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1.
Pathologica ; 111(2): 58-61, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31388196

RESUMO

INTRODUCTION: The 2011 WHO Classification for lung adenocarcinoma enlightened the need for a wise use of immunohistochemistry to preserve tissue for both diagnosis and molecular studies. The current recommendation is to use a panel comprising TTF1 and p40 to classify tumors with no clear squamous or glandular differentiation as many studies have showed the higher specificity of p40 over p63 as marker of squamous differentiation. However, the co-expression of both markers opens a new scenario with subsequent classification and potentially treatment issues. MATERIALS AND METHODS: We report a case of a non-small lung cell carcinoma (NSCLC) with coexistent expression of TTF1 and p40 in the same tumour cells. To our knowledge, this peculiar immunohistochemical profile is very rare, and thus a review of the clinical and molecular features including molecular variances of the tumour was performed. Review of the pertinent literature was also carried out. RESULTS: Two additional articles describing unusual cases of NSCLC with coexistent expression of TTF1 and p40 were found and compared to our case. Interestingly, they all carried out aberrant mutation in TP53 oncogene and were of advance stage. CONCLUSION: The positivity for both "squamous" and "adenocarcinomatous" markers and mutations of TP53 could be the expression of a not fully recognized variant of NSCLC with possible implications for classification, diagnosis and therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Epitopos Imunodominantes/genética , Neoplasias Pulmonares/genética , Mutação/genética , Fragmentos de Peptídeos/genética , Fatores de Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Humanos , Epitopos Imunodominantes/biossíntese , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/biossíntese , Fatores de Transcrição/biossíntese
2.
Head Neck Pathol ; 9(1): 79-84, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24969705

RESUMO

Polymorphous low grade adenocarcinoma (PLGA) is a tumor of minor salivary glands that exhibits considerable morphologic overlap with adenoid cystic carcinoma and cellular pleomorphic adenoma, especially in small biopsy specimens. Unlike these other tumor types. PLGAs do not harbor a myoepithelial component, yet their frequent positivity for p63 diminishes the usefulness of this particular myoepithelial marker as a discriminating immunostain. p40 is an antibody that recognizes ΔNp63, a p63 isoform that is more specific for true myoepithelial differentiation. As such, p40 immunostaining could help distinguish PLGAs from adenoid cystic carcinomas and pleomorphic adenomas. In this study, p63 and p40 immunohistochemistry was performed on paraffin embedded, formalin fixed tissue from 11 PLGAs, 101 adenoid cystic carcinomas, and 31 pleomorphic adenomas. All 11 PLGAs (100 %) were positive for p63 but completely negative for p40. Among adenoid cystic carcinomas, 91 of 101 (90 %) were positive for p63 and 90/101 (89 %) were positive for p40. The single discordant p63+/p40- adenoid cystic carcinoma exhibited solid architecture and high grade features not typically seen in PLGA. Among pleomorphic adenomas, 21/31 (68 %) were positive for p63 and 13/31 (42 %) were positive for p40. For the pleomorphic adenomas, the discordant p63+/p40- staining pattern was seen only in the overtly mesenchymal chondromyxoid stroma. The cellular epithelial component of the pleomorphic adenomas demonstrated concordant p63+/p40+ or p63-/p40- immunophenotypes. PLGA consistently exhibits a p63+/p40- immunophenotype that can help distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma, tumors that characteristically demonstrate concordant p63 and p40 immunostaining patterns. A p63/p40 immunohistochemical panel can provide a valuable tool for making the distinction between these morphologically similar but clinically divergent entities.


Assuntos
Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/análise , Neoplasias das Glândulas Salivares/diagnóstico , Adenoma Pleomorfo/diagnóstico , Carcinoma Adenoide Cístico/diagnóstico , Diagnóstico Diferencial , Humanos , Epitopos Imunodominantes/biossíntese , Imuno-Histoquímica , Imunofenotipagem , Proteínas de Membrana/biossíntese , Gradação de Tumores , Fragmentos de Peptídeos/biossíntese , Glândulas Salivares Menores/patologia
3.
J Proteomics ; 77: 176-86, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22959960

RESUMO

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. To identify new vaccine candidates a protein microarray was constructed by expressing the open reading frames (ORFs) from Chlamydia mouse pneumonitis (MoPn). C57BL/6, C3H/HeN and BALB/c mice were immunized either intranasally or intravaginally with live MoPn elementary bodies (EB). Two additional groups were immunized by the intramuscular plus subcutaneous routes with UV-treated EB, using CpG and Montanide as adjuvants to favor a Th1 response, or Alum, to elicit a Th2 response. Serum samples collected from the three strains of mice were tested in the microarray. The array included the expression of 909 proteins from the 921 ORFs of the MoPn genome and plasmid. A total of 530 ORFs were recognized by at least one serum sample. Of these, 36 reacted with sera from the three strains of mice immunized with live EB. These antigens included proteins that were previously described as immunogenic such as MOMP and HSP60. In addition, we uncovered new immunogens, including 11 hypothetical proteins. In summary, we have identified new immunodominant chlamydial proteins that can be tested for their ability to induce protection in animal models and subsequently in humans.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Pneumonia por Clamídia/imunologia , Epitopos Imunodominantes/imunologia , Animais , Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/patologia , Chlamydia trachomatis/metabolismo , Pneumonia por Clamídia/metabolismo , Pneumonia por Clamídia/patologia , Imunização , Epitopos Imunodominantes/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Análise Serial de Proteínas/métodos
4.
J Immunol ; 188(11): 5283-92, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22544926

RESUMO

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.


Assuntos
Endotélio Vascular/imunologia , Antígeno HLA-A2/fisiologia , Linfócitos T Citotóxicos/imunologia , Ligação Competitiva/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Antígeno HLA-A2/biossíntese , Antígeno HLA-A2/metabolismo , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/metabolismo , Epitopos Imunodominantes/fisiologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Ligação Proteica/imunologia , Espectrometria de Massas por Ionização por Electrospray , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia
5.
J Immunol ; 186(7): 3927-33, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21357536

RESUMO

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (ß), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ∼84 HSV-1 proteins are recognized by CD8(+) T cells, and most (∼80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 ß and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Herpesvirus Humano 1/imunologia , Epitopos Imunodominantes/imunologia , Doença Aguda , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Células Cultivadas , Epitopos de Linfócito T/isolamento & purificação , Feminino , Herpes Simples/imunologia , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/isolamento & purificação , Ceratite Herpética/imunologia , Ceratite Herpética/metabolismo , Ceratite Herpética/virologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/biossíntese , Gânglio Trigeminal/imunologia , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/virologia , Latência Viral/imunologia
6.
Carbohydr Res ; 346(2): 348-51, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21176892

RESUMO

This work reports the synthesis and the biological validation of a trisaccharide analogue of the HNK-1 epitope. The 3-O-sulfo-ß-d-GlcpA-(1→3)-ß-d-Galp-(1→4)-ß-d-Glcp-allyl has been prepared by enzymatic glucuronylation of allyl lactoside by an engineered recombinant Escherichia coli strain followed by a chemoselective sulfation. Subsequent covalent attachment of the ozone-oxidised trisaccharide to bovine serum albumin provided a neo-glycoconjugate, which has been interrogated with antibodies specific to the human natural killer carbohydrate epitope HNK-1. ELISA assays confirmed the absolute requirement of the sulfate group for protein recognition and the potential application of this synthetic oligosaccharide as HNK-1 surrogate.


Assuntos
Compostos Alílicos/metabolismo , Antígenos CD57/biossíntese , Escherichia coli/enzimologia , Epitopos Imunodominantes/biossíntese , Oligossacarídeos/biossíntese , Trissacarídeos/biossíntese , Compostos Alílicos/química , Compostos Alílicos/imunologia , Encéfalo/imunologia , Antígenos CD57/química , Antígenos CD57/imunologia , Sequência de Carboidratos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/imunologia , Soroalbumina Bovina/química , Trissacarídeos/química , Trissacarídeos/imunologia
7.
J Immunol ; 184(7): 3866-77, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20194724

RESUMO

Triptolide is a biologically active component purified from Chinese herbal plant Tripterygium wilfordii Hook F. It is widely used in East Asia for treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, Bechect's disease, psoriasis, atopic dermatitis, and asthma. However, its immunological mechanisms are poorly understood. IL-12 and IL-23 are closely related heterodimeric cytokines that share the common subunit p40. They are produced by APCs and are key factors in the generation and effector functions of Th1 and Th17 cells, respectively. They have been strongly implicated in the pathogenesis of several autoimmune disorders. In this study, we investigated the molecular mechanism whereby triptolide inhibits the expression of the p40 gene in APCs. We demonstrate that triptolide does so at the transcriptional level in part through targeting CCAAT/enhancer-binding protein-alpha (C/EBPalpha), which directly interacts with the p40 promoter and inhibits its transcription in inflammatory macrophages. Triptolide can activate the transcription of C/EBPalpha, and phosphorylation of Ser21 and Thr222/226 critical for C/EBPalpha inhibition of p40. Further, activation of C/EBPalpha by triptolide is dependent on upstream kinases ERK1/2 and Akt-GSK3beta. This study provides mechanistic insights into the immunomodulatory capacity of triptolide and has strong implications for its therapeutic applications in autoimmune diseases.


Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Proteína alfa Estimuladora de Ligação a CCAAT/efeitos dos fármacos , Diterpenos/farmacologia , Imunossupressores/farmacologia , Interleucina-12/biossíntese , Interleucina-23/biossíntese , Fenantrenos/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/imunologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Compostos de Epóxi/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/efeitos dos fármacos , Epitopos Imunodominantes/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/imunologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
8.
PLoS Pathog ; 6(12): e1001237, 2010 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-21203487

RESUMO

M. tuberculosis (MTB) species-specific antigenic determinants of the human T cell response are important for immunodiagnosis and vaccination. As hypoxia is a stimulus in chronic tuberculosis infection, we analyzed transcriptional profiles of MTB subject to 168 hours of hypoxia to test the hypothesis that upregulation by hypoxia might result in gene products being recognized as antigens. We identified upregulation of two region of difference (RD) 11 (Rv2658C and Rv2659c), and one RD2 (Rv1986) absent from commonly used BCG strains. In MTB infected persons, the IL-2 ELISpot response to Rv1986 peptides was several times greater than the corresponding IFN-γ response to the reference immunodominant ESAT-6 or CFP-10 antigens. The IL-2 response was confined to two epitopic regions containing residues 61-80 and 161-180. The biggest population of IL-2 secreting T cells was single cytokine positive central memory T cells. The IL-2 response to live MTB bacilli lacking Rv1986 was significantly lower than the response to wild type or mutant complemented with Rv1986. In addition, the IL-2 response to Rv1986 was significantly lower in HIV-TB co-infected persons than in HIV uninfected persons, and significantly increased during antiretroviral therapy. These findings demonstrate that Rv1986 is an immunodominant target of memory T cells and is therefore of relevance when considering the partial efficacy of currently used BCG vaccines and provide evidence for a clinical trial comparing BCG strains.


Assuntos
Hipóxia/imunologia , Epitopos Imunodominantes/genética , Linfócitos T/imunologia , Ativação Transcricional , Tuberculose/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Perfilação da Expressão Gênica , Humanos , Epitopos Imunodominantes/biossíntese , Memória Imunológica , Especificidade do Receptor de Antígeno de Linfócitos T
9.
Intervirology ; 52(1): 35-42, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19372702

RESUMO

BACKGROUND: Antibodies induced from glycoprotein B (gB) by antigenic domain (AD)-1 demonstrate broad neutralizing activity across different human cytomegalovirus (HCMV) types. This study aimed to prepare a novel HCMV vaccine using the modified adenoviral vector Ad5F35 to direct the expression of the conserved HCMV epitope AD-1 and to determine its transfer and expression in peripheral blood mononuclear cells (PBMCs). METHODS: AD-1 genes were amplified from AD169 HCMV strain and cloned into the Ad5F35 vector. Ad5F35-AD-1 virus vaccine was prepared by packaging Ad5F35-AD-1 into HEK293 cells. RT-PCR and fluorescence detection were used to detect the expression of AD-1 in HEK293 cells. PBMCs were stimulated in vitro with Ad5F35-AD-1 virus vaccine. The AD-1 expression in PBMCs was determined with immunocytochemistry and cell viability was measured to observe the possible adverse effects of AD-1 on PBMCs. RESULTS: We constructed an Ad5F35-AD-1 vector and transferred it into HEK293 cells to prepare the Ad5F35-AD-1 virus vaccine successfully. The AD-1 gene was proved to be expressed in HEK293 cells. In vitro stimulation of PBMCs with Ad5F35-AD-1 showed the highly efficient expression of AD-1 and low cytopathic activity in PBMCs. CONCLUSION: The novel vaccine Ad5F35-AD-1 is a promising candidate for clinical trials and may be of utility in prime-boost strategies for HCMV prevention and control.


Assuntos
Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/imunologia , Epitopos Imunodominantes/imunologia , Proteínas do Envelope Viral/imunologia , Adenoviridae/genética , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/uso terapêutico , DNA Recombinante/metabolismo , Vetores Genéticos/metabolismo , Humanos , Epitopos Imunodominantes/biossíntese , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Estrutura Terciária de Proteína , Transfecção , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
10.
J Immunol ; 182(3): 1526-32, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19155500

RESUMO

Conventional CD8(+) T cell responses against intracellular infectious agents are initiated upon recognition of pathogen-derived peptides presented at the cell surface of infected cells in the context of MHC class I molecules. Among the major MHC class I loci, HLA-B is the swiftest evolving and the most polymorphic locus. Additionally, responses restricted by HLA-B molecules tend to be dominant, and most associations with susceptibility or protection against infectious diseases have been assigned to HLA-B alleles. To assess whether the differences in responses mediated via two major HLA class I loci, HLA-B and HLA-A, may already begin at the Ag presentation level, we have analyzed the diversity and binding affinity of their peptide repertoire by making use of curated pathogen-derived epitope data retrieved from the Immune Epitope Database and Analysis Resource, as well as in silico predicted epitopes. In contrast to our expectations, HLA-B alleles were found to have a less diverse peptide repertoire, which points toward a more restricted binding motif, and the respective average peptide binding affinity was shown to be lower than that of HLA-A-restricted epitopes. This unexpected observation gives rise to new hypotheses concerning the mechanisms underlying immunodominance of CD8(+) T cell responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica , Antígenos HLA-A/metabolismo , Antígenos HLA-B/metabolismo , Epitopos Imunodominantes/metabolismo , Alelos , Motivos de Aminoácidos , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/virologia , Citotoxicidade Imunológica/genética , Genoma Bacteriano/genética , Genoma Bacteriano/imunologia , Genoma Viral/genética , Genoma Viral/imunologia , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/fisiologia , Ligantes , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Proteoma/genética , Proteoma/metabolismo
11.
J Immunol ; 181(9): 6361-70, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18941227

RESUMO

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.


Assuntos
Apresentação de Antígeno/imunologia , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/química , Regulação da Expressão Gênica/imunologia , Hepacivirus/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígeno HLA-B35/biossíntese , Antígeno HLA-B35/química , Antígeno HLA-B35/imunologia , Antígeno HLA-B35/metabolismo , Hepacivirus/genética , Hepatite C Crônica/imunologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Dados de Sequência Molecular , Proteoma/biossíntese , Proteoma/síntese química , Proteoma/imunologia , Proteoma/metabolismo , Linfócitos T Citotóxicos/virologia , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/síntese química , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo
12.
Infect Immun ; 76(6): 2642-50, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18391007

RESUMO

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Epitopos Imunodominantes/imunologia , Leptospira/imunologia , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Animais , Especificidade de Anticorpos , Proteínas da Membrana Bacteriana Externa/genética , Clonagem Molecular , Colágeno Tipo IV/química , Feminino , Fibronectinas/química , Gelatina/farmacologia , Heparina/farmacologia , Humanos , Soros Imunes/imunologia , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/genética , Leptospira/genética , Leptospira/metabolismo , Leptospirose/sangue , Leptospirose/imunologia , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia
14.
Protein Expr Purif ; 59(2): 309-13, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18406167

RESUMO

The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGERA179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.


Assuntos
Antígenos Virais/biossíntese , Antígenos Virais/imunologia , Glicoproteínas/biossíntese , Glicoproteínas/imunologia , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Antígenos Virais/genética , Escherichia coli/genética , Vetores Genéticos/genética , Glicoproteínas/genética , Humanos , Imunoquímica , Epitopos Imunodominantes/isolamento & purificação , Imunoglobulinas/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/genética
15.
Vet Microbiol ; 128(1-2): 136-47, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18006251

RESUMO

Ehrlichia ruminantium, a tick-transmitted pathogen, is the causative agent of heartwater in ruminants. In this study, a proteomic approach was used to identify host cell-specific E. ruminantium proteins encoded by the map1 multigene family, expressed in vitro in bovine endothelial and tick cell cultures. Two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to establish the identities of immunodominant proteins. Proteins extracted from E. ruminantium-infected endothelial cells were shown to be products of the map1 gene, whereas tick cell-derived E. ruminantium proteins were products of a different gene, map1-1. The expressed proteins were found to be glycosylated. Differential expression of MAP1 family proteins in vitro in mammalian and tick cell cultures indicates that the map1 multigene family might be involved in the adaptation of E. ruminantium to the mammalian host and vector tick.


Assuntos
Proteínas de Bactérias/genética , Doenças dos Bovinos/microbiologia , Ehrlichia ruminantium/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Hidropericárdio/microbiologia , Peptídeos/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bovinos , Células Cultivadas , Ehrlichia ruminantium/genética , Células Endoteliais/citologia , Células Endoteliais/microbiologia , Glicosilação , Interações Hospedeiro-Patógeno/fisiologia , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Ixodidae/citologia , Ixodidae/microbiologia , Peptídeos/metabolismo , Proteômica , Proteínas Recombinantes/biossíntese , Alinhamento de Sequência , Ovinos
16.
Vet Microbiol ; 124(3-4): 256-63, 2007 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-17548173

RESUMO

Mycobacterium tuberculosis heat shock protein70 (HSP70) is a major antigen with both chaperone and cytokine functions. It has been used as an adjuvant to induce or potentiate humoral and cellular immunity, both in the form of a mixture with peptide antigens, and as a fusion protein. We have evaluated the effects of HSP70 on foot and mouth virus (FMDV) subunit vaccines. FMDV VP1, and a synthetic multi-epitope FMDV (EG), and VP1-HSP70 and EG-HSP70 fusion proteins were all heterologously expressed in the yeast Pichia pastoris, and used as antigen in mice. The recombinant VP1 and EG alone was able to induce both humoral and marginal cell-mediated immune responses, while the HSP70 fusions markedly enhanced both the humoral and cell-mediated immune responses. The most prominent immune responses arose from vaccination with the EG-HSP70 fusion product. Both fusion protein-induced Th1-like cytokine (IFN-gamma) and Th2-like cytokine (IL-4) were identified.


Assuntos
Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Epitopos Imunodominantes/biossíntese , Pichia/genética , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Feminino , Febre Aftosa/prevenção & controle , Imunização , Interferon gama/sangue , Interleucina-4/sangue , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Pichia/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia
17.
J Immunol ; 178(11): 7235-41, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17513772

RESUMO

Both human CMV and murine CMV (MCMV) elicit large CD8 T cell responses, despite the potent effects of viral genes that interfere with the MHC class I (MHC I) pathway of Ag presentation. To investigate the impact of immune evasion on CD8 T cell priming, we infected mice with wild-type (wt) MCMV or a mutant lacking its MHC I immune evasion genes, Deltam4+m6+m152 MCMV. In acute infection, the two viruses elicited a CD8 T cell response to 26 peptide epitopes that was virtually identical in total size, kinetics, and immunodominance hierarchy. This occurred despite results demonstrating that primary DCs are susceptible to the effects of MCMV's MHC I immune evasion genes. Eight months later, responses to both wt and mutant MCMV displayed the same CD8 T cell "memory inflation" and altered immunodominance that characterize the transition to chronic MCMV infection in C57BL/6 mice. Taken together, these findings suggest either that cross-priming dominates over direct CD8 T cell priming in both acute and chronic MCMV infection, or else that the MHC I immune evasion genes of MCMV are unable to alter direct CD8 T cell priming in vivo. At 2 years postinfection, differences in CD8 T cell immunodominance emerged between individual mice, but on average there were only slight differences between wt and mutant virus infections. Overall, the data indicate that the presence or absence of MHC I immune evasion genes has remarkably little impact on the size or specificity of the MCMV-specific CD8 T cell response over an entire lifetime of infection.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Epitopos Imunodominantes/metabolismo , Muromegalovirus/imunologia , Interferência Viral/imunologia , Doença Aguda , Animais , Antígenos Ly/biossíntese , Antígenos Ly/metabolismo , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Doença Crônica , Citotoxicidade Imunológica , Imunidade Inata , Epitopos Imunodominantes/biossíntese , Lectinas Tipo C/biossíntese , Lectinas Tipo C/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Receptores Semelhantes a Lectina de Células NK
18.
Vopr Virusol ; 51(3): 46-8, 2006.
Artigo em Russo | MEDLINE | ID: mdl-16826757

RESUMO

The study was undertaken to comparatively examine the efficiency of HIV antibody detection by enzyme immunoassay using recombinant antigens and synthetic peptides containing the most immunodominant region of the human immunodeficiency virus envelop protein gp41-cytotoxic T-lymphocytic (CTL) epitope. The application of the synthetic peptides that mimic this region has been to be inadequately effective. The recombinant proteins that contain the CTL epitope may be successfully used for the diagnosis of HIV-1, by using enzyme immunoassay as they show a higher sensitivity in detecting antibodies than do the synthetic peptises.


Assuntos
Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/diagnóstico , HIV-1/imunologia , Técnicas Imunoenzimáticas/métodos , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Anticorpos Anti-HIV/sangue , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Peptídeos/síntese química , Peptídeos/imunologia , Valor Preditivo dos Testes , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia
19.
J Immunol ; 176(12): 7308-16, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16751374

RESUMO

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.


Assuntos
Antígenos de Neoplasias/análise , Epitopos de Linfócito T/análise , Proteínas de Membrana/análise , Fragmentos de Peptídeos/análise , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/patologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/metabolismo , Antígenos de Superfície , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/metabolismo , Células HCT116 , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Epitopos Imunodominantes/análise , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/metabolismo , Imunossupressores/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
20.
Infect Immun ; 74(1): 711-20, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16369028

RESUMO

Ehrlichia canis major immunoreactive proteins of 36 and 19 kDa elicit the earliest detectable antibody responses during the acute phase of canine monocytic ehrlichiosis. Genes encoding the major immunoreactive 36-kDa protein of E. canis and the corresponding ortholog of E. chaffeensis (47 kDa) were identified and the proteins characterized. The molecular masses of the strongly immunoreactive recombinant proteins were larger than predicted (26.7 and 32.9 kDa, respectively) but were consistent with those of the corresponding native proteins (36 and 47 kDa). Similar to other reported ehrlichial immunoreactive glycoproteins, carbohydrate was detected on the recombinant expressed proteins, indicating that they were glycoproteins. Both glycoproteins (gp36 and gp47) have carboxy-terminal serine/threonine-rich tandem repeat regions containing repeats that vary in number (4 to 16 repeats) and amino acid sequence among different isolates of each species. E. canis gp36 was recognized by early acute-phase antibodies (day 14), and species-specific antibody epitopes were mapped to C-terminal nonhomologous repeat units of gp36 and gp47. Periodate treatment of recombinant gp36 reduced the antibody reactivity, and nonglycosylated synthetic peptide repeat units from E. canis gp36 and E. chaffeensis gp47 were substantially less immunoreactive than corresponding recombinant peptides, demonstrating that glycans are important epitope determinants that are structurally conserved on the recombinant proteins expressed in Escherichia coli. E. canis gp36 and E. chaffeensis gp47 were differentially expressed only on the surface of dense-cored ehrlichiae and detected in the Ehrlichia-free supernatants, indicating that these proteins are released extracellularly during infection.


Assuntos
Ehrlichia canis/imunologia , Ehrlichia chaffeensis/imunologia , Epitopos Imunodominantes/biossíntese , Sequências de Repetição em Tandem/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Cães , Ehrlichiose/imunologia , Glicoproteínas/biossíntese , Glicoproteínas/imunologia , Glicosilação , Epitopos Imunodominantes/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular
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