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1.
Braz. j. med. biol. res ; 48(12): 1095-1100, Dec. 2015. graf
Artigo em Inglês | LILACS | ID: lil-762920

RESUMO

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.


Assuntos
Animais , Masculino , Camundongos , Linfócitos B/imunologia , Proteínas de Choque Térmico/imunologia , Imunomodulação/genética , /genética , RNA Mensageiro/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos B/metabolismo , Citometria de Fluxo , Expressão Gênica/genética , Proteínas de Choque Térmico/uso terapêutico , Memória Imunológica/fisiologia , Imunofenotipagem/classificação , Mediadores da Inflamação/análise , Interferon gama/análise , /imunologia , /análise , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/genética , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/classificação , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
2.
Braz J Med Biol Res ; 48(12): 1095-100, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26397973

RESUMO

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.


Assuntos
Linfócitos B/imunologia , Proteínas de Choque Térmico/imunologia , Imunomodulação/genética , Interleucina-10/genética , RNA Mensageiro/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos B/metabolismo , Citometria de Fluxo , Expressão Gênica/genética , Proteínas de Choque Térmico/uso terapêutico , Memória Imunológica/fisiologia , Imunofenotipagem/classificação , Mediadores da Inflamação/análise , Interferon gama/análise , Interleucina-10/imunologia , Interleucina-12/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/classificação , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
3.
Arch Dermatol Res ; 301(2): 151-7, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18633632

RESUMO

Increase in the number of patients with atopic dermatitis (AD) has been recently reported. T helper (Th) cells that infiltrate AD skin lesions are Th2-type dominant; reduced exposure to environmental Th1-cytokine-inducing microbes is believed to contribute to the increased number of AD patients. Regulatory type immune responses have been also associated with the occurrence of AD. It has been reported that antigen 85B (Ag85B) purified from mycobacteria is a potent inducer of Th1-type immune response in mice as well as in humans. In this study, we have examined the effect of plasmid DNA encoding Ag85B derived from Mycobacterium kansasii on AD skin lesions induced by oxazolone (OX) application. Th2-cytokine mediated mouse AD model with immediate type response followed by a late phase reaction was developed by repeated applications of low-dose OX to sensitized mice. Mice were immunized with plasmid DNA encoding cDNA of Ag85B before OX sensitization or during repeated elicitation phase. Both therapies were associated with significant suppression of immediate type response, clinical appearance, dermal cell infiltration, reduced IL-4 production, and augmented IFN-gamma mRNA expression compared to placebo-treated mice. Additionally, increased number of Foxp3(+) regulatory T cells were observed in the skin sections in Ag85B treated mice. The results of this study suggest that Ag85B DNA vaccine is a potential therapy for Th2 type dermatitis.


Assuntos
Reação de Fase Aguda/patologia , Antígenos de Bactérias/farmacologia , Citocinas/metabolismo , Dermatite Atópica/patologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/patologia , Células Th2/patologia , Reação de Fase Aguda/induzido quimicamente , Reação de Fase Aguda/tratamento farmacológico , Animais , Antígenos de Bactérias/uso terapêutico , DNA/farmacologia , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxazolona/efeitos adversos , Plasmídeos/farmacologia , Vacinas de DNA/uso terapêutico
4.
Am J Respir Crit Care Med ; 172(11): 1452-6, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16151038

RESUMO

RATIONALE: Priorities for developing improved regimens for treatment of latent tuberculosis (TB) infection include (1) developing shorter and/or more intermittently administered regimens that are easier to supervise and (2) developing and evaluating regimens that are active against multidrug-resistant organisms. OBJECTIVES AND METHODS: By using a previously validated murine model that involves immunizing mice with Mycobacterium bovis bacillus Calmette-Guérin to augment host immunity before infection with virulent Mycobacterium tuberculosis, we evaluated new treatment regimens including rifapentine and moxifloxacin, and assessed the potential of the Mycobacterium leprae heat shock protein-65 DNA vaccine to augment the activity of moxifloxacin. MEASUREMENTS: Quantitative spleen colony-forming unit counts, and the proportion of mice with culture-positive relapse after treatment, were determined. MAIN RESULTS: Three-month, once-weekly regimens of rifapentine combined with either isoniazid or moxifloxacin were as active as daily isoniazid for 6-9 mo. Six-month daily combinations of moxifloxacin with pyrazinamide, ethionamide, or ethambutol were more active than pyrazinamide plus ethambutol, a regimen recommended for latent TB infection after exposure to multidrug-resistant TB. The combination of moxifloxacin with the experimental nitroimidazopyran PA-824 was especially active. Finally, the heat shock protein-65 DNA vaccine had no effect on colony-forming unit counts when given alone, but augmented the bactericidal activity of moxifloxacin. CONCLUSIONS: Together, these findings suggest that rifapentine, moxifloxacin, and, perhaps, therapeutic DNA vaccination have the potential to improve on the current treatment of latent TB infection.


Assuntos
Antibióticos Antituberculose/uso terapêutico , Compostos Aza/uso terapêutico , Quinolinas/uso terapêutico , Rifampina/análogos & derivados , Tuberculose/tratamento farmacológico , Vacinas de DNA/uso terapêutico , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Fluoroquinolonas , Seguimentos , Camundongos , Camundongos Endogâmicos BALB C , Moxifloxacina , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/uso terapêutico , Baço/microbiologia , Resultado do Tratamento , Tuberculose/microbiologia
5.
Immunology ; 113(1): 130-8, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15312144

RESUMO

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.


Assuntos
Proteínas de Bactérias/genética , Linfócitos T CD8-Positivos/imunologia , Chaperoninas/genética , Interferon gama/biossíntese , Tuberculose Pulmonar/terapia , Vacinas de DNA/uso terapêutico , Animais , Antígenos CD18/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Chaperonina 60 , Proteína Ligante Fas , Feminino , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Regulação para Cima , Receptor fas/metabolismo
6.
Infect Immun ; 71(4): 2192-8, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12654841

RESUMO

The use of DNA constructs encoding mycobacterial proteins is a promising new approach to vaccination against tuberculosis. A DNA vaccine encoding the hsp60 molecule of Mycobacterium leprae has previously been shown to protect against intravenous infection of mice with Mycobacterium tuberculosis in both the prophylactic and immunotherapeutic modes. It is shown here, however, that this vaccine was not effective in a more realistic aerosol infection model or in a model of latent tuberculosis in the lungs. Moreover, when given in an immunotherapeutic model the immunized mice developed classical Koch reactions characterized by multifocal discrete regions of cellular necrosis throughout the lung granulomas. Similar and equally severe reactions were seen in mice given a vaccine with DNA coding for the Ag85 antigen of M. tuberculosis. This previously unanticipated safety problem indicates that DNA vaccines should be used with caution in individuals who may have already been exposed to tuberculosis.


Assuntos
Pulmão/patologia , Mycobacterium tuberculosis/patogenicidade , Vacinas contra a Tuberculose/efeitos adversos , Tuberculose Pulmonar/prevenção & controle , Vacinas de DNA/efeitos adversos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Chaperonina 60/genética , Chaperonina 60/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Necrose , Organismos Livres de Patógenos Específicos , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose Pulmonar/patologia , Tuberculose Pulmonar/terapia , Vacinação , Vacinas de DNA/uso terapêutico
7.
Infect Immun ; 68(6): 3090-6, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10816448

RESUMO

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.


Assuntos
Antígenos de Bactérias , Vacina BCG/uso terapêutico , Proteínas de Bactérias/uso terapêutico , Mycobacterium avium/imunologia , Tuberculose/prevenção & controle , Vacinas de DNA/uso terapêutico , Animais , Proteínas de Bactérias/genética , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Feminino , Receptores de Hialuronatos/isolamento & purificação , Interferon gama/biossíntese , Antígenos Comuns de Leucócito/isolamento & purificação , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Linfócitos T Auxiliares-Indutores , Vacinação
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
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