eds., Academic Press, New York, v.1, p.1-34, 1976.

24. Wallace, F.G. Exper. Parasitology, 18:124-193, 1966.

MITOCHONDRIA, HYDROGENOSOMES AND THE EARLY EVOLUTION OF EUKARYOTES

T. M. Embley, Dept. Zoology, The Natural History Museum, London SW7 5BD, UK

email: [e-mail]; tel: [phone]; fax: [phone]

Mitochondria are often seen as a defining feature of eukaryotic cells, serving to provide efficient energy generation using oxygen as terminal electron acceptor. However, Diplomonads, Trichomonads and Microsporidia lack functional mitochondria and are hypothesised to have diverged from the main eukaryotic stock prior to the mitochondrion endosymbiosis. They have thus assumed importance as putative relics of a phase of pre-mitochondrion eukaryote evolution. Recent data have now revealed that Diplomonads and Trichomonads contain genes which probably originated from the mitochondrion endosymbiont, leaving Microsporidia as the chief candidates for an extant primitively-amitochondriate eukaryote group. In the first part of my talk I will discuss recent molecular data from our laboratory which suggests that Microsporidia probably once contained mitochondria and may in fact be highly derived fungi rather than primitive deep-branching eukaryotes. Although they lack functional mitochondria trichomonads still retain a double membraned organelle called a hydrogenosome. Hydrogenosomes produce hydrogen through a brief pathway comprising two enzymes: pyruvate ferredoxin oxidoreductase (PFOR) and hydrogenase, linked by an electron transfer protein: ferredoxin. Hydrogenosomes occur in a number of phylogenetically distinct groups the best characterised being trichomonads, ciliates and chytrid fungi, so it appears that they have evolved repeatedly and independently. In the second part of my talk I will discuss recent progress towards elucidating the possible evolutionary origins of hydrogenosomes, and also discuss the current consensus that they may be biochemically modified mitochondria.

RT12 - ^{IMMUNOLOGY III}

EARLY IMMUNE RESPONSE IN HUMAN LEISHMANIASIS

Aldina Barral; Gloria Bomfim; M. C. Almeida, E.M. Carvalho & M. Barral-Netto.

Serviço de Imunologia HUPES-Universidade Federal da Bahia and Centro de Pesquisas Gonçalo Moniz-FIOCRUZ. R. Waldemar Falcão, 121; 40.295-001 Salvador - Bahia, Brazil ([e-mail])

Development of cell-mediated immunity to the parasite is elemental in controlling infection in all forms of leishmaniasis. Factors involved in disease outcome are, therefore, of intense interest in leishmaniasis. Considerable effort has been employed to investigate the dichotomy of Th1 and Th2 cell subsets in leishmaniasis. Aspects such as the type of antigen-presenting cell, the pattern of surface molecules of these cells (B7, ICAM, e.g.), MHC class II haplotypes, cytokines and other products, such as prostaglandins, present during maturation of CD4+ precursors may be of utmost importance in determining the future result of leishmania infection. An initial response that allow Leishmania unimpeded establishment is likely to allow a better multiplication, and stimulation of a Th2 type of response. On the other hand a response that has a predominance of macrophage activating cytokines will keep the parasite in check, and will be linked to a Th1-type of protective response.

Our initial approach to evaluate initial responses in man was to study the responses of lymph nodes (LN) from leishmaniasis patients. We have reported that 66% of untreated CL patients early in their disease have a palpable LN. Patients with enlarged LN had higher specific immune responses than patients without such involvement both at the IgG antibody level and at skin-test responses emphasizing the importance of LN involvement. We had also access to patients with LN previous to any other clinical manifestation of leishmaniasis .Some of these patients cured without treatment, whereas other develop ulcerations. When the cytokine pattern was determined by RT-PCR in cells obtained from the LN all of them exhibited a mixed pattern. LN cells from all patients exhibited a signal for IFN-g and IL-4 mRNA.

Since LN enlargement occurred many days after infection, the next step was to explore the initial response of the human macrophage upon infection by Leishmania. Monocyte-derived human macrophages were infected by L. amazonensis and the production of TNF-a, PGE₂ as well as the expression of co-stimulatory molecules assessed. Uninfected macrophages produced low amounts of TNF-a, with a mean of 18.6 pg/ml (ranging from .13 to 41.9 pg/ml; n = 5) whereas infected cells produced a mean of 282 pg/ml (range: 108.8 to 428.6 pg/ml.

Expression of HLA-Dr, B7, CD11b and ICAM-1 were evaluated by cytofluorimetry on the surface of Leishmania-infected human macrophages, and compared to paired cultures of uninfected cells. Leishmania infection lead to a marked decrease of ICAM-1 expression and less markedly decreases of HLA-Dr and B-7; CD11b levels did not change significantly.

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

CHEMOKINE GENE EXPRESSION IN TRYPANOSOMA CRUZI INFECTED MICE

¹Aliberti, J. C.; ²Talvani, A.; ²Gazzinelli, R. T. & ¹Silva, J. S.

¹Faculdade de Medicina de Ribeirão Preto-USP, Depto de Imunologia, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brasil and ²Depto de Bioquímica e Imunologia, UFMG, Belo Horizonte, MG, Brasil

Although the morphological findings of Chagas' disease have been well described, its pathogenic mechanism has not been completely elucidated. Inflammatory reaction is detected in the vicinity of ruptured pseudocysts, in the parasite inoculation site or in target organs, such as the heart. In the acute phase of infection, the cellular infiltrated consists initially of neutrophils with a gradual increase of monocytes and lymphocytes. Several hypotheses have been formulated to explain the occurrence of myocarditis in the acute and chronic phases of T. cruzi infection. An increasing amount of evidence suggests that the cellular infiltrate is mainly due to the presence of parasites in the inflammatory site. Whereas CD4 T cells are important in the triggering the inflammation in the heart, CD8 T cells appear to be the dominant cells in cardiac tissue from chronically infected animals.

We have previously shown that after the early interaction parasite-macrophages there is production of pro-inflammatory (e.g. IL-1, IL-12 and TNF-a) and regulatory cytokines (e.g. IL-10 and TGF-b) that may modulate the specific immune response as well as the inflammatory infiltrate. Other molecules that certainly play a role mediating or regulating the cell infiltrate are the chemokines, a complex superfamily comprising more than 30 small secreted proteins (8-12 kDa) that exhibit chemotactic effects. To evaluate the role of chemokines in T. cruzi infection, we first studied the expression of the C-X-C chemokines as well as the C-C chemokines by macrophages exposed to T. cruzi or parasite products. The C-X-C chemokines, Crg-2 and KC as well as C-C chemokines, MIP-1a, MIP-1b and RANTES were all induced by infection of macrophage with trypomastigotes. In contrast, expression of the chemokines TCA-3, SDF1-a and SDF1-b were not altered by macrophage exposure to the parasite.

Since we agreed that chemokine gene expression by macrophages exposed to T. cruzi trypomastigotes might help to explain, at least in part, the complex cell-chemokine interaction and, therefore, the inflammatory reaction at the inoculation site of parasites, we have examined the chemokines mRNA expression in cells from the inflammatory exudate formed after intraperitoneal (ip) infection with T. cruzi. The results revealed expression of KC, MIP-2, MIP-1a, MIP-1b, RANTES and JE mRNAs at 12, but not at 24 hs, post-infection (pi). On the other hand, the IFN-g-induced chemokines Crg-2 and Mig, peaked only 72 to 96 hours pi. Accordingly, 12 and 24 h pi we found high neutrophil counts and around 4 to 5 days pi the infiltrate cells were predominantly lymphocytes. Therefore, the pattern of chemokine mRNA expression correlated with the cell type present in peritoneum after parasite injection. Similar pattern of chemokine mRNA expression was observed in the spleen, but high levels chemokine mRNA was maintained until 15 days pi. In the cardiac tissue, expression of MIP-1a and MIP-1b mRNA was not detected, while RANTES mRNA was found 5 to 15 days pi. JE mRNA was detected only on days 13 to 15 pi. KC, MIP-2, LIX, Crg-2 and Mig mRNA expression was detected in the heart tissue on days 5 to 15 pi, concomitantly with the presence of most severe acute mycarditys in T. cruzi infected mice.

We also examined T cells derived cytokines, monokines and chemokines gene expression in heart tissue of mice chronically infected with the Colombian strain of T. cruzi. A clear dominance of IFN-g, TNF-a and IL-10 mRNAs were observed at the different stages of infection. Consistent with these observations, the IFN-g-induced chemokines Mig, Crg-2, JE and RANTES mRNAs were all expressed in high levels in the cardiac tissues of infected animals. The monokines IL-1 and IL-12, and the chemokines MIP-1a, MIP-1b and MIP-2, were expressed mainly at the initial phase, prior to day 30 pi. Altogether, these results suggest that chemokines play a key role in triggering and maintaining of the inflammatory reactions, both at the inoculation site and myocardium.

However, the regulatory role of cytokines controlling chemokine gene expression are poorly understood. Thus, we decide to study the role of different cytokines as modulators of T. cruzi-induced chemokine expression gene expression, both in vivo and in vitro. Since early after infection there is IL-12 secretion, which induces IFN-g production by NK cells, we evaluated how IFN-g influences chemokine mRNAs expression by macrophages exposed to trypomastigotes. Our results results show that IFN-g increases expression Mig, MIP1-b, JE and Crg-2. In contrast, KC and MIP-1a expression was completely blocked. We also investigated the role of other pro-inflammatory or regulatory cytokines which are induced by macrophage exposure to T. cruzi trypomastigotes. The pro-inflammatory cytokines, IL-1b and TNF-a, potentate the expression of the chemokines KC, Crg-2, MIP-1a and JE. The regulatory cytokines, IL-10 and TGF-b inhibited the expression of almost all chemokine tested. The only exception observed was the induction of KC in macrophages by IL-10.

To investigate the regulatory role of IFN-g and TNF-a in vivo, we used IFN-g- (GKO) or TNFRp55-deficient (p55-/-) mice following ip infection with 5.000 trypomastigote forms of T. cruzi (Y or CL strain). The cellular infiltrates observed in the inoculation site in control animals (wt) consisted primarily of neutrophils at the first day pi, and mainly lymphocytes at the seventh day pi. The chemokine expression pattern correlated with the cell type observed. Thus, when neutrophils predominated, the expression of chemokine that attract these cells (KC) was detected and, seven days pi, the expression of chemokines Mig, Crg-2 and RANTES (lymphocytes chemoattractants) was predominant. A distinct inflammatory response was observed in the GKO mice. In these, a predominant neutrophilic and a discrete lymphocytic infiltrate were observed only after five days pi (peaking at seven days pi). The infected GKO mice exhibited strong expression of KC and MIP-2, which may explain the neutrophil migration, RANTES expression at 5 days pi and only a basal expression of Mig and Crg-2. Similar results were found in the cardiac tissue of GKO mice acutely infected with T. cruzi. Interestingly, p55-/- mice did not presented neither neutrophilic infiltrate, or expression of chemokines that attract neutrophis during acute infection. In contrast, a large lymphocytic infiltrate was observed in these animals at second day pi. The early appearance of lymphocytes was associated with high level expression of Crg-2 mRNA. In the heart tissue of infected wt mice, there was expression of Mig, Crg-2 and KC at days seventh pi. However, infected GKO and p55-/- mice did not presented Crg-2 and KC expression, respectively. Conversely, GKO mice expressed only MIP-1a while p55-/- mice expressed MIP-1a, JE and RANTES (after seven days pi). Finally, spleen cells of infected mice exhibited a chemokine expression pattern similar to that observed in the peritoneal cavity cells. In the latter, lymphotactin, SDF-1a and -b were detected after 5 days pi. Altogether, these results suggest that IFN-g, TNF-a and chemokines, play a crucial role in the modulation of the inflammatory response observed during T. cruzi infection and lead us to speculate that a modulation of cytokines and chemokines could result in susceptibility or resistance to the parasite.

Supported by FAPESP, FAPEMIG, CAPES and CNPq.

IDENTIFICATION AND ISOLATION OF ADJUVANT MOLECULES FROM TOXOPLASMA GONDII TACHYZOITES

Ricardo T. Gazzinelli,^1,2 Yasu S. Morita,³ Sara Hieny,⁴ Paul T. Englund,³ and Alan Sher⁴

¹Departament of Biochemistry and Immunology, UFMG; ²Laboratory of Chagas' Disease, CPqRR-FIOCRUZ, Belo Horizonte, MG, Brazil; ³Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD; and Laboratory of Parasitic Diseases, NIH, Bethesda, MD, USA

Toxoplasma gondii is an intracellular pathogen that, as part of its normal life cycle induces a potent cell mediated immunity (CMI) response leading to host resistance (1). However, excessive CMI elicited by microorganisms can cause severe host-tissue damage and death (2). Therefore, studies on the interaction of this parasite with the host immune system therefore provide a unique approach for identifying events that selectively lead to the triggering of host protective CMI. Recent studies indicate that interleukin-12(IL-12) is a key cytokine for initiation of CMI as well as important mediator in regulating disease outcome (3,4). Our studies have demonstrated that T. gondii induce IL-12 synthesis by macrophages both in vivo and in vitro. Upon stimulation with IL-12 and other co-stimulatory monokines (e.g. TNF-a, IL-1b and IL-15) (5,6,7), NK cells produce high levels of IFN-g, which further enhances IL-12 synthesis by macrophages previously stimulated by interaction with this protozoa. The early IL-12 and IFN-g synthesis by macrophages and NK cells, respectively, appear to have an important role in directing the development of CMI. Thus, these cytokines will favor the differentiation of T helper (Th) precursor cells into Th1 lymphocytes (8,9), which mediate CMI. The IFN-g produced by NK cells or Th1 lymphocytes stimulates macrophage effector function forming an important barrier against the replication of tachyzoite forms of T. gondii. A major interest of our groups is to identify, isolate and characterize putative molecules from T. gondii tachyzoites that trigger the synthesis of monokines by macrophages. Defining the chemical nature and structure of such protozoan products may allow us to design artificial compounds that could be used as selective adjuvant for inducing CMI.

Hydrophobic nature of tachyzoite molecules that trigger cytokine synthesis by inflammatory macrophages. In our studies we described a cytokine inducing activity on inflammatory macrophages present on soluble tachyzoite antigens (STAg) or live tachyzoites (5,10). The activity present on STAg was shown to be destroyed by treatment with periodate or proteinase k (10). A more recent experiment suggest a hydrophobic nature of tachyzoite molecules that activate macrophages. Briefly, STAg was submitted to treatment with Proteinase K folowed by butan-1-ol:water partition (2:1). As control, untreated STAg was submitted to similar partition with water saturated butan-1-ol. Our results show that all cytokine inducing activity of the untreated STAg is present in the aqueous phase. In contrast, after digestion with Proteinase K most of the cytokine inducing activity is found in the organic phase. These results were our first indication that glycosylphosphatidylinositol (GPI) anchors from T. gondii tachyzoites could be involved on initiation of cytokine synthesis by inflammatory macrophages.

A protocol that allows purification of GPI anchors (or GPI-linked molecules) has been recently developed by McConville and Blackwell (11). This method is based on a chromathography of hydrophobic interaction, using the resin octyl-Sepharose. The octyl-Sepharose binds to the lipidic part of GPI anchor , and the material bound to the column is eluted in a gradient of propan-1-ol. Similar procedure has been recently used to purify GPI-mucins from T. cruzi trypomastigotes, which are also potent inducers of pro-inflammatory cytokine synthesis by macrophages (12,13). Thus, STAg preparation was loaded into a octyl-Sepharose column. The column was then washed followed by elution with linear gradient of propan-1-ol (15-60%). The flow through as well as 30 ml fractions obtained from propan-1-ol gradient and the final wash were collected, analyzed for propan-1-ol gradient by a reflectometer, protein content by Bradford assay, and for their ability to induce cytokine synthesis by either unprimed or IFN-g primed inflammatory macrophages.

First, our results indicate that there is a total overlap of STAg fractions eluted from octyl-Sepharose that trigger the synthesis of IL-12 and TNF-a. The peak of cytokine inducing activity was eluted between the propan-1-ol concentrations of 28% and 51%, respectively. Although, there was a certain overlap between cytokine inducing activity and protein peaks eluted from ocytl-Sepharose, the protein peak did elute a little earlier between 19 and 40% of propan-1-ol in the 0.1 M ammonium acetate buffer. Although the cytokine responses by IFN-g primed macrophages are higher (specially for IL-12/p40), the same fractions also induced the cytokine synthesis by unprimed inflammatory macrophages.

The cytokine inducing activity present on STAg fractions eluted at from octyl-Sepharose 28 to 51% of propan-1-ol have similar properties of STAg molecules responsible for triggering IL-12(p40) and TNF-a synthesis by inflammatory macrophages. In order to test if similar properties were kept by STAg fractions eluted from octyl-Sepharose, we pooled fractions eluted between 28 and 51% of propan-1-ol (STAg-oS) and submitted them to treatment with Proteinase K followed by butan-1-ol:water partition . As control, untreated STAg-oS were submitted to similar partition with water saturated butan-1-ol. Both organic and aqueous phase were tested in macrophage cultures. The results show that most cytokine inducing activity of the untreated STAg-oS is present in the aqueous phase. In contrast, after digestion with Proteinase K most of the cytokine inducing activity is found in the organic phase.

In our next experiments, we compared the susceptibility to different chemical treatments of IL-12(p40) and TNF-a inducing activity present in STAg, STAg-oS and STAg-oS treated with proteinase K (STAg-oS-PK). Each of the different STAg preparations (i.e. STAg, STAg-oS or STAg-oS-PK) were submitted to either periodate oxidation, nitrous acid deamination and alkaline hydrolysis and tested for their ability to induce IL-12(p40) and TNF-a synthesis by IFN-g primed macrophages. As previously shown (5,13), mild treatment periodic acid that destroys vicinal carbohydrates and myo-inositol resulted in complete abrogation of cytokine inducing activity present in STAg. Identical results were observed in STAg-oS and STAg-oS-PK.

The nitrous acid deamination that cleaves the linkage of glucosamine and myo-inositol from GPI anchor, was also completely effective in destroying any IL-12(p40) or TNF-a inducing activity present on STAg, STAg-oS or STAg-oS-PK. In order to confirm, the results with nitrous acid deamination we supplied to intracellular tachyzoites complete medium containing radiolabeled glucosamine. A STAg preparation was obtained from purified tachyzoites and loaded into octyl-sepharose column. Our results indicate that the peak of glucosamine incorporation in the parasite material bound to the octyl-Sepharose column, coincides with the peak of cytokine inducing activity. In our next experiments we intend to check if the peaks of radioactivity an cytokine inducing activity of STAg-oS-PK also present same hydrofobicity in a Thin Layer Chromatography (TLC).

Finally we submitted each one of these preparations to alkaline hydrolysis that would cleave any acyl linkage of the lipid tails to the phosphoinositol. The alkaline hydrolysis had only a partial effect in destroying both IL-12(p40) and TNF-a inducing activity on IFN-g primed macrophages.

Cytokine inducing activity of STAg co-migrates with a known GPI anchor (Lipid C) from Trypanosoma brucei In our next set of experiments we tried to define the degree of hydrofobicity of STAg glycolipid that displays the cytokine inducing activity on inflammatory macrophages. So we run the STAg-oS and STAg-oS-PK in a TLC plate using the solvent system chloroform:methanol:water 10:3:3. As known markers we used radiolabeled Lipid A and Lipid C GPI anchors from T. brucei as well as myristic acid. The TLC plate run for a total lengh of 8 cm and was devided into 9 fractions of 1 cm; fraction 1 started 0.5 cm before loading spot and fraction 9 finished 0.5 cm after front of TLC running. Each fraction was scraped individualy and extracted three times with buthanol and water, dried and tested in their ability to trigger the synthesis of TNF-a by inflammatory macrophages primed with IFN-g. Before scraping the 9 fractions from STAg-oS-PK we stained the TLC plate with iodine. In different experiments we consistently observed four major spots stained by iodine in the STAg-oS-PK and two spots on the STAg-oS preparations. Although not visible by iodine staining, most of the cytokine inducing activity present on STAg-oS-PK was observed on fraction 4 and co-migrated with Lipid C. In different experiments the cytokine inducing activity was seen in fractions 4 and 5 from STAg-oS-PK run in a TLC. In contrast, most of the cytokine inducing activity was present on fraction 1 from TLC plates run with STAg-oS.Previous studies (13,14,15) suggest that GPI anchors extracted from Plasmodium falciparum and Trypanosoma cruzi trypomastigotes are potent inducers of cytokine synthesis by murine inflammatory macrophages. Our data are consistent with the hypothesis that the glycolipids present on STAg that trigger the cytokine synthesis by macrophages are GPI anchors covalently linked to tachyzoite proteins.

References

1) Gazzinelli, R.T, Denkers, E.Y. and Sher, A. Infect. Agents Dis 2:139-149.

2) Gazzinelli, R.T., Wysocka, M., Hieny, S., Scharton-Kersten, T., Cheever, A., Kuhn, R., Muller, W., Trinchieri, G. and Sher, A. 1994. J. Immunol.157:798-805.

3) Biron, C.A. and Gazzinelli, R.T. 1995. Curr. Opinion Immunol. 7:485-496.

4) Gazzinelli, R.T. 1996. Mol. Med. Today 2:258-267.

5) Gazzinelli, R.T., Hieny, S., Wynn, T., Wolf, S., Sher, A. 1993. Proc. Natl. Acad. Sci. USA. 90:6115- 6119.

6) Hunter, C.A., Chizzonte, R., Remington, J. 1995. J. Immunol. 155:4347-4354.

7) Carson, W.E., Ross, M.E., Baiocchi, R.A., Marien, M.J., Boiani, N., Grabstein, K., Caligiuri, M.. 1995. J. Clin. Invest. 96:2578-2582.

8) Seder, R.A., Gazzinelli, R.T., Sher, A. and Paul, W. 1993. Proc. Natl. Acad. Sci. USA. 90:10188-10192.

9) Hsieh, C.S., Macatonia, S.E., Tripp, C.S., Wolf,S., O'Garra, A. and Murphy, K.M. 1993. Science 260:547- 549.

10)Grunvald E, Chiaramonte M, Hieny S, Wysocka M, Trinchieri G, Vogel S, Gazzinelli RT and Sher A. 1996. Infect. Immun. 64:2010-2018.

11)McConville, M.J. and Blackwell J.M. 1991. J. Biol. Chem. 266:15170-15179.

12)Almeida I.C., Ferguson M.A., Schenkman S. and Travassos L.R. 1994. Biochem. J. 304:793-802.

13)Camargo, M.M., Almeida, I.C., Pereira, M.E.S., Ferguson, M.A.J., Travassos, L.R., and Gazzinelli, R.T. 1997. J. Immunol.; 158: 5980-5991.

14) Camargo, M.M., Andrade, A.C., Almeida, I.C., Travassos, L.R., and Gazzinelli, R.T. 1997. J. Immunol. (In press).

15) Schofield, L. and Hackett, F. 1993. J. Exp. Med. 177:145-153.

Supported b CNPq/PADCT (#62.0106/95-6-SBIO), CNPq (#522.056/95-4) and NIH (#AI27608)

CYTOKINE AND ANTI-CYTOKINE THERAPY IN THE INDUCTION OF A TH2 TO TH1 SWITCH IN EXPERIMENTAL MURINE CUTANEOUS LEISH-MANIASIS

J. Farrel

Abstract not received.

RT13 - ^{MOLECULAR BIOLOGY II}

PROTEASOME AND ITS GENES IN TRYPANOSOMA CRUZI

Martins de Sá, C., Bartholomeu, D. C., Alves, S. A., Faria, L. O., Batista,J. A. N., Vainstein, M and Lima, B. D.

Depto de Biologia Celular, Universidade de Brasilia, 70910.900 Brasilia-DF. BRASIL

Supported by CNPq , FAP-DF

The 20S Proteasome is the major celular proteinase complex of all eukaryotic cells so far analysed. The complex is a dimer consisting of four stacked rings, each contaning seven subunits. In eukaryotes several different proteasomal subunits have molecular masses ranging from 21 to 35 kDa. The eukaryotic proteasome subunits can be divided into two classes: alpha-type or beta-type based on their homology to the two differents subunits alpha and beta, of the 20 S proteasome of the archeon Thermoplasm acidophilum (1). The unique arrangement of the 28 subunits is determined by specific amino acid sequences following a multistep assembly pathway (2). This particle play a major role in nonlysosomal proteolysis via the selective degradation of intracellular proteins, mostly by ATP/ubiquitin-dependent as a catalytic core of the 26S proteinase complex and also ubiquitin-independent pathways (1). The proteasomes are involved in the turnover or activation of transcription factors (3), apoptosis (4), in cell cycle control (5) and the generation of antigenic peptides which are presented at the cell surface by MHC class I molecules (6). In this last process, two beta-subunits, LMP2 and LMP7, which are encoded in the MHC locus and are induced by gamma-interferon, a lymphokine which plays a crucial role in the cellular immune response towards pathogens, are implicated in antigens processing (7). Aiming to learn about the structure and biological function of 20S-proteasome in low eukaryotic systems a genetic and molecular study of proteasome em Trypanosoma cruzi has been undertaken in our lab. Previously, a systematic analysis of this particle was made in various species of trypanosomatids. We have shown that the trypanosomatids proteasomes are relatively simple as compared with their mammalian counterpart. Additional works in our lab led to the cloning of one cDNA family encoding T. cruzi proteasomal alpha type subunit. Here, we repport on the characterization of their genomic clones, delimitation of the transcripts and deduced amino acids sequences. T. cruzi proteasome subunit contain most of the proteasomes boxes sequences described for every proteasomal proteins. These observations allowed us to test for the possibility of the human counterpart subunits to be assembled in T. cruzi proteasome. To test this, we have transfected T. cruzi with two human alpha type subunits, HsPROS27 and HSPROS30. Expression of these subunits did not result in the formation of proteasome-like particles, but HsPROS27 was found as a 13S intermediate complex. These results would be in agreement with the notion that proteasome assembly in eukaryotic cell is more complex than in archaebacteria (8).

Another topic of interest in our lab concerns the proteasome expression in host cells upon T. cruzi infection and gamma-interferon treatment. These preliminary results will be also discussed.

References

1- Coux, et al. (1996) Annu. Rev. Biochem. 65, 80-847

2- Schmidtke, et al. (1997) J. Mol. Biol. 268, 95-06

3- Ciechanover, (1994) Cell 79, 13-21

4- Drexler, (1997) Proc. Natl. Acad. Sci. USA. 94, 885-860

5- Murray, (1995) Cell 81, 149-152

6- Rock, et al (1994) Cell 78, 761-771

7- Fruh, et al (1994) EMBO J. 13 (14), 3236-3244

8- Zwickl, et al (1994) Nat. Struct. Biol. 1, 765-770

THE Ca²⁺-SIGNALING AND INVASION PHENOTYPE OF A TRYPANOSOMA CRUZI OLIGOPEPTIDASE B NULL MUTANT

Barbara Burleigh, Elisabet Caler and Norma Andrews

Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT. 06520 USA

Infective forms of Trypanosoma cruzi are able to induce transient increases in the intracellular free calcium concentration [Ca²⁺]i in non-phagocytic mammalian cells (Tardieux et al. 1994). Parasite-induced [Ca²⁺]i-transients, which are due to inositol-3-phosphate mediated release of Ca²⁺ from intracellular stores (Rodriguez et al. 1995), are important for T. cruzi invasion. Prevention of intracellular [Ca²⁺]i transients in host cells by pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, or by buffering intracellular Ca²⁺ with MAPTA-AM, results in a significant decrease in the levels of invasion by trypomastigotes (Tardieux et al. 1994; Rodriguez et al. 1995).

A T. cruzi Ca²⁺-signaling activity demonstrated in soluble trypomastigote extracts following disruption of the parasites by sonication was found to be heat labile and sensitive to proteolytic degradation (Tardieux et al. 1994). Further analysis revealed that the Ca²⁺-signaling activity was tightly coupled to the activity of a parasite peptidase. Ca²⁺-transients in NRK fibroblasts were blocked by preincubation of trypomastigote soluble extracts with specific protease inhibitors, including synthetic peptide inhibitors containing arginine in the P1 position (Burleigh and Andrews, 1995). This information was used to identify and purify a candidate peptidase, and a full length cDNA clone encoding the peptidase was subsequently isolated (Burleigh et al. 1997). The nucleotide sequence predicted an 81 kDa polypeptide which contained the GxSxGGzz consensus sequence common to members of the prolyl oligopeptidase family of serine hydrolases (Barrett and Rawlings, 1992). Since the T. cruzi enzyme is most similar to the oligopeptidase B enzymes from Escherichia coli and Moraxella lacunata, with respect to sequence homology (~30%) and substrate specificity (cleavage after basic residues), it has been named the T. cruzi oligopeptidase B (Burleigh et al. 1997; Burleigh and Andrews, in press).

Direct evidence for T. cruzi oligopeptidase B involvement in the generation of host cell Ca²⁺-transients came from antibody inhibition experiments. IgG purified from polyclonal antisera raised to purified recombinant oligopeptidase B (rPEP) inhibited activity of the enzyme in soluble parasite extracts. Immunoinhibition of oligopeptidase B activity resulted in a significant reduction and/or delay in the onset of Ca²⁺-transients in NRK fibroblasts by treated trypomastigote extracts when compared to extracts treated with pre-immune IgG (Burleigh et al. 1997).

Oligopeptidase B alone is insufficient to generate Ca²⁺-transients in mammalian cells: addition of purified enzyme or rPEP to NRK fibroblasts does not lead to signaling. In addition, this enzyme is expressed in all life cycle stages of T. cruzi and not restricted to infective stages that are able to generate Ca²⁺-transients in host cells. Therefore, it has been postulated that oligopeptidase B generates an active Ca²⁺-agonist by processing a stage-specific (invasive stages) precursor molecule. Based on substrate preferences reported for other members of the oligopeptidase B family, this putative precursor is likely to be a peptide of <30 amino acids (Barrett and Rawlings, 1992).

Oligopeptidase B is a cytosolic enzyme (Burleigh et al, 1997) and there is no evidence to suggest that it is constitutively secreted (B. Burleigh, unpublished observations). Thus, in order to address the question of the possible involvement of intracellular oligopeptidase B in Ca²⁺-signaling and invasion by live parasites, oligopeptidase B gene knockout parasites (pep-/-) were generated by targeted gene replacement. The null mutants are viable and capable of undergoing metacyclogenesis. Analysis of soluble extracts prepared from pep-/- metacyclics revealed Ca²⁺-signaling activity. However, this activity was abolished by pretreatment with Z-Phe-Ala-fluoromethyl ketone (Z-Phe-Ala-FMK), a protease inhibitor which does not affect either oligopeptidase B or the soluble Ca²⁺-signaling activity of wild type extracts. Reconstitution of Ca²⁺-signaling activity in Z-Phe-Ala-FMK treated pep-/- metacyclic extracts was achieved by the addition of active recombinant oligopeptidase B. These results confirm the role of T. cruzi oligopeptidase B in the generation of Ca²⁺-signaling in mammalian cells and suggests redundancy whereby an alternate protease/peptidase is able to generate a Ca²⁺-agonist in parasite extracts.

Metacyclics generated from the null mutants were not impaired in motility or attachment to cells, however, they exhibited reduced (~50%) infectivity for NRK fibroblasts. The residual infection by (pep-/-) metacyclics was not reduced by pre-treatment of NRK cells with thapsigargin which depletes intracellular Ca²⁺ stores. This treatment reduced invasion levels of wild type and partial gene knockout (single allele replacement; pep-/-) metacyclics to the level observed for the double knockout. These results support the idea that oligopeptidase B plays an important role in the mechanism by which T. cruzi signals host cells and triggers mobilization of Ca²⁺ from intracellular stores. If this mechanism of Ca²⁺-signaling is impaired, the ability of T. cruzi to invade host cells is also impaired.

Pre-treatment of wild type trypomastigotes with Z-Phe-Arg-FMK, a membrane permeant inhibitor of oligopeptidase B (and cruzipain), resulted in a significant reduction in invasion (Burleigh and Andrews, 1995). If this reduction was due to the inhibition of intracellular oligopeptidase B, it is predicted that pep-/- metacyclics should be refractory to Z-Phe-Arg-FMK pre-reatment, whereas the wild type or partial knockout parasites should be inhibited. When these predictions were tested, the following results were observed: residual invasion of null mutant metacyclics was not reduced by pre-incubation with Z-Phe-Arg-FMK, but invasion of the partial knockout parasites was reduced to the level of the null mutants. In addition, pre-treatment of pep-/- metacyclics with 50 uM Z-Phe-Ala-FMK failed to reduce the level of residual invasion observed by these parasites, suggesting that the Z-Phe-Ala-FMK sensitive enzyme detected in parasite soluble extracts does not contribute to the ability of T. cruzi to generate a Ca²⁺-agonist in intact parasites.

In summary, the Trypanosoma cruzi oligopeptidase B is a key component in the mechanism by which infective stages of this parasite generate a Ca²⁺-signaling activity for host cells. When oligopeptidase B is deleted, parasite viability, motility, growth rate, ³⁵S-methionine incorporation and progression through the life cycle in vitro are unaffected. However, metacyclics generated from oligopeptidase B null mutants are significantly less invasive than the partial knockout or wild type parasites. Our evidence suggests that this reduction in invasive capacity observed in pep-/- metacyclics is linked to the inability of these parasites to induce intracellular Ca²⁺-transients in host cells. This, in turn, suggests that the residual invasion of NRK cells by null mutant metacyclics is independent of host cell intracellular Ca²⁺ stores. Work is in progress to determine if influx of Ca²⁺ from the extracellular medium accounts for the residual invasivenes of pep-/- parasites or if an alternative pathway is involved.

Barrett, A.J. and N.D. Rawlings. 1992. Biol. Chem. Hoppe-Seyler 373:353-360.

Burleigh, B.A. and N.W. Andrews. 1995. J.Biol. Chem. 270:5172-5180.

Burleigh, B.A., Caler, E.V., Webster, P. and N.W. Andrews. 1997. J. Cell Biol. 136:609-620.

Burleigh, B.A. and N.W. Andrews. Handbook of Proteolytic Enzymes. Barrett, A.J., Rawlings, N.D. and J.F. Woessner eds. in press.

Tardieux, I., Nathanson, M.H., and N.W. Andrews. 1994. J. Exp.Med. 179:1017-1022.

Rodriguez, A., Rioult, M.G., Ora, A., and N.W. Andrews. 1995. J. Cell Biol. 129:1263-1273.

This work has been supported by the American Heart Association, CT Affiliate and the NIH.

MOLECULAR EPIDEMIOLOGY OF AMERICAN TRYPANOSOMIASIS IN BRAZIL BASED ON DIMORPHISMS OF rRNA AND MINI-EXON GENE SEQUENCES

Fernandes, O.^{1, 2}; Mangia, R.H.³; Lisboa, C.V.⁴; Souto, R.P.⁵; Pinho, A.P.⁴; Zingales, B.⁵; Coura, J.R.¹; Campbell, D.A.⁶; Jansen, A.M.⁴

1.Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil, 4365, Rio de Janeiro, Brasil, CEP 21045-900, 2. Departamento de Patologia, Universidade do Estado do Rio de Janeiro, 3. Departamento de Bioquímica e Biologia Molecular, IOC; 4. Departamento de Protozoologia, IOC; 5. Departamento de Bioquímica, Instituto de Química, Unversidade de São Paulo, Brasil; 6. Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California, USA.

American trypanosomiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. The domestic cycle results from human-vector contact, due to factors that enable the colonization of artificial ecotopes by the insects that use small rodents, marsupials, and additionally dogs and cats, as food sources. In this latter cycle, the parasite has the possibility of interacting with the human host, causing eventually Chagas disease.

Trypanosomacruzi is not a homogeneous population of parasites; it is composed rather by a pool of sub-populations that circulate in both the domestic and sylvatic cycles. Isolates of T. cruzi derived from different hosts indicate a high heterogeneity in biological parameters and genetic characteristics, reflected both in the nuclear and kinetoplast genome. The initial studies on enzyme eletrophoretic analysis of T. cruzi isolates have determined three different T. cruzi populations: two that predominate in the sylvatic environment (Z1 and Z3) and one that was mainly found in the domestic cycle (Miles et al., Trans. Royal Soc. Trop. Med. Hyg.,71: 217 - 225, 1977; Miles et al., Trans. Royal Soc. Trop. Med. Hyg., 74: 221 - 237, 1980). Recently, increasing the number of studied gene loci revealed by characteristic enzyme electrophoretic profiles, a broad genetic diversity was evidenced among T. cruzi stocks (Tibayrenc & Ayala, Evolution, 42: 277 - 292, 1988). In contrast to the heterogeneity suggested by the isozymic patterns, PCR amplification of sequences from the 24Sa ribosomal RNA (rRNA) gene and from the non-transcribed spacer of the mini-exon gene indicated dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages (Souto et al., Mol. Biochem. Parasitol., 83: 141 - 152, 1996).

In the present study, 157 T. cruzi isolates obtained from humans, triatomines and sylvatic mammalian reservoirs from twelve Brazilian States were analyzed by the rRNA and mini-exon gene typing approaches. The stocks were classified into the two proposed lineages and according to the domestic or sylvatic cycle of the parasite. Data presented provide evidence for a strong association of T. cruzi lineage 1 with the domestic cycle, while in the sylvatic cycle both lineages circulate equally.

Thirty-two T. cruzi stocks from the sylvatic cycle were isolated from captured animals (26 from Golden Lion Tamarins - Leontopithecus rosalia, 2 from opossums, 3 from rodents and 1 from a three-toed sloth) from Poço das Antas Biological Reserve, Rio de Janeiro, where no Chagas disease has been described so far. Interestingly, while lineage 2 is found exclusively among the opossums, rodents and sloth, lineage 1 isolates were all evidenced in the primate isolates. The unequal distribution of the stocks from Poço das Antas Biological Reserve is due to the higher prevalence of T. cruzi infection in the primates (47%) in comparison to the other mammals (rodents - 13% and opossums - 6%).

Molecular typing of human parasites isolates from three well characterized endemic regions of Chagas disease (Minas Gerais, Paraíba and Piauí) and from the Amazonas State, where T. cruzi is enzootic, suggests that in some endemic areas in Brazil there is a preferential linkage between both cycles mediated by lineage 1-stocks.

Our results strongly suggest that the preferential distribution of lineage 1 among humans can be explained by the pre-adaptation of this given sub-population to primates and the possibility for lineage 1 to evade from the sylvatic to the domestic cycle. Further studies on the T. cruzi-primate interaction is still necessary to address the question of the parasitism phenomena regarding American tripanosomiasis.

KINETOPLAST DNA STRUCTURE AND REPLICATION IN T. Cruzi

Lys Guilbride and Paul T. Englund, Dept. of Biological Chemistry, Johns Hopkins Medical School, Baltimore, MD 21205, U.S.A.

Kinetoplastid parasites replicate their kDNA by one of two very different mechanisms, exemplified by those of C. fasciculata and T. brucei. In C. fasciculata, the newly synthesized minicircles are distributed uniformly around the network periphery whereas in T. brucei they are found only in antipodal positions at the network edge. We have recently developed a vivid, rapid, and accurate method for determining the mode and stage of replication of each network within large populations of isolated kDNA from any parasite. The technique involves selective incorporation of a fluorescent nucleotide into the gaps specific to newly replicated minicircles. The pattern and distribution of fluorescence obtained reflects the distribution of newly replicated minicircles within the network and is diagnostic of the underlying replicative mechanism. Using this method we have shown that T.cruzi epimastigotes replicate kDNA by a mechanism very similar to that of C. fasciculata. In other studies we found that the structure of networks in different stages of replication are very similar in amastigotes and epimastigotes. Unexpectedly, the structure of networks in trypomastigotes was very different. Using the fluorescence labeling technique, all the networks incorporate label only very weakly, but uniformly over the entire surface area, showing that the minicircles are almost all covalently closed. EM analysis also showed that trypomastigote networks are much smaller in surface area than those of amastigotes and epimastigotes, and that the minicircle density within trypomastigote networks is very much higher. Studies are in progress to determine whether these differences are the result of changes in network topology or in minicircle copy number.

(Supported by NIH grant GM27608)

RT14 - ^EPIDEMIOLOGY

MALARIA: ANALYSIS OF THE EPIDEMIOLOGICAL PROFILES OF THREE DISTINCT POPULATIONS OF RONDONIA, WESTERN AMAZON, BRAZIL

Luis Marcelo Aranha Camargo

Department of Parasitology, University of São Paulo, Montenegro, RO

The concept of malaria as an epidemiological monomorphic disease is no longer valid. Actually, malaria has a polymorphic epidemiological structure exhibiting region-specific profiles. Accordingly measures for the control of malaria should take into consideration its regional characteristics. The failure of past "universal" control models is a corollary to this assertive. Thus, the planing and adopting of malaria control measures heavily depends on the correct knowledge about its regional and even infra-regional epidemiological profiles.

For 6 years we have been studying and comparing the epidemiological structure of malaria in 3 different populations in Rondonia.

Candeias do Jamary is a 7,000 people village located not far from Porto Velho (25 km) on the banks of the Candeias River. Its economy depends on Porto Velho where most of the Candeias male residents have their jobs either as public servants or construction workers. There is also a small agricultural activity in the surroundings of Candeias. In addition, some adult males work seasonally at various garimpos. Most inhabitants of Candeias are migrants from South- and Northeastern Brazilian States who came to Rondonia in the 1970s.

Fazenda Urupá is a small agro-industrial settlement (~173 inhabitants), in the middle of the forest on the banks of the Preto River. Its population is mainly made up by migrants from the South who came quite recently to Rondonia and who work on the extraction and processing of lumber.

Portochuelo is a riverine village of 200 inhabitants on the banks of the very large Madeira River. In contrast with Candeias and Urupá most of the inhabitants of Portochuelo (94.5%) were born in the Amazonian region. They live on fishing and agriculture. Commerce is negligible and depends on Porto Velho, which can be reached only by boat throughout most of the year.

Malaria in Candeias is hypoendemic and seasonal. Most cases (180-330 new cases/1,000 inhabitants) occur mainly in the dry season, between June and September. There is a predominance of P.vivax over P.falciparum, which varies, according to the year, from 1.22 to 2.08 times. Anopheles darlingi is the almost sole vector. Males over 10 years are the main risk group, which is suggestive of extra-domiciliary transmission. There is a considerable component of imported malaria, that is, malaria acquired elsewhere and brought to Candeias, mainly by workers returning from garimpos. The high degree of genetic polymorphism of P.falciparum strains points in the same direction.

Malaria in Urupá may reach an annual incidence of 970 cases per 1 000 inhabitants, most of them (60%) in the dry season (June-September). P.vivax (60%) predominates over P.falciparum. Although its high incidence rate, only a small segment of the population is at risk: adult males. Malaria in woman is relatively rare and in children is negligible. Most of the infected adult males work in the lumber processing plant, close to the river and to the Anopheles darlingi breeding places. Thus, malaria in Urupá can be regarded as a professional disease. There are very few cases of imported malaria, and the genetic polymorphism of P.falciparum is accordingly low.

Malaria in Portochuelo has an annual incidence of 363 cases per 1 000 inhabitants, most of them occurring in the dry season. P.vivax (60%) predominates over P. falciparum. There is no imported malaria. Most of the cases occur among boys and girls under 15 years, including chlidren less than 1 year old, which is suggestive of intra-domiciliary transmission. Anopheles darlingi seems to be the main vector although there are many other Anopheles spp. in the area.

We have designed, "custom-made", control measures for each one of the localities (they will be presented). Under the supervision of Dr. Juan Salcedo, we have initially tested our protocols in Urupá. In the first year the number of malaria cases dropped 75%. During the same period, the prevalence of P.falciparum went down from 40 to 5%.

Control measures for Portuchuelo are planned to start next year.

As to Candeias, due to the larger size of its population, we will be unable to carry out control measures with our own means.

EPIDEMIOLOGY OF HUMAN VISCERAL LEISHMANIASIS IN THE ENDEMIC AREA OF JEQUIÉ, BAHIA, BRAZIL

Edson Moreira, CPqGM, Fiocruz, Salvador, BA, Brazil.

Abstract not received.

MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS OF TRYPANOSOMA CRUZI, LEISHMANIA, AND OTHER PATHOGENIC MICROORGANISMS

Michel Tibayrenc, ORSTOM/CNRS, Montpellier, France.

Abstract not received.

LEISHMANIA/HIV CO-INFECTIONS, AN EMERGING PROBLEM, A WHO RESPONSE

P. Desjeux, WHO/TDR, Geneva, Switerzland

Abstract not received.

*

To whom the correspondence should be sent.

Publication Dates

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