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1.
Am J Trop Med Hyg ; 66(4): 409-15, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12164297

RESUMO

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.


Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias , Hipersensibilidade Tardia/microbiologia , Hanseníase Dimorfa/microbiologia , Mycobacterium leprae/isolamento & purificação , Nervos Periféricos/microbiologia , Pele/microbiologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Biópsia , Chaperonina 60 , Chaperoninas/análise , Chaperoninas/imunologia , Humanos , Imuno-Histoquímica , Hanseníase Dimorfa/imunologia , Lipopolissacarídeos/análise , Lipopolissacarídeos/imunologia , Macrófagos/microbiologia , Mycobacterium leprae/imunologia , Nervos Periféricos/imunologia , Células de Schwann/microbiologia , Pele/imunologia
4.
J Bacteriol ; 182(12): 3590-2, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10852894

RESUMO

The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.


Assuntos
Mycobacterium leprae/metabolismo , Processamento de Proteína , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Mycobacterium leprae/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Recombinação Genética
5.
Immunol Lett ; 75(1): 69-76, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11163869

RESUMO

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Citocinas/biossíntese , Eritema Nodoso/imunologia , Hanseníase Virchowiana/imunologia , Diferenciação Celular , Citocinas/genética , Humanos , Leucócitos Mononucleares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo
6.
Lepr Rev ; 71 Suppl: S130-7, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11201870

RESUMO

In order to increase our understanding of the immunological basis of erythema nodosum leprosum (ENL), we studied Th-like cytokine profiles in 130 leprosy patients, employing both the conventional and a novel, real-time, fluorogenic reverse transcriptase-based PCR (RT-PCR). The concomitant expression of both Th-like cytokines, interferon-gamma and IL-4, and the regulatory cytokines, IL-10 and IL-12, was studied in the peripheral blood cells of leprosy patients with and without ENL. In the conventional RT-PCR, varied cytokine profiles were observed in individual patients of all clinical types. Fifty-three percent of lepromatous patients without ENL and 59% of tuberculoid leprosy patients showed co-expression of IFN gamma and IL-4, indicating a non-polarized Th 0 pattern. Of the 36 patients with ENL, 58% demonstrated a polarized Th 1 pattern, with only 30% expressing both cytokines. Semiquantitative RT-PCR indicated a lower expression of IL-4 compared to that of IFN gamma in the lepromatous patients without ENL; the difference was even greater among those with ENL. The sensitive, real-time PCR confirmed the down-regulation of IL-4 and IL-10, with absence of IL-4 in half of the patients, resulting in skewing of the cytokine response toward a Th 1-like profile.


Assuntos
Regulação para Baixo , Eritema Nodoso/diagnóstico , Interleucina-4/fisiologia , Hanseníase Virchowiana/diagnóstico , Hanseníase Tuberculoide/diagnóstico , Citocinas/fisiologia , Ensaio de Imunoadsorção Enzimática , Eritema Nodoso/complicações , Feminino , Humanos , Hanseníase Virchowiana/complicações , Hanseníase Tuberculoide/complicações , Masculino , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
8.
Nature ; 400(6741): 269-71, 1999 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-10421369

RESUMO

Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.


Assuntos
Proteínas de Bactérias , Tuberculose/terapia , Vacinas de DNA/uso terapêutico , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antituberculosos/uso terapêutico , Chaperonina 60 , Chaperoninas/genética , Terapia Combinada , Feminino , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Isoniazida/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Plasmídeos , Pirazinamida/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tuberculose/imunologia , Vacinas de DNA/imunologia
9.
Int. j. lepr. other mycobact. dis ; 67(2): 177-178, Jun., 1999. ilus
Artigo em Inglês | SES-SP, HANSEN, SESSP-ILSLACERVO, SES-SP | ID: biblio-1226877
10.
Int. j. lepr. other mycobact. dis ; 66(4): 597-598, Dec. 1998.
Artigo em Inglês | SES-SP, HANSEN, SESSP-ILSLACERVO, SES-SP | ID: biblio-1226828
12.
Vaccine ; 15(8): 834-8, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9234527

RESUMO

Past attempts to use fractions of mycobacteria as an alternative to BCG have given disappointing results. The availability of cloned genes and suitable vectors has now opened a new avenue in which individual mycobacterial protein antigens are synthesised within transfected mammalian cells. In an ex vivo transfection approach with a retroviral vector we found that even a single antigen (hsp65) could evoke strong protection when expressed as a transgene and that expression of protection was largely a function of antigen specific cytotoxic T cells. We now find that intramuscular injection of plasmid DNA expressing the antigen from either a viral or a murine promoter can also give protection equivalent to Bacillus Calmette-Guérin (BCG). Plasmids expressing some other mycobacterial antigens, hsp70, 36 kDa and 6 kDa, are also effective, suggesting that this approach may lead to a new vaccine.


Assuntos
Vacina BCG/imunologia , Proteínas de Bactérias , Tuberculose/prevenção & controle , Vacinas de DNA/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacina BCG/genética , Chaperonina 60 , Chaperoninas/genética , Chaperoninas/imunologia , Células Clonais/imunologia , Injeções Intramusculares , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Plasmídeos/genética , Plasmídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem
13.
Infect Immun ; 65(4): 1395-401, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9119479

RESUMO

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.


Assuntos
Antituberculosos/farmacologia , Resistência Microbiana a Medicamentos/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Isoniazida/farmacologia , Mycobacterium tuberculosis/enzimologia , Oxirredutases/biossíntese , Peroxidases , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Oxirredutases/genética , Peroxirredoxinas , Virulência/genética
16.
Immunology ; 86(1): 97-103, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7590888

RESUMO

Cytokine profiles of circulating mononuclear cells were studied with the aim of delineating T-cell subsets in leprosy patients with active disease. Using reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokine mRNA and enzyme-linked immunoassay (ELISA) for the secreted products, interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied. Three antigens, native Mycobacterium leprae, a recombinant antigen LSR/A15 of M. leprae and peptide 624 spanning 58-77 amino acids of the latter, were used to induce cytokine expression and release. Half of the subjects, irrespective of the clinical type or antigen used, showed a mixed T-helper type 0 (Th0)-like cytokine pattern, with evidence of the concomitant presence of IFN-gamma and IL-4. The remainder showed a polarized pattern based on the type of leprosy. Lepromatous patients with disseminated disease had Th2-type cytokines, with IL-4 but not IFN-gamma. In contrast, tuberculoid leprosy patients with localized disease showed a Th1-like profile, with the presence of IFN-gamma but not IL-4. Of interest was the stability of the Th phenotype for M. leprae-related antigens. Both the recombinant and the peptide antigens induced the same phenotype as the natural M. leprae bacillus in all except four of 45 leprosy patients.


Assuntos
Antígenos de Bactérias/imunologia , Citocinas/análise , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Subpopulações de Linfócitos T/química , Linfócitos T Auxiliares-Indutores/química , Sequência de Bases , Citocinas/genética , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Humanos , Interferon gama/análise , Interleucina-4/análise , Interleucina-6/análise , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo
17.
Int J Lepr Other Mycobact Dis ; 63(1): 35-41, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7730717

RESUMO

Skin biopsy and slit-skin smears from 46 leprosy patients and 4 nonleprosy patients were tested for the presence of Mycobacterium leprae by the polymerase chain reaction (PCR) using primers based on the sequence of the LSR/15 kD gene. The PCR was found to be specific and sensitive, with a detection level of 10 and 100 bacilli. PCR using skin biopsies gave a higher detection rate than did slit-skin smears, probably due to the higher density of bacilli in a 4-mm punch biopsy. Dot blot hybridization with radioactive probes was 10-fold more sensitive than the ethidium bromide staining. Eight patients who did not show acid-fast bacilli in tissues by the conventional methods were shown to have PCR-amplified M. leprae DNA. False-negative results were obtained in 3 cases even though formal evidence for tissue inhibitors was absent.


Assuntos
DNA Bacteriano/análise , Genes Bacterianos , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase , Pele/microbiologia , Sequência de Bases , Sondas de DNA/química , Eletroforese em Gel de Ágar , Reações Falso-Negativas , Humanos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Dados de Sequência Molecular , Mycobacterium leprae/isolamento & purificação , Valor Preditivo dos Testes , Sensibilidade e Especificidade
18.
Clin Exp Immunol ; 99(2): 196-202, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7851011

RESUMO

Leprosy may be complicated by episodes of increased cell-mediated immunity towards Mycobacterium leprae (reversal reactions) which result in severe local immunopathology in skin lesions and peripheral nerves. Using in situ hybridization and MoAb techniques we have demonstrated TNF-alpha mRNA and TNF-alpha protein in macrophages infiltrating leprosy skin and peripheral nerve. Levels of TNF-alpha mRNA are significantly increased in reactional skin and nerve, particularly in borderline tuberculoid patients. TNF-alpha mRNA and TNF-alpha protein levels are higher in reactional nerves then reactional skin. In both reactional skin and nerve TNF-alpha mRNA is more abundant than TNF-alpha protein; this may reflect the rapid turnover of TNF-alpha protein in an immunologically dynamic situation, such as is seen in reversal reaction. Our findings emphasize the importance of documenting both mRNA and protein production when assessing the role of cytokines in pathology. The leprosy reversal reaction may be regarded as a useful model of tissue immunopathology in which TNF-alpha is generated as part of the host response to infection, but also produces local tissue damage.


Assuntos
Hanseníase/imunologia , Hanseníase/patologia , Nervos Periféricos/patologia , Pele/patologia , Fator de Necrose Tumoral alfa/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mycobacterium leprae/imunologia , Nervos Periféricos/imunologia , Nervos Periféricos/microbiologia , RNA Mensageiro/biossíntese , Pele/imunologia , Pele/microbiologia , Fator de Necrose Tumoral alfa/biossíntese
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