T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques...

Detection of intracytoplasmic cytokines by flow cytometry

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.

Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

The beginning and expansion of flow cytometry in Brazil

The author describes and make comments on the favorable conditions that made possible the creation and organization of the first laboratory of flow cytometry in Brazil and South America installed at the Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro, in November 1988.

Atypical Mucocutaneous Leishmaniasis Caused by Leishmania braziliensis in an Acquired Immunodeficiency Syndrome Patient: T-cell Responses and Remission of Lesions Associated with Antigen Immunotherapy

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5 percent) most of them with CD8+ phenotype (33 percent). Very low CD4+ cells (2.2 percent) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells

Leishmanioses: reaçäo de imunofluorescência indireta para pesquisa de anticorpos IgM em fraçöes de soros separados por cromatografia de gel filtraçäo / Leishmaniasis: indirect immunofluorescent test to detect IgM antibodies in serum fractions separated by gel filtraction cromatography

Um total de 143 soros de pacientes (120 da forma cutânea localizada, seis da mucocutânea e 17 com leishmaniose visceral), provenientes de ambulatórios ou de hospitais do Grande Rio de Janeiro, suspeitos de leishmaniose tegumentar ou visceral americanas, foi submetido às reaçöes de imunofluorescência indireta (RIFI-IgM e IgG). Estes soros foram selecionados porque se apresentavam com RIFI-IgG de títulos elevados ou eram RIFI-IgM reagentes no soro. Como existe a possibilidade de falsos resultados de IgM näo reagentes na presença de títulos elevados de IgG e falsos IgM reagentes, devido à presença de fator reumatóide (autoanticorpos IgM anti-IgG), utilizou-se a separaçäo das fraçöes IgM e IgG do soro destes pacientes. Para isro, procedeu-se a separaçäo destas imunoglobulinas em coluna de Sephacryl S-300, nos casos em que os soros eram IgM negativo e IgG maior ou igual a 360, com a finalidade de se detectar falsos negativos e, em soros IgM reagentes, falsos positivos. Nestes últimos, também realizou-se a prova do látex para fator reumatóide. Deste modo a RIFI-IgM da fraçäo IgM separada no Sephacryl permitiu evidenciar apenas um soro - de paciente da forma cutânea localizada (0,7 por cento) - falso negativo por provável competiçäo com títulos de anticorpos IgG elevados. Por outro lado, permitiu o encontro de 23 (16,1 por cento) soros RIFI-IgM falso-reagentes para leishmanioses, devido à presença de fator reumatóide (seis soros eram de leishmaniose mucocutânea e os 17 restantes de leishmaniose visceral). Em outros indivíduos da forma cutânea localizada (8,4 por cento) a RIFI-IgM do soro e a RIFI da fraçäo IgM eram reagentes, embora também tivessem positivos (maior ou igual a 20 U/ml) à prova do látex, como os outros 23 indivíduos anteriores. Os 107 indivíduos restantes (74,8 por cento) foram RIFI-IgM reagentes no soro e na fraçäo IgM, mas, entretanto, todos eram negativos para fator reumatóide. Todas as RIFI-IgM das fraçöes IgM eram näo-reagentes. Os resultados demonstram a utilidade da presente metodologia na obtençäo de maior confiabilidade nos testes de RIFI-IgM para leishmanioses

Chagas'disease and HIV co-infection: genotypic characterization of the Trypanosoma cruzi strain

In the past few years, new aspects of the immunopathology of Chagas' disease have been described in immunosuppressed patients, such as fatal central system lesions related to the reactivation of the parasite. This article is the first description of the genotypic characterization, at the strain level, of Trypanosoma cruzi isolated from a patient with Chagas' disease/AIDS co-infection. The presence of four hypodense lesions was observed in the cranial compute tomographic scan. The diagnosis of AIDS was assessed by the detection of anti-HIV antibodies using enzyme-linked immunosorbent assay (ELISA) and Western blot techniques. The CD4+ lymphocyte counts were maintained under 200 cells/mm cube number during one year demonstrating the severity of the state of immunosuppression. Chagas' disease was confirmed by serological and parasitological methods. Trypomastigote forms were visualized in a thick blood smear. The parasite isolated is genotypically similar to the CL strain. The paper reinforces that cerebral Chagas' disease can be considered as another potencial opportunistic infection in AIDS resulting from the reactivation of a dormant T. cruzi infection acquired years earlier.

Tumor necrosis factor-æ in human American tegumentary leishmaniasis

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine produced by activated macrophages and other cells. In order to verify whether the serum levels of TNF-alpha in American tegumentary leishmaniasis patients are associated with the process of cure or aggravation of the disease, 41 patients were studied: 26 cases of cutaneous leishmaniasis (CL) and 15 of mucocutaneous leishmaniasis (MCL). During active disease the serum levels of TNF-alpha of MCL patients were significantly higher than those of CL patients and control subjects (healthy individuals and cutaneous lesions from other etiologies). The MCL patients had serum titers of TNF-alpha significantly lower at the end of antimonial therapy than before therapy. After a six-month follow-up, the MCL patients had serum levels of TNF-alpha similar to those observed at the end of the therapy as well as to those of Cl patients and control subjects. No significant variation in the serum levels of TNF-alpha was observed in CL patients throughout the study period (before, at the end of therapy and after a six-month follow-up). The possible relationship between the high TNF-alpha serum levels and severity of the disease is discussed.

Imunofluorescência (IF) para toxoplasmose em fraçöes IgM de soro humano / Imunofluorescent antibody test (IF) for toxoplasmosis in human serum IgM fractions

Um total de 221 soros de pacientes procedentes de ambulatórios de hospitais do Rio de Janeiro, cuja suspeita clínica era a de infecçåo recente pelo Toxoplasma gondii, foi submetido à reaçåo de imunofluorescéncia indireta (IF) para a pesquisa de anticorpos das classes de imunogloobulinas M e G. Os soros dos 221 indivíduos foram diluídos ao quádruplo, a partir de 1:16. Já que existe a possibilidade de falsos resultados IgM negativos na presença de títulos elevados de IgG e falsos resultados IgM positivos na presença de fator reumatóide (auto-anticorpos IgM anti IgG), utilizou-se a separaçåo das fraçöes IgM e IgG do soro de pacientes. Este procedimento, usando-se coluna de sephacryl S-300, foi realizado em 187 soros IF-IgM nåo reagentes e IF-Ig maior ou igual a 1:1024, com a finalidade de detectar falsos IF-IgM negativos, e em 34 soros IF-IgM reagentes para detectar falsos If-IgM positivos. Nestes últimos também realizou-se a prova látex para a pesquisa dos chamados fatores reumatóides. A IF, realizada na fraçåo IgM separada do soro, permitiu evidenciar 21 soros (11,2 por cento) falso - IgM nåo reagentes devido à possivel competiçåo com anticorpos anti-T. gondii em títulos elevados nas fraçöes IgC. Permitiu, inclusive, o encontro de 5 soros (14,7 por cento) IF-IgM falso reagentes para T. gondii devido à presença de auto-anticorpos IgM anti IgG (fator reumatóide). Os resultados demonstraram a utilidade do presente procedimento no sentido de obter-se maior acurácia nos testes IF-IgM para toxoplasmose

Disseminated American muco-cutaneous leishmaniasis caused by Leishmania brasiliensis brasiliensis in a patient with AIDS: a case report

The authors report a case of culture-proven disseminated American muco-cutaneous leishmaniasis caused by Leishmania brasiliensis brasiliensis in an HIV positive patient. Lesions began in the oropharynx and nasal mucosa eventually spreading to much of the skin surface. The response to a short course of glucantime therapy was good

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally inffected mice, using limiting dilution analysis

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57 B1/10 mice. The results showed that, in Swiss and B1/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a grater number of parasites were observed in heart tissue from Swiss mice than from B1/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and B1/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores

Indirect immunofluorescence test in new world Leishmaniasis: serological and clinical relation-ship

O teste de imunofluorescência indireta (IF) para a detecçäo de anticorpos anti-Leismania nas classes IgG e IgM foi realizado em soros de indivíduos dos seguintes grupos: 214 pacientes com leismaniose cutânea, quatro pacientes com leismaniose mucocutânea, 28 indivíduos sadios com intradermorreaçäo de Montenegro (IDRM) positiva, 29 indivíduos sadios com IDRM negativa e 16 pacientes com leishmaniose visceral. Os indivíduos dos quatro primeiros grupos eram provenientes de uma área da periferia da cidade do Rio de Janeiro (Jacarepaguá) onde a leishmaniose tegumentar causada por Leishmania braziliensis brasiliensis é endêmica. Entre os pacientes com leishmaniose cutânea foi observado que os títulos de IF-IgM foram significantemente mais altos nos casos com menos de quatro meses de evoluçäo do que em pacientes com períodos mais longos de evoluçäo da doença e que os títulos de IF-IgG foram significantemente mais altos em pacientes com lesöes múltiplas do que nos portadores de lesäo única. Os pacientes com leishmaniose visceral tiveram títulos de IF-IgG significantemente superiores aos dos pacientes com leishamiose cutânea. zum grupo de 28 indivíduos selecionados entre os 214 pacientes com leishmaniose cutânea tiveram seus títulos de IF (IgG e IgM) comparados aos de dois grupos controles constituídos de indivíduos sadio, moradores na área endêmica com IDRM respectivamente positiva e negativa. Títulos de IF-IgG e IF-IgM significantemente superiores foram encontrados no grupo com doença ativa. O mesmo grupo de pacientes apresentou título de IF-IgG significantemente mais baixos após a terapêutica antimonial do que durante a mesma

Experimental canine mucocutaneous leishmaniasis (Leishmania braziliensis braziliensis)

Quatro cäes foram inoculados por via intredérmica com 3 x 10**6 promastigotos de Leishmania brasiliensis braziliensis. Três deles desenvolveram lesöes no sítio do inóculo, mas uma úlcera mucosa foi detectada 16 meses após o inóculo. Os achados foram similares áqueles verificados na infecçäo canina natural, bem como na doença humana. Estes resultados sugerem que o cäo pode representar um modelo experimental para a infecçäo causada pela L. b. brasiliensis