The effect of glucagon-like peptide 2 on intestinal permeability and bacterial translocation in acute necrotizing pancreatitis.

BACKGROUND: Acute pancreatitis (AP) initiates a generalized inflammatory response that increases intestinal permeability and promotes bacterial translocation (BT). Impairment of the intestinal epithelial barrier is known to promote BT. Glucagon-like peptide 2 (GLP-2), a 33 residue peptide hormone, is a key regulator of the intestinal mucosa by stimulating epithelial growth. The purpose of this study was to determine whether GLP-2 decreases intestinal permeability and BT in AP. METHODS: To examine whether GLP-2 can decrease intestinal permeability and thereby decrease BT in acute necrotizing pancreatitis, 34 male Sprague-Dawley rats (200 to 300 g) were studied. AP was induced in group I and group II by pressure injection of 3% taurocholate and trypsin into the common biliopancreatic duct (1 mg/kg of body weight). The potent analog to GLP-2 called ALX-0600 was utilized. Group I rats received GLP-2 analog (0.1 mg/kg, SQ, BID) and group II rats received a similar volume of normal saline as a placebo postoperatively for 3 days. Group III and group IV received GLP-2 analog and placebo, respectively. At 72 hours postoperatively, blood was drawn for culture of gram-negative organisms. Specimens from mesenteric lymph nodes (MLN), pancreas and peritoneum were harvested for culture of gram-negative bacteria. Intestinal resistance as defined by Ohm's law was determined using a modified Ussing chamber to measure transepithelial current at a fixed voltage. A point scoring system for five histologic features that include intestinal edema, inflammatory cellular infiltration, fat necrosis, parenchymal necrosis, and hemorrhage was used to evaluate the severity of pancreatitis. Specimens from MLN, pancreas, jejunum, and ileum were taken for pathology. RESULTS: All group I and group II rats had AP. The average transepithelial resistance in group I was 82.8 Omega/cm(2) compared with 55.9 Omega/cm(2) in group II (P <0.01). Gram-negative BT to MLN, pancreas, and peritoneum was 80%, 0%, and 0%, respectively in group I compared with 100%, 30%, and 20% translocation in group II. CONCLUSION: GLP-2 treatment significantly decreases intestinal permeability in acute pancreatitis.

Optimizing fluorescent labeling of endothelial cells for tracking during long-term studies of autologous transplantation.

BACKGROUND: The fluorescent marker PKH26 has been demonstrated to be useful for the tracking of endothelial cells in short-term studies; however, the optimal labeling conditions for long-term implants have not been determined. This study was designed to evaluate the effects of PKH26 on endothelial cell proliferation and to identify labeling conditions that would yield the greatest fluorescence over time without adversely affecting cell viability. MATERIALS AND METHODS: Canine jugular vein endothelial cells (CJVECs) were labeled with 0. 04 microM PKH26. Proliferation of labeled and control cells was assessed for 8 consecutive days by [(3)H]thymidine uptake. In a second experiment, CJVECs were labeled at concentrations of 0, 5, 8, 10, and 20 micromol/L. Cells were maintained in culture for 60 days. The fluorescence intensity of each cell population was measured using two techniques. At baseline and at 60 days, fluorescence was measured using a fluorescence-activated cell sorter. On days 14, 28, 45, and 60 fluorescence was measured by constructing gray-scale histograms from photomicrographs taken of each flask under rhodamine illumination. Mean viable cell number for each concentration was determined after 60 days. RESULTS: In the first experiment, PKH26-labeled and unlabeled CJVECs demonstrated nearly identical growth curves, suggesting that PKH26 had no adverse effect on proliferation. In the second experiment, after 60 days, the 10 and 20 microM groups displayed greater fluorescence by histogram than the 0, 5, or 8 microM groups; however, they were not significantly different from each other (mean intensity 8.2 vs 9.1, P > 0.05, Student-Newman-Keuls test for multiple comparisons). Over 60 days, the cells labeled with 20 microM PKH26 experienced the only significant decrease in viable cells compared to the unlabeled group (5.5 x 10(5) vs 9.6 x 10(5) cells/flask, P < 0.05). Importantly, we observed no significant differences in cell number between the 10 microM group and the lower concentrations compared to the unlabeled cells (P > 0.05). CONCLUSIONS: We conclude that a concentration of 10 microM PKH26 provides the optimal labeling condition for endothelial cells when long-term tracking is desired.