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The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. We have previously characterized a T. cruzi virulence factor named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family. Single mutant parasite clones (Tc52(+/-)) exhibiting low virulence in vitro and in vivo were obtained by targeted Tc52 gene replacement. In this report, we have extended our study to analyze the immune response and the disease phenotype in Tc52(+/-)-infected BALB/c mice, during the acute and chronic phases of the disease. Significantly lower parasitemia were found in Tc52(+/-)-infected mice, as compared to wild-type parasite (WT)-infected ones. However, the expansion of all classes of lymphocytes and macrophages was similar for both clones. Furthermore, except for IgG2b levels which were higher in the case of WT-infected mice, all classes of Ig presented no significant difference for WT and Tc52(+/-)-infected animals. Interestingly, a lack of suppression of IL-2 production and of T-cell proliferation inhibition was observed in the case of spleen cells from Tc52(+/-)-infected mice. Finally, the pattern of inflammation process was different and characterized as diffused in the case of Tc52(+/-)-infected mice, or presenting numerous foci in the case of WT-infected mice. Localization of the Tc52 protein in tissue sections and infected heart cell primary cultures by immunofluorescence and immunogold labeling, respectively, revealed the presence of Tc52 at the amastigote surface and associated to aggregates within host cell vesicles. Taken together, these results reinforce the notion of Tc52 being a virulence factor playing a role in the phenotype of the immune response associated to the infection and on the course of the disease.

The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. We have recently identified a T. cruzi-released protein related to thiol-disulfide oxidoreductase family, called Tc52, which is crucial for parasite survival and virulence. In vitro, Tc52 in combination with IFN-gamma activates human macrophages. In vivo, active immunization with Tc52 relieves the immunosuppression associated to acute infection and elicits a specific immune response. As dendritic cells (DC) have a central role in the initiation of immune responses, we investigated whether Tc52 may modulate DC activity. We show that Tc52 induces human DC maturation. Tc52-treated immature DC acquire CD83 and CD86 expression, produce inflammatory chemokines (IL-8, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1 alpha), and present potent costimulatory properties. Tc52 binds to DC by a mechanism with the characteristics of a saturable receptor system and signals via Toll-like receptor 2. While Tc52-mediated signaling involves its reduced glutathione-binding site, another portion of the molecule is involved in Tc52 binding to DC. Finally, we report that immunization with Tc52 protects mice in vivo against lethal infection with T. cruzi. Together these data evidence complex molecular interactions between the T. cruzi-derived molecule, Tc52, and DC, and suggest that Tc52 and related class of proteins might represent a new type of pathogen-associated molecular patterns. Moreover, the immune protection data suggest that Tc52 is among candidate molecules that may be used to design an optimal multicomponent vaccine to control T. cruzi infection.

We have recently characterized a novel Leishmania major gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). In the present study we have extended our approach to the analysis of LmS3arp activity on T- and B-cell functions in a murine model. The results presented in this report show that LmS3arp plays a dual role in the regulation of T- and B-cell reactivity. Indeed, we found that injection of the LmS3arp recombinant protein (rLmS3arp) into BALB/c mice induces preferential activation of B cells, as shown by the following criteria: (i) increased expression of CD69 molecules on immunoglobulin M (IgM)-secreting spleen cells, (ii) a considerable increase of IgM-secreting B cells, and (iii) elevated levels of IgM antibodies in the sera of injected animals. Moreover, the IgM antibodies are not specific to the Leishmania antigens but preferentially recognize heterologous antigens like myosin, thyroglobulin, DNA, and keyhole limpet hemocyanin. Furthermore, the strong polyclonal expansion of nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is concomitant with a marked inhibition of T-cell proliferation. Analysis of cytokine production revealed a significant downregulation of gamma interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together, our data suggest that rLmS3arp, through direct or indirect action toward B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response.

An intense suppression of T cell proliferation to mitogens and to antigens is observed in a large number of parasitic infections. The impairment of T cell proliferation also occurred during the acute phase of Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi. A wealth of evidence has accumulated that illustrates the ability of T. cruzi released molecules to influence directly a variety of diverse immunological functions. In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen) and Tc52 (an immunosuppressive protein) released molecules on the host immune system. The gene targeting approach developed to further explore the biological function(s) of Tc52 molecule, revealed interesting unexpected functional properties. Indeed, in addition to its immunusuppressive activity a direct or indirect involvement of Tc52 gene product alone or in combination with other cellular components in T. cruzi differentiation control mechanisms have been evidenced. Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo. Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression. It is hoped that such approaches might allow rational design of tools to control T. cruzi infections.

We have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi-associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild-type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52+/-) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild-type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52+/- single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.

Reports of a wide variation in space and time of the frequency of autogeny in Aedes (Ochlerotatus) detritus (Haliday, 1833) of the Camargue (Delta of the Rhône, Bouches-du-Rhône, Gard, France) led us to make an enzymatic analysis of male and female adults from different larval biotopes. The study showed the existence of two genetically distinct, sympatric populations which are morphologically indistinguishable. By diagnostic enzymes monomorph Got-2, Gpd, the grouping of the individual into two subgroups satisfies a Hardy-Weinberg equilibrium for the polymorphic enzymes. It is concluded that Ae. detritus is composed of a complex of two sibling species provisionally designated by the letters A (Got-2RR, GpdCC) and B (Got-2LL, GpdBB). In the present article, we retrace the history of the binomen Ae. detritus since the original description (sub nom. Culex detritus) to the split into the detritus complex. Certain ecophysiological (steno-eurygamy) and chorological (bioclimatic gradients N-S) criteria show that the sibling species B should be assigned to the taxon described in UK (Holywood, County Down, Ireland) by A.H. Haliday. Species A is here named Aedes (Ochlerotatus) Coluzzii n. sp.

The wing morphometry of a laboratory-bred population of Phlebotomus perniciosus was studied using a semi-automatic, measurement system. The methods used were found to be precise, reproducible and valid for morphometrical studies. Each of several, different wing indices was found to be similar whether the wing was mounted in Hoyer's medium, Euparal or chloral balm. All samples showed similar differences between the wings of males and females, the wings of the males being smaller than those of the females.

In a Moroccan focus of cutaneous leishmaniasis caused by Leishmania tropica, 7,907 female sandflies captured with CDC traps were dissected from summer to autumn 1989. Among species of the genus Phlebotomus, only P. sergenti harbored promastigotes. Eighty-nine strains belonging to the complex L. tropica were isolated. The frequency of vector infection was zero in June, rose to 1.3% in August, and reached 9.9% in October, which indicates that the period of high risk is at the end of the hot season. Out of 89 strains isolated, 74 were completely typed and corresponded to the following four zymodemes: MON-102 (one strain), MON-107 (56 strains), MON-122 (two strains), and MON-123 (15 strains). Only the first two were observed in humans. The distribution of zymodemes MON-102 and MON-107 was very different in humans, dogs, and the vector. In one of the sites surveyed, which was strongly dominated by MON-107, the absence of human cases involving this zymodeme suggests the existence of a wild reservoir.

Ecoepidemiological analysis of a Moroccan focus of leishmaniasis caused by Leishmania tropica revealed considerable enzymatic diversity. Seven zymodemes belonging to the complex were identified in 149 strains isolated from humans, dogs, and the vector Phlebotomus sergenti. Three distinct subgroups were identifiable, two of which were in turn, composed of three "small variant" zymodemes. The diversity appears to be related to the age of the focus, which may have allowed colonization by zymodemes of different geographic origins. Diversification into "small variants" is apparently the result of recent mutation, possibly associated with genetic exchange.

In anautogenous females of the Dipteran Culicidae Aedes detritus (Haliday, 1833), the neurosecretory products synthesized by the A cells of the pars intercerebralis are stored in the pericarya in absence of blood meal; on the contrary, in the autogenous females they are steadily released from emergency till the stages 3-4 of vitellogenesis; a slight storage occurs by the end of vitellogenesis. These facts suggest the important meaning of these cells in the regulation processes of vitellogenesis in Mosquitoes.

The presence of an anautogenous and stenogamic population of Culex pipiens pipiens in Languedoc, France, is reported. The anautogeny was confirmed through 10 generations. A Callot morphogram corresponded to the anautogenous form. These results raise again the question of the type of relationships, genetic or ecological, of the characters classically associated with autogeny.

Ecdysteroid and juvenile hormone titers were determined in the whole body of females of Aedes detritus and A. caspius. Since both hormones were assayed from the same extract, this method allowed determination of their simultaneous variations during egg formation, i.e., from the time the females emerged until the onset of oviposition. A drastic hormonal increase was observed at the beginning of vitellogenesis. This increase occurred as two high and sharp peaks, the first of ecdysteroids and the second, which took place 8 hr later, of juvenile hormones. The two peaks together lasted less than 12 hr, with the highest level at about 3 X 10(-7) mumol/mg fresh tissue. After the juvenile hormone peak, the oocytes entered into stage III/b, the time at which the intensive phase of vitellin accumulation in the eggs begins.

The study of allozymic variations at four enzymatic loci (Est-2, alpha-Gpd, Got-1 and Got-2) in populations of Aedes detritus (Hal.) from 27 localities in Southern France has shown that this species is composed of two kinds of sympatric populations which do not interbreed in nature. Single specimens of Aedes detritus can be attributed to one or other type of population (sibling species) by the genotype at the alpha-Gpd or Got-2 loci (type A populations are homozygous for Got-2R allele and the frequency of the alpha-GpdC allele is higher than 98%; type B populations are homozygous for Got-2L allele and the frequency of the alpha-GpdB allele is higher than 98%). Moreover, allelic frequencies at the Est-2 and Got-1 loci are different in both types of populations. The fact that both kinds of populations coexist in the same pond shows that the isolating mechanism is not an adaptation to the larval environment, but rather involves mechanisms pecular to adults (precopulatory mechanism or interpopulation sterility).

The authors have made the first discovery of Phlebotomus mascittii in Spain. The species was caught in Catalonia in the provinces of Gerona and Barcelona. Altogether 7 individual sandflies (4 male, 3 female) were collected (using oiled paper traps) in four separate sub-mediterranean sites (between 650 and 780 m).

The Phycomycete, Coelomomyces psorophorae Couch, 1945, was found in the south of France in the salt marsh mosquito Aedes detritus (Haliday, 1833). The fungus invades the ovarioles but leaves the follicules intact. At high magnification, (using a scanning electron microscope), the crater-form punctuations adorning the sporangium were seen to result from openings (ostioles) of the network of small canals that traverse the external wall.

The study of the morphology of eggs, larvae, pupae and adults of Aedes excrucians from Germany, Italy and France has led the authors to rise again the binomen Aedes surcoufi (Theobald, 1912) to particularize the european populations. At the same time, the characteristics of the different species of the Aedes excrucians complex are discussed.

The number of gonotrophic cycles of phlebotomine sandflies in a leishmaniasis focus in the Cévennes was determined by the examination of the ovaries. The method was to count the number of dilatations on the ovariolar stalks which showed how many times the females had laid eggs. It was found that females of P. ariasi undergo at least three gonotrophic cycles. From mid-June at the beginning of the season, until the middle of August the proportion of parous females was low. After this time until the end of the season in mid-September, the frequency of 2 parous and 3 parous individuals steadily rose. It is concluded that the end of the summer is the period of maximum risk for the transmission of leishmaniasis in this focus.

Using two sibling species of the Aedes detritus (Haliday, 1833) complex, one autogenous (A) and the other anautogenous (B), the role played by the larval environment on vitellogenesis was confirmed. A poor larval diet reduced the fecondity equally of both autogenous and anautogenous females. With the latter however blood meal ensured the maturation of a not negligible number of ovocytes.