Encapsulation of human islets in novel inhomogeneous alginate-ca2+/ba2+ microbeads: in vitro and in vivo function

Microencapsulation may allow for immunosuppression-free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3+/-2.5 and 87.5+/-2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4+/-0.5 and 6.3+/-0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30-55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca(2+)/Ba(2+) microbeads allow for long-term in vivo human islet graft function, despite long-distance shipment.     

Chronic effects of caerulein and secretin on the endocrine pancreas of the rat

Secretin and caerulein increase pancreatic somatostatin content when administered chronically to rats. We examined whether this change occurs in vitro and results in altered islet hormone secretion. Pancreatic somatostatin content was increased from 0.25 +/- 0.01 (mean +/- SE) to 0.41 +/- 0.03 nmol/pancreas (P less than 0.001, n = 8) in rats treated for 10 days with caerulein (1 microgram/kg) and secretin (100 micrograms/kg) every 8 h. Somatostatin content in isolated rat pancreatic islets cultured for 10 days in medium containing caerulein and secretin (10(-9) M) was also increased (2.5 +/- 1.0 to 3.6 +/- 1.3 fmol/islet, P less than 0.02, n = 7), although islet DNA content was unchanged. Small increases in glucagon content were observed in both systems, but insulin content was not changed. Isolated perfused pancreases from peptide-treated rats and islets cultured in medium containing the two peptides exhibited significantly greater somatostatin responses to 5 mM glucose and 20 mM theophylline. Insulin responses to glucose and theophylline stimulation were not altered, although basal accumulation of insulin was greater in islet cultures with added caerulein and secretin. These results suggest that caerulein and secretin have direct actions on islet hormone synthesis with effects on hormone responses to stimulation.     

胰淀素对人胰岛β细胞凋亡的影响及其分子机制的初步研究

目的：初步探讨胰淀素（amylin）对人胰岛β细胞凋亡的影响及其分子机制。方法： 分离培养人胰岛细胞，免疫组化鉴定β细胞后分为对照组（培养液中含56 mmol/L葡萄糖）、胰淀素组（培养液中含10μmol/L胰淀素+56 mmol/L葡萄糖）及氨基胍组（培养液中含10 μmol/L胰淀素+05 mmol/L氨基胍+56 mmol/L葡萄糖），于37 ℃、5%CO2 培养24 h后做胰岛素释放实验，测定培养液上清胰岛素、一氧化氮（NO2-/NO3-）、还原型谷胱甘肽(GSH)水平。原位末端核苷酸标记法（TUNEL）和胰岛素免疫组化双染色法及ELISA检测胰岛β细胞凋亡， RT-PCR检测胰岛细胞p53和bcl-2 mRNA表达水平。结果：胰淀素组胰岛β细胞凋亡小体富计系数（217±021）、β细胞凋亡百分数（13%）、NO2-/NO3-（2013±173）μmol/L和p53 mRNA表达水平（034±004）显著高于氨基胍组和对照组(P<001)，而胰岛素（334±131）mU·L-1/1×106 cells、GSH［（56±08） mg/L］和bcl-2 mRNA（007±001）表达水平则显著低于氨基胍组和对照组(P<005)。结论： 胰淀素可诱导人胰岛β细胞凋亡，其机制可能与胰岛β细胞抗氧化能力降低引起p53高表达有关。     

Perfluorodecalin-enriched fibrin matrix for human islet culture

Disruption of microenvironment and decrease in oxygen supply during isolation and culture lead to pancreatic islet injury and their poor survival after transplantation. This study aimed to create a matrix for culturing islets, using fibrin as scaffold and perfluorodecalin as oxygen diffusion enhancing medium. Human pancreatic islets were divided in four groups: control, islets cultured in fibrin, islets in fibrin containing non-emulsified perfluorodecalin, and finally islets in fibrin supplemented with emulsified perfluorodecalin. After an overnight culture, cell damage (viability, proinsulin and insulin unregulated release, apoptosis (caspase-3 activation), secretory function, and presence of hypoxia markers (HIF-1a and VEGF expression) were assessed. Islets cultured in a matrix, had similar islet viability to controls (no matrix) but decreased levels of active caspase-3 and unregulated hormone release, but high level of hypoxia markers expression. Although the supplementation of fibrin with non-emulsified perfluorodecalin improves secretory response, there was no decrease in hypoxia markers expression. In contrast, emulsified perfluorodecalin added to the matrix improved islet function, islet viability and maintained level of hypoxia markers similar to control. Fibrin matrix supplemented with emulsified perfluorodecalin can provide a beneficial physical and chemical environment for improved pancreatic human islet function and viability in?vitro.     

Influence of pancreatic islets on spheroid formation and functions of hepatocytes in hepatocyte-pancreatic islet spheroid culture

Hepatotrophic stimulation of hepatocytes is necessary to preserve long-term function of hepatocytes in hepatocyte transplantation and bioartificial liver system. The main source of hepatotrophic factors in portal venous blood seems to be the pancreatic islets. It was also reported that hepatocyte spheroids, tightly packed multicellular aggregates, showed enhanced liver-specific activities and a prolonged differentiated state compared with cells that were maintained as a monolayer. On the basis of these two facts, the authors tried to form hepatocyte-pancreatic islet spheroids and to evaluate the influence of pancreatic islets on spheroid formation and functions of hepatocytes in spheroid culture. Hepatocytes and pancreatic islet cells were harvested from adult male Sprague-Dawley rats weighing 200-250 g. Hepatocytes were cultured in spinner flasks with either basic nonstimulated medium (hepatocytes only [group BH] and cocultures with islet cells [group BI]) or hormone-stimulated medium (hepatocytes only [group HH] and cocultures with islet cells [group HI]). The size and morphology of spheroids, as determined by phase-contrast microscopy, and liver-specific functions, such as albumin secretion, urea synthesis, and ammonia removal, were compared among groups. The results were as follows: the size of spheroids, 66 +/- 53.4 microm, in group BH on day 2 was smaller than in group BI (179 +/- 66.2 microm on day 2, p < 0.05). In group BI, group HH, and group HI, smooth spheroids were observed on culture day 2. However, in group BH rugged incomplete aggregates were observed on the same day. In groups with basal medium, group BI showed better results in terms of hepatocyte-specific function such as albumin secretion, urea synthesis, and ammonia removal compared with group BH on days 2 and 3 (p < 0.05). In groups with hormone-defined medium, cocultures had no impact on albumin secretion rate, urea synthetic rate, and ammonia removal rate. In conclusion, we made a new type of hepatocyte-pancreatic islet spheroid, using a rotational culture method. Pancreatic islets in a spheroid culture system stimulated hepatocyte spheroid formation and some hepatocyte-specific functions in vitro.     

Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation

Functional poly(ethylene glycol) (PEG) derivatives, including monosuccinimidyl PEG (MSPEG) with molecular weight (MW) of 2000 (2 kDa) as well as 5 kDa and disuccinimidyl PEG (DSPEG) with MW of 3 and 6 kDa, were synthesized and characterized. They were used to modify the surface of adult porcine islets for cytoprotection. The islets were isolated, purified and modified with functional PEG. Untreated porcine islets were used as control. An in vitro human antibody/complement-mediated cytotoxicity test based on the release of intracellular lactate dehydrogenase was used to evaluate cytotoxicity of human serum to the modified islets. In vitro cell viability was assessed using membrane-integrity straining and islet metabolism in culture. In vitro islet functionality was evaluated by glucose-stimulated insulin release of islets in static incubation with human serum. In vivo islet functionality was evaluated by monitoring non-fasting blood glucose level in streptozotocin-induced diabetic (SCID) immunocompromized mice after intraportal transplantation of porcine islets. Results show that all the PEG derivatives used in the study showed significant in vitro and in vivo cytoprotections against cytotoxic effects elicited by human serum and diabetic SCID mice, respectively, to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to MSPEG ones, due to the capacity of the succinimidyl groups to selectively react with amino groups of the albumin under physiological conditions. The effects of both MW and concentration of the PEG derivatives on cytoprotection were significant. It appears that this novel biotechnology will be an attractive approach for improved xenotransplantation of islets.     

Xenogeneic islet re-transplantation in mice triggers an accelerated, species-specific rejection

Xenogeneic islets could provide an unlimited source of tissue for the treatment of diabetes, and could in theory be transplanted repeatedly in a recipient. However, little is known on the consequences of islet re-transplantation in a recipient who has rejected a first graft. In this study, we investigated the functional consequence of xeno islet re-transplantation in mice sensitized with islets from different species. Sprague-Dawley (SD)-rat islets transplanted in sensitized C57/Bl6 mice that rejected either SD- or Lewis-rat islets underwent accelerated rejection. However, accelerated rejection was not found in mice sensitized with pig or human islets, suggesting that accelerated rejection was species specific. Immunohistochemistry showed increased binding of antibodies and accelerated leucocyte infiltration on re-grafted islets in sensitized mice. In situ apoptosis detection indicated that islet cell apoptosis was correlated with the time of leucocyte infiltration, but not with the time of antibody binding. In vitro experiments with cultured islet cells showed that although antibody binding was increased after incubation with sensitized mouse serum, islet cell cytotoxicity was not increased, suggesting that humoral immunity did not play a direct role in islet destruction. These results indicate that there is a cell-mediated, species-specific accelerated rejection after re-transplantation of xenogeneic islets.     

β-cell loss and β-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition

Amyloid deposition and reduced β-cell mass are pathological hallmarks of the pancreatic islet in type 2 diabetes; however, whether the extent of amyloid deposition is associated with decreased β-cell mass is debated. We investigated the possible relationship and, for the first time, determined whether increased islet amyloid and/or decreased β-cell area quantified on histological sections is correlated with increased β-cell apoptosis. Formalin-fixed, paraffin-embedded human pancreas sections from subjects with (n = 29) and without (n = 39) diabetes were obtained at autopsy (64 ± 2 and 70 ± 4 islets/subject, respectively). Amyloid and β cells were visualized by thioflavin S and insulin immunolabeling. Apoptotic β cells were detected by colabeling for insulin and by TUNEL. Diabetes was associated with increased amyloid deposition, decreased β-cell area, and increased β-cell apoptosis, as expected. There was a strong inverse correlation between β-cell area and amyloid deposition (r = -0.42, P < 0.001). β-Cell area was selectively reduced in individual amyloid-containing islets from diabetic subjects, compared with control subjects, but amyloid-free islets had β-cell area equivalent to islets from control subjects. Increased amyloid deposition was associated with β-cell apoptosis (r = 0.56, P < 0.01). Thus, islet amyloid is associated with decreased β-cell area and increased β-cell apoptosis, suggesting that islet amyloid deposition contributes to the decreased β-cell mass that characterizes type 2 diabetes.     

Glucose sensitivity of porcine and human islets in vitro

Preliminary experiments about the suitability of different commonly used culture media in our laboratory indicated, that prolonged exposure to high glucose concentrations during low temperature culture (LTC) impairs the viability of long term cultured human islets. As a consequence of the heterogenity of tested media the present study was aimed to evaluate the influence of different glucose concentrations on survival, viability and in-vitro function of cultured human islets in order to optimize islet survival until transplantation and to compare species dependent differences in glucose sensitivity. Quantified aliquots of freshly isolated (digestion-filtration, ficoll gradient purification) islets from consecutively processed human (n=6) and porcine (n=11) pancreata were subjected to different glucose concentrations (human islets: 500, 750, 1000 and 2000 mg/l; porcine islets: 1000 and 2000 mg/l) in CMRL (22°C) for 8–10 days. After LTC survival, viability and glucose-stimulated insulin release of incubated tissue was assessed. A reduction of glucose concentration promotes survival and viability of human islets but impairs in vitro function at the same time, presumably due to a reduced glucose oxidation as expressed by the significantly reduced stimulation index. In contrast to these findings in the human, elevated glucose concentration in porcine islet culture increases survival but reduces the glucose-stimulated insulin release and the viability of cultured islets. The contradiction of the results in regard to islet survival related to islet viability are still unclear in the pig and needs further evaluation.     

Beta-cell expression of 65-kDa heat-shock protein mRNA is function- and age-dependent.

This study examined the expression of mRNA coding for the 65-kDa heat-shock protein (HSP) in rat islet cells of different functional states and different ages. In addition, beta cells and non-beta cells purified by fluorescence-activated cell sorting were studied. Total RNA from islet cells and insulin-producing RINm5F cells was isolated and analyzed by Northern blotting using a cDNA probe coding for the human homologue to the mycobacterial 65-kDa HSP, after which blots were quantified by densitometric scanning. Isolated beta cells were found to express 65-kDa HSP mRNA. The expression was increased in Lewis islet cells exposed to heat shock or high glucose concentration, four- and three-fold, respectively (p < 0.01). In isolated beta cells cultured at high glucose concentration a doubling in the content of 65-kDa HSP mRNA was seen compared with islets cultured at low glucose concentration (p < 0.05). In islets from Lewis rats fasted for 24 h, the content of 65-kDa HSP mRNA was 42% lower than in islets isolated from normally fed Lewis rats (p < 0.01). Both in BB rats and Wistar Furth rats the content of 65-kDa HSP mRNA was found to be higher in the 30- and the 60-day-old rats compared with the neonatal animals (p < 0.01). The expression of 65-kDa HSP mRNA was increased in RINm5F cells following heat shock, while no induction was seen after stimulation with glucose, TPA or IBMX. It is concluded that the 65-kDa heat-shock protein belongs to the family of inducible functional antigens in beta cells, which strengthens the interest in 65-kDa HSP as an antigen possibly involved in the initiation of autoimmune beta-cell destruction.     

Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice

Glucagon-like peptide 1 (GLP-1) amplifies glucose-induced insulin release in vivo and in vitro. Activation of GLP-1 receptor (GLP-1R) signaling leads to differentiation of exocrine cells towards a beta-cell phenotype in vitro and stimulation of islet cell proliferation in vitro and in vivo, suggesting a potential role for GLP-1 in the modulation of islet growth and differentiation. To determine whether basal levels of GLP-1R signaling are essential for islet development, we examined islet cell composition and topography in GLP-1R-/- mice. Total beta-cell volume and number are not altered, but the topography of beta cells is markedly different in GLP-1R-/- mice compared with GLP-1R+/+ controls. The distribution of beta cells is shifted from large to small and medium-sized islets in the absence of GLP-1R signaling (large islets: 50 +/- 3% in GLP-1R+/+ vs 28 +/- 4% in GLP- 1R-/-, P < 0.01 and medium islets: 32 +/- 2% in GLP- 1R+/+ vs 48 +/- 3% in GLP-1R-/-, P < 0.001). Furthermore, GLP-1R-/- islets exhibit abnormalities in cell topography, with two to threefold more centrally located alpha cells detected in GLP-1R-/- islets. These alterations in alpha- and beta-cell topography indicate that basal levels of GLP-1 signaling in the normal rodent are involved in the normal cellular organization of the endocrine pancreas.     

MKK3 signalling plays an essential role in leukocyte-mediated pancreatic injury in the multiple low-dose streptozotocin model

In vitro studies have implicated activation of the p38 mitogen-activated protein kinase (MAPK) signalling pathway in cytokine-mediated pancreatic beta-cell injury. Activation of the p38 MAPK occurs through two different upstream kinases, mitogen-activated protein kinase kinase 3 (MKK3) and MKK6. This study examined the role of MKK3 signalling in an in vivo model of cytokine-dependent pancreatic injury induced by multiple low doses of streptozotocin (MLD-STZ). Groups of wild-type (WT) or Mkk3-/- C57BL/6J mice received 5 daily injections of STZ (40 mg/kg) and were killed on day 5, week 2 or week 4. MLD-STZ in WT mice exhibited two distinct phases of pancreatic damage: islet cell apoptosis (immunostaining for cleaved caspase-3) on day 5 in the absence of leukocyte infiltration, and this was followed by islet inflammation (leukocyte infiltration and cytokine production) and further islet cell apoptosis on day 14 resulting in a loss of insulin-producing beta-cells and an 80% incidence of hyperglycaemia. Mkk3-/- mice were not protected from the initial phase of STZ-induced islet cell apoptosis day 5. However, Mkk3-/- mice were completely protected from the induction of hyperglycaemia. This was attributed to inhibition of leukocyte infiltration, production of pro-inflammatory cytokines and islet cell apoptosis at day 14 of MLD-STZ. In vitro studies showed that cultured islets from Mkk3-/- and WT mice are equally susceptible to STZ and cytokine-induced apoptosis. In conclusion, MKK3 signalling plays an essential role in the development of islet inflammation leading to destruction of beta-cells and hyperglycaemia in MLD-STZ-induced pancreatic injury.     

Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.Abbreviations DMSO Dimethyl sulfoxide - FBS Fetal bovine serum - FDA Fluorescein diacetate - IEQ Islet equivalent - IL-1 Interleukin 1-beta - INF- Interferon gamma - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - NG Newport green - PARP Poly (ADP-ribose) polymerase - PI Propidium iodide - TNF- Tumor necrosis factor alpha     

Macrophage migration inhibitory factor deficiency protects pancreatic islets from cytokine-induced apoptosis in vitro

During pathogenesis of diabetes, pancreatic islets are exposed to high levels of cytokines and other inflammatory mediators that induce deterioration of insulin-producing beta cells. Macrophage migration inhibitory factor (MIF) plays a key role in the onset and development of several immunoinflammatory diseases and also controls apoptotic cell death. Because the occurrence of apoptosis plays a pathogenetic role in beta cell death during type 1 diabetes development and MIF is expressed in beta cells, we explored the influence of MIF deficiency on cytokine-induced apoptosis in pancreatic islets. The results indicated clearly that elevated MIF secretion preceded C57BL/6 pancreatic islets death induced by interferon (IFN)-γ + tumour necrosis factor (TNF)-α + interleukin (IL)-1β. Consequently, MIF-deficient [MIF-knock-out (KO)] pancreatic islets or islet cells showed significant resistance to cytokine-induced death than those isolated from C57BL/6 mice. Furthermore, upon exposure to cytokines pancreatic islets from MIF-KO mice maintained normal insulin expression and produced less cyclooxygenase-2 (COX-2) than those from wild-type C57BL6 mice. The final outcome of cytokine-induced islet apoptosis in islets from wild-type mice was the activation of mitochondrial membrane pore-forming protein Bcl-2-associated X protein and effector caspase 3. In contrast, these apoptotic mediators remained at normal levels in islets from MIF-KO mice suggesting that MIF absence prevented initiation of the mitochondrial apoptotic pathway. Additionally, the protection from apoptosis was also mediated by up-regulation of prosurvival kinase extracellular-regulated kinase 1/2 in MIF-KO islets. These data indicate that MIF is involved in the propagation of pancreatic islets apoptosis probably via nuclear factor-κB and mitochondria-related proteins.     

Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas

In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans ('insulitis') results in destruction of insulin-producing beta cells. Cytokines released from islet-infiltrating mononuclear cells are known to be cytotoxic both directly and by upregulating Fas for FasL-induced apoptosis. To investigate the role of caspase-3, a major effector of apoptosis in beta-cell death, we asked whether cytokine- and/or FasL-induced apoptosis was associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Measurement of caspase-3 activity using a fluorogenic cleavage assay was validated in NOD mouse thymocytes undergoing dexamethasone (Dex)-induced apoptosis. For cytokine-induced apoptosis, NIT-1 cells or islets were exposed to IL-1 beta and IFN-gamma for 24 h. Caspase-3-like activity was increased 2.1+/-0.7 and 2.4+/-0.9-fold in lysates of cytokine-treated NIT-1 cells and NOD mouse islets, respectively. However, NIT-1 cells exhibited 2.1% (4.7 pg active caspase-3/microg protein) and islets 0.8% (1.9 pg active caspase-3/microg protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/microg protein). After 24 h cytokine-exposure, the percentage of Fas-positive NIT-1 cells increased from 1.4+/-1.1 to 29.7+/-11.6%. Addition of FasL for a further 3 h increased caspase-3-like activity an additional 1.8-fold in cytokine-treated NIT-1 cells. In summary, exposure of NOD mouse insulinoma cells or islets to IL-1 beta and IFN-gamma for 24 h induced caspase-3-like activity that, in the case of insulinoma cells at least, can be further enhanced by interaction of cytokine-induced Fas receptor with FasL. Compared to thymocytes, insulinoma cells and islets from NOD mice were characterised by low basal and cytokine-induced caspase-3 activity.     

Increased glucagon-stimulated insulin secretion of cryopreserved rat islets transplanted into nude mice

Cryopreservation is the only available technique for long-term storage of pancreatic islets. The freezing/thawing protocol may cause considerable loss of viable islet tissue and impair its function in vivo. The aim of this study was to investigate glucose and insulin levels after transplantation of fresh and cryo/thawed rat islets. Rat pancreatic islets were isolated following intraductal collagenase injection and Ficoll gradient purification. After isolation, islets were cultured for 24 h and then either transplanted or frozen after stepwise addition of DMSO according to Rajotte et al. and stored in liquid nitrogen. After rapid thawing islets were stepwise transferred into RPMI medium and cultured for another 24 h. The recipients were athymic mice with streptozotocine-induced diabetes. Two hundred fresh (n=13) or cryo/thawed (n=15) islets were transplanted beneath the renal capsule. Glucose levels were measured for 14 days and blood samples for insulin determination were obtained 15 min after i.p. glucagon (10 mg/kg) administration on day 14. Glucose levels were normalized (<9 mmol/l) in all recipients within 3 days since transplantation. On day 14, mean fasting values±SE in fresh and cryo/thawed islet groups were 4.0±0.6 and 4.4±0.4 mmol/l, respectively (P>0.05). Fasting insulin levels were higher in the cryo/thaw than in the fresh islet group (1.67±0.33 vs 0.57±0.13 ng/ml; P<0.01). Post-glucagon levels did not differ significantly (1.45±0.24 vs 0.86±0.24 ng/ml; P=0.06). While glucagon significantly increased insulin levels (P<0.01) in the fresh islet group, no change in insulin levels was observed (P>0.05) in the cryo/thaw group. Immunohistochemical staining demonstrated fragmentation of viable islet tissue which was more apparent in the cryo/thaw group. We conclude that in a short-term study cryo/thawed rat islets produce higher insulin levels than fresh islets transplanted into nude mice. This may be due to better islet survival or loss of feed-back regulation.     

Fructokinase activity in rat liver, ileum, parotid gland, pancreas, pancreatic islet, B and non-B islet cell homogenates

The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/- 0.11; n = 11) and pancreatic islets (0.46 +/- 0.07; n = 6). The paired ratio between fructokinase and hexokinase isoenzyme activity was also highest in liver (548 +/- 45%; n = 8) and lowest in parotid gland (0.93 +/- 0.52%; n = 3). Such a ratio was not significantly different in pancreas, islets and purified B or non-B islet cells, with an overall mean value of 2.57 +/- 0.46% (n = 12). The present findings thus unambiguously document the presence of fructokinase activity in all cell types under consideration, except possibly parotid cells, with the following hierarchy: liver > ileum > pancreas. Relative to paired hexokinase activity, no obvious difference was found for fructokinase activity in B versus non-B islet cells.     

Prolonged controlled hemorrhagic shock in a swine model: is there a role for mechanical circulatory assistance?

Outcomes of mechanical circulatory assistance during hemorrhagic shock were evaluated in a swine model. Pigs were bled to a mean arterial pressure of 35 mm Hg (group I, n = 3) or 40 mm Hg (group II, n = 5; group III, n = 5), maintained there for 30 minutes, and then resuscitated with fluids alone (groups I and II) or fluids plus mechanical circulatory assistance (group III). Mean blood loss was greater in group I than in groups II or III (1,037 +/- 212 vs. 862 +/- 387 ml vs. 681 +/- 117 ml, respectively; I vs. III, p < 0.05) and survival was shorter (230 +/- 25.5 min vs. 709 +/- 251 min vs. 662 +/- 428 min, respectively; I vs. II or III, p < 0.05). Cardiac arrhythmia caused death in most cases. Mean biochemical parameters increased progressively in all cases. Left anterior descending coronary artery flow stayed relatively constant in group II but increased in group III. Superior mesenteric artery flow returned to baseline in group II but increased in group III. Cardiac output was similar in groups II and III, but SGOT levels significantly differed (750 +/- 135 U/L vs. 359 +/- 157 U/L; p < 0.005). These results suggest that the swine model will be useful for studying ways to improve outcomes after prolonged hemorrhagic shock.     

Suitability of cellulose molecular dialysis membrane for bioartificial pancreas: in vitro biocompatibility studies

The success of immunoisolation devices for islet transplantation depends on the properties and biocompatibility of semipermeable immunobarrier membranes. In the present study, we have evaluated the in vitro biocompatibility of the cellulose membrane Spectra/Por 2 (MW no larger than 12- 14,000) for its possible application in islet immunoisolation. The membrane was found to be hydrophilic (octane contact angle: 153.2+/-0.66 degrees) and exhibited decreased protein adsorption. It showed mechanical stability after 1 month of storage in PBS (pH 7.4) with tensile strength, percent elongation, and Young's modulus of 88.88 MPa, 36.22, and 291.8 MPa, respectively. It allowed regulated transport of glucose and insulin in an in vitro diffusion assay. The high viability of NIH3T3 fibroblasts and the inability of lymphocytes to proliferate in vitro on exposure to the membrane leach-out products suggested its noncytotoxic and nonimmunogenic nature. Macrophages, when cultured on membranes, did not show increased expression of inflammatory surface marker such as CD11b/CD18, CD45, CD14, and B 7.2. Image analysis studies showed integrity and intact morphology of mouse islets cultured on and inside the membranes with high viability (91%, 89.7%). These islets also retained their functionality, as judged by insulin secretion. The present study provides sufficient documentation to consider cellulose molecular dialysis membrane Spectra/Por 2 (MW no larger than 12-14,000) as a potential candidate for immunoisolation of islets.     

Apoptosis in cultured rat islets of langerhans and occurrence of Bcl-2, Bak, Bax, Fas and Fas ligand

Isolated Langerhans islets are widely used for diabetic transplantation experiments and investigations of the mechanisms leading to the death or survival of insulin-producing cells in cultured islets. The present study was aimed at investigating programmed cell death and the role of apoptosis-associated peptides in insulin and glucagon cells of islets isolated from untreated rats and held in cultured suspension. Islets were removed from medium on days 0, 7, 14, 21 and 29, embedded in Epon, and semi-thin serial sections were prepared. At designated intervals, histologic sections were treated with the direct fluorescein-labelled TUNEL method and immunostained for pancreatic hormones (glucagon, insulin) and apoptotic peptides [Bak, Bax, Fas, Fas ligand (FasL)], as well as for the anti-apoptotic peptide Bcl-2. All tissue sections were investigated using confocal laser scanning microscopy under identical setting for semiquantitative estimation of staining intensity. The percentage of apoptotic cells was between 1.6 and 2.1% and most apoptotic cells were beta-cells. Corresponding cells often contained Bak and Bax. Fas and FasL were mostly detected in islet cells within the first week after preparing the cultured suspension. The insulin content was low (1.1 +/- 0.22 ng per islet) directly after isolation. It then increased progressively up to day 14, after which it began to decrease. Glucagon expression, on the other hand, remained high for the entire duration of the investigation. In conclusion, the islet beta-cells may recover after the isolation procedure, but after 4 weeks in culture, both the insulin content and Bcl-2 staining decrease. Moreover, apoptosis is mediated by different mechanisms after the isolation procedure and after culturing the islets for 1 month. The present data may be important for further studies on isolated, cultivated or transplanted islets.