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Background: In vitro studies show that Leishmania infection decreases the adhesion of inflammatory phagocytes to connective tissue by a mechanism dependent on the modulation of integrin function. However, we know little about the influence of this reduction in leukocyte adhesion on parasite dissemination from the infection site., Methods: In this work, we used a model of chronic peritonitis induced by thioglycollate to study the effect of L. amazonensis infection on the ability of inflammatory phagocyte populations to migrate from an inflammatory site to the draining lymph node. Uninfected or Leishmania-infected thioglycollate-elicited peritoneal exudate cells were transferred from C57BL/6 to BALB/c mice or from Ly5.1+ to Ly5.1- mice. The transferred cells were injected into the peritoneal cavity and tracked to the draining lymph node., Results: Migrating cells corresponded to approximately 1% of the injected leukocytes. The proportion of migrating CD11b+CD11c+ (myeloid dendritic cell) was lower after incubation with Leishmania (1.3 to 2.6 times lower in the experiments using C57BL/6 to BALB/c animals and 2.7 to 3.4 times lower in the experiments using Ly5.1+ to Ly5.1- animals) than after leukocyte incubation with medium alone (P < 0.01). There was no consistent decrease in the migration of CD11b+F4/80+ (macrophage) or SSChi GR-1+ (neutrophil) populations., Conclusions: Coincubation with Leishmania changes the migratory pattern of dendritic cells in vivo. Such changes in dendritic cell migration may be associated with immunological events that maintain inflammation at the sites of infection.

Most inbred strains of mice, like the BALB/c strain, are susceptible to Leishmania amazonensis infections and resistant to Leishmania braziliensis infections. This parasite-related difference could result from the activity of an L. amazonensis-specific virulence factor. In agreement with this hypothesis, it is shown here that the intravenous injection of BALB/c mice with L. amazonensis amastigote extract (LaE) but not the L. braziliensis extract confers susceptibility to L. braziliensis infection. This effect was associated with high circulating levels of IgG1 anti-L. amazonensis antibodies and with an increase in interleukin-4 (IL-4) production and a decrease in gamma interferon production by draining lymph node cells. Moreover, the effect was absent in IL-4-knockout mice. The biological activity in the LaE was not mediated by amphiphilic molecules and was inhibited by pretreatment of the extract with irreversible serine protease inhibitors. These findings indicate that the LaE contains a virulence-related factor that (i) enhances the Leishmania infection by promoting Th2-type immune responses, (ii) is not one of the immunomodulatory Leishmania molecules described so far, and (iii) is either a serine protease or has an effect that depends on that protease activity. In addition to being Leishmania species specific, the infection-enhancing activity was also shown to depend on the host genetic makeup, as LaE injections did not affect the susceptibility of C57BL/6 mice to L. braziliensis infection. The identification of Leishmania molecules with infection-enhancing activity could be important for the development of a vaccine, since the up- or downmodulation of the immune response against a virulence factor could well contribute to controlling the infection.

Spleen or spleen plus bone marrow cells from (BALB/cxC57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4+ and CD8+ T cells 7 and 21 days after donor cell transfer. The populations of CD8+CD45RBlow and CD8+CD44high cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8+ T cells leading to enhanced graft survival.

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.

The production and partial characterization of a monoclonal antibody, the IgG1 IH1, which recognizes an antigen distributed in canine monocytes/macrophages, is reported here. The distribution and apparent molecular weight of the antigen recognized by the IH1 MAb was determined in peripheral blood leukocytes, peripheral blood monocyte-derived macrophages and tissue sections of spleen, liver and skin, using Western blotting, immunocytochemistry, immunohistochemistry and flow cytometry. The IH1 MAb-recognized antigen was detected in Western blotting under non-reducing conditions spread out as a large band covering the position corresponding to the migration of molecules with molecular weights from 55 to 73 kDa. The IH1 MAb labeled blood monocytes, tissue macrophages in lymph nodes, and in the mantle zone of the spleen, and Kupffer cells in the liver. It did not react with human cells. In flow cytometric analysis, the IH1 MAb reacted with a subpopulation of monocytes. The MAb described herein may become a valuable tool for diagnosis and research on canine diseases.

Steroids hormones modify the hematological features of homozygous sickle cell disease, including the levels of fetal hemoglobin. We used semi-quantitative RT-PCR analysis of GATA-1, GATA-2, NF-E2, and gamma-globin mRNA levels in a two-phase liquid culture system of human adult erythroid cells in order to assay the effect of progesterone upon gene expression. The levels of expression of GATA-1 and gamma-globin mRNA were significantly increased in cells treated with progesterone compared to untreated cells (1.7- to 2.0-fold). Progesterone treatment did not produce any stimulatory effect upon GATA-2 and NF-E2 mRNA expression. Differences in the synthesis of HbF protein could not be detected by flow cytometry, although we observed a small difference in mean intensity fluorescence between cells treated and cells untreated with progesterone on days 7 and 9. Using anti-transferrin receptor and anti-glycophorin A antibodies, we verified that addition of progesterone did not cause any change in erythroid proliferation and differentiation. In conclusion, it is possible that the increased expression of gamma-globin mRNA after progesterone treatment observed in this study may be related to the increased GATA-1 mRNA expression. Interactions of the steroid receptors with the basal transcriptional machinery and with transcription factors might mediate their transcriptional effects.

To study the role of autoreactive T cells in the pathogenesis of cardiomyopathy in Chagas' disease, we generated a cell line by repeated in vitro antigenic stimulation of purified splenic CD4+ T lymphocytes from a chronically Trypanosoma cruzi-infected mouse. Cells from this line were confirmed to be CD4+ CD8- and proliferated upon stimulation with soluble heart antigens from different animal species, as well as with T. cruzi antigen, in the presence of syngeneic feeder cells. In vitro antigen stimulation of the cell line produced a Th1 cytokine profile, with high levels of IFNgamma and IL-2 and absence of IL-4, IL-5 and IL-10. The cell line also terminated the beating of fetal heart clusters in vitro when cocultured with irradiated syngeneic normal spleen cells. In situ injection of the cell line into well established heart transplants also induced the cessation of heart beating. Finally, adoptive transfer of the cell line to heart-immunized or T. cruzi-infected BALB/c nude mice caused intense heart inflammation.

Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8(+) T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8(+) T cells in both mouse strains. The number of pulmonary CD4(+) T cells and immunoglobulin-positive cells was independent of the number of CD8(+) T cells. In susceptible mice, the loss of CD8(+) T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8(+) T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8(+) T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8(+) T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8(+) T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

Bacterial products have served as important immunological tools to study lymphocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW > or = 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 kDa were highly toxic for mice and had no mitogenic activity. The possibility that LPS was present as a contaminant in the Cb mitogen preparations was excluded because spleen cells from the LPS non-responder C3H/HeJ mice responded well to the Cb mitogen, and the antibiotic polymyxin B, which is an inhibitor of LPS, had no effect on the Cb-mitogen activity. However, an anti-lipoteichoic acid monoclonal antibody (3-1 mAb) inhibited to a great extent the proliferation of spleen cells induced by the Cb mitogen but had no effect on the LPS or concanavalin A stimulation of these cells. Moreover, the Cb mitogen was specifically adsorbed and eluted from a protein G Sepharose column to which the anti-lipoteichoic acid 3-1 mAb had been conjugated. These results support the view that lipoteichoic acid is a selective B cell mitogen.

The pathogenesis of chronic chagasic myocarditis remains incompletely understood. Several hypotheses have been proposed: (1) direct tissue destruction by Trypanosoma cruzi; (2) neurogenic theory; (3) anti-heart immune reactions; and (4) microvascular disease. We present herein a dynamic alternative hypothesis. We believe that the development of myocarditis is related to progressive and additive focal cellular necrosis, and associated reactive and reparative myocardial fibrosis and surrounding myocytes hypertrophy. These processes may be initiated and perpetuated by autoimmune factors and alterations in the myocardial microcirculation. This could imply future therapeutic strategies in the management of chronic chagasic patients to optimize the medical treatment and hopefully improve the prognosis.

The biological activities previously described for a crude extract derived from seeds of Artocarpus integrifolia (jack fruit) are shown in the present work to be assigned to two distinct fractions present in this extract. One fraction is the D-galactose binding lectin, jacalin, obtained by affinity purification on a D-galactose Sepharose column. The other fraction is a D-mannose-binding protein which we propose to call 'Artocarpin'. As is well documented, jacalin binds to human IgA1 and is a useful tool for the purification of this immunoglobulin. We show here that the remaining biological activities consisting of the proliferative response of mouse spleen cells and human peripheral blood mononuclear cells and polyclonal activation of human and mouse B cells for the secretion of immunoglobulin are mediated by artocarpin. Artocarpin is unique in its capacity to induce polyclonal activation of B cells in the absence of proliferation. BALB/c nu/nu spleen cells failed to proliferate which indicates that this lectin is a T cell-dependent B cell polyclonal activator.