RESUMO
It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL-17A, IFN-γ and IL-10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR- (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine-producing cells. Lesions from TT patients were found to have more CD4+ IL-17A+ cells than those from LL patients. Higher concentrations of IL-17A and IL-1ß were observed in serum from PB than MB patients. The highest serum IFN-γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+ ). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.
Assuntos
Mediadores da Inflamação/imunologia , Hanseníase Paucibacilar/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Células Th1/imunologia , Células Th17/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Criança , Busca de Comunicante , Feminino , Citometria de Fluxo , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Interferon gama/sangue , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/sangue , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/sangue , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Hanseníase/sangue , Hanseníase/microbiologia , Hanseníase Multibacilar/sangue , Hanseníase Multibacilar/imunologia , Hanseníase Multibacilar/microbiologia , Hanseníase Paucibacilar/sangue , Hanseníase Paucibacilar/microbiologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Mycobacterium leprae/fisiologia , Células Th1/metabolismo , Células Th17/metabolismo , Adulto JovemRESUMO
The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO2) were stimulated with: 10 µg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 µg/ml), PHA (1 µg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.
Assuntos
Interferon gama/metabolismo , Hanseníase Paucibacilar/diagnóstico , Mycobacterium leprae/imunologia , Adulto , Idoso , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Testes de Liberação de Interferon-gama , Hanseníase Paucibacilar/sangue , Hanseníase Paucibacilar/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Leprosy is a dermato-neurological disease caused by Mycobacterium leprae infection that manifests across a wide range of clinical and immunological outcomes. Diagnosis is still currently based on clinical manifestations and simple tests are needed. This study investigated whether biomarkers induced by defined M. leprae proteins in 24-h whole blood assays (WBA) could discriminate active leprosy patients from at-risk contacts. Newly diagnosed, untreated paucibacillary (PB; tuberculoid leprosy/borderline tuberculoid [TT/BT]) and multibacillary (MB; borderline lepromatous/lepromatous leprosy [BL/LL]) leprosy patients, as well as healthy household contacts (HHC) of MB patients, were recruited in central western Brazil (Goiânia/Goiás). Cell-based responses to the ML0276, ML1623, ML0405, ML1632, 92f, and ML1011 antigens were measured by Luminex 14-plex assays detecting eotaxin, IFNγ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-15, IL-17A, IL-23, IL-31, IP-10, and TNFα. Our data reinforce that IFNγ is currently the best indicator of the antigen-specific cellular immune response of TT/BT leprosy and demonstrate that the same antigens promote the secretion of IL-4 in blood from BL/LL leprosy patients. While none of the biomarkers tested could discriminate leprosy patients from HHC, our data indicate that, although most HHC antigen-specific responses are qualitatively similar to TT/BT patients, some HHC can respond similarly to BL/LL patients.
Assuntos
Antígenos de Bactérias/imunologia , Citocinas/metabolismo , Hanseníase/diagnóstico , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Brasil , Saúde da Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Although curable, leprosy requires better diagnostic and prognostic tools to accompany therapeutic strategies. We evaluated the serum samples of leprosy patients from Venezuela and Brazil for reactivity against the specific recombinant proteins, ML0405 and ML2331, and the LID-1 fusion protein that incorporates both of these antigens. Antigen-specific IgG was highest in lepromatous leprosy patients (LL) and decreased across the disease spectrum, such that only a small subset of true tuberculoid patients (TT) tested positive. The impact of multidrug therapy (MDT) on these antibody responses was also examined. Several years after treatment, the vast majority of Venezuelan patients did not possess circulating anti-LID-1, anti-ML0405, and anti-ML2331 IgG, and the seropositivity of the remaining cases could be attributed to irregular treatment. At discharge, the magnitude and proportion of positive responses of Brazilian patients against the proteins and phenolic glycolipid (PGL)-I were lower for most of the clinical forms. The monthly examination of IgG levels in LL patient sera after MDT initiation indicated that these responses are significantly reduced during treatment. Thus, responses against these antigens positively correlate with bacillary load, clinical forms, and operational classification at diagnosis. Our data indicate that these responses could be employed as an auxiliary tool for the assessment of treatment efficacy and disease relapse.
Assuntos
Anticorpos Antibacterianos/sangue , Monitoramento de Medicamentos/métodos , Imunoglobulina G/sangue , Hanseníase/diagnóstico , Antibacterianos/uso terapêutico , Antígenos de Bactérias , Brasil , Humanos , Hanseníase/tratamento farmacológico , Estudos Longitudinais , Proteínas Recombinantes , Recidiva , Fatores de Tempo , Resultado do Tratamento , VenezuelaRESUMO
Immunotherapy has been proposed as a method to treat mucosal leishmaniasis for many years, but the approach has been hampered by poor definition and variability of antigens used, and results have been inconclusive. We report here a case of antimonial-refractory mucosal leishmaniasis in a 45 year old male who was treated with three single injections (one per month) with a cocktail of four Leishmania recombinant antigens selected after documented hypo-responsiveness of the patient to these antigens, plus 50 microg of GM-CSF as vaccine adjuvant. Three months after treatment, all lesions had resolved completely and the patient remains without relapse after two years. Side effects of the treatment included only moderate erythema and induration at the injection site after the second and third injections. We conclude that carefully selected microbial antigens and cytokine adjuvant can be successful as immunotherapy for patients with antimonial-refractory mucosal leishmaniasis.
Assuntos
Antígenos de Protozoários/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Imunoterapia , Úlcera da Perna/tratamento farmacológico , Leishmania/imunologia , Leishmaniose Mucocutânea/tratamento farmacológico , Adjuvantes Imunológicos/uso terapêutico , Animais , Humanos , Leishmaniose Mucocutânea/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Serological tests to detect Trypanosoma cruzi antibodies have been used for screening blood donors, for epidemic studies, and for diagnosis of probably infected persons. Among different tests, the enzyme-linked immunosorbent assay (ELISA) with total, semipurified, or synthetic antigens has been widely used, mainly due to its easy automation. Aiming to improve serological studies concerning Chagas' disease, we have developed and evaluated a new test, the TcF-ELISA, using an artificially engineered recombinant antigen, which contains tandem sequences of different T. cruzi-specific peptides. The sensibility of the TcF-ELISA was determined with 101 serum samples from chagasic patients well-defined by clinical and epidemiological criteria. The specificity was determined with 39 serum samples from leishmaniasis or kala-azar patients and 150 serum samples from nonchagasic blood donors from Sao Paulo, Brazil. The TcF-ELISA showed 100% sensitivity and 98.94% of specificity. Compared with conventional ELISA (with semipurified T. cruzi epimastigote antigens), the TcF-ELISA showed advantages; for example, it distinguishes better between reagent and nonreagent serum and provides better precision and a lower occurrence of leishmaniasis cross-reactions. Our studies demonstrate high reproducibility between two different lots of the TcF ELISA and its applicability for the serological diagnosis of Chagas' disease.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/genética , Doença de Chagas/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Peptídeos/genética , Peptídeos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.
Assuntos
Imunidade Inata/imunologia , Leishmania braziliensis/imunologia , Fatores de Iniciação de Peptídeos/imunologia , Proteínas de Protozoários , Animais , Citocinas/biossíntese , Citocinas/imunologia , Citotoxicidade Imunológica , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos SCID , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Recombinantes/imunologia , Baço/citologia , Baço/imunologiaRESUMO
A tetrapeptide and a recombinant protein, each representing 4 immunodominant epitopes of Trypanosoma cruzi, were tested by use of ELISA for the detection of serum antibodies. Sera from individuals with Chagas' disease, including persons untreated and successfully or unsuccessfully treated, were tested. These assays detected antibody in 100% of the parasitemias. The antibody reactivity decreased based on the success of treatment. Higher sensitivity was observed for tetrapeptide/recombinant protein assays than for lysate-based ELISA, and specificity was improved, particularly with Leishmania sera. The results indicate that multiepitope antigens provide a more sensitive and specific alternative to lysate for detection of anti-T. cruzi antibodies, as required for developing blood screening assays.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Oligopeptídeos , Proteínas Recombinantes , Brasil/epidemiologia , Humanos , Epitopos Imunodominantes , Parasitemia/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Five cases of Trypanosoma cruzi meningoencephalitis in HIV-infected patients are reported. All patients presented with mass lesions on head computed tomographic scan, trypanosomes in the cerebrospinal fluid and failure to respond to antitoxoplasmosis therapy. Benznidazole therapy was associated with clinical improvement in 1 patient. Another 4 patients had T cruzi identified in a peripheral smear. T cruzi needs to be considered in the differential diagnosis of HIV-infected patients with central nervous system mass lesions if they have a history of appropriate exposure.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Meningoencefalite/parasitologia , Trypanosoma cruzi , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico por imagem , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Adulto , Animais , Encéfalo/diagnóstico por imagem , Feminino , Humanos , Masculino , Meningoencefalite/diagnóstico por imagem , Meningoencefalite/tratamento farmacológico , Radiografia , Tomógrafos ComputadorizadosRESUMO
An enzyme-linked immunosorbent assay (ELISA) has been developed to detect IgG and IgM antibodies in human sera against a synthetic tripeptide derived from a hybrid peptide containing 3 specific epitopes from Trypanosoma cruzi. This assay was compared in Brazil with one using conventional antigen, the alkaline crude extract. Serum samples were divided into positive (40 samples) or negative (107 samples) for Chagas disease. Positive samples included 9 serum samples from patients with acute Chagas disease, while negative samples included 57 samples from patients suffering from viral diseases. The total percentages of IgG positive samples from patients with chronic Chagas disease for alkaline extract and synthetic tripeptide were 93.5% and 100%, respectively. All samples from patients with acute Chagas disease were confirmed positive for IgM antibodies by using both the tripeptide and the alkaline extract. However, the results for anti-T. cruzi IgM in the group of chronic Chagas disease patients demonstrated that 41.9% were positive for IgM with the alkaline extract, while the synthetic peptide showed a significantly lower number of positive samples (12.9%). The serum samples from healthy people showed similar results for both antigens. However, 40% of the serum samples from patients presenting with viral diseases were IgM positive for Chagas disease when assayed with conventional antigen; with the synthetic tripeptide as antigen, 100% of this group of samples were found to be negative. Thus, as the results of ELISA with synthetic tripeptide showed higher rates of sensitivity and specificity than ELISA with conventional antigen, the former should be included as a laboratory tool in the serodiagnosis of Chagas disease.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Doença de Chagas/diagnóstico , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/química , Epitopos/imunologia , Humanos , Peptídeos/síntese química , Peptídeos/imunologia , Sensibilidade e EspecificidadeRESUMO
Diffuse cutaneous leishmaniasis (DCL) is characterized by the presence of numerous nonulcerated nodules and plaques containing large numbers of Leishmania amazonensis parasites and few lymphoid elements. The immune responses of DCL patients reflect severe antigen-specific T-cell deficiencies, while the antibody response to Leishmania antigens is often accentuated. We report herein on the Leishmania antigen-specific antibody subclass distribution in DCL patients and demonstrate that a dominant antigen contributing to the biased immunoglobulin G4 antibody subclass in sera of DCL patients is Leishmania heat shock protein 83.
Assuntos
Antígenos de Protozoários/imunologia , Proteínas de Choque Térmico/imunologia , Imunoglobulina G/sangue , Leishmania/imunologia , Leishmaniose Tegumentar Difusa/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Brasil/epidemiologia , Humanos , Imunoglobulina G/imunologia , Leishmaniose Tegumentar Difusa/sangue , Leishmaniose Tegumentar Difusa/epidemiologiaRESUMO
Two well-defined synthetic peptides TcD and PEP2 were used in a sero-epidemiological study for the detection of Trypanosoma cruzi infections in an indigenous group in the Amazon region of Ecuador. Of the 18 communities studied along the Rio Napo, province of Napo, 15 (83.3%) were found to be positive for T. cruzi infection. Of the 1,011 individuals examined 61 (6.03%) resulted positive. A prevalence of infection of 4.8% was found in children aged 1-5 years. The prevalence of infection increased with age, with adults 50 years or older showing a maximum prevalence of 18.8%. Autochthonous transmission of T. cruzi is present among this isolated indigenous population.
Assuntos
Doença de Chagas/epidemiologia , Doença de Chagas/transmissão , Indígenas Sul-Americanos , Adolescente , Criança , Pré-Escolar , Equador/epidemiologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos SoroepidemiológicosRESUMO
The diagnosis of visceral leishmaniasis (VL), a serious and often fatal parasitic disease caused by members of the Leishmania donovani complex, remains problematic. Current methods rely on clinical criteria, parasite identification in aspirate material, and serology. The latter methods use crude antigen preparations lacking in specificity. A previously described cloned antigen, rK39, of Leishmania specific for all members of the L. donovani complex (L. chagasi, L. donovani, L. infantum) was very useful in the serodiagnosis by ELISA of both human and canine VL. The present study demonstrated that rK39 seroreactivity correlated with active disease. The sera from early or self-healing infected subjects reacted with leishmanial lysate and were generally nonreactive with rK39. These data demonstrate the utility of rK39 in the serodiagnosis of VL and as an indicator of active disease.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Proteínas de Protozoários/imunologia , Animais , Especificidade de Anticorpos , Antígenos de Protozoários/genética , Biomarcadores , Brasil/epidemiologia , Criança , Clonagem Molecular , Reações Cruzadas , Cães , Ensaio de Imunoadsorção Enzimática , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Proteínas de Protozoários/genética , Testes SorológicosRESUMO
Gamma interferon (IFN-gamma) plays an important role in experimental Trypanosoma cruzi infections, presumably by controlling the early replication of parasites in host macrophages. In this work, we show that NK cells represent an important cell type responsible for the production of most of the IFN-gamma in the early stage of T. cruzi infection and that the in vivo treatment of mice with anti-NK1.1 monoclonal antibody made resistant animals susceptible to the infection. Through in vitro experiments, we demonstrate that normal splenocytes from euthymic or athymic nude mice cultivated for 48 h with live T. cruzi trypomastigotes produced elevated levels of IFN-gamma. In addition, NK-depleted splenocytes show a drastic reduction of IFN-gamma production in response to live T. cruzi trypomastigotes. We also demonstrated that IFN-gamma production is dependent on a factor secreted by adherent cells. Supernatants of spleen cells from athymic nude mice are able to induce IFN-gamma production by normal splenocytes when cultured with trypomastigotes. The addition of anti-interleukin-10 to these cultures resulted in a marked increase in IFN-gamma production. On the other hand, the absence of NK cells led to an increased secretion of interleukin-10 upon in vitro stimulation with T. cruzi. Taken together, these results suggest that NK cells are the major source of IFN-gamma that could be involved in limiting the replication of T. cruzi in host macrophages during the early acute phase of the infection.
Assuntos
Doença de Chagas/parasitologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Células Matadoras Naturais/metabolismo , Animais , Adesão Celular , Células Cultivadas , Doença de Chagas/metabolismo , Suscetibilidade a Doenças , Feminino , Imunidade Inata , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Parasitemia/metabolismo , Baço/citologia , Baço/metabolismo , Baço/parasitologiaRESUMO
The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.
Assuntos
Regulação da Expressão Gênica , Genes de Protozoários , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Leishmania donovani/genética , Macrófagos/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/genética , Feminino , Antígenos de Histocompatibilidade Classe II/biossíntese , Leishmania/classificação , Leishmania/genética , Leishmania/imunologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Fases de Leitura Aberta , Fragmentos de Peptídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas de Protozoários/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
In 1991, a community cross-sectional study was conducted in a village situated near the beach and close to Salvador, the capital city of Bahia, in Brazil, to determine the prevalence of visceral leishmaniasis since 1989. A serological survey was made of human and canine reservoirs and an intradermal skin test for leishmaniasis was used to assess cellular immune responses. Nearly 30% of the 243 individuals in the study area had positive skin tests and 14% had positive serology, the latter being compatible with recent infection; 29 of 460 dogs examined were seropositive. A possible association was observed between human infection and the presence of dogs in or near residences, but not between human infection and malnutrition. This report describes the evolution of a new focus of visceral leishmaniasis, its expansion toward a metropolitan area, and current measures taken to control the epidemic.
Assuntos
Leishmaniose Visceral/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Antiprotozoários/análise , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , Cães , Humanos , Imunidade Celular , Lactente , Recém-Nascido , Leishmaniose Visceral/imunologia , Pessoa de Meia-Idade , Estado Nutricional , Estações do Ano , Testes CutâneosRESUMO
CD8+ T cells and lysis of parasitized macrophages seem to be important in the resistance to murine leishmaniasis. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) from patients with either cutaneous (CL) or mucosal (ML) leishmaniasis in cell lysis assays using 51-Cr-labeled Daudi or K562 cells, or autologous antigen-pulsed macrophages as targets. Results are reported as lytic units (number of cells required for 30% lysis) per million PBMC. Exposure of patient PBMC (n = 12) to lysate from Leishmania amazonensis promastigotes led to an increase in cytotoxic activity compared to unstimulated patient cells against Daudi (81.8 +/- 14.9 vs 13.6 +/- 5 lytic units (LU) per million PBMC; mean +/- SEM) and K562 (65.7 +/- 8.4 vs 13.1 +/- 5 LU/10(6) PBMC). ML had higher responses than CL in both targets (80.4 +/- 11.0 vs 46.4 +/- 11.6 LU/10(6) PBMC for K562, and 104.3 +/- 23.8 vs 59.3 +/- 14.3 LU/10(6) PBMC for Daudi). Normal control PBMC, stimulated with L. amazonensis antigen had 6.32 +/- 3.72 LU/10(6) PBMC against Daudi cells and 9.06 +/- 2.78 LU/10(6) PBMC against K562. The cell responsible for lysis of the K562 cells was characterized as NK, by means of cell separation employing magnetic beads coupled to antibodies. Addition of recombinant TGF-beta or recombinant human IL-10 reduced L. amazonensis-induced cytotoxicity by 90% and 70%, respectively. Cytotoxicity of antigen-stimulated PBMC was also demonstrated against autologous L. amazonensis antigen-pulsed macrophages in the range of 6.7 to 41.7 LU/10(6) PBMC.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Relação Dose-Resposta Imunológica , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Leishmaniose Cutânea/parasitologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Camundongos , Linfócitos T Citotóxicos , Fator de Crescimento Transformador beta/imunologia , Células Tumorais Cultivadas/imunologiaRESUMO
Twenty-four patients with acute visceral leishmaniasis and leukopenia (< 1500 neutrophils/mm3) due to Leishmania chagasi were studied, 4 in an open-label pilot study and 20 in a double-blind, placebo-controlled trial. Patients received granulocyte-macrophage colony-stimulating factor (GM-CSF), 5 micrograms/kg daily, or placebo for 10 days, plus 10-20 mg/kg pentavalent antimony daily for 20 days. In GM-CSF recipients, neutrophil counts increased threefold and fourfold over baseline at 5 and 10 days, respectively, and were significantly higher than those in placebo recipients (P < .02). Eosinophil and monocyte counts were significantly increase in GM-CSF recipients at 10 days (P < or = .03). Secondary infections occurred in 3 GM-CSF and in 8 placebo recipients (P = .04). All patients had complete resolution of their leishmaniasis at 3 months. Few adverse events were recorded. GM-CSF, 5 micrograms/kg daily for 10 days, was safe, rapidly reversed neutropenia, and reduced the number of secondary infections in patients with leishmaniasis.
Assuntos
Infecção Hospitalar/prevenção & controle , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Neutropenia/tratamento farmacológico , Adolescente , Adulto , Antimônio/economia , Antimônio/uso terapêutico , Antiprotozoários/economia , Antiprotozoários/uso terapêutico , Medula Óssea/patologia , Criança , Pré-Escolar , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/economia , Custos de Cuidados de Saúde , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/complicações , Contagem de Leucócitos , Masculino , Meglumina/economia , Meglumina/uso terapêutico , Antimoniato de Meglumina , Neutropenia/complicações , Neutropenia/etiologia , Compostos Organometálicos/economia , Compostos Organometálicos/uso terapêutico , Projetos Piloto , Proteínas Recombinantes/economia , Proteínas Recombinantes/uso terapêutico , Índice de Gravidade de DoençaRESUMO
Visceral leishmaniasis is associated with a marked depression of T cell responses, which has been characterized by the absence of IL-2 and IFN-gamma production by lymphocytes on in vitro stimulation with Leishmania Ag. The aim of this study was to evaluate both the mechanism of these immunologic abnormalities and the restoration of in vitro T cell responses to Leishmania Ags. A total of 15 untreated visceral leishmaniasis patients were evaluated. Although IFN-gamma and IL-4 levels in the supernatants of lymphocyte cultures were very low or absent, mRNA for these cytokines and for IL-10 were observed in PBMCs. Addition of IFN-gamma plus IL-2 enhanced lymphocyte proliferation by 158%. Restoration of T cell proliferative responses and IFN-gamma production was also observed by the addition of a neutralizing mAb alpha-IL-10. Neutralizing mAb alpha-IL-4 did not restore T cell responses but alpha-IL-10 and alpha-IL-4 mAbs had a synergistic effect on lymphocyte proliferation. The IFN-gamma levels in supernatants of lymphocyte cultures stimulated with Leishmania chagasi Ag or L. chagasi Ag plus alpha-IL-4, alpha-IL-10, or alpha-IL-4 plus alpha-IL-10 mAbs were 26 +/- 30 pg/ml, 41 +/- 18 pg/ml, 146 +/- 73 pg/ml, and 174 +/- 106 pg/ml, respectively. These data indicate that Th2 cell activation occurs in visceral leishmaniasis and that in vitro production of IFN-gamma and lymphocyte proliferation can be restored by blocking the inhibitory effect of the Th2 cytokines on mononuclear cells.
Assuntos
Interferon gama/biossíntese , Leishmaniose Visceral/imunologia , Ativação Linfocitária , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Protozoários , Humanos , Tolerância Imunológica , Técnicas In Vitro , Interferon gama/genética , Interferon gama/farmacologia , Interleucina-10/antagonistas & inibidores , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-2/farmacologia , Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Interleucina-4/metabolismo , Leishmania/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.