Peritransplant and long-term secretion of interleukin-1beta in cyclosporine treated syngeneic rats allografted with islets of langerhans.

Interleukin-1beta (IL-1beta) is one of the proinflammatory cytokines that may mediate primary nonfunction of islets of Langerhans, limiting the success of allogeneic transplantation. The aim of this study was to assess differences between the secretion of IL-1beta as well as glycemia in peri- and long-term periods of intraportal islet allo-transplantation with or without cyclosporine (CyA) immunosuppression. Inbred Wistar albino rats were transplanted intraportally with rat islets isolated by collagenase digestion. The two recipient groups (6 rats/group) were: group 1, control, islet transplantation (ITX) without any treatment and group 2, CyA-treated via the femoral muscle on days -1, 0, +1, and +2. Serum IL-1beta (pg/mL) levels were measured by ELISA on days 0 (pre-ITX), +1, +2, and +195. Tail vein blood was used to evaluate glycemia (mg/dL). No major differences were observed in IL-1beta secretion on days 0, +1, or +195 between the groups. Immunosuppressive treatment produced significantly lower secretion in group 2 (P < .002) on day +2. Significantly greater secretions were detected at days +195, +1, and +195 compared to days 0, +2, and +2, respectively (P < .002; P < .008; P < .002). Positive correlations were observed between IL-1beta levels on days +1 and +2 (r = 0.845, P < .034). The mean values in groups 1 and 2 on days 0, +1, and +2 were 140.6 +/- 4.62 vs 119.1 +/- 12.12, 73.1 +/- 19.59 vs 88.3 +/- 14.08, 106.5 +/- 13.79 vs 92.5 +/- 15.8, respectively. No animal in group 1 displayed glycemia while three group 2 animals did at day +195. However, a negative correlation was found between IL-1beta on day 0 and glycemia on day +195 (r = -0.999, P < .026). Our results suggest that IL-1beta secretion, which is detrimental for islet engraftment, decreases at peritransplant day +2, but is upregulated during long-term graft survival both in controls and in CyA-treated recipients.

A modified automated method for isolation of viable pancreatic islets in laboratory animals.

To achieve successful islet transplantation, a high viability is required. For this reason an automated method including two chambers: one for islets isolation and one for recirculation and collection was developed. Recently, we produced a modified version of this work by building a similar system of glass where marbles were not used for disaggregation, and the pancreatic tissue had to be disrupted mechanically before the digestion phase. By using the reconfigured system, we obtained 260 +/- 20 islets from each Wistar albino rat (weighing 220 to 240 g) pancreas. Islets were observed at 35 minutes after the start of perfusion (closed circuit) and the optimum time to stop the isolation determined to be 40 minutes based upon islets viability. Although the present system is configured for islet isolation from small laboratory animals (rat, mouse), we have also obtained thousands of islets at 25 minutes after treatment of a 0.5-g piece of pig pancreas. Compared to the time-consuming manual method usually used for islet isolation from small laboratory animals, the new technique is economic, easy to use, and does not reduce islets viability.

Effect of human somatotropin hormone on cultured rat islets.

Because growth hormone (GH) improves the insulin secretion capacity of isolated human fetal islets in vitro, we sought to show that it positively influences isolated rat islets. Islets isolated from Wistar albino rats by a modified automated system were cultured in media containing 87% RPMI 1640, 10% FCS, 2% antibiotic-antimycotic, and 1% L-glutamine for 12 +/- 2 days. The cultured islets were divided into two groups: growth hormone negative (Group I) and growth hormone positive (Group II). On the 5th day we observed a decrease in the islet cell counts in both groups (Group I 28% versus Group II 45%). On the 10th day, the decrease continued in the GH-negative group (59%), while the count remained stable in the GH-positive group. The viability of rat islets was determined by fluorescein diacetate (FDA) plus propidium iodide (PI) staining. In comparison to the peripheral green, central orange-red staining pattern of Group I islets upon fluorescent microscopy, Group II showed more compact islets. Cultured islets seemed to be brighter than those without GH in the cultured islets. In conclusion, we observed that 2 weeks of incubation in the presence of GH acts positively on cultured rat islets for both their amount and their viability.