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2.
Vaccine ; 37(1): 19-24, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30497835

RESUMO

In the current antibiotic resistance scenario, vaccines may provide best defense against lethal bacterial diseases. So far, there is no idealvaccine available against plague. Despite providing complete protection in small animal models, F1/LcrV based vaccine failed to provide ideal protection in non human primates. Here, we cloned, expressed and purified YopE of Yersinia pestis and flagellin C (FliC) of Salmonella Typhi. However the best possible protection needs the significant induction of IFN-γ and TNF-α. To determine the protective potential of the recombinant YopE alone or in formulation with FliC, Balb/C mice were immunized subcutaneously. The formulations were prepared with alum, a human compatible adjuvant. In our studies, the combination of YopE + FliC induced significantly strong humoral and cellular immune responses. A combination of YopE + FliC provided 83% protection whereas YopE alone provided only 50% against 100LD50 of Y. pestis in a mouse model.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacina contra a Peste/imunologia , Peste/prevenção & controle , Proteínas Recombinantes/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Flagelina/genética , Flagelina/imunologia , Imunidade Celular , Imunidade Humoral , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Salmonella typhi , Vacinação , Yersinia pestis/imunologia
3.
Front Microbiol ; 7: 1053, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27458447

RESUMO

Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1-LcrV and F1-LcrV-HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1-LcrV and F1-LcrV-HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it's isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1-LcrV-HSP70(II) sera in comparison to anti-F1-LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1-LcrV-HSP(II) immunized mice in comparison to F1-LcrV. Both F1-LcrV and F1-LcrV-HSP70(II) provided 100% protection. Our research findings suggest that F1-LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

4.
PLoS Negl Trop Dis ; 8(12): e3322, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25474358

RESUMO

No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Vacina contra a Peste/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/química , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico HSP70/química , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Peste/patologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Vacinação
5.
J Biol Chem ; 288(43): 30815-31, 2013 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-24025335

RESUMO

To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation.


Assuntos
Movimento Celular/fisiologia , Macrófagos/metabolismo , Monócitos/metabolismo , Trombina/metabolismo , Quinases Ativadas por p21/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Ativação Enzimática/fisiologia , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Humanos , Macrófagos/citologia , Camundongos , Monócitos/citologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Trombina/genética , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
6.
Virus Genes ; 46(2): 219-30, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23188193

RESUMO

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.


Assuntos
Dengue/virologia , Escherichia coli/genética , Expressão Gênica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Dengue/diagnóstico , Vírus da Dengue/química , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Escherichia coli/metabolismo , Humanos , Dados de Sequência Molecular , Redobramento de Proteína , Alinhamento de Sequência , Proteínas não Estruturais Virais/metabolismo
7.
J Biol Chem ; 287(43): 36291-304, 2012 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22893700

RESUMO

Toward understanding the mechanisms of vascular wall remodeling, here we have studied the role of NFATc1 in MCP-1-induced human aortic smooth muscle cell (HASMC) growth and migration and injury-induced rat aortic wall remodeling. We have identified PKN1 as a novel downstream target of NFATc1-cyclin D1/CDK6 activity in mediating vascular wall remodeling following injury. MCP-1, a potent chemoattractant protein, besides enhancing HASMC motility, also induced its growth, and these effects require NFATc1-dependent cyclin D1 expression and CDK4/6 activity. In addition, MCP-1 induced PKN1 activation in a sustained and NFATc1-cyclin D1/CDK6-dependent manner. Furthermore, PKN1 activation is required for MCP-1-induced HASMC growth and migration. Balloon injury induced PKN1 activation in NFAT-dependent manner and pharmacological or dominant negative mutant-mediated blockade of PKN1 function or siRNA-mediated down-regulation of its levels substantially suppressed balloon injury-induced smooth muscle cell migration and proliferation resulting in reduced neointima formation. These novel findings suggest that PKN1 plays a critical role in vascular wall remodeling, and therefore, it could be a promising new target for the next generation of drugs for vascular diseases, particularly restenosis following angioplasty, stent implantation, or vein grafting.


Assuntos
Divisão Celular , Movimento Celular , Ciclina D1/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteína Quinase C/metabolismo , Animais , Ciclina D1/genética , Quinase 6 Dependente de Ciclina/genética , Ativação Enzimática , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Humanos , Músculo Liso Vascular/patologia , Mutação , Miócitos de Músculo Liso/patologia , Fatores de Transcrição NFATC/genética , Neointima/genética , Neointima/metabolismo , Neointima/patologia , Proteína Quinase C/genética , Ratos
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