These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.
Transplantation of pancreatic islets has great potential for treating Type I diabetes. Ex vivo gene therapy may promote re-vascularization or inhibit apoptosis of the islets and promote graft. In this study, we investigated the feasibility of non-viral gene delivery using Enhanced Green Fluorescent Protein (EGFP) and human Vascular Endothelial Growth Factor (hVEGF(165)) expression plasmids as model reporter and therapeutic genes. LipofectAMINE/pDNA and Superfect/pDNA complexes showed high transfection efficiency in rapidly dividing Jurkat cells, but low transfection in non-dividing human islets. LipofectAMINE/pCAGGS-hVEGF transfected islets showed relatively higher levels of hVEGF than in those transfected with LipofectAMINE/pCMS-EGFP complexes or 5% glucose. To exclude endogenously secreted hVEGF, real time RT-PCR experiment was repeated using pCAGGS vector-specific forward primer and hVEGF gene-specific reverse primer. In this case, both non-transfected islets and the islets transfected with LipofectAMINE/pCMS-EGFP complexes showed negligible amplification of hVEGF. On glucose challenge, insulin release from LipofectAMINE/pCAGGS-hVEGF transfected human islets increased from 10.78 +/- 4.56 to 65 +/- 5 ng/ml, suggesting little adverse effect on islet beta cell response to glucose challenge. The low transfection efficiency is due to the islets being a cluster of approximately 1000 non-dividing cells. This underscores the importance of experimentation with the actual human islets.
In AP, the production of inflammatory cytokines precedes up-regulation of P- and E-selectin, whose expression coincided with the increased infiltration of CD18-positive cells and neutrophil sequestration in lung tissue. Temporally, these events correlate with evidence of histologic pulmonary injury and underscore the role of adhesion molecules as mediators of pathophysiologic events. This mechanistic pathway may afford novel therapeutic interventions in clinical disease by using blocking agents to ameliorate the systemic manifestations of AP.
Acute pancreatitis (AP) is characterized by release of proteolytic enzymes from the pancreas and a powerful inflammatory cytokine cascade that mediates the systemic manifestations and contributes to the mortality of the disease. The purpose of this study was to examine a potential link between pancreatic proteolytic enzymes, which are increased in AP, and cytokine production. To evaluate this, we incubated rat peritoneal macrophages (PMO) with increasing concentrations of trypsin and measured cytokine production. Supernatants from the cell cultures were assayed for TNF-alpha and IL-1beta, and the PMO were collected for the evaluation of cytokine mRNA by polymerase chain reaction (PCR). Further to evaluate the role of pancreatic proteases in triggering the cytokine cascade in AP, trypsin was injected into the peritoneal cavity of Sprague-Dawley rats, and the production of cytokines was measured in the peritoneal fluid. Controls included injection of inactivated trypsin. Incubation of PMO with trypsin in vitro resulted in a dose-dependent increase in TNF-alpha production with maximal response (2,660.5+/-748.8 pg/mL) at 10 microg/mL protease. Peak TNF-alpha and IL-1beta release was noted 16 h after stimulation of the PMO (2,759.5+/-698.0 pg/mL and 160,596+/-4,065 cpm, respectively). Trypsin-induced TNF-alpha production was not due to release of cell-associated cytokine, inasmuch as activation of PMO with this protease causing an increase in TNF-alpha mRNA by 30 minutes, reaching a 14-fold increase at 4 h. Trypsin-injected animals produced TNF-alpha-containing ascitic fluid in a dose-dependent manner with peak TNF-alpha at 2 h (371.3+/-180 pg/mL) versus control (53.8+/-11.2 pg/mL; p < 0.022). No TNF-alpha was found in ascites of rats injected with heat-inactivated trypsin. Histologic examination of trypsin-injected animals revealed evidence of pulmonary inflammation at 2 and 4 hours. We conclude that the proteolytic enzyme trypsin stimulates cytokine production from macrophages in vitro and in vivo. This model demonstrates for the first time that trypsin is a potential mediator of the cytokine response seen during AP.
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