Virmondes Rodrigues

Th1/Th2 immune responses are associated with active cutaneous leishmaniasis and clinical cure is associated with strong interferon-γ production

In leishmaniasis, Th1-related cytokines production seems to be crucial for host control of parasite burden and clinical cure. Visceral and diffuse cutaneous leishmaniasis are characterized by negative skin test for parasite antigens and failure to produce Th1 cytokines, whereas tegumentary leishmaniasis is characterized by positive skin test and the ability of peripheral blood mononuclear cells (PBMCs) to produce Th1 cytokines. In this study, specific antibody plasma levels and cytokine production in PBMC culture supernatants were evaluated by enzyme-linked immunoabsorbent assay in patients with active or cured cutaneous leishmanial lesions and in subjects without disease history living in the same endemic area. Higher tumor necrosis factor-alpha, interferon (IFN)-gamma, interleukin (IL)-12, IL-4, and IL-10 levels were observed in patients with active lesions, whereas cured subjects produced only IFN-gamma at elevated levels. Analysis of specific antibody isotypes correlate with cellular immune response observed in vitro, as the production of IgG1 and IgG3 was higher in patients with active lesions. Our results suggest the presence of a mixed Th1/Th2 response during active disease and that clinical cure is associated with a sustained Th1 response characterized by elevated IFN-gamma levels and down-modulation of IL-4 and IL-10 production.

T-Helper Cell Type 17/Regulatory T-Cell Immunoregulatory Balance in Human Radicular Cysts and Periapical Granulomas

INTRODUCTION Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop to contain a periapical infection. This study analyzed the nature of the inflammatory infiltrate, presence of mast cells, and in situ expression of cytokines (interleukin [IL]-17 and transforming growth factor [TGF]-beta), chemokines (macrophage inflammatory protein [MIP]-1beta and monocyte chemotactic protein [MCP]-1), and nuclear transcription factor (FoxP3) in human periapical granulomas and cysts compared with a control group. METHODS Fifty-five lesions (25 periapical cysts, 25 periapical granulomas, and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by hematoxylin-eosin staining, and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines, and FoxP3. RESULTS The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates (P = .04). Mixed inflammatory infiltrates were significantly more frequent in patients with sinus tract (P = .0001). The number of mast cells was significantly higher in granulomas than in cystic lesions (P = .02). A significant difference in the expression of IL-17 (P = .001) and TGF-beta (P = .003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with sinus tract (P = .03). CONCLUSIONS We observed that chronic periapical lesions might experience a reagudization process that is correlated with an increased leukocyte infiltration, with the predominance of neutrophils attracted by a chemokine milieu, as well as the increased presence of IL-17.

Interleukin-13 in the skin and interferon-gamma in the liver are key players in immune protection in human schistosomiasis.

Summary:  Immunity against schistosomes includes anti‐infection immunity, which is mainly active against invading larvae in the skin, and anti‐disease immunity, which controls abnormal fibrosis in tissues invaded by schistosome eggs. Anti‐infection immunity is T‐helper 2 (Th2) cell‐dependent and is controlled by a major genetic locus that is located near the Th2 cytokine locus on chromosome 5q31‐q33. Mutations in the gene encoding interleukin (IL)‐13 that decrease or increase IL‐13 production account, at least in part, for that genetic control. In contrast, protection against hepatic fibrosis is dependent on interferon (IFN)‐γ and is controlled by a major genetic locus that is located on 6q23, near the gene encoding the IFN‐γ receptor β chain. Mutations that modulate IFN‐γ gene transcription are associated with different susceptibility to disease. These data indicate that IL‐13 in the skin and IFN‐γ in the liver are key players in protective immunity against schistosomes. These roles relate to the high anti‐fibrogenic activities of IFN‐γ and to the unique ability of IL‐13 in Th2 priming in the skin and in the mobilization of eosinophils in tissues. The coexistence of strong IFN‐γ and IL‐13‐mediated immune responses in the same subject may involve the compartmentalization of the anti‐schistosome immune response between the skin and the liver.

Infection and disease in human schistosomiasis mansoni are under distinct major gene control

INSERM U399, Immunology and Genetic of Parasitic Diseases/Laboratory of Parasitology-Mycology, Faculty of Medicine, Universite de la Mediterranee, Marseille, France Institute of Nuclear Medicine and Molecular Biology, University of Gezira, Gezira, Sudan Laboratorio De Imunologia, Faculdade de Medicina do Triangulo Mineiro, Mineiro, Brazil Alzaiem Alazihri University, Omdurman, Sudan Faculty of Medicine, University of Gezira, Gezira, Sudan INSERM U436 Paris, France

Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules

Despite the clear need to control tuberculosis, the diagnosis and prevention of this serious disease are poorly developed and have remained fundamentally unchanged for more than 50 years. Here, we introduce an innovative approach to directly identify Mycobacterium tuberculosis antigens produced in vivo in humans with tuberculosis. We combined reversed phase high performance liquid chromatography and mass spectrometry and categorize four distinct M. tuberculosis proteins produced presumably in lung lesions and excreted in the urine of patients with pulmonary tuberculosis. The genes (MT_1721, MT_1694, MT_2462 and MT_3444) coding for these proteins were cloned and the recombinant molecules were produced in Escherichia coli. The proteins were recognized by immunoglobulin G antibodies from tuberculosis patients but not from non‐diseased subjects. In addition, the recombinant proteins were recognized strongly by peripheral blood mononuclear cells from healthy purified protein derivative of tuberculin‐positive individuals and to a lesser extent from patients with tuberculosis. These molecules are the only proteins reported to date that are derived directly from bodily fluids of tuberculosis patients, therefore are interesting candidate antigens for the development of vaccine and/or antigen detection assay for accurate diagnosis of active tuberculosis.

Selective inability of spleen antigen presenting cells from Leishmania donovani infected hamsters to mediate specific T cell proliferation to parasite antigens

Summary Golden hamsters (Mesocricetus auratus) infected with Leishmania donovani develop a disease similar to human kala‐azar. There is conspicuous hypergammaglobulinaemia and their T cells do not respond to stimulation by parasite antigens. The impairment of the cellular immune response seems to be restricted to parasite antigens since infected animals are able to develop a T cell response to the mitogen Concanavalin A (Con‐A) and, after sensitization, to the antigens keyhole limpet haemocyanin (KLH) and human serum albumin (DNP‐HSA). In the present investigations we studied the role played by infected macrophages in the development of the cellular unresponsiveness present in visceral leishmaniasis. Adherent spleen cells from infected hamsters were unable to present L. donovani antigens to antigen specific T cells, however they were able to present KLH. Conversely. T cells from infected animals did not respond to parasite antigens even when these antigens were presented by normal syngeneic macrophages. Interestingly, lymphocytes from inguinal lymph nodes of infected animals sensitized in their foot pad with parasite antigens proliferated well when stimulated in vitro with L. donovani antigens. These results suggest that the defect in the cellular immune response of the L. donovani infected hamsters is a consequence of a selective inability of their antigen presenting cells to process and present parasite antigens to T cells.

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

BACKGROUND Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. METHODS Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. RESULTS TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). CONCLUSIONS Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Identification of Mycobacterium tuberculosis Ornithine Carboamyltransferase in Urine as a Possible Molecular Marker of Active Pulmonary Tuberculosis

ABSTRACT Although the antigen detection assay has the potential to discriminate active tuberculosis from latent infection, development of such a test for the accurate diagnosis of this serious disease has only recently become a matter of interest. Here we present evidence that a Mycobacterium tuberculosis protein (ornithine carboamyltransferase, coded for by MT_1694; Rv1656 [argF]) is an interesting candidate molecule for this test development. The protein was initially discovered by mass spectroscopy in urine of patients with pulmonary tuberculosis and shown by Western blot analysis to be present in M. tuberculosis crude cell extract as well as in the culture supernatant (“secreted” protein). In addition, a recombinant ornithine carboamyltransferase (rMT1694) produced in Escherichia coli was recognized by immunoglobulin G (IgG) antibodies from patients with active tuberculosis but not by IgG from uninfected healthy subjects. Moreover, rMT1694 was strongly recognized by peripheral blood mononuclear cells from both healthy tuberculin purified protein derivative (PPD)-positive individuals and patients with pulmonary tuberculosis. More importantly, a capture enzyme-linked immunosorbent assay formatted with rabbit IgG antibodies specific to rMT1694 was able to identify the presence of this antigen in urine samples from 6 of 16 patients with pulmonary tuberculosis and in none of 16 urine samples collected from healthy PPD+ controls. These results indicate that an improved antigen detection assay based on M. tuberculosis ornithine carboamyltransferase may represent an important new strategy for the development of a specific and accurate diagnostic test for tuberculosis.

Genetic and Functional Role of TNF-alpha in the Development Trypanosoma cruzi Infection

TNF-alpha plays an important role in trypanocidal mechanisms and is related to tissue injury. This cytokine has been detected in the heart of human chagasic patients where it is associated with tissue damage. This study investigated whether TNF-alpha levels and the presence of genetic polymorphisms are associated with the presence of T. cruzi infection and/or with the development of the cardiac form in chronic chagasic patients. Genomic DNA of 300 subjects from an endemic area was extracted and analyzed by PCR using specific primers. TNF-alpha was assayed in culture supernatants by ELISA. An association was observed between the absence of the TNF-238A allele and negative serology. Furthermore, seropositive individuals carrying the TNF-238A allele produced significantly higher TNF-alpha levels without stimulation (p = 0.04) and after stimulation with LPS (p = 0.007) and T. cruzi antigens (p = 0.004). The present results suggest that the polymorphism at position -238 influences susceptibility to infection and that this allele is associated with higher TNF-alpha production in seropositive individuals.

Antileishmania Immunological Tests for Asymptomatic Subjects Living in a Visceral Leishmaniasis-Endemic Area in Brazil

The objective of this study was to evaluate the behavior of different tests used for the diagnosis of visceral leishmaniasis (VL) in asymptomatic subjects living in an endemic area. No gold standard is available for the diagnosis of asymptomatic infection with Leishmania. In continuation of a previous study, 1,017 subjects living in a VL-endemic area were clinically reevaluated. Of these, 576 had at least one positive serological test in a first assessment. About 3 years after the first evaluation, none of the subjects had progressed to clinical VL. Among this group, 246 subjects were selected, and five serological tests (enzyme-linked immunosorbent assay p [ELISAp], ELISArK39, ELISArK26, indirect immunofluorescence test [IIFT] using L. amazonensis promastigote antigen, and an immunochromatographic test using rK39 antigen [TRALd]) and the Montenegro skin test (MST) were repeated. There was a significant increase in the number of subjects who tested positive in the MST, IIFT, ELISAp, and ELISArK39 in the second evaluation. For all tests, there were subjects who tested positive in the first evaluation and negative in the second evaluation. A positive result in the serological tests and MST in subjects from the endemic area studied did not indicate a risk of progression to VL and may only be temporary.