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BMC Immunol ; 9: 39, 2008 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-18652679


BACKGROUND: Human B cells and plasmacytoid dendritic cells (pDC) are the only cells known to express both TLR7 and TLR9. Plasmacytoid dendritic cells are the primary IFN-alpha producing cells in response to TLR7 and TLR9 agonists. The direct effects of TLR7 stimulation on human B cells is less understood. The objective of this study was to compare the effects of TLR7 and TLR9 stimulation on human B cell function. RESULTS: Gene expression and protein production of cytokines, chemokines, various B cell activation markers, and immunoglobulins were evaluated. Purified human CD19+ B cells (99.9%, containing both naïve and memory populations) from peripheral blood were stimulated with a TLR7-selective agonist (852A), TLR7/8 agonist (3M-003), or TLR9 selective agonist CpG ODN (CpG2006). TLR7 and TLR9 agonists similarly modulated the expression of cytokine and chemokine genes (IL-6, MIP1 alpha, MIP1 beta, TNF alpha and LTA), co-stimulatory molecules (CD80, CD40 and CD58), Fc receptors (CD23, CD32), anti-apoptotic genes (BCL2L1), certain transcription factors (MYC, TCFL5), and genes critical for B cell proliferation and differentiation (CD72, IL21R). Both agonists also induced protein expression of the above cytokines and chemokines. Additionally, TLR7 and TLR9 agonists induced the production of IgM and IgG. A TLR8-selective agonist was comparatively ineffective at stimulating purified human B cells. CONCLUSION: These results demonstrate that despite their molecular differences, the TLR7 and TLR9 agonists induce similar genes and proteins in purified human B cells.

Linfócitos B/imunologia , Ativação Linfocitária/efeitos dos fármacos , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Memória Imunológica , Ativação Linfocitária/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Receptor Toll-Like 9/agonistas
J Clin Pharmacol ; 48(6): 755-62, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18401016


852A is a specific toll-like receptor 7 (TLR7) agonist. Thirty-two healthy adults (8 subjects per group) received two 1-g topical applications over 400 cm(2), separated by >or= 5 days, of 852A 0.01% followed by vehicle, vehicle followed by 852A 0.1%, 852A 0.3% followed by vehicle, or vehicle followed by 852A 1.0%. Systemic absorption was minimal as 852A was not quantifiable in any serum sample up to 24 hours postadministration and was only quantifiable at 24 hours in the urine of 4 of 8 subjects after application of 852A 1.0%. No systemic adverse events were associated with drug treatment. Gene expression analysis from application site biopsies showed a >or=2-fold increase in expression for 40 genes in at least 2 subjects. CXCL9/MIG (8/32 subjects), CCL2/MCP1 (7/32), and OAS3 (5/32) were most frequently increased, followed by other type I interferon-inducible genes. Cluster analysis of the genes with a >or=2-fold increase did not reveal a definitive pattern with respect to 852A concentration or time of biopsy. Overall, single topical application of 852A up to 1.0% was well tolerated. Data gathered from these subjects are suggestive that 852A can produce increases in local gene expression consistent with TLR7 stimulation.

Regulação da Expressão Gênica/efeitos dos fármacos , Quinolinas/administração & dosagem , Sulfonamidas/administração & dosagem , Receptor 7 Toll-Like/agonistas , Administração Cutânea , Adulto , Estudos de Coortes , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pomadas , Quinolinas/efeitos adversos , Quinolinas/farmacocinética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Sulfonamidas/efeitos adversos , Sulfonamidas/farmacocinética , Fatores de Tempo
BMC Immunol ; 8: 26, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17935622


BACKGROUND: Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011. RESULTS: Global gene expression was evaluated using the Affymetrix U133A GeneChip(R) and selected genes were confirmed using real time TaqMan(R) RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNalpha subtypes, IFNalpha2, alpha5, alpha6, alpha8, alpha1/13, alpha10, alpha14, alpha16, alpha17, alpha21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program. CONCLUSION: Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival.

Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Receptor 7 Toll-Like/agonistas , Apresentação do Antígeno/genética , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Receptor 8 Toll-Like/agonistas , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
J Transl Med ; 5: 7, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17257431


BACKGROUND: The objective of this study was to identify the molecular processes responsible for the anti-lesional activity of imiquimod in subjects with actinic keratosis using global gene expression profiling. METHODS: A double-blind, placebo-controlled, randomized study was conducted to evaluate gene expression changes in actinic keratosis treated with imiquimod 5% cream. Male subjects (N = 17) with > or = 5 actinic keratosis on the scalp applied placebo cream or imiquimod 3 times a week on nonconsecutive days for 4 weeks. To elucidate the molecular processes involved in actinic keratosis lesion regression by imiquimod, gene expression analysis using oligonucleotide arrays and real time reverse transcriptase polymerase chain reaction were performed on shave biopsies of lesions taken before and after treatment. RESULTS: Imiquimod modulated the expression of a large number of genes important in both the innate and adaptive immune response, including increased expression of interferon-inducible genes with known antiviral, anti-proliferative and immune modulatory activity, as well as various Toll-like receptors. In addition, imiquimod increased the expression of genes associated with activation of macrophages, dendritic cells, cytotoxic T cells, and natural killer cells, as well as activation of apoptotic pathways. CONCLUSION: Data suggest that topical application of imiquimod stimulates cells in the skin to secrete cytokines and chemokines that lead to inflammatory cell influx into the lesions and subsequent apoptotic and immune cell-mediated destruction of lesions.

Aminoquinolinas/administração & dosagem , Aminoquinolinas/uso terapêutico , Ceratose Actínica/tratamento farmacológico , Ceratose Actínica/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/genética , Adjuvantes Imunológicos/farmacologia , Administração Tópica , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Quimiocinas/genética , Quimiocinas/metabolismo , Demografia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Formas de Dosagem , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imiquimode , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Interferon Tipo I/farmacologia , Ceratose Actínica/genética , Ceratose Actínica/patologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Reconhecimento de Padrão/metabolismo , Reprodutibilidade dos Testes , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
J Med Chem ; 48(10): 3481-91, 2005 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-15887957


1H-Imidazo-[4,5-c]quinolines were prepared while investigating novel nucleoside analogues as potential antiviral agents. While these compounds showed no direct antiviral activity when tested in a number of cell culture systems, some demonstrated potent inhibition of virus lesion development in an intravaginal guinea pig herpes simplex virus-2 assay. We have determined that the in vivo antiviral activity can be attributed to the ability of these molecules to induce the production of cytokines, especially interferon (IFN), in this model. Subsequently, we found that the compounds also induce in vitro production of IFN in human peripheral blood mononuclear cells (hPBMCs). The in vitro results reported herein and the in vivo results reported previously led to the discovery of imiquimod, 26, which was developed as a topical agent and has been approved for the treatment of genital warts, actinic keratosis, and superficial basal cell carcinoma.

Aminoquinolinas/síntese química , Antivirais/síntese química , Imidazóis/síntese química , Indutores de Interferon/síntese química , Interferons/biossíntese , Quinolinas/síntese química , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Efeito Citopatogênico Viral/efeitos dos fármacos , Cobaias , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imiquimode , Indutores de Interferon/química , Indutores de Interferon/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Relação Estrutura-Atividade