Development of cell mediated immunity to flagellar antigens and acquired resistance to infection by Trypanosoma cruzi in mice

Modulation by BCG and/or cyclophosphamide of sensitization of mice with flagellar fraction (a tubulin-enriched fraction) prevented death of mice challenged with T. cruzi CL strain trypomastigotes recovered from Vero cells. A methodology was ceveloped to assay specific antigens and to determine optimal doses for sensitization and elicitation of DTH in mice. CL strain is predominantly myotropic strain which does not produce important parasitism of mononuclear phagocyte cells; these cells appear to control infection when activated in vivo. Maximum protection was seen in this study when BCG and cyclophosphamide were associated, but protection was observed also when cyclophosphamide, that prevents supressor T cells, was applied 2 days before flagellar fraction sensitization in normal mice. These experiments suggested that the macrophage may have an important role in the early phases of infection particularly when nonspecific stimulation is associated with specific sensitization. A correlation betwen delayed hypersensitivity to parasite antigens and protection was observed.

Interactions between Leishmania mexicana mexicana promastigotes and amastigotes and murine peritoneal macrophages in vitro

Unstimulated adherent mouse peritoneal cells were cultured in vitro and infected with equal numbers of a single strain of Leishmania m. mexicana amastigotes (AM), virulent promastigotes (VP), avirulent promastigotes (AVP) and fixed promastigotes (FP). Duplicate May-Grünwald-Giemsa stained coverslips were examined at time intervals up to 13 days. By 3 hr post infection, the number of macrophages containing parasites varied between 60.5% (VP) and 84% (AM) for macrophages exposed to living parasites, compared to 6.5% for macrophages exposed for FP. However, variable numbers of parasites showed degenerative changes by 3 hr, and the number of macrophages containing morphologically intact parasites varied significantly between cells infected with AM (84%) and those infected with VP (42%) or AVP(40%). The mean number on intacte parasites/macrophage also differed significantly between AM-infected cells and living or fixed promastigotes-infected cells. Quantitation of intact and degenerated parasites indicated parasite multiplication, as well as destruction, in VP-infected cells and parasite survival and multiplication in AM-infecte monolayers; in contrast no evidence of parasite multiplication was seen in AVP-infected cells. Changes in the mono layer itself (cell loss and macrophage vacuolization) were also evaluated. These results suggest that crucial events determining the outcome of infection occur in the host-parasite relationship during the fist 24 hours of infection. These events are apparently influenced not only by parasite or host strain but by environmentally induced variation within a given strain.

Lymph node cell responsiveness in BALB/C mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human Schistosomiasis: I. Granuloma formation and modulation around polyacrylamide antigen-conjugated beads

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.

Prospects for a nonliving vaccine against Schistosomiasis based on cell-mediated immune resistance mechanisms

We have designed a vaccine model based on induction of cell-mediated immunity and shown that it protects mice against Schistosoma mansoni infection. Mice are immunized by intradermal injection with schistosome antigens plus BCG. Resistance is dependent on the route of antigen presentation and the adjuvant chosen. The pattern of resistance correlates with sensitization of T lymphocytes for production of gamma interferon, a macrophage activating lymphokine that stimulates the cellular effector mechanism of protection. Purified schistosome paramyosin, a muscle cell component present in soluble parasite antigenic preparations, is immunogenic for T lymphocytes and induces resistance when given intradermally with BCG. It is likely that this protein, and possibly other soluble molecules that are released by the parasites of a challenge infection, induce a cellular inflammatory response resulting in larval trapping and/or killing by activated macrophages. These results verify the feasibility of a vaccine against schistosomiasis based on induction of cell-mediated immune resistance mechanisms.

Effect of the host specific treatment in the phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

Single doses of drugs active aginst Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearence of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treatment animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.

T cell derived cytokines in lung-phase immunity to Schistosoma mansoni

In C57Bl/6 strain mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni immune elimination of challenge parasites occurs in the lungs. Leococytes were recovered from the lungs of such mice by bronchoalveolar lavage and cultured in vitro with larval antigen; the profile of cytokines released was then analyzed. From 14 days after vaccination, BAL cultures contained infiltrating lymphocytes wich produced abundant quantitties of IFN-g and IL-3. Challenge of vaccinated mice resulted in a second influx of IFN-g nd IL-3- producing cells, earlier than after vaccination or in the appropriate contropls. Ablation studies revealed that CD4+ T cells were the source of IFN-g. The timing of cytokine production after vaccination, and challenge was coincident with the phases of macrophage activation previously reported. At no time could lymphocytes in BAL cultures to stimulated to proliferate with either larval Ag or mitogen, in contrast to splenocytes from the same mice. Furthermore, T cell growth factor activity was not detected in BAL cultures stimulated with Ag. We suggest that the lymphocytes recruited to the lungs are memory/effector cells, When Ag. released challenge schistosomula is presented to these cells, they respond by secreting cytokines wich mediate the formation of cellular aggregates around the parasites, blocking their onward migration.

Trypanosoma cruzi recognition by macrophages and muscle cells: perspectives after a 15-year study

Macrophages and muscle cells are the main targets for invasion of Trypanosoma cruzi. Ultrastructural studies of this phenomenon in vitro showed that invasion occurs by endocytosis, with attachment and internalization being mediated by different components capable of recognizing epi-or trypomastigotes (TRY). A parasitophorus vacuole was formed in both cell types, thereafter fusing with lysosomes. Then, the mechanism of T. cruzi invasion of host cells (HC) is essentially similar (during a primary infection in the abscence of a specific immune response), regardless of wether the target cell is a professional or a non-professional phagocytic cell. Using sugars, lectins, glycosidases, proteinases and proteinase inhibitors, we observed that the relative balance between exposed sialic acid and galactose/N-acetyl galactosamine (GAL) residues on the TRY surface, determines the parasite's capacity to invade HC, and that lectin-mediated phagocytosis with GAL specificity is important for internalization of T. cruzi into macrophages. On the other hand, GAL on the surface to heart muscle cells participate on TRY adhesion. TRY need to process proteolytically both the HC and their own surface, to expose the necessary ligands and receptors that allow binding to, and internalization in the host cell. The diverse range of molecular mechanisms which the parasite could use to invade the host cell may correspond to differences in the available "receptors"on the surface of each specific cell type. Acute phase components, with lectin or proteinase inhibitory activities (a-macroglobulins), may also be involved in T. cruzi-host cell interaction.

Immunity to intracellular bacteria

Immunity to intracellular bacteria including Mycobacterium tuberculosis. Mycobacterium leprae, and Listeria monocytogenes depends on specific T cells. Evidence to be described suggests that CD4 (alpha/beta)T cells which interact with each other and with macrophages contribute to acquired resistence against as well as pathogenesis of intracellular bacterial infections.

Evaluation of spleen cell population and effect of splenectomy on granuloma modulation in BALB/c mice infected with Schistosoma mansoni

A kinetic study of the cells present in the spleen of BALB/c mice infected with Schistosoma mansoni was carried out. The lymphocytes were evaluated phenotypically with monoclonal antibodies and the effect of splenectomy on the modulation of periovular granuloma was also investigated. The infected mice had proportional increases in the numbers of neutophils, plasma cells, macrophages and eosinophils in the spleen. The largest number of neutrophil, plasma cells and macrophage were found between the 8th and the 12th week of infection, while the amount of eosinophils were higher later on, around the 20th week. The lymphocytes phenotipically characterized as Thy 1.2, Lyt 1.2 (CD4) increased mildly in proportional numbers. However, the percentage of lymphocytes with the Lyt 2.2 (CD8) phenotype, which is characteristic of supressor and cytotoxic T cells, increased significantly with the progress of the disease. The numbers of B lymphocytes, which comprise 50% of the mononuclear cells present in the spleen, increased significantly till the 16th week they began to decrease. The mean diameters of periovular granulomas were comparatively similar in both experimental groups (splenectomized and non-splenectomized mice). However, the evolutive types of granuloma (exudative, intermediate and fibrous) in splenectomized mice were proprtionally different from those of non splenectomized mice in the 16th and 24th week of infection. It is inferred that lymphonodes or other secondary lymphoide organs, in the abscence of the spleen, assume a modulating action on periovular granulomas, although the evolution of the granulomas is somehow delayed in splenectomized mice.

Parasite and egg burden, hepatic collagen and histologic pattern of liver granulomas in selection III high and low antibody responder mice infected with Schistosoma mansoni

Selection III mice have particular immunological characteristics: they are high (H III) or low (L III) antibody producer animals, yet both lines display similar T cell responses and macrophage activities. We submittedthese mice to infection with Schistosoma mansoni to assess in vivo parasite and egg burden, hepatic collagen and cellular composition of granulomas in both lines. Titration of anti-Schistosoma IgG by ELISA showed remarkably higher values inH III line, at both studied periods (8th and 12th weeks post-infection). Nevertheless, the number of adult worms recovered from the portal system was similar inboth lines, being not associated with anti-Schistosoma antibody levels. There isan increase in hepatic collagen from the 8th to the 12th weeks post-infection, which is paralleled by an increase in the number of eggs in the liver. This association apparently occurs at the same radio in H III and L III animals. The most important difference found between the two lines was the outstanding contrast interms of volume and eosinophil counts in the granulomas, with lesions from H IIImice clearly being larger and containing more of these cells than LIII lesions.

Calomys callosus: an alternative model to study fibrosis in Schistosomiasis mansoni: the pathology of the acute phase

Twenty Calomys callosus, Rengger, 1830 (Rodentia-Cricetidae) were studied in the early stage of the acute schistosomal mansoni infection (42nd day). The same number of Swiss Webster mice were used as a comparative standard. Liver and intestinal sections, fixed in formalin-Millonig and embedded in paraffin, were stained with hematoxilin and eosin, PAS-Alcian Blue, pH = 1.0 and 2.5, Lennert's Giemsa, Picrosirius plus polarization microscopy, Periodic acid methanamine silver, Gomori's silver reticulin and resorcin-fuchsin. Immunohistological study (indirect immunofluorescence and peroxidase labeled extravidin-biotin methods) was done with antibodies specific to pro-collagen III, fibronectin, elastin, condroitin-sulfate, tenascin, alpha smooth muscle actin, vimentin and desmin. The hepatic granulomas were small, reaching only 27 of the volume of the hepatic Swiss Webster granuloma. They were composed mainly by large immature macrophages, often filled by schistosomal pigment, characterizing an exsudative-macrophage granuloma type. The granulomas were situated in the parenchyma and in the portal space. They were often intravascular, poor of extracellular matrix components, except fibronectin and presented, sometimes alpha smooth muscle actin and vimentin positive cells. The C. callosus intestinal granulomas were similar to Swiss Webster, showing predominance of macrophages. Therefore, the C. callosus acquire very well the Schistosoma mansoni infection, without developing strong hepatic acute granulomatous reaction, suggesting lack of histopathological signs of hypersensitivity.

Impact of Antifibrotic Treatment on the Course of Schistosoma mansoni Infection in Murine Model

Administration of an antifibrotic agent as an adjunct to antihelmintic treatment with the objective of morbidity reduction was investigated in the murine schistosomiasis mansoni model. Antifibrotic, ß-aminopropionitrile treatment has a profound effect on the cellular matrix composition of the liver granuloma of Schistosoma mansoni infected mice when given alone, resulting in increase macrophage infiltration. These macrophages, in response to stimulation with soluble egg antigen or lipopolysaccharide produced elevated levels of nitric oxide but low levels of tumor necrosis factor alpha compared to untreated infected mice. This also correlated with reduced liver granuloma size. In spite of low numbers of eggs in the liver, mice receiving a combine treatment had a high level of resistance to a challenge infection compared with mice receiving only praziquantel. Those mice also exhibited a reduced lymphocyte proliferative response, similar to that of infected untreated mice. Antifibrotic treatment has an impact on the dynamic of the cellular nature of granulomas and impacts on the host immunity to infection

Eokines: synthesis, storage and release from human eosinophils

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil´s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have recently discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of Sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer an attractive target for therapeutic intervention.