Identificação e purificação de prostaglandinas e leucotrienos produzidos por Paracoccidioides brasiliensis

Pós-graduação em Doenças Tropicais - FMB

Determinação molecular da viabilidade do Mycobacterium leprae: uma comparação com outras abordagens metodológicas

Pós-graduação em Doenças Tropicais - FMB

Molecular detection of Leishmania spp. in road-killed wild mammals in the Central Western area of the State of São Paulo, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hypersensitivity induced in dogs by nymphal extract of Amblyomma cajennense ticks (Acari:Ixodidae)

In general, hosts develop resistance to ticks after repeated infestations; nevertheless, several studies on naturally occurring host-tick interactions were unable to detect resistance of hosts to ticks even after repeated infestations. The purpose of this investigation was to study the type of cutaneous hypersensitivity to unfed nymphal extract of A. cajennense in dogs, which, unlike guinea pigs, do not develop resistance. A first, but no second, peak in skin reaction was observed, suggesting that cellular immunity is an important mechanism of resistance to ticks. This may partially explain why guinea pigs, but not dogs, develop resistance against ticks.

Long-term evaluation of oral gavage with periodontopathogens or ligature induction of experimental periodontal disease in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estrutura da comunidade parasitária de duas espécies de peixes autóctones da RPPN Foz do rio Aguapeí, Alto rio Paraná

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of human TGF-beta on the gene expression profile of Schistosoma mansoni adult worms

Schistosoma mansoni is responsible for schistosomiasis, a parasitic disease that affects 200 million people worldwide. Molecular mechanisms of host-parasite interaction are complex and involve a crosstalk between host signals and parasite receptors. TGF-beta signaling pathway has been shown to play an important role in S. mansoni development and embryogenesis. In particular human (h) TGF-beta has been shown to bind to a S. mansoni receptor, transduce a signal that regulates the expression of a schistosome target gene. Here we describe 381 parasite genes whose expression levels are affected by in vitro treatment with hTGF-beta. Among these differentially expressed genes we highlight genes related to morphology, development and cell cycle that could be players of cytokine effects on the parasite. We confirm by qPCR the expression changes detected with microarrays for 5 out of 7 selected genes. We also highlight a set of non-coding RNAs transcribed from the same loci of protein-coding genes that are differentially expressed upon hTCF-beta treatment. These datasets offer potential targets to be explored in order to understand the molecular mechanisms behind the possible role of hTGF-beta effects on parasite biology. (C) 2012 Elsevier B.V. All rights reserved.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that-at its mature stage-consists of an inner germinal and an outer laminated layer (GL ; LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host-parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta+CD4+T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8+T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels,

Cyclic nucleotide specific phosphodiesterases of Leishmania major

Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. Conclusion The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

Human complement component C3 -binding proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that uses the host mononuclear phagocyte as a niche for survival and replication during infection. Complement component C3 has previously been shown to enhance the binding of M. tuberculosis to mononuclear phagocytes. Using a C3 ligand affinity blot protocol, we identified a 30 kDa C3-binding protein in M. tuberculosis as heparin-binding hemagglutinin (HbhA). HbhA was found to be a hydrophobic protein that localized to the cell membrane/cell wall fraction of M. tuberculosis, and this protein has previously been shown by others to be located on the surface of M. tuberculosis. The C3-binding activity of HbhA was localized to the C-terminus of the protein, which consists of lysine-alanine repeats. Full-length recombinant HbhA coated onto latex beads was shown to mediate the adherence of the beads to murine macrophage-like cells in both a C3-dependent and a C3-independent manner. An in-frame 576 by deletion in the hbhA gene was created in a virulent strain of M. tuberculosis using a PCR technique known as gene splicing by overlap extension (SOEing). Using the ΔhbhA mutant, HbhA was found not to be necessary for growth of M. tuberculosis in laboratory media or in macrophage-like cells, nor is HbhA required for adherence of M. tuberculosis to macrophage-like cells. HbhA is, however, required for infectivity of M. tuberculosis in mice. Mice infected with the ΔhbhA mutant show decreased growth in the lungs, liver, and spleen compared to mice infected with the wild-type strain. Using the ΔhbhA mutant strain, we were able to purify and identify a second 30-kDa C3-binding protein, HupB. These data demonstrate that HbhA is required for the in vivo but not the in vitro survival of M. tuberculosis and that HbhA is not necessary for the adherence of M. tuberculosis to the macrophage-like cells used in these studies. The expression of two proteins that bind human C3 may aid in the efficient binding of M. tuberculosis to complement receptors for uptake into mononuclear cells, or may influence other aspects of the host-parasite interaction. ^

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 3 - On the interaction of vector species and parasite strain in the reaction of bugs to infection by Trypanosoma cruzi

The reaction of nine vector species of Chagas' disease to infection by seven different Trypanosoma cruzi strains; Berenice, Y, FL, CL, S. Felipe, Colombiana and Gávea, are examined and compared. On the basis of the insects' ability to establish and maintain the infection, vector species could be divided into two distinct groups which differ in their reaction to an acute infection by T. cruzi. While the proportion of positive bugs was found to be low in Triatoma infestans and Triatoma dimidiata it was high, ranging from 96.9% to 100% in the group of wild (Rhodnius neglectus, Triatoma rubrovaria)and essentially sylvatic vectors in process of adaptation to human dwellings, maintained under control following successful insecticidal elimination of Triatoma infestans (Panstrongylus megistus, Triatoma sordida and Triatoma pseudomaculata). An intermediate position is held by Triatoma brasiliensis and Rhodnius prolixus. This latter has been found to interchange between domestic and sylvatic environments. The most important finding is the strikingly good reaction between each species of the sylvatic bugs and practically all T. cruzi strains herein studied, thus indicating that the factors responsible for the excellent reaction of P.megistus to infection by Y strain, as previously reported also come into operation in the reaction of the same vector species to acute infections by five of the remaining T.cruzi strains. Comparison or data reported by other investigators with those herein described form the basis of the discussion of Dipetalogaster maximus as regards its superiority as a xenodiagnostic agent.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and,to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxity between anti-trypanossomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypothesis for the mechanism of action of phenothiazines are discussed.

Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite

The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection.