RESUMO
Infection with influenza virus induces severe pulmonary immune pathology that leads to substantial human mortality. Although antiviral therapy is effective in preventing infection, no current therapy can prevent or treat influenza-induced lung injury. Previously, we reported that influenza-induced pulmonary immune pathology is mediated by inflammatory monocytes trafficking to virus-infected lungs via CCR2 and that influenza-induced morbidity and mortality are reduced in CCR2-deficient mice. In this study, we evaluated the effect of pharmacologically blocking CCR2 with a small molecule inhibitor (PF-04178903) on the entry of monocytes into lungs and subsequent morbidity and mortality in influenza-infected mice. Subcutaneous injection of mice with PF-04178903 was initiated 1 d prior to infection with influenza strain H1N1A/Puerto Rico/8/34. Compared with vehicle controls, PF-04178903-treated mice demonstrated a marked reduction in mortality (75 versus 0%) and had significant reductions in weight loss and hypothermia during subsequent influenza infection. Drug-treated mice also displayed significant reductions in bronchoalveolar lavage fluid total protein, albumin, and lactose dehydrogenase activity. Administration of PF-04178903 did not alter viral titers, severity of secondary bacteria infections (Streptococcus pneumoniae), or levels of anti-influenza-neutralizing Abs. Drug-treated mice displayed an increase in influenza nucleoprotein-specific cytotoxic T cell activity. Our results suggest that CCR2 antagonists may represent an effective prophylaxis against influenza-induced pulmonary immune pathology.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Antivirais/administração & dosagem , Pulmão/imunologia , Pulmão/patologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/imunologia , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Receptores CCR2/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Antivirais/uso terapêutico , Inibição de Migração Celular/efeitos dos fármacos , Inibição de Migração Celular/imunologia , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Pulmão/microbiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/mortalidade , Piperazinas/uso terapêutico , Piperidinas/administração & dosagem , Piperidinas/uso terapêutico , Pneumonia Pneumocócica/tratamento farmacológico , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/patologia , Piridinas/uso terapêutico , Análise de SobrevidaRESUMO
PURPOSE: We have investigated the effects of BRCA1 over-expression and knockdown on 5F-203-induced gene expression and cytotoxicity in human breast cancer cells. 5F-203 is a chemotherapeutic prodrug that both induces a p450 enzyme, CYP1A1, and is metabolically activated by CYP1A1. METHODS: We used several molecular biological techniques to confirm our findings. BRCA1 regulates sensitivity to 5F-203 by regulating the expression of CYP1A1 mRNA and its EROD activity. XRE-Luc reporter assays, semi-quantitative RT-PCR, Western blot analysis, EROD activity measurements, gene knockdown and MTT cell survival assays were used for this study. RESULTS: Our results show that the ability of 5F-203 treatments to increase CYP1A1 mRNA level and CYP1A1 enzymatic activity (EROD activity) are affected by BRCA1 protein levels. In addition, the ability of 5F-203 treatments to induce proteins, P53 and P53 target genes such as P21, is significantly decreased in BRCA1 knockdown cells, suggesting that BRCA1-related effects could at least partially explain why BRCA1 knockdown increases resistance to 5F-203-mediated cytotoxicity. We also observed altered expression of the two major transcription factors (AhR and ARNT) that affect CYP1A1 expression when BRCA1 protein levels are altered. CONCLUSION: BRCA1 is an important protein, which affects 5F-203-mediated cytotoxicity. Our findings are potentially clinically significant; they suggest that those patients most likely to respond to this new prodrug will have tumors containing normal amounts of BRCA1.