Perfil imunológico associado à susceptibilidade, resistência e cura na infecção por Leishmania ( Leishmania ) infantum

Visceral Leishmaniasis (VL) is endemic in Brazil and the northeast region had the highest incidence of the disease , despite, in the last 30 years, it has spread to all geographic regions of the country. Leishmania infantum is the m ain etiological agent of VL in Latin America, Europe and North Africa. However, not all infected individuals develop the disease; in fact, the majority present spontaneous re solution of infection without symptoms. The evaluation of the immunological profil e has been mostly conducted stimulating, with Leishmania spp. antigen, peripheral blood mononuclear cells isolated from subjects with VL. These studies showed that VL patients had an inhibition of both, lymphocyte proliferation and proinflammatory response to Leishmania spp. antigen. Our study aimed to evaluate the immune response in active LV, cured post treatment and asymptomatic infection. To reach this aim, we analyzed immunophenotypic features related to activation, Treg and memory lymphocytes, by flow cytometry, as well as, evaluation of cytokine production, in ex vivo or in whole blood culture. In active VL volunteers, a longitu dinal study was conducted with reassessment at 4 and 14 months after clinical cure. The control group included individuals th at live d in endemic region and were either Positive Control, consisting of individuals with positive anti - L eishmania spp. serology and/or positive PCR for Leishmania  spp. and Negative Control composed by individuals with negative anti - Leishmania antibodie s serology and negative PCR for Leishmania . During VL, CD4 lymphocytes showed greater activation and memory profile s and were the major source of cytokines in culture when compared to CD8 lymphocytes , and these were not Leishmania specific. There were act ivated lymphocytes during VL (CD4 + CD69 + :4.9%) when compared to control groups, Positive (CD4 + CD69 + :1.96%, p=0.0045) and Negative (CD4 + CD69 + :1.35%, p=0.006), on the other hand, this was non - specific activation. The lymphocyte activation profile remain ed el evated even 14 months post treatmen t. A fter clinical cure , the activation was Leishmania specific (CD4 + CD25 + absence of SLA: 8.4%, and presence of SLA: 10.7% p=0.0279). CD8 + CD25 + lymphocytes were able to produce Leishmania specific IFN - γ in both, Positive Controls (absence of SLA 5.2% and presence of SLA: 9.5%, p=0.0391) and Cured 4 month (absence of SLA: 3.9%; presence of SLA: 10.7% p=0.0098). Whole blood culture cells, of VL patients, were able to produce IFN - γ, by SLA stimulation (absence of SLA: 28.0 pg ∕mL, and presence: 44.3 pg∕mL p=0.0020) as well as recovered groups (absence of SLA 2.3 pg∕mL and presence of SLA 139.8 pg∕mL, p=0.0005). However, the high level of IL - 10 seem ed to inhibit pro - inflammatory activity of IFN - γ and TNF - α during symptomatic dis ease . Unlike other pro - inflammatory cytokines, active VL group d id not produce Leishmania specific IL - 2 (absence of SLA 2.4 pg∕mL and presence of SLA: 2.6 pg∕mL). Based on these data we conclude that the restoration of lymphocyte activation and decreased i n IL - 10 Leishmania specific production were related to a protective immune profile.

Efeito de glucanas do fungo Caripia montagnei em modelo de inflamação intestinal induzida por tnbs em ratos Wistar e em células de carcinoma de cólon humano HT-29

Compounds derived from fungi has been the subject of many studies in order to broaden the knowledge of their bioactive potential. Polysaccharides from Caripia montagnei have been described to possess anti-inflammatory and antioxidant properties. In this study, glucans extracted from Caripia montagnei mushroom were chemically characterized and their effects evaluated at different doses and intervals of treatment. It was also described their action on colonic injury in the model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and its action on cells of the human colon carcinoma (HT-29). Compounds extracted of C. montagnei contain high level of carbohydrates (96%), low content of phenolic compounds (1.5%) and low contamination with proteins (2.5%). The (FT-IR) and (NMR) analysis showed that polysaccharides from this species of mushroom are composed of α- and β-glucans. The colonic damage was evaluated by macroscopic, histological, biochemical and immunologic analyses. The results showed a reduction of colonic lesions in all groups treated with the glucans of Caripia montagnei (GCM). GCM significantly reduced the levels of IL-6 (50 and 75 mg/kg, p < 0.05), a major inflammatory cytokine. Biochemical analyses showed that such glucans acted on reducing levels of alkaline phosphatase (75 mg/kg, p < 0.01), nitric oxide (p < 0.001), and myeloperoxidase (p < 0.001). These results were confirmed microscopically by the reduction of cellular infiltration. The increase of catalase activity suggest a protective effect of GCM on colonic tissue, confirming their anti-inflammatory potential. GCM displayed cytostatic activity against HT-29 cells, causing accumulation of cells in G1 phase, blocking the cycle cell progression. Those glucans also showed ability to modulate the adhesion of HT-29 cells to Matrigel® and reduced the oxidative stress. The antiproliferative activity against HT-29 cells displayed by GCM (p <0.001) can be attributed to its cytostatic activity and induction of apoptosis by GCM

O papel modulatório do ferro sobre a resposta imune na Leishmaniose Visceral

Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-γ, TNF-α, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 μM supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-γ, TNF-α and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-α, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-γ, TNF-α e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-γ, TNF-α and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs

Determinantes envolvidos na resposta imune celular humana à infecção por Leishmania infantum chagasi

Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-γ (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-γ produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients

Receptores de citocinas proinflamatórias na pré-eclâmpsia

Preeclampsia is a disease specific of human pregnancy that affects 3-8% of pregnant women, and it is one of the three leading causes of maternal mortality and morbidity. The disease is characterized by hypertension and proteinuria after the 20th week of gestation. The risk factors for this disease are not completely understood but appear to include dysregulation of the immune response arising from defects in placentation, environmental and genetic factors. This study aimed to determine whether the variation in the amount of proinflammatory cytokine receptors IL-1R2, IL-6R and TNF-αR1 would be involved in preeclampsia. They were recruited women with preeclampsia (n=24) and women who evolved during pregnancy without changes in blood pressure (n=12) were recruited. Clinical and laboratory data were collected. The cytokine receptors (IL-1R2, TNF-αR1 and IL-6R) were assessed in mononuclear cells isolated from peripheral blood using flow cytometry (Control = 8; PE = 24). C-reactive protein (CRP) was determined by CRP ultrasensitive method (Control = 7; PE = 18) was performed using sera pregnant women. Women with preeclampsia had higher weight at the beginning of the pregnancy (p=0.0171) and lower gestational age at delivery (0.0008). Classical monocytes were decreased in preeclampsia but not intermediate or non-classical monocytes. The frequency of IL-1R2 pro inflammatory cytokine receptors is decreased in women with PE only in the subpopulation of non-classical monocytes (p = 0.0011). TNF-αR1 receptor and IL-6R, had a decreased frequency in the three subpopulations of monocyte (classic, intermediate and non-classical) when compared to women with normal pregnancy. An increase in IL-1R2 receptor in TCD4+ lymphocytes, but a decrease in TNF-receptor and IL-6R in women with preeclampsia were found. No differences in the frequency of those receptors in CD3+/CD8+ in preeclampsia. There was no difference in C-reactive protein in preeclampsia. The reduction in the amount of IL-1R2, TNF- αR1 and IL-6R monocytes and lymphocytes can be involved in the regulation of inflammation observed in preeclampsia, contributing to disease.

Efeito de glucanas do fungo Caripia montagnei em modelo de inflamação intestinal induzida por tnbs em ratos Wistar e em células de carcinoma de cólon humano HT-29

Compounds derived from fungi has been the subject of many studies in order to broaden the knowledge of their bioactive potential. Polysaccharides from Caripia montagnei have been described to possess anti-inflammatory and antioxidant properties. In this study, glucans extracted from Caripia montagnei mushroom were chemically characterized and their effects evaluated at different doses and intervals of treatment. It was also described their action on colonic injury in the model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and its action on cells of the human colon carcinoma (HT-29). Compounds extracted of C. montagnei contain high level of carbohydrates (96%), low content of phenolic compounds (1.5%) and low contamination with proteins (2.5%). The (FT-IR) and (NMR) analysis showed that polysaccharides from this species of mushroom are composed of α- and β-glucans. The colonic damage was evaluated by macroscopic, histological, biochemical and immunologic analyses. The results showed a reduction of colonic lesions in all groups treated with the glucans of Caripia montagnei (GCM). GCM significantly reduced the levels of IL-6 (50 and 75 mg/kg, p < 0.05), a major inflammatory cytokine. Biochemical analyses showed that such glucans acted on reducing levels of alkaline phosphatase (75 mg/kg, p < 0.01), nitric oxide (p < 0.001), and myeloperoxidase (p < 0.001). These results were confirmed microscopically by the reduction of cellular infiltration. The increase of catalase activity suggest a protective effect of GCM on colonic tissue, confirming their anti-inflammatory potential. GCM displayed cytostatic activity against HT-29 cells, causing accumulation of cells in G1 phase, blocking the cycle cell progression. Those glucans also showed ability to modulate the adhesion of HT-29 cells to Matrigel® and reduced the oxidative stress. The antiproliferative activity against HT-29 cells displayed by GCM (p <0.001) can be attributed to its cytostatic activity and induction of apoptosis by GCM

O papel modulatório do ferro sobre a resposta imune na Leishmaniose Visceral

Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-γ, TNF-α, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 μM supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-γ, TNF-α and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-α, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-γ, TNF-α e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-γ, TNF-α and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs