RESUMEN
Cell-free antigens (CFAg) derived from Paracoccidioides brasiliensis have typically been used in immunodiffusion reactions for serodiagnosis or therapeutic follow-up of paracoccidioidomycosis patients. Thus, we investigated the usefulness of CFAg obtained from cultures at different ages, to evaluate cellular immunity by the footpad test, in experimental murine paracoccidioidomycosis. Male mice infected with P. brasiliensis 265 strain were challenged in the footpad with CFAg obtained from four- (4d CFAg) or 11-day-old cultures (11d CFAg). The increase in footpad swelling provoked by 4d CFAg and 11d CFAg was similar and showed significant difference in relation to control groups. However, the infiltrate pattern was strikingly different: 4d CFAg induced a predominant mononuclear infiltrate whereas 11d CFAg provoked a predominant polymophonuclear infiltrate. These different inflammatory patterns were associated with distinct electrophoretic characteristics. By comparison with 11d CFAg, 4d CFAg showed more numerous and intense bands, including a strong one of 43 kDa (gp43). These results suggest that CFAg derived from Pb 265 isolate can be used as a reagent to evaluate cellular immunity; however, the culture's age is critical because only young cultures are able to induce a typical mononuclear infiltrate. The efficacy of this new paracoccidioidin to assay the cellular immunity in infections caused by other P. brasiliensis isolates is under investigation.
Asunto(s)
Animales , Masculino , Ratones , Hipersensibilidad Tardía , Paracoccidioides , ParacoccidioidomicosisRESUMEN
In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), studies have focused on the role of neutrophils that are involved in primary response to the fungus. Neutrophil functions are regulated by pro- and anti-inflammatory cytokines. The molecular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll-like receptors (TLR). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated with GM-CSF, IL-15, TNF-alpha or IFN-gamma and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H(2)O(2) and IL-6, IL-8, TNF-alpha and IL-10 production by activated and challenged cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression inducing an additional effect to that of cytokines. On the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H(2)O(2) production, but this process was not associated with TLR2 or TLR4. Neutrophils activation with GM-CSF and TNF-alpha resulted in a significative increase in IL-8 production, while IL-15 and IFN-gamma have no effect. Pb18 alone also increased IL-8 production. None of the cytokines activated neutrophils for IL-10 release. This cytokine was only detected after Pb18 challenge. Interestingly, IL-8 and IL-10 production involved TLR2 and mainly TLR4 modulation. Our data suggest that Pb18 uses TLR4 to gain access to human neutrophils. This interaction results in IL-8 and IL-10 production that may be considered as a pathogenic mechanism in paracoccidioidomycosis.
Asunto(s)
Activación Neutrófila/inmunología , Neutrófilos/inmunología , Paracoccidioidomicosis/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Adulto , Citocinas/biosíntesis , Citocinas/inmunología , Humanos , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Interleucina-8/biosíntesis , Interleucina-8/inmunología , Persona de Mediana Edad , Neutrófilos/metabolismo , Paracoccidioides/inmunología , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 4/biosíntesis , Adulto JovenRESUMEN
Leishmaniasis, a zoonosis of worldwide distribution, presents a significant impact on immunosupressed patients. This study aimed to evaluate Leishmania chagasi infection in BALB/c mice immunosuppressed with dexamethasone. Spleen cells stimulated or not with L. chagasi were cultured for cytokine quantification (IFN-gama, IL-2, IL-4 and IL-10) by sandwich ELISA. Parasite loads in the spleen and liver were determined by means of culture microtitration. Immunosuppressed groups showed statistically lower spleen weight and CD4-cell percentage in blood on the day of infection and produced Th1 and Th2 cytokines on other days of the study. The other infected groups, weather immunosupressed or not, also produced Th1 and Th2 cytokines. Parasite loads in the spleen and liver were not statistically different among the groups. It was concluded that L. chagasi infection was not affected by dexamethasone-induced immunosuppression, probably due the reversible effect of the treatment.
Asunto(s)
Animales , Masculino , Ratones , Terapia de Inmunosupresión , Inmunidad Celular , Leishmaniasis Visceral , Ratones Endogámicos BALB C , DexametasonaRESUMEN
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production.
Asunto(s)
Apoptosis , Interleucina-8/fisiología , Neutrófilos/fisiología , Paracoccidioides/fisiología , Adulto , Anticuerpos Monoclonales/inmunología , Humanos , Interleucina-8/metabolismo , Persona de Mediana Edad , FagocitosisRESUMEN
The immune response in leishmaniasis may result in a polarization of the T lymphocyte subpopulation, altering cell phenotype and resulting in immune protection or disease exacerbation. Leishmania may persist in the body either during asymptomatic infections or after treatment, which represents high risk under immunosuppression. The objective of this study was to evaluate the effect of infection with immunosuppression by dexamethasone associated with pentoxifylline on animal weight, spleen weight, spleen and hepatic parasitic load and immunopathology, as well as the IFN-ã and IL-10 production in spleen cell culture of Balb/c mice infected with Leishmania chagasi. The infection did not cause body weight gain in animals, but both the weight and size of the spleen were increased. The immunosuppression using dexamethasone associated with pentoxifylline affected body weight gain and spleen weight and size in both infected and non-infected animals. The immunosuppression did not significantly alter the course of the splenic or hepatic parasite burden. Dexamethasone and pentoxifylline significantly affected cytokine production, but did not influence the Th1/Th2 ratio in infected animals.(AU)
Asunto(s)
Animales , Ratones , Leishmaniasis , Citocinas , Terapia de Inmunosupresión , Infecciones Asintomáticas , Carga de Parásitos , LeishmaniaRESUMEN
Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against the fungus and several papers show the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, the studies focusing on polymorphonuclear neutrophils (PMNs) antifungal functions are scarcer. Thus, the objective of the present paper was to assess the capacity of human PMNs to kill virulent P. brasiliensis strain in vitro, before and after priming with different cytokines. Moreover, the involvement of oxygen metabolites in this activity was evaluated. Nonactivated cells failed to exhibit antifungal activity. However, when these cells were IFN-gamma, TNF-alpha or GM-CSF activated, a significative fungicidal activity was detected. This process was significantly inhibited when P. brasiliensis challenge occurred in presence of catalase (CAT - a scavenger of H2O2) and superoxide dismutase (SOD - a scavenger of superoxide anion). From these results it is concluded that cytokines activation is required for P. brasiliensis killing by human PMNs, and that H2O2 and superoxide anion participate as effectors molecules in this process.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Peróxido de Hidrógeno/metabolismo , Interferón gamma/farmacología , Neutrófilos/inmunología , Paracoccidioides/inmunología , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Catalasa/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Peróxido de Hidrógeno/inmunología , Interferón gamma/inmunología , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Paracoccidioides/aislamiento & purificación , Paracoccidioides/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Superóxido Dismutasa/farmacología , Superóxidos/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Human monocytes activated by recombinant tumor necrosis factor alpha (TNF-alpha) exhibited significant fungicidal activity on the yeast cells of a highly virulent strain of Paracoccidioides brasiliensis. This process was significantly inhibited in the presence of catalase (CAT - a scavenger of H2O2), but not in the presence of superoxide-dismutase (SOD - a scavenger of superoxide anion) or NG-monomethyl-L-arginine (NG-MMLA - a nitric oxide inhibitor). Furthermore, there was a direct association between the intracellular killing of the fungus and the production of H2O2 by activated cells. These results strongly suggest a role for H2O2 in the killing of highly virulent strains of P. brasiliensis by TNF-alpha-activated human monocytes.