RESUMO
The arene-supported cationic nickel allyl complexes serve as good catalysts for olefin hydrosilylation at room temperature. Detailed mechanistic studies based on experiments and DFT calculations support the novel mechanism, which includes the facile Si-H bond cleavage and Si-C bond formation, assisted by a non-innocent allyl ligand.
RESUMO
A cDNA clone encoding the Na+, K(+)-Atpase gamma-subunit polypeptide was cloned from a human fetal liver cDNA library. The deduced amino acid sequence of the protein comprised 58 amino acids with a calculated molecular weight of 6400 Da, and showed about 86% homology when compared with those of the bovine, rat and mouse Na+, K(+)-ATPase gamma-subunits published elsewhere. Northern blot analysis showed that the cDNA hybridized to a 0.7 kb mRNA which was expressed in human kidney, pancreas and fetal liver.
Assuntos
DNA Complementar/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Feto/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Pâncreas/metabolismo , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/química , Distribuição TecidualRESUMO
cDNA clones encoding two different subtypes of histone macroH2A1, macroH2A1.1 and macroH2A1.2, have been isolated from human liver tissue. The open reading frames in the isolated clones predicted proteins comprising 368 and 371 amino acids respectively. Estimated molecular masses of the two proteins were 39.0 kDa and 39.4 kDa. Human histone macroH2A1.1 and macroH2A1.2 showed about 98% identity with their counterparts isolated from rat. The features of the nucleotide sequences of the two macroH2A1 subtypes in human were the same as in the rat system. Northern blot analysis showed that the macroH2A1.1 and macroH2A1.2 subtypes were expressed as mRNA species with a size of 1.5 and 4.4 kb, respectively. They were expressed in all human tissues examined.
Assuntos
DNA Complementar/isolamento & purificação , Histonas/genética , Fígado/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sondas de DNA , DNA Complementar/química , Humanos , Dados de Sequência Molecular , RNA Mensageiro/análise , Alinhamento de SequênciaRESUMO
The D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct cytolysis of pancreatic beta-cells. cDNA covering the major outer capsid protein (VP1) of the EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J mice immunized with live recombinant BCG-VP1 (rBCG-VP1) became diabetic when challenged with the highly diabetogenic EMC-D virus, but the control mice inoculated with normal BCG developed diabetes during the same challenge. VP1-specific antibodies (including neutralizing antibodies) were markedly increased over time and reached the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than 4 weeks. Mice and guinea pigs immunized with live rBCG-VP1 showed strong delayed-type hypersensitivity to the VP1 of the EMC-D virus. The preventive immunity still worked effectively 10 months after the primary immunization. At that time, the VP1-specific antibody was almost undetectable in the bloodstream, but a large number of VP1-specific lymphocytes was found in the spleen of the immunized mice. Our results show that live rBCG-VP1 elicits effective humoral and long-lasting cellular immune responses against EMC-D virus infection that results in the prevention of virus-induced diabetes in susceptible mice.
Assuntos
Vacina BCG/uso terapêutico , Proteínas do Capsídeo , Capsídeo/imunologia , Infecções por Cardiovirus/complicações , Diabetes Mellitus Tipo 1/prevenção & controle , Diabetes Mellitus Tipo 1/virologia , Vírus da Encefalomiocardite , Mycobacterium bovis/imunologia , Vacinas Sintéticas/uso terapêutico , Animais , Sequência de Bases , Capsídeo/genética , Clonagem Molecular , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/imunologia , Genoma Viral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Mycobacterium bovis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti-inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)-12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose- and time-dependent manner as it was measured by determining annexin V-positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex-mediated apoptosis. The Dex-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex-induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.
Assuntos
Apoptose/fisiologia , Antígenos CD40/fisiologia , Caspases/metabolismo , Células Dendríticas/citologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Anti-Inflamatórios/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Antígenos CD40/imunologia , Inibidores de Caspase , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Dexametasona/antagonistas & inibidores , Ativação Enzimática , Expressão Gênica , Glucocorticoides/antagonistas & inibidores , Antagonistas de Hormônios/farmacologia , Humanos , Mifepristona/farmacologia , Receptores de Glucocorticoides/antagonistas & inibidores , Transdução de Sinais/fisiologiaRESUMO
To understand modulation of a novel immune-related cytokine, interleukin-18, by human papillomavirus type (HPV) 16 oncogenes, HaCaT, normal keratinocyte cell line, and C-33A, HPV-negative cervical cancer cell line, were prepared to establish stable cell lines expressing E6, E6 mutant (E6m), E6E7, or E7 constitutively. Expressions of various HPV oncogene transcripts were identified by RT-PCR. Expression of HPV oncogene E6 was reversely correlated to the expression of interleukin-18, a novel pro-inflammatory cytokine. The expression of E6 in C-33A, independent of E6 splicing, resulted in decreased IL-18 expression and that of IL-18 was also significantly reduced in HaCaT cells expressing E6. The level of p53 was reduced in C-33A cells expressing E6 whereas not altered in HaCaT cells expressing E6, suggesting that E6 downregulated IL-18 expression via an independent pathway of p53 degradation in HaCaT cells which have a mutated p53 form. However, E7 did not affect IL-18 expression significantly in both C-33A and HaCaT cells. Cotransfection experiments showed that E6 oncogene did not inhibit the activities of IL-18 promoter P1 and P2, suggesting that E6 oncogene indirectly inhibited IL-18 expression. Taken together, E6, E6m and E6/E7 inhibited IL-18 expression with some variation, assuming that cells expressing E6 oncogene can evade immune surveillance by downregulating the expression of immune stimulating cytokine gene, IL-18, and inhibiting the cascade of downstream effects that follow activation of the IL-18 receptor.
Assuntos
Interleucina-18/metabolismo , Proteínas Oncogênicas Virais/farmacologia , Papillomaviridae , Proteínas Repressoras , Proteína Supressora de Tumor p53/metabolismo , Ligação Competitiva , Regulação para Baixo , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Células HeLa/virologia , Humanos , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-18/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is a well established animal model for human multiple sclerosis (MS). Identification of pathogenic epitopes may be helpful in understanding the pathogenesis of this immune-mediated disease. In order to analyze the viral epitopes, we have generated approx. 150 recombinant lambda gt11 clones expressing various capsid areas of TMEV. Six predominant areas, ranging from 13-26 amino acid residues, (3 in VP1, 2 in VP2 and 1 in VP3) are readily recognized by conformation-independent antibodies from virus-infected mice. These areas have been designated as A-1A (VP1 13-27th residues), A-1B (VP1 145-167), A-1C (VP1 251-276), A-2A (VP2 2-14), A-2B (VP2 165-179), and A-3A (tentatively VP3 24-43). Antibodies from TMEV-infected susceptible SJL/J mice strongly react with A-1B, A-2A and A-2B, in contrast to antibodies from resistant BALB/c mice which mainly recognize A-1A and A-2A. Interestingly, the reactivity pattern of antibodies from TMEV-infected mice are somewhat different from that of antibodies from TMEV-immunized mice. Although the majority of antibodies in TMEV-infected mice recognizes conformation-dependent epitopes, the differential recognition of the conformation-independent antibody epitopes by susceptible mice may play a role in TMEV-induced demyelination.
Assuntos
Antígenos Virais , Vírus Elberfeld do Camundongo/imunologia , Animais , Anticorpos Antivirais , Antígenos Virais/química , Doenças Desmielinizantes/imunologia , Infecções por Enterovirus/imunologia , Epitopos/química , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologiaRESUMO
1. Interleukin-12 (IL-12) may play a central role in the development and progression of rheumatoid arthritis by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN-gamma and low IL-4 production. In this study we investigated the effect of auranofin (AF), an anti-rheumatic gold compound, on IL-12 production in mouse macrophages and dendritic cells, and studied whether AF-mediated inhibition of IL-12 production could regulate a cytokine profile of antigen (Ag)-primed CD4(+) Th cells. 2. Treatment with AF significantly inhibited IL-12 production in lipopolysaccharide (LPS)-stimulated macrophages and also in CD40L-stimulated dendritic cells. AF-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4(+) T cells. AF did not influence the cell surface expression of the class II MHC molecule and the costimulatory molecules CD80 and CD86. 3. Addition of recombinant IL-12 to cultures of AF-pretreated macrophages and CD4(+) T cells restored IFN-gamma production in Ag-primed CD4(+) T cells. 4. The in vivo administration of AF resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with LPS or heat-killed Listeria monocytogenes (HKL), leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in Ag-primed CD4(+) T cells. 5. These findings may explain some known effects of AF including anti-rheumatic effects and the inhibition of encephalitogenicity, and point to a possible therapeutic use of AF in the Th1-mediated immune diseases such as autoimmune diseases.
Assuntos
Antirreumáticos/farmacologia , Auranofina/farmacologia , Citocinas/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-12/biossíntese , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Relação Dose-Resposta Imunológica , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Interleukin-12 (IL-12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN-gamma and low IL-4 production. In this study we investigated whether retinoid-mediated inhibition of interleukin-12 production in mouse macrophages could regulate cytokine profile of antigen (Ag)-primed CD4(+) Th cells. Pretreatment with retinoids (9-cis-RA, all-trans-RA, TTNPB) significantly inhibited IL-12 production by mouse macrophages stimulated with lipopolysaccharide (LPS) or heated-killed Listeria monocytogenes (HKL). Retinoid-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4(+) T cells. Addition of recombinant IL-12 to cultures of retinoid-pretreated macrophages and CD4(+) T cells restored IFN-gamma production in CD4(+) T cells. The in vivo administration of 9-cis-RA resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in CD4(+) T cells. These findings may explain some known effects of retinoids including the inhibition of encephalitogenicity, and point to a possible therapeutic use of retinoids in the Th1-mediated immune diseases such as autoimmune diseases.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Citocinas/sangue , Interleucina-12/biossíntese , Macrófagos/metabolismo , Retinoides/farmacologia , Animais , Antígenos de Bactérias/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Interferon gama/biossíntese , Interleucina-12/farmacologia , Interleucina-4/biossíntese , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/imunologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteínas Recombinantes/farmacologiaRESUMO
1 Interleukin-12 (IL-12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses which are characterized by high IFN-gamma and low IL-4 production. In this study we investigated the effects of curcumin, a natural product of plants obtained from Curcuma longa (turmeric), on IL-12 production by mouse splenic macrophages and the subsequent ability of these cells to regulate cytokine production by CD4+ T cells. 2 Pretreatment with curcumin significantly inhibited IL-12 production by macrophages stimulated with either lipopolysaccharide (LPS) or head-killed Listeria monocytogenes (HKL). 3 Curcumin-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4+ T cells. Addition of recombinant IL-12 to cultures of curcumin-pretreated macrophages and CD4+ T cells restored IFN-gamma production in CD4+ T cells. 4 The in vivo administration of curcumin resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in CD4+ T cells. 5 These findings suggest that curcumin may inhibit Th1 cytokine profile in CD4+ T cells by suppressing IL-12 production in macrophages, and points to a possible therapeutic use of curcumin in the Th1-mediated immune diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Curcumina/farmacologia , Interleucina-12/antagonistas & inibidores , Interleucina-12/biossíntese , Macrófagos/metabolismo , Células Th1/metabolismo , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Interferon gama/biossíntese , Interleucina-4/biossíntese , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/imunologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos DBA , Células Th1/efeitos dos fármacosRESUMO
The cDNA clones, which were highly expressed in liver tissues of hepatocellular carcinoma (HCC) patients, were identified using dot hybridization and reconfirmed by Northern blot analysis. One of the clones, ninjurin (nerve injury induced protein), showed a much higher expression level in the liver tissue of HCC patients than in normal liver tissue. Interestingly, the presence of ninjurin mRNA transcripts was detected with high intensity in HCC tissues when combined with viral infection and cirrhosis, but not with a normal liver or HCC tissue unrelated with viral infection and cirrhosis. We produced a N-terminal part of recombinant ninjurin protein, as well as a monoclonal antibody specific to this polypeptide. The intensity of immunohistochemical staining of the liver tumor tissue, and regenerating tissue for the ninjurin protein, was stronger than that of normal liver tissue. These results suggest that ninjurin may play an important role in the development of HCC combined with cirrhosis and viral infection.
Assuntos
Carcinoma Hepatocelular/metabolismo , Moléculas de Adesão Celular Neuronais/biossíntese , Hepatite Viral Humana/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Fatores de Crescimento Neural/biossíntese , Animais , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular Neuronais/genética , Hepatite Crônica/complicações , Hepatite Crônica/genética , Hepatite Crônica/metabolismo , Hepatite Viral Humana/complicações , Hepatite Viral Humana/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Fígado/anatomia & histologia , Fígado/patologia , Fígado/fisiologia , Fígado/fisiopatologia , Cirrose Hepática/complicações , Cirrose Hepática/genética , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/genética , Fatores de Crescimento Neural/genética , RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The Mycobacterium bovis bacilli Calmette-Guerin (BCG) pcp gene that encodes the pyrrolidone carboxyl peptidase (Pcp) was cloned from a lambdagtll genomic library and sequenced. The nucleotide sequence contains a 669 bp open reading frame coding for a protein of 222 amino acid residues with a calculated molecular mass of 23,209 Da. The deduced amino acid sequence is highly homologous to the Pcps from Bacillus amyloliquefaciens, Pseudomonas fluorescens, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. A multiple sequence alignment revealed highly conserved domains. The BCG pcp gene was overexpressed in Escherichia coli. The Pcp was purified to homogeneity. The recombinant protein was further confirmed by an enzymatic assay.
Assuntos
Mycobacterium bovis/genética , Piroglutamil-Peptidase I/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Mycobacterium bovis/enzimologia , Estrutura Terciária de Proteína , Piroglutamil-Peptidase I/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
KRM-1648 resistant Mycobacterium tuberculosis strains were identified from a collection of rifampicin-resistant strains. Several strains had novel rpoB gene mutations in codons 512, 529 and 533 of the rpoB gene. The strains with mutations in codons 526 or 531, major mutation sites in rifampicin-resistant M. tuberculosis, were resistant to KRM-1648. Also, the strains with other mutations in the rpoB gene that were initially susceptible to KRM-1648 were prone to developing KRM-1648 resistance after further mutation. Thus, KRM-1648 is unlikely to be useful for the treatment of rifampicin-resistant tuberculosis.
Assuntos
DNA Bacteriano/análise , RNA Polimerases Dirigidas por DNA/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Rifamicinas/farmacologia , Alelos , Sequência de Bases , RNA Polimerases Dirigidas por DNA/análise , Resistência a Múltiplos Medicamentos/genética , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Linfócitos T Citotóxicos/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/prevenção & controle , Adenocarcinoma/terapia , Animais , Apresentação de Antígeno , Células da Medula Óssea/citologia , Técnicas de Cocultura , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Citotoxicidade Imunológica , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunização , Memória Imunológica , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-4/farmacologia , Neoplasias Renais/patologia , Ativação Linfocitária , Depleção Linfocítica , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Células Tumorais Cultivadas/imunologia , VacinaçãoRESUMO
A major problem with air calorics appears to be one of technique. A pilot study led to the design of irrigating tips which allow consistant air presentation and simultaneous measurements of irrigating temperature at the delivery orifice (adding a second sensor). Preset temperatures of 24 C and 50 C in our system yielded air equilibration mean temperatures of 27.4 C and 45 C at the delivery orifice during testing. A normal study was carried out at these temperatures with an air flow of six liters per minute for 60 seconds. The range of caloric responses, mean maximum speed, and standard deviation are comparable to values previously reported with water stimulations. Statistical testing indicated no difference between ears for either warm or cold. Also, there was no significant difference for warm versus cold responses. We have performed over 2000 clinical examinations that incorporate this technique with satisfactory results and remarkable acceptance by the patients. The normal or "standard" probe size has been found to be adequate for the majority of the clinic population.
Assuntos
Testes Calóricos/métodos , Testes de Função Vestibular/métodos , Ar , Testes Calóricos/instrumentação , Humanos , Projetos Piloto , TemperaturaRESUMO
Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.
Assuntos
Anticorpos Antineoplásicos/imunologia , Dendritos/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Células da Medula Óssea/imunologia , Células Cultivadas , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Indicadores e Reagentes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Células Tumorais CultivadasRESUMO
Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3.
Assuntos
Catepsina B/metabolismo , Cistatina C/metabolismo , Cistatinas Salivares/metabolismo , Animais , Western Blotting , Catepsina B/genética , Linhagem Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cistatina C/genética , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cistatinas Salivares/genéticaAssuntos
Bacteriólise , Biotecnologia/métodos , DNA Bacteriano/isolamento & purificação , Mycobacterium/genética , Acetilglucosaminidase , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese em Gel de Campo Pulsado , Temperatura Alta , Muramidase , Mycobacterium avium/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Sefarose , Fatores de TempoRESUMO
Using synthetic peptides, we have defined the major linear antibody epitopes of Theiler's murine encephalomyelitis virus (TMEV), i.e., A1A (VP1(12-25)), A1Ba (VP1(146-160)), A1Cb (VP1(262-276)), A2A (VP2(2-16)), A2B (VP2(165-179)), and A3A (VP3(24-37)). A time course study with either pooled or individual sera indicates that susceptible SJL mice intracerebrally infected with TMEV strongly and selectively recognize the A1Cb epitope of VP1, compared with resistant BALB/c or C57BL/6 mice, which broadly recognize most of the epitopes on the different capsid proteins. However, antibodies from SJL mice subcutaneously immunized with TMEV recognize primarily A1Ba, A1Cb, and A2A epitopes. A similar predominant recognition of the A1Cb epitope was found with antibodies from the cerebrospinal fluid of intracerebrally virus-infected SJL mice. Interestingly, a substantial level of antibodies against the A1Cb epitope in virus-infected SJL mice is of the immunoglobulin G2a subclass, in contrast to an undetectable level of this immunoglobulin G subclass in virus-immunized SJL mice. The level of in vitro viral neutralization by antibodies did not correlate with the clinical signs. Antibodies to A1Cb, A2A, and A2B were able to neutralize viral plaque formation in vitro, while antibodies to A3A, A1A, and A1Ba were not.
Assuntos
Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Capsídeo/imunologia , Epitopos/imunologia , Poliomielite/imunologia , Theilovirus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Proteínas do Capsídeo , Doenças Desmielinizantes/imunologia , Isotipos de Imunoglobulinas/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Ensaio de Placa Viral , Proteínas Estruturais Virais/imunologiaRESUMO
The pyrG gene encoding cytidine 5'-triphosphate (CTP) synthetase which catalyzes the terminal reaction for de novo synthesis of pyrimidine nucleotides was cloned and sequenced from Mycobacterium bovis BCG. The BCG pyrG was screened in lambda ZAPII library employing a DNA probe which was constructed from PCR based on a consensus pattern of glutamine amidotransferase type I. The BCG CTP synthetase deduced from a nucleotide sequence consisting of 586 amino acid residues with a calculated molecular weight of 63,642 daltons. Alignment of BCG CTP synthetase with all sequences available in GenBank indicates that the residues suggested as a catalytic triad for glutamine hydrolysis at the carboxyl terminal domain and an amino terminal segment are completely conserved among all CTP synthetases.