The fate of intraportally transplanted islets in diabetic rats. A morphologic and immunohistochemical study.

Streptozotocin-induced diabetes in the rat can be reversed by the transplantation of isogenic islets of Langerhans from neonatal donors. We studied the morphology of intraportally transplanted islets with the aid of the immunoperoxidase staining technique to identify insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-containing cells at 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 39 weeks, and 65 weeks after transplant. Embolized pancreatic tissue, composed of approximately 80% acini and 20% islets, is initially distributed throughout the liver mainly to terminal branches of the portal system. Endothelialization and organization occur rapidly with the smaller fragments and within the first 4 weeks for larger thrombi. Exocrine pancreatic elements largely disappear as islet cells move into the hepatic lobules from the portal spaces. At 65 weeks after transplant, all islet cell types can be identified within large complex islet structures. The results of this study establish the survival and continued function of all known rat pancreatic islet cell types long after transplantation and support the theory that islet transplantation may represent the most physiologic replacement of hormonal deficiencies in the diabetic recipient.     

The role of pancreatic islets in experimental pancreatic carcinogenicity.

Our previous studies have suggested that the presence of intact islets is essential for the induction of pancreatic exocrine tumors in the Syrian hamster model. To validate this, we investigated the effect of the carcinogen, N-nitrosobis(2-oxo-propyl)amine (BOP) in hamsters, in which homologous isolated intact islets were transplanted into the submandibular gland (SMG). Freshly isolated pure islets from hamster donors were transplanted into the left SMG of 20 female host hamsters. Ten of these hamsters (group 1) received BOP (40 mg/kg) weekly for 3 weeks. Another 10 hamsters (group 2) were kept untreated. In groups 3 and 4 (10 hamsters each) the salt solution or isolated pancreatic ductal cells, respectively, was injected into the gland. In other groups (10 hamsters each) islets were transplanted into the peri-SMG connective tissue (group 5) or into the renal subcapsular space (group 6). Hamsters of group 1 (40 mg/kg, weekly for 3 weeks) as were group 7 hamsters, which served as BOP-treated controls. All BOP-treated hamsters developed pancreatic lesions. Similar hyperplastic and atypical ductal/ductular proliferation and in situ carcinoma were found in the SMG of many group 1 hamsters. No such lesions were found in the SMG, peri-SMG, or renal subcapsular space of the other groups. Islets appear to be involved in carcinogenicity of BOP. The mechanism is obscure.     

组织工程预血管化处理对胰岛移植的影响

BACKGROUND: Pancreatic islet transplantation via portal vein system leads to the apoptsis of a number of islet cells due to local hypoxia, thereby affecting transplant outcomes. OBJECTIVE: To explore the effect of pre-micrvascularization network of tissue-engineered constructs on the survival of transplanted islets and the feasibility of xenogenic islet transplantation. METHODS: A 5-mm-long cylindrical silicone tube filled with Matrigel TM matrix surrounding the superficial epigastric vessel was placed in the groin of diabetic mice. After the syngeneic islets with 300 islet equivalents (IEQ) were transplanted into the silicone chamber on days 0, 14 and 28 post-chamber implantation, respectively, the recovery time of blood glucose was observed. The islets with the quantity of 100 IEQ, 200 IEQ and 300 IEQ, respectively, were transplanted on day 28 post-implantation and then the blood glucose level was determined. Moreover, the survival of human pancreatic islets with 1 000 IEQ transplanted into the pre-vascularizated chamber or under the renal capsule of diabetic mice, followed by the treatment of anti-CD45RB and/or anti-CD40L (MR-1) was analyzed. RESULTS AND CONCLUSION:An abundant micro-vascularized network was established in the silicone chamber on day 28 post-implantation. The time of the blood glucose returning to normal level in diabetic mice was negatively correlated with the time required for pre-vascularization and the number of implanted islets. No islet grafts implanted in the silicone chamber and treated by anti-CD45RB survived for long term. However, one of seven (14.3%) grafts survived for long term, which was not significantly different from the transplantation under the renal capsule group (n=8, MST > 71 days, P > 0.05). The tissue-engineered pre-vascularization network markedly extends the survival time of the islet grafts before transplantation. The transplantation of the xenogenic pancreatic islets into the vascularized silicone chamber might be a promising method in the future clinical application. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Dual origin,development, and fate of bovine pancreatic islets

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo‐acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of ‘perilobular giant islets’ are distinguishable from larger numbers of ‘intralobular small islets’. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin‐synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette‐like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the ‘interlobular small islets’ persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T‐cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo‐acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy.     

Ultrastructural evidence for blood microvessels devoid of an endothelial cell lining in transplanted pancreatic islets.

The aim of the present study was to investigate, at the ultrastructural level, the process of revascularization of freshly isolated islets or cultured islets after transplantation under the kidney capsule of syngeneic mice. Native islets in adult pancreases from mice, pigs, and humans contained only capillaries with fenestrated endothelium. However, the endothelial cell lining was disrupted in both freshly isolated and cultured mouse islets. Shortly after transplantation (6 weeks) approximately 80% of graft microvessels contained no endothelial cell lining. Similar data on microvessel morphology were found when fetal porcine islet-like cell clusters were implanted into athymic nude mice. Re-endothelialization was a slow process, with 25% of the microvessels still lacking endothelium 6 months after transplantation of cultured mouse islets or islet-like cell cluster. However, when freshly isolated mouse islets are used only 25% of microvessels within the islet graft lacked endothelium 6 weeks after implantation. We suggest that capillaries damaged during islet isolation may provide a preformed channel, serving as a scaffold for newly formed islet graft blood vessels. The presence of non-endothelialized microvessels, with an associated lack of barrier function, might make transplanted islets more prone to thrombosis or an attack by the immune system. This provides a tentative explanation for the increased vulnerability of islet grafts when compared with whole pancreas transplants.     

MRI of transplanted surface-labeled pancreatic islets with heparinized superparamagnetic iron oxide nanoparticles

Transplantation of insulin-secreting pancreatic islets can provide real-time regulation of blood glucose in patients with type 1 diabetes mellitus. Currently, noninvasive and repetitive monitoring of islet engraftment and function is an emerging and promising modality for successful islet transplantation. Here we report a new technique for highly sensitive in?vivo magnetic resonance (MR) imaging of transplanted pancreatic islets. To this end, heparinized superparamagnetic iron oxide (heparin-SPIO) nanoparticle was newly synthesized for chemical conjugation onto islet surface. Compared to typical cellular labeling of Feridex(?) via random endocytosis, chemical conjugation of heparin-SPIO was stable and improved the hypointensity of transplanted islets due to surface modification of every islet. These heparin-SPIO-conjugated islets showed normal viability and insulin secretion, and were quantified by spin echo T(2)-weighted MR imaging with linear correlation depending on transplanted islet mass in?vitro and in?vivo for 30 days. Also, from the immunohistochemistry, we confirmed the existence of heparin-SPIO and insulin biosynthesis in transplanted islets. However, Feridex-uptake islets showed late glucose responsiveness according to changing glucose concentration although they could normally control the blood glucose levels in diabetic mouse. Thus, we anticipate that this surface labeling with heparin-SPIO can be directly applicable for MR imaging of transplanted islets.     

Function of rat pancreatic islets exposed to interleukin-18 in vitro.

The recently cloned cytokine interleukin-18 (IL-18) has been shown to promote a Th1-cell immune response, which may be a prerequisite for development of Type 1 diabetes. In this study we examined the effects of IL-18 on the function of isolated rat pancreatic islets. The islets were cultured in medium RPMI 1640 + 10% fetal calf serum and exposed for 48 h to recombinant human IL-18 (0, 0.1, 1 and 10 nM). In some experiments IL-18 (l0 nM) was combined with interleukin-12 (10 ng/ml), since these cytokines may act synergistically. IL-18 alone, or in combination, with IL-12 did not affect the islet DNA content suggesting absence of cytotoxicity. However, both cytokines induced an increased islet insulin content compared to non-cytokine exposed control islets. A slight increase in the medium insulin accumulation was observed when 1.0 nM IL-18 was added, but not in other experimental groups. Glucose-stimulated insulin release, glucose oxidation and (pro)insulin biosynthesis rates were not affected by the cytokines after culture. In acute experiments IL-18 had a small stimulatory effect on glucose-stimulated insulin secretion. It was also tested if IL-18 (10 nM) could affect IL-1beta (25 U/ml) induced suppression of the glucose oxidation rate, but this was not the case. We conclude that IL-18 has minor stimulatory effects on beta-cell function, and no clear synergistic effect is observed when IL-12 is added together with IL-18. If IL-18 is involved in beta-cell destruction in Type 1 diabetes, it is likely that this effect is secondary to an influence on the action of other cytokines.     

Somatostatin inhibition of phospholipase C activity in isolated rat pancreatic islets.

We have assessed the effect of somatostatin on the phospholipase C activity in isolated rat pancreatic islets. The phospholipase C activity was measured as the generation of inositol 1,4,5-trisphosphate and its metabolite inositol 1,3,4-trisphosphate from the hydrolysis of polyphosphoinositides. Inositol phosphates were measured using anion-exchange fast protein liquid chromatography analysis of extracts from islets prelabelled with myo-[3H]inositol. Somatostatin (1-1000 nmol l-1) significantly inhibited the glucose-induced (12 mmol l-1) phospholipase C activity in a concentration-dependent manner. The Ca2+ channel blocker verapamil (25 mumol l-1) also inhibited the glucose-induced (12 mmol l-1) phospholipase C, whereas the combination of somatostatin and verapamil did not induce any additional inhibition. At 3.3 mmol l-1 glucose, the hypoglycaemic sulphonylurea, tolbutamide (1 mmol l-1), increased the phospholipase C activity. This effect was reversed by somatostatin (100 nmol l-1). Tolbutamide did not further increase the glucose-induced (12 mmol l-1) phospholipase C activity. However, the somatostatin inhibition of glucose-induced (12 mmol l-1) phospholipase C was reversed by tolbutamide. The activator of adenylyl cyclase, forskolin (20 mumol l-1), did not exert any effect on the PLC-inhibition of somatostatin, whereas forskolin alone inhibited the phospholipase C activation at 12 mmol l-1 glucose. Our study demonstrates that somatostatin inhibits the hydrolysis of polyphosphoinositides in pancreatic islets, apparently via a mechanism dependent on Ca2+ and not on cAMP.     

The fate of transplanted tails in frog larvae

Frog larvae recieved transplants of autografts, allografts, and xenografts using Rana brevipoda and Rana japonica. Tail tip autografts heal rapidly, continue to grow but undergo degeneration at metamorphosis. Allografts and xenoqrafts also heal but show signs of rejection by the host's immune system. Onset of rejection depends upon the stage when grafts were performed. If transplants were made during early stages, onset was late; if performed later, onset was early. Because frog larvae will eventually undergo metamorphosis as a result of thyroxine effects, they are excellent experimental models for correlating the immune and endocrine systems with differentiation, growth, and aging.     

Treatment of rat pancreatic islets with reactive PEG

Covalent attachment of polymers to cells and tissues could be used to solve a variety of problems associated with cellular therapies. Insulin-dependent diabetes mellitus is a disease resulting from the autoimmune destruction of the beta cells of the islets of Langerhans in the pancreas. Transplantation of islets into diabetic patients would be an attractive form of treatment, provided that the islets could be protected from the host's immune system in order to prevent graft rejection. If reaction of polyethylene glycol (PEG) segments with the islet surface did not damage function, the immunogenicity and cell binding characteristics of the islet could be altered. To determine if this process damages islets, rat islets have been isolated and treated with protein-reactive PEG-isocyanate (MW 5000) under mild reaction conditions. An assessment of cell viability using a colorimetric mitochondrial activity assay showed that treatment of the islets with PEG-isocyanate did not reduce cell viability. Insulin release in response to secretagogue challenge was used to evaluate islet function after treatment with the polymer. The insulin response of the PEG-treated islets was not significantly different than untreated islets in a static incubation secretagogue challenge. In addition, PEG-isocyanate-treated islets responded in the same manner as untreated islets in a glucose perifusion assay. Finally, the presence of PEG on the surface of the islets after treatment with the amine-reactive N-hydroxysuccinimide-PEG-biotin (not PEG-isocyanate) was confirmed by indirect fluorescence staining. These results demonstrate the feasibility of treating pancreatic islets with reactive polymeric segments and provide the foundation for further investigation of this novel means of potential immunoisolation.     

Ontogenetic appearance of immunoreactive endocrine cells in rat pancreatic islets

Summary Chronological development of immunoreactive, pancreatic endocrine cells was immunohistochemically studied in rats. The first immunoreaction occurs for glucagon on day 11.5 and for insulin on day 12.5 of gestation, respectively, in the cells located within the cap-like or tubular pancreatic primordium derived from the gut wall. Immunoreactive somatostatin cells appear first at the periphery of primitive islets on day 15.5. On day 18.5, the cells of the primitive islets obtain their definitive arrangement and the islets are now separated from the tissue of the exocrine pancreas. Decapitation or encephalectomy performed on day 16.5 embryos fails to influence the ensuing further development of endocrine pancreas. This suggests that the hypothalamus or pituitary does not play an essential role in the histogenesis of the pancreatic islets.     

The effect of physical training on insulin secretion of rat pancreatic islets

Rats were either physically trained by a 12-wk swimming program or were freely eating or weight matched, sedentary controls. Islets of Langerhans were isolated and incubated at various glucose concentrations. Within the range of physiological glucose concentrations the rate of insulin release from islets of trained rats was lower than that from islets of sedentary controls. The DNA content of the islets was similar in the different groups. The demonstrated decreased glucose sensitivity of the insulin secretory mechanism within the β-cells of trained rats may partly explain the finding of lower plasma insulin concentrations during intravenous glucose tolerance test in these rats compared with sedentary rats. Epididymal fat pads of trained rats were smaller than those of weight matched controls which in turn were smaller than those of freely eating controls, these differences being due to differences in fat cell size. The lower glucose sensitivity of the β-cells in trained rats was probably not a consequence of the low body weight and small fat depots in these rats.     

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.     

Distribution and developmental changes of annexin V in rat pancreatic islets

We investigated the expression patterns and the dynamic changes of annexin V in the rat pancreatic islets during postnatal development by both Western blot analysis and immunohistochemistry using polyclonal anti-annexin V antibodies. Pancreatic islets were exclusively shown to express annexin V, while exocrine tissues were not. Pancreatic duct cells and capillary endothelial cells in the pancreas were also stained. Annexin V was present in both A and B cells in islets. Strong staining was detected on the cell membrane and in peri-membranous areas of islet cells. The expression level of annexin V in pancreatic islets gradually increased with postnatal development to remain constant after the 12th week of life. The present study suggested that annexin V may be concerned with the maturation of islet functions.     

Somatostatin inhibition of phospholipase G activity in isolated rat pancreatic islets

We have assessed the effect of somatostatin on the phospholipase C activity in isolated rat pancreatic islets. The phospholipase C activity was measured as the generation of inositol 1,4,5-trisphosphate and its metabolite inositol 1,3,4-trisphosphate from the hydrolysis of polyphosphoinositides. Inositol phosphates were measured using anion-exchange fast protein liquid chromatography analysis of extracts from islets prelabelled with myo-[³H]inositol. Somatostatin (1–1000 nmol l^-1) significantly inhibited the glucose-induced (12 mmol l^-1) phospholipase C activity in a concentration-dependent manner. The Ca²⁺ channel blocker verapamil (25 μmol l^-1) also inhibited the glucose-induced (12 mmol l^-1) phospholipase C, whereas the combination of somatostatin and verapamil did not induce any additional inhibition. At 3.3 mmol l^-1 glucose, the hypoglycaemic sulphonylurea, tolbutamide (1 mmol l^-1), increased the phospholipase C activity. This effect was reversed by somatostatin (100 nmol l^-1). Tolbutamide did not further increase the glucose-induced (12 mmol l^-1) phospholipase C activity. However, the somatostatin inhibition of glucose-induced (12 mmol l^-1) phospholipase C was reversed by tolbutamide. The activator of adenylyl cyclase, forskolin (20 μmol l^-1), did not exert any effect on the PLC-inhibition of somatostatin, whereas forskolin alone inhibited the phospholipase C activation at 12 mmol l^-1 glucose. Our study demonstrates that somatostatin inhibits the hydrolysis of polyphosphoinositides in pancreatic islets, apparently via a mechanism dependent on Ca²⁺ and not on cAMP.