Inhibition of mouse and rat lymphoproliferation by gangliosides

Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mug/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [³H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a beta-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mug/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mug/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.UNIFESP-EPM Departamento de CirurgiaUNIFESP-EPM Disciplina de Técnica Operatória e Cirurgia ExperimentalUNIFESP-EPM Disciplina de ParasitologiaUNIFESP, EPM, Depto. de CirurgiaUNIFESP, EPM Disciplina de Técnica Operatória e Cirurgia ExperimentalUNIFESP, EPM Disciplina de ParasitologiaSciEL

Neutrophils Reduce the Parasite Burden in Leishmania (Leishmania) amazonensis-Infected Macrophages

Background: Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L) major, whereas less information is available for L. (L) amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L.) amazonensis (C3H/HePas). in contrast, the susceptible strain (BALB/c) displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L.) amazonensis-infected macrophages in vitro.Methodology/Principal Findings: Mouse peritoneal macrophages infected with L. (L.) amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. the main findings were 1) intracellular parasites were efficiently destroyed in the co-cultures; 2) the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas) or susceptible (BALB/c) to L. (L.) amazonensis; 3) parasite destruction did not require contact between infected macrophages and neutrophils; 4) tumor necrosis factor alpha (TNF-alpha), neutrophil elastase and platelet activating factor (PAF) were involved with the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-alpha, as reported for other Leishmania species.Conclusions/Significance: the present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

In Vitro and In Vivo Activity of a Palladacycle Complex on Leishmania (Leishmania) amazonensis

Leishmaniasis is an important public health problem with an estimated annual incidence of 1.5 million of new human cases of cutaneous leishmaniasis and 500,000 of visceral leishmaniasis. Treatment of the diseases is limited by toxicity and parasite resistance to the drugs currently in use, validating the need to develop new leishmanicidal compounds. We evaluated the killing by the palladacycle complex DPPE 1.2 of Leishmania (Leishmania) amazonensis, an agent of human cutaneous leishmaniasis in the Amazon region, Brazil. DPPE 1.2 destroyed promastigotes of L. (L.) amazonensis in vitro at nanomolar concentrations, whereas intracellular amastigotes were killed at drug concentrations 10-fold less toxic than those displayed to macrophages. L. (L.) amazonensis-infected BALB/c mice treated by intralesional injection of DPPE 1.2 exhibited a significant decrease of foot lesion sizes and a 97% reduction of parasite burdens when compared to untreated controls. Additional experiments indicated the inhibition of the cathepsin B activity of L. (L.) amazonensis amastigotes by DPPE 1.2. Further studies are needed to explore the potential of DPPE 1.2 as an additional option for the chemotherapy of leishmaniasis

Controle da síntese de uma exoprotease de Blastocladiella emersonii

BV UNIFESP: Teses e dissertaçõe

Caracterização de proteínas de superfície em tripanosomatídeos: valor taxonômico e avaliação de sua antigenicidade

BV UNIFESP: Teses e dissertaçõe

Cytokine secretion in supernatants from the co-cultures of neutrophil/<i>L. (L.) amazonensis</i>-infected macrophages.

<p>Inflammatory neutrophils from BALB/c mice were co-cultured with <i>L. (L.) amazonensis</i>-infected macrophages (10∶1). Supernatants from 24 and 72 h cultures were tested at 1∶2 dilution for Cytometric Bead Array immunoassay as described in Section <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013815#s4" target="_blank">Materials and Methods</a>. Cytokine concentrations were determined from standard curves plotted using a four-parameter logistic curve fitting model. A value of 0 was assigned when the concentration of a cytokine was below the detection limit for the assay. Bars represent the SD. *<i>P</i><0.05 and **<i>P</i><0,001.</p

Activation of <i>L. (L.) amazonensis</i> and <i>L</i>. (<i>L</i>.) <i>chagasi</i> infected macrophages.

<p>Infected macrophages were cultured in the presence of TNF-α plus LPS. Four days later, cultures were fixed and stained for infection index determination. <i>L</i>. (<i>L</i>.) <i>chagasi</i> was used as a positive control for activation (A). Nitric oxide was assayed in the culture supernatants (B). Bars represent the SD. ^*<i>P</i><0.001.</p

Effect of NE inhibitor (A), and PAF antagonist (B) on the leishmanicidal activity in <i>L. (L.) amazonensis</i>-infected macrophages co-cultured with neutrophils and secretion of nitric oxide in the supernatants from these co-cultures (C and D).

<p>Bars represent the SD. ^*<i>P</i><0.001.</p

Effect of NO, superoxide anion and hydrogen peroxide depletion on parasite load in <i>L. (L.) amazonensis</i>-infected macrophages co-cultured with neutrophils (A) and secretion of nitric oxide in the supernatants from these co-cultures (B).

<p>Bars represent the SD. ^*<i>P</i><0.001.</p