Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Cancer Res ; 77(5): 1119-1126, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27879265

RESUMO

IL6 is a pleiotropic cytokine with both pro- and anti-inflammatory properties, which acts directly on cancer cells to promote their survival and proliferation. Elevated serum IL6 levels negatively correlate with survival of cancer patients, which is generally attributed to the direct effects of IL6 on cancer cells. How IL6 modulates the host immune response in cancer patients is unclear. Here, we show the IL6 signaling response in peripheral blood T cells is impaired in breast cancer patients and is associated with blunted Th17 differentiation. The mechanism identified involved downregulation of gp130 and IL6Rα in breast cancer patients and was independent of plasma IL6 levels. Importantly, defective IL6 signaling in peripheral blood T cells at diagnosis correlated with worse relapse-free survival. These results indicate that intact IL6 signaling in T cells is important for controlling cancer progression. Furthermore, they highlight a potential for IL6 signaling response in peripheral blood T cells at diagnosis as a predictive biomarker for clinical outcome of breast cancer patients. Cancer Res; 77(5); 1119-26. ©2016 AACR.


Assuntos
Neoplasias da Mama/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-6/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/sangue , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-6/sangue , Pessoa de Meia-Idade , Transdução de Sinais , Resultado do Tratamento , Adulto Jovem
2.
PLoS One ; 8(8): e72789, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24023646

RESUMO

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Lactobacillus/imunologia , Adulto , Idoso , Antígenos CD36/metabolismo , Doença Crônica , Estudos de Coortes , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/enzimologia , Infecções por HIV/microbiologia , Humanos , Imunidade/imunologia , Inflamação/sangue , Inflamação/imunologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Fosforilação , Receptores Imunológicos/metabolismo , Receptor 2 Toll-Like/metabolismo , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Cytometry A ; 83(8): 752-62, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23765858

RESUMO

We have recently added three new fluorophores-BD Horizon™ V450, BD Horizon V500, and BD Horizon V550 (V450, V500, and V550; BD Biosciences, San Jose, CA) to our existing AmCyan product, forming a group of four violet-excitable dyes from which we have produced functional antibody conjugates. These conjugates, with emission maxima that range from 450 to 535 nm, are compatible with multilaser flow cytometry (FCM) and can be used for polychromatic FCM in three-color or two-color combinations; in fact, V500 fills a spectral opening that has thus far not been exploited by other manufacturers of FCM reagents. We here report that conjugates based on BD Horizon dyes performed well within a useful sensitivity range, established by testing a representative group of violet-excitable FCM reagents currently available, and that V500 has better compatibility with FITC in multicolor applications than does AmCyan.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Memória Imunológica , Fosforilação , Processamento de Proteína Pós-Traducional , Coloração e Rotulagem , Linfócitos T/imunologia , Linfócitos T/metabolismo
4.
Clin Cancer Res ; 18(2): 499-509, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22128302

RESUMO

PURPOSE: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody-based immunotherapy that includes the activation of natural killer (NK) cells. EXPERIMENTAL DESIGN: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP). RESULTS: VTX-2337 selectively activates TLR8 with an EC(50) of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158). CONCLUSIONS: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Benzazepinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Receptor 8 Toll-Like/agonistas , Aminoquinolinas/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Sinergismo Farmacológico , Células HEK293 , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Polimorfismo de Nucleotídeo Único , Receptores de IgG/genética , Receptores de IgG/metabolismo , Rituximab , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo
5.
Methods Mol Biol ; 717: 155-69, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21370030

RESUMO

In recent years, techniques that combine the use of phospho-specific antibodies and multiparameter flow cytometry have been developed for the detection of protein phosphorylation at the single cell level. Flow cytometry is uniquely suited for this type of analysis, as it can measure functional and phenotypic markers in the context of complex cell populations. Phosphorylation can be assessed simultaneously in multiple cell subsets, and due to the small sample sizes required, and the rapid analyses of large numbers of cells in this approach, rare cell analysis is possible without the ex vivo expansion of cells.In this chapter, we detail flow cytometric protocols for the detection of intracellular phospho-proteins in samples derived from whole blood and peripheral blood mononuclear cell preparations. These protocols define steps for cell activation, fixation, permeabilization, and staining by phospho-specific and phenotyping antibodies. We discuss technical difficulties inherent to this technique and suggest solutions to commonly encountered problems. Additionally, we show examples of phospho-protein detection in lymphocyte subsets, dendritic cells, and monocytes activated with various stimuli, including mitogens, cytokines, and superantigens. Finally, we highlight a potential clinical trial application for this flow cytometric assay as a platform for pharmacodynamic monitoring of kinase inhibitors.


Assuntos
Anticorpos Fosfo-Específicos/análise , Citometria de Fluxo/métodos , Leucócitos Mononucleares/citologia , Fosfoproteínas/análise , Fosfoproteínas/sangue , Animais , Humanos , Transdução de Sinais , Coloração e Rotulagem/métodos
6.
J Immunol ; 179(4): 2627-33, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17675526

RESUMO

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8(+) T cells, and a lower proportion of IFN-gamma-producing CD4(+) and/or CD8(+) T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8(+) T cells in breast cancer patients was almost completely CD28(+)CD45RA(-) (memory phenotype). CMV-responsive CD8(+) T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27(-)CD28(-)CD45RA(+)) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.


Assuntos
Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Adulto , Antígenos Virais/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/patologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Feminino , Humanos , Imunoterapia , Influenza Humana/imunologia , Influenza Humana/patologia , Masculino , Pessoa de Meia-Idade
7.
Curr Opin Immunol ; 17(4): 434-40, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15950444

RESUMO

Our ability to analyze T-cell function in vitro has progressed in recent years to include analysis of early signaling events, such as specific protein phosphorylation, intermediate functions, such as degranulation and cytokine production, and later functions, such as proliferation. Many assays are now available to monitor these events, and comparative studies of some of these assays have been published. Major recent developments in this area include the ability to measure T-cell degranulation via cell surface exposure of CD107 and the use of polychromatic flow cytometry to examine multiple phenotypes and functions of responding T cells.


Assuntos
Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Cálcio/metabolismo , Proliferação de Células , Humanos , Fosforilação , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/metabolismo
8.
BMC Immunol ; 6: 13, 2005 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-15978127

RESUMO

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.


Assuntos
Citocinas/sangue , Citometria de Fluxo/normas , Linfócitos T/química , Preservação de Sangue , Criopreservação , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo/métodos , Liofilização , Humanos , Indicadores e Reagentes , Laboratórios , Linfócitos/química , Fosfoproteínas/sangue , Reprodutibilidade dos Testes , Manejo de Espécimes , Proteínas da Matriz Viral/sangue
9.
BMC Immunol ; 4: 9, 2003 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-12952557

RESUMO

BACKGROUND: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. RESULTS: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. CONCLUSION: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.


Assuntos
Citocinas/análise , Citometria de Fluxo/métodos , Algoritmos , Automação , Criopreservação , Citometria de Fluxo/instrumentação , Citometria de Fluxo/normas , Corantes Fluorescentes , Humanos , Software , Linfócitos T/imunologia , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...