Pancreatic islet and stem cell transplantation in diabetes mellitus: results and perspectives.
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Diabetes Mellitus, Type 1↗
Biomedical subjects
Reinhard G Bretzel
Publications and source records attributed to Reinhard G Bretzel.
High prevalence of steatorrhea in 101 diabetic patients likely to suffer from exocrine pancreatic insufficiency according to low fecal elastase 1 concentrations: a prospective multicenter study.
Impaired exocrine pancreatic secretion has been frequently observed in diabetic patients by different methods, including direct function tests. However, the clinical importance remained unclear. In the present study, the fecal fat excretion in patients with type 1 or type 2 diabetes mellitus and exocrine dysfunction according to fecal elastase 1 concentrations <100 microg/g was investigated. Subjects with a history of gastrointestinal cancer, gastrointestinal surgery, alcohol abuse, or inflammatory diseases were excluded. In 101 patients the mean (+/- SD) fat excretion was 9.19 +/- 5.39 g. Only 41 patients (40.6%) had normal fat excretion <7 g/day. In 40 patients (39.6%), it was higher than 10 g/day, indicating relevant steatorrhea. The fat excretion did not correlate with diabetes type, duration, or clinical symptoms. This finding is of some clinical importance and might influence pathophysiological concepts and the management of diabetic patients.
Celiac Disease↗
Protein kinase C delta activation and translocation to the nucleus are required for fatty acid-induced apoptosis of insulin-secreting cells.
Insulin resistance as well as pancreatic beta-cell failure can be induced by elevated free fatty acid (FFA) levels. We studied the mechanisms of FFA-induced apoptosis in rat and human beta-cells. Chronic treatment with high physiological levels of saturated fatty acids (palmitate and stearate), but not with monounsaturated (palmitoleate and oleate) or polyunsaturated fatty acids (linoleate), triggers apoptosis in approximately 20% of cultured RIN1046-38 cells. Apoptosis restricted to saturated FFAs was also observed in primary cultured human beta-cells, suggesting that this mechanism is potentially relevant in vivo in humans. To further analyze FFA-induced signaling pathways leading to apoptosis, we used RIN1046-38 cells. Apoptosis was accompanied by a rapid (within 15 min) nuclear translocation of protein kinase C (PKC)-delta and subsequent lamin B1 disassembly. This translocation was impaired by the phospholipase C inhibitor U-73122, which also substantially reduced apoptosis. Furthermore, lamin B1 disassembly and apoptosis were decreased by cell transfection with a dominant-negative mutant form of PKC-delta. These data suggest that nuclear translocation and kinase activity of PKC-delta are both necessary for saturated fatty acid-induced apoptosis.
Animals↗
Successful human islet isolation utilizing recombinant collagenase.
The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 +/- 0.8 vs. 85.6 +/- 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.
Animals↗
Polymers for induction of revascularization in the rat fascial flap: application of vascular endothelial growth factor and pancreatic islet cells.
One of the major obstacles in transplanting avascular tissue or metabolically active cells for ischemic diseases is the loss of transplanted cells due to lack of oxygen and nutrients in the early posttransplantation period. Biodegradable polymeric tissue engineering scaffolds and hydrogels have a potential to incorporate cells or cellular organoids such as islets of Langerhans and growth factors. In this study, we tested the efficiency of two types of polymeric materials to carry recombinant human vascular endothelial growth factor (rhVEGF) or pancreatic tumor cell lines, namely Ins-1 and AR42J, for the induction of new vessels. Chitosan hydrogel fibers with micropores were prepared and molded into a cylinder construct (5 mm phi; 8 mm height). Macroporous PLGA scaffolds with a pore size of 250-400 microm were prepared and cut into cylinders (6 mm phi; 3 mm height). Both chitosan and PLGA constructs were loaded with rhVEGF (3 microg) or seeded with the cell lines (5 x 10(5) cells and 3 x 10(5) cells/construct, respectively, for AR42J and INS-1 cells), and transplanted into the fascial flaps of Wistar rats. At distinct time points up to 4 weeks postimplantation, polymers were explanted, fixed, and vessel density was counted on sections stained with anti-Factor-VIII antibody. Additionally, the kinetics of rhVEGF release from PLGA microspheres (phi of 50-80 microm) was determined using VEGF Elisa. Endogenous VEGF release from pancreatic rat cell lines was also determined. Light microscopy study was performed on H&E-stained paraffin sections of the islet-polymer samples. The vascular density of rhVEGF-loaded chitosan constructs was increased fourfold 2 weeks after subcutaneous transplantation compared with rhVEGF-unloaded controls (465 +/- 144 vs. 104 +/- 80 vessels per mm2, p < 0.05). Protein leakage occurred, but was not observed after 2 weeks. Higher insulin content was detected in rat islet grafts transplanted following VEGF application. More than 50% of total rhVEGF was released on the first day of in vitro culture of PLGA microspheres. rhVEGF secretion had another, but smaller, peak on the third day followed by a constant release. By comparison, endogeneous VEGF secretion of pancreatic tumor cells was measured within a 3-day culture period. Biodegradable polymer scaffolds and hydrogels may have potential use as solid supports to induce angiogenesis for pancreatic cell transplantation.
Animals↗
Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.
The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43 degrees C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1beta, IFN-gamma, or TNF-alpha was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.
Animals↗
Different role of saturated and unsaturated fatty acids in beta-cell apoptosis.
It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human beta-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in beta-cells. RIN1046-38 cells and isolated human beta-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24-72 h with 1mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human beta-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.
Apoptosis↗
Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes.
Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted beta(2)-integrin and urokinase receptor mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2 3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented beta(2)-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.
Animals↗
High-dosed nicotinamide decreases early graft failure after pig to nude rat intraportal islet transplantation.
BACKGROUND: Clinical and experimental data indicate that early failure of intraportally grafted islets is mediated by inflammation. Although nicotinamide (NA) has the potential to protect rodent islets it is unknown whether large mammalian islets can be protected in an inflammatory environment. Therefore, we investigated NA-mediated protection of pig islets intraportally transplanted into diabetic nude rats characterized by involvement of NO in intrahepatic graft failure. METHODS: Nonfasting serum glucose levels were evaluated after intraportal transplantation (TX) of 4000 pig islets or intraportal sham-TX in diabetic nude rats infused for 7 days with saline (0 mg), 500 mg, or 1000 mg NA/kg/day. The nitrate/nitrite serum levels were colorimetrically quantified 0, 24, and 48 hr posttransplant. RESULTS: Graft survival after 21 days was not improved (2/13) by 500 mg NA compared to 0 mg NA (1/22) and 500 mg sham-TX (0/7). A total of 1000 mg NA promoted sustained normoglycemia in transplanted rats (10/18, P<0.001 vs. 0 mg NA, P<0.05 vs. 500 mg NA) but deteriorated hyperglycemia in 1000 mg NA sham-TX (P<0.01 vs. 0 mg sham-TX). Regeneration of endogenous islets determined as pancreatic insulin content was only measured in islet recipients receiving 1000 mg NA (P<0.001). Posttransplant NO levels were not affected by NA and increased in all recipients two-fold (P<0.05 vs. day 0). CONCLUSIONS: Compared with efficient administration in syngeneic rodent models NA has to be applied in significant higher doses for protection of xenografted pig islets implying that protection of islets from large mammalians after transplantation into a proinflammatory organ seems feasible. In contrast to other observations graft survival was not mediated by interference of NA with hepatic NO generation.
Animals↗
Further reports on diabetes mellitus after the exstirpation of the pancreas.
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Animals↗
Successful single donor islet allotransplantation in the streptozotocin diabetes rat model.
The objective of this study was to define pretransplant islet culture conditions for optimum tissue engraftment in the rat islet allotransplantation model. Lewis rat islets were cultured in TCM 199/5% fetal calf serum for I day at 37 degrees C, followed by 1 day of culture at 22 degrees C. When islets from single donors were allotransplanted intraportally into single streptozotocin-diabetic Wistar-Furth rats, complete normoglycemia was restored within 1 day after transplantation in seven out of seven rats, and persisted up to immunological rejection about 1 week later. In five out of six rats receiving a posttransplant injection of antilymphocyte serum, plasma glucose was normalized for >100 days. These data demonstrate, for the first time, successful single-donor-to-single-recipient transplantation of allogeneic rat pancreatic islets. Because islet engraftment may still be regarded as a main problem for clinical islet transplantation, the pretransplant islet culture regimen outlined in this article may lead to a more efficient use of donor pancreatic islet tissue in the clinical setting, as well.
Animals↗