Quantification of Basal and Stimulated ROS Levels as Predictors of Islet Potency and Function

We have developed a luminol-based assay using intact islets, which allows for quantification of reactive oxygen species (ROS). In addition, an index capable of characterizing metabolic and mitochondrial integrity prior to transplantation was created based on the capacity of islets to respond to high glucose and rotenone (mitochondrial respiratory chain complex I inhibitor) by production of ROS. To validate this assay, lipid peroxidation and antioxidative defense capacity were evaluated by detection of malondialdehyde (MDA) levels and glutathione peroxidase activity (GPx), respectively. Also, flow cytometric analyses of ROS (dihydroethidine), apoptosis (Annexin V, active caspases), necrosis (Topro3), and mitochondrial membrane potential (JC-1) were done in parallel to correlate with changes in luminol-measured ROS. ATP/ADP ratios were quantified by HPLC and the predictive value of ROS measurement on islet functional potency was correlated with capacity to reverse diabetes in a streptozotocin-induced diabetic NOD.scid mouse model as well as in human transplant recipients. Our data demonstrate that levels of ROS in islets correlate with the percentage of apoptotic cells and their functional potency in vivo. The ROS indices following glucose and rotenone exposure are indicative of metabolic potency and mitochondrial integrity and can be used as surrogate markers to evaluate the quality of islets prior to transplantation.     

Nitric oxide is not involved in the initiation of insulin secretion from rat islets of Langerhans

Summary The involvement of nitric oxide as an intracellular messenger in the control of insulin secretion from pancreatic Beta cells was studied in rat islets of Langerhans by measuring: (i) nitric oxide generation in response to physiological insulin secretagogues; (ii) the effects of inhibitors of nitric oxide synthesis on insulin secretory responses to physiological secretagogues, and on insulin synthesis; (iii) changes in islet cyclic guanosine monophosphate in response to secretagogues; (iv) the effects of exogenous cyclic guanosine monophosphate and dibutyryl cyclic guanosine monophosphate on insulin secretion from electrically permeabilised islets and from intact islets, respectively. These studies produced no evidence that nitric oxide generation is required for the initiation of insulin secretion by common secretagogues. However, the results of our experiments suggest that the generation of nitric oxide may be involved in long-term, glucose-dependent increases in cyclic guanosine monophosphate content of islet cells, although the physiological relevance of these changes requires further investigation.     

Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet Isolation

Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.     

阴囊前外侧岛状皮瓣再造尿道的解剖学研究及临床应用

目的 为阴囊前外侧岛状皮瓣的设计提供解剖学基础。方法 对20具成人男尸采用少量氨水经股动脉冲洗后，灌注红色乳胶逐段解剖阴部外动脉及其分支。结果 阴囊前外侧皮瓣的血供来自阴囊前动脉，阴囊前动脉的发起点恒定，血供范围大，故该皮瓣有充足的营养及较大的范围，也便于皮瓣的松解和转移。结论 利用此皮瓣重建尿道，适用于阴茎型尿道下裂病例。     

Fine-needle aspiration of pancreatic extramedullary plasmacytoma: Possible confusion with islet cell tumor

We report two cases of extramedullary plasmacytoma (EMP) of the pancreas, diagnosed by fine-needle aspiration (FNA). Plasmacytoma of the pancreas is a rare neoplasm with only 12 cases recorded in the literature. Because of its scarcity and the cytomorphologic similarity between plasma cells and endocrine cells, EMP of the pancreas may be confused with neuroendocrine (islet cell) tumors of the pancreas. Immunohistochemical staining for light chain and/or neuroendocrine markers will prevent diagnostic error when interpreting plasmacytoid neoplasms of any site susceptible to endocrine tumors, including the pancreas. © 1994 Wiley-Liss, Inc.     

人胎阑尾IAPP-、SS-及5-HT免疫反应性细胞的免疫组织化学研究

为了探讨胰岛淀粉样多肽，在胎阑尾中的个体发及其与其他生物活性物质的关系，用免疫组织ＰＡＰ法，对３６例１２－３８周人胎阑尾中ＩＡＰＰ免疫反应性细胞，生称抑素ＩＲ细胞５－羟色胺－ＩＲ细胞进行定位研究。结果表明，肥１２周，阑尾上皮中已可见到ＳＳ－ＩＲ细胞，而ＩＡＰＰ－及５－ＨＴ－ＩＲ细胞于１４周才见到。在整个胎期，阑尾中的ＩＡＰＰ－及ＳＳ－ＩＲ细胞始络分散存在，数量较少，而５－ＨＴ－ＩＲ细胞数量随胎龄增     

C-reactive protein concentration is not related to islet autoimmunity status in offspring of parents with type 1 diabetes

Autoimmunity may be associated with acute or chronic inflammation. In order to determine whether the inflammatory marker C-reactive protein (CRP) was an indicator of inflammatory events that precede, predict, or associate with islet autoimmunity or type 1 diabetes, CRP was measured in sequential antibody-negative, seroconversion, and follow-up-positive samples from 65 prospectively studied islet autoantibody-positive children. Although changes in CRP concentrations were observed in some children, overall CRP concentrations were similar in antibody-negative samples (median, 0.21 mg/L), antibody-positive samples (median, 0.26 mg/L), and samples at seroconversion (median, 0.26 mg/L). CRP concentrations at diabetes onset (median, 0.59 mg/L) were not significantly increased over antibody-negative samples (P = 0.07). CRP concentrations did not predict diabetes development. CRP concentrations were related to age (r = 0.26; P < 0.001) and were increased in samples obtained from October to January (P < 0.001). These findings suggest that CRP concentrations are not a valuable marker of progression to type 1 diabetes and highlight the importance of correcting analyses for seasonal variations.     

Interaction of hepatocytes and pancreatic islets cotransplanted in polymeric matrices

Heterotopic hepatocyte transplantation (HcTx) in polymeric matrices may become an alternative to liver transplantation for metabolic disorders. Hepatotrophic stimulation by means of a portocaval shunt operation is an established, but invasive, procedure used to optimize hepatocyte engraftment in matrices. We evaluated hepatocyte and pancreatic islet cotransplantation (ICT) as an alternative noninvasive approach to hepatotrophic stimulation. Lewis rats served as donors and recipients. Hepatocytes and islets were isolated using collagenase digestion and seeded into polyvinylalcohol matrices. HcTx and ICT were compared with HcTx plus portocaval shunt and HcTx without stimulation. Matrices were investigated at 1, 3, and 6 months after implantation: the test methods applied were trichrome staining, PAS, immunohistochemistry for insulin, glucagon and incorporated BrdU, and in situ hybridization for albumin RNA. Hepatocytes expressed albumin RNA and formed conglomerates without atypias in all animals. ICT and portocaval shunting increased the number of hepatocytes and BrdU uptake. Alpha cells migrated into the islet-surrounding hepatocytes, whereas beta cells remained immobile. It is concluded that ICT and portocaval shunting supported engraftment of hepatocytes in polymeric matrices equally well. ICT did not interfere with recipient glucose metabolism and did not induce hyperproliferative premalignant foci within the transplanted hepatocytes. The technique is an attractive approach to hepatotrophic stimulation of bioartificial liver equivalents. Received: 10 February 1999 / Accepted: 22 April 1999