Mouse anti-Human NKRF monoclonal antibody, clone 2G7

Background: Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells. Results: The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 mu M), and pulmonary surfactant (100 mu g mL(-1)), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-alpha, il-1 beta) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-alpha (TNF-alpha). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction. Conclusion: P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation.

The aims of the present study were to investigate the roles and underlying mechanisms of microRNA-124-3p (miR-124-3p) in the progression of acute myocardial infarction (AMI). The expression of miR-124-3p was determined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). TargetScan analysis and a luciferase reporter assay were conducted to reveal the association between miR-124-3p and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) repressing factor (NKRF). To investigate the role of miR-124-3p in AMI, a cell model of myocardial hypoxic/ischemic injury was established by subjecting H9c2 cardiac cells to hypoxia for 48 h. The viability of cells was determined using an MTT assay, and cell apoptosis was analyzed by flow cytometry. Additionally, the expression levels of inflammatory factors [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6] were measured via ELISA. Furthermore, gene and protein expression levels were determined by performing RT-qPCR and western blot analyses, respectively. It was revealed that the expression of miR-124-3p was significantly increased in the blood of patients with AMI and hypoxia-treated H9c2 cells. Additionally, it was demonstrated that NKRF was a direct target of miR-124-3p. The hypoxia-induced decrease in the viability of H9c2 cells and increase in cell apoptosis were eliminated by the downregulation of miR-124-3p. Furthermore, hypoxia significantly increased the levels of TNF-alpha, IL-1 beta and IL-6, whereas miR-124-3p downregulation eliminated these effects. Downregulated expression of B-cell lymphoma 2, pro-caspase 3 and pro-caspase 9 protein, and upregulated expression of cleaved caspases 3 and 9 was observed in hypoxic H9c2 cells; the altered expression of these proteins was suppressed by miR-124-3p inhibitor. Additionally, miR-124-3p inhibitor suppressed the hypoxia-induced activation of the NF-kappa B signaling pathway in H9c2 cells. Furthermore, it was demonstrated that the various effects of miR-124-3p inhibitor on H9c2 cells were eliminated by the small interfering RNA-mediated downregulation of NKRF. In conclusion, the results of the present study indicated that miR-124-3p downregulation protected against AMI via inhibition of inflammatory responses and the apoptosis of cardiomyocytes by regulating the NKRF/NF-kappa B pathway.