Aldina Barral

Treatment of Visceral Leishmaniasis with Pentavalent Antimony and Interferon Gamma

Acute visceral leishmaniasis is associated with an antigen-specific immunosuppression of mononuclear cells as evidenced by defective in vitro production of interferon gamma. We evaluated treatment with recombinant human interferon gamma in combination with conventional pentavalent antimony therapy in patients with visceral leishmaniasis. Six of eight patients with visceral leishmaniasis (mean duration, 17 months) that had been unresponsive to multiple courses of pentavalent antimony responded to treatment with recombinant human interferon gamma (100 to 400 micrograms per square meter of body-surface area per day) in addition to pentavalent antimony (20 mg per kilogram of body weight per day) for 10 to 40 days. The other two patients improved initially but then relapsed and required treatment with amphotericin B. Eight of nine additional patients with previously untreated severe visceral leishmaniasis were also successfully treated with the combination of interferon gamma and pentavalent antimony. The 14 patients who responded to this regimen had marked improvement in symptoms and in measures of anemia and leukopenia, as well as weight gain, a decrease in spleen size, and an absence or reduction of leishmanias in splenic aspirates. These patients had no recurrence of illness after a mean (+/- SE) follow-up of 8 +/- 1 months. Fever was the only major side effect of interferon gamma. We conclude that the combination of interferon gamma and pentavalent antimony is effective in treating seriously ill patients with refractory or previously untreated visceral leishmaniasis.

Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance

BackgroundDespite clinical descriptions of severe vivax malaria cases having been reported, data regarding immunological and inflammatory patterns are scarce. In this report, the inflammatory and immunological status of both mild and severe vivax malaria cases are compared in order to explore immunopathological events in this disease.Methods and ResultsActive and passive malaria case detections were performed during 2007 in Buritis, Rondônia, in the Brazilian Amazon. A total of 219 participants enrolled the study. Study individuals were classified according to the presence of Plasmodium vivax infection within four groups: non-infected (n = 90), asymptomatic (n = 60), mild (n = 50) and severe vivax infection (n = 19). A diagnosis of malaria was made by microscopy and molecular assays. Since at present no clear criteria define severe vivax malaria, this study adapted the consensual criteria from falciparum malaria. Patients with severe P. vivax infection were younger, had lived for shorter time in the endemic area, and recalled having experienced less previous malaria episodes than individuals with no malaria infection and with mild or asymptomatic infection. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP), serum creatinine, bilirubins and the graduation of disease severity. Plasma levels of tumour necrosis factor (TNF), interferon-gamma(IFN-gamma) and also IFN-gamma/interleukin-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity. Both laboratory parameters of organ dysfunction and inflammatory cytokines were reduced during anti-parasite therapy in those patients with severe disease.ConclusionDifferent clinical presentations of vivax malaria infection present strong association with activation of pro-inflammatory responses and cytokine imbalance. These findings are of utmost importance to improve current knowledge about physiopathological concepts of this serious widespread disease.

CD4+CD25+ T Cells in Skin Lesions of Patients with Cutaneous Leishmaniasis Exhibit Phenotypic and Functional Characteristics of Natural Regulatory T Cells

Endogenous regulatory T (Treg) cells are involved in the control of infections, including Leishmania infection in mice. Leishmania viannia braziliensis is the main etiologic agent of cutaneous leishmaniasis (CL) in Brazil, and it is also responsible for the more severe mucocutaneous form. Here, we investigated the possible involvement of Treg cells in the control of the immune response in human skin lesions caused by L. viannia braziliensis infection. We show that functional Treg cells can be found in skin lesions of patients with CL. These cells express phenotypic markers of Treg cells--such as CD25, cytotoxic T lymphocyte-associated antigen 4, Foxp3, and glucocorticoid-induced tumor necrosis factor receptor--and are able to produce large amounts of interleukin-10 and transforming growth factor- beta . Furthermore, CD4+CD25+ T cells derived from the skin lesions of 4 of 6 patients with CL significantly suppressed in vitro the phytohemagglutinin-induced proliferative T cell responses of allogeneic peripheral-blood mononuclear cells (PBMCs) from healthy control subjects at a ratio of 1 Treg cell to 10 allogeneic PBMCs. These findings suggest that functional Treg cells accumulate at sites of Leishmania infection in humans and possibly contribute to the local control of effector T cell functions.

Seroconversion against Lutzomyia longipalpis Saliva Concurrent with the Development of Anti–Leishmania chagasi Delayed-Type Hypersensitivity

Antibody responses to salivary gland sonicate (SGS) from Lutzomyia longipalpis were investigated using serum samples from individuals living in an area where visceral leishmaniasis is endemic. Individuals were classified into 2 groups, according to the alteration of their responses to Leishmania chagasi antigen over the course of 6 months. Group 1 included children who experienced anti-L. chagasi seroconversion from negative to positive; group 2 included children who experienced delayed-type hypersensitivity (DTH) response to L. chagasi antigen conversion from negative to positive. Individuals who experienced seroconversion against L. chagasi antigens did not have increased anti-saliva antibody response, whereas those who developed a positive anti-L. chagasi DTH response had increased immunoglobulin (Ig) G, IgG1 and IgE anti-SGS antibody levels. Despite wide variation, serum samples from individuals in group 2 recognized more bands in SGS than did those from individuals in group 1. This simultaneous appearance of anti-saliva humoral response and anti-L. chagasi cell-mediated immunity supports the hypothesis that induction of immune response against SGS can facilitate induction of a protective response against leishmaniasis.

Clinical and immunopathological aspects of disseminated cutaneous leishmaniasis

The clinico-pathological and immunological findings in eight patients from Bahia, Brazil with disseminated cutaneous leishmaniasis are described. This condition differs from anergic diffuse cutaneous leishmaniasis (DCL) and from classical American cutaneous leishmaniasis (ACL). The number of lesions in these patients ranged from 75 to 800 and were characterized by papules and an acneiform type of lesion with a few ulcers rather than nodules that are the main characteristic of DCL. On the other hand the high incidence of mucosal disease (38%) in patients with disseminated cutaneous leishmaniasis make the prevalence of mucosal involvement in this condition higher than that observed in ACL. L. amazonensis (five cases) and L. braziliensis (two cases) were the causal agents in the patients where the infecting agent was characterized. Antibody titers in disseminated cutaneous leishmaniasis were higher than those observed in ACL and patients with the highest antibody titers had mucosal involvement. Abnormalities in cellular immunity that are not observed in ACL such as decrease in CD4+ cells and absence of T cell response to leishmania antigen were observed in several patients with disseminated cutaneous leishmaniasis but restoration of these abnormalities occurred after treatment. In spite of the great number of lesions, the therapeutic response was good in six patients with disappearance of the lesions in a period shorter than that observed in ACL. In the two patients that presented therapeutic failure the causal agent was L. amazonensis. In such patients there was a predominance of ulcerated lesions, and a high titer of antibody was detected.

A simple method for human peripheral blood monocyte Isolation

We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

Antigen-specific immunosuppression in visceral leishmaniasis is cell mediated.

Visceral leishmaniasis is associated with an antigen-specific immunosuppression during the acute disease. Patients become responsive to Leishmania antigen in both in vivo and in vitro assays after successful antimony therapy. The cell type involved in the suppression of lymphocyte reactivity to Leishmania antigen was studied by selective depletion of mononuclear cell (MNC) populations and in co-cultivation experiments. Adherent cells were depleted on plastic and by passage on nylon wool columns. High-avidity Fc+ cells were depleted by adherence to BSA-anti-BSA complexes and OKT4+ and OKT8+ cells were depleted by treatment with monoclonal antibody (anti-OKT4+ and OKT8+) and complement. Depletion of MNC preparations of adherent cells, high-avidity Fc+ cells, OKT4+ cells and OKT8+ cells failed to restore the lymphocyte reactivity to Leishmania antigen. Antimony therapy was associated with restoration of the proliferative responses of unseparated MNC (before treatment 460 +/- 76 cpm and after treatment 4,293 +/- 1,442 cpm). Co-culture of frozen cells obtained before chemotherapy with autologous MNC obtained after treatment reduced the response of posttreatment cells to Leishmania antigen by 80%. We conclude that the antigenic specific suppression of lymphocyte proliferation in visceral leishmaniasis is cell mediated.

Balance of IL-10 and Interferon-γ plasma levels in human visceral leishmaniasis: Implications in the pathogenesis

BackgroundLeishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis.MethodsTo explore immunoregulatory mechanisms, 20 visceral leishmaniasis (VL) patients were evaluated during active disease and at different periods up to one year after treatment determining their plasma cytokine levels, clinical parameters (palpable spleen and liver) and antibody levels.ResultsElevated plasma levels of IFN-γ and of IL-12 p40 were observed during active disease, significantly decreasing after treatment whereas in vitro Leishmania antigen-stimulated IFN-γ production by PBMC exhibited an inverse pattern being low during disease and increasing steadily thereafter. Absence of IFN-γ activity is a hallmark of VL. The main candidate for blunting IFN-γ activity is IL-10, a cytokine highly elevated in plasma with sharp decrease after treatment. Activity of IL-10 is inferred by high levels of anti-Leishmania specific IgG1 and IgG3. TGF-β had elevated total, but not of active, levels lessening the likelihood of being the IFN-γ counterpart. Spleen or liver size presented a steady decrease but return to normal values at only 120 days after treatment. Anti-Leishmania IgG (total and subclasses) levels and DTH or Leishmania-stimulated lymphocyte proliferation conversion to positive also present a slow decrease after treatment. IL-6 plasma levels were elevated in only a few patients.ConclusionTaken together our results suggest that IFN-γ and IL-10 are the molecules most likely involved in determining fate of disease. After treatment, there is a long delay before the immune profile returns to normal what precludes using plasma cytokine levels as criteria of cure as simpler clinical evaluations, as a palpable spleen or liver, can be used.

Human mucosal leishmaniasis: neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines.

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL‐17‐type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL‐17 and IL‐17‐inducing cytokines (IL‐1β, IL‐23, IL‐6 and TGF‐β) were detected by immunohistochemistry in ML patients. IL‐17+ cells exhibited CD4+, CD8+ or CD14+ phenotypes, and numerous IL‐17+ cells co‐expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17‐mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP‐9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL‐17 is involved in ML pathogenesis.

Neutrophils and Macrophages Cooperate in Host Resistance against Leishmania braziliensis Infection

Neutrophils play an active role in the control of infections caused by intracellular pathogens such as Leishmania. In the present study, we investigated the effect of neutrophil depletion at the time of Leishmania braziliensis infection of BALB/c mice and how neutrophils interact with the infected macrophage to promote parasite elimination. The in vivo depletion of neutrophils led to a significant increase in parasite load and enhanced the Th1-Th2 immune response in this experimental model of infection. BALB/c mice coinoculated with both parasites and live neutrophils displayed lower parasite burdens at the site of infection and in the draining lymph nodes. In vitro, we observed that live neutrophils significantly reduced the parasite load in L. braziliensis-infected murine macrophages, an effect not observed with Leishmania major. L. braziliensis elimination was dependent on the interaction between neutrophils and macrophages and was associated with TNF-α as well as superoxide production. Furthermore, cooperation between neutrophils and macrophages toward parasite elimination was also observed in experiments performed with L. braziliensis-infected human cells and, importantly, with two other New World Leishmania species. These results indicate that neutrophils play an important and previously unappreciated role in L. braziliensis infection, favoring the induction of a protective immune response.