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Abstract: The P2X7 nucleotide receptor is an adenosine 5′-triphosphate (ATP)-gated ion channel, which induces cation channel opening imparting significant permeability to Ca2+, and is widely expressed in cells of hematopoietic origin. Our previous report showed that P2X7-mediated calcium response was absent in three Epstein-Barr virus (EBV)-positive and P2X7 positive cell lines. In this report, we detected the cell surface ATPase activity, which contributes to the hydrolysis of extracellular ATP, and the expression of CD39, which is the main source of ATPase on hematopoietic cells, in these cell lines. Then, we tried to restore the P2X7-mediated calcium response in LCL-H and J6-1 cells by either increasing the concentration of agonist or suppressing the ATPase activity by βγMeATP, a synthetic poorly metabolizable ATP analogue. The results showed that LCL-H and J6-1 cells had higher levels of ATPase activity and CD39 expression. The treatment of 300μM βγMeATP efficiently inhibited the ATPase activity on LCL-H and J6-1 cells. Both elevation of agonist concentration (10mM ATP or 1mM BzATP) and pretreatment with 300μM βγMeATP followed by stimulation with normal concentration of agonists (1mM ATP or 0.1mM BzATP) could cause P2X7-mediated calcium response in LCL-H but neither in J6-1 cells. These results suggested that multiple mechanisms contributed to the loss of the P2X7-mediated calcium response. CD39-associated high ATPase activity contributed to the loss of the P2X7-mediated calcium response in LCL-H cells, while additional mechanism(s) existed in J6-1 cells. [Copyright &y& Elsevier]

Abstract: DNA vaccine against M-CSFRJ6-1 (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFRJ6-1 DNA vaccines, we constructed genetically fused vaccines encoding M-CSFRJ6-1 and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFRJ6-1), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFRJ6-1 tumor cells, and inducing IFN-γ and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFRJ6-1 tumor cells. Our results suggest that when genetically fused with M-CSFRJ6-1, LL-37 could enhance adaptive immune response against M-CSFRJ6-1 in a murine model challenged with tumor cells bearing M-CSFRJ6-1. [Copyright &y& Elsevier]