Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010.

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999-2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p<0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007-2010 when compared to 1999-2002 (445.4±156.8 vs. 421.3±155.4×0(3) IEQ; p<0.05). Islet purity and total number of ß cells significantly improved over the study period (p<0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999-2010, and these parallel improvements in clinical outcomes over the same period.

Pretreatment of donor pigs with a diet rich in soybean oil increases the yield of isolated islets.

INTRODUCTION: The pig is considered the donor species of choice for islet xenotransplantation. However, isolation of porcine islets is difficult, particularly from young pigs. Early life exposure to a high-fat diet (HFD) reportedly encourages islet ß-cell expansion in neonatal rodents and improves islet viability in culture from pretreated weanling pigs. In this study, we examined the influence of young donor pretreatment with a soybean oil-enriched HFD on porcine islet mass and yield after islet isolation. MATERIALS AND METHODS: Postweaning and between days 70 and 250, pigs were fed either a standard diet (control group; n = 5) or an HFD (experimental group; n = 6). Biochemical blood parameters and acute C-peptide response to intravenous glucose were monitored before pancreas procurement. The study was blinded to objectively evaluate the influence of treated diet. After procurement, pancreas biopsy samples were taken from control and pretreated donor pigs to assess islet number by using a dithizone scoring method and histologic islet area fraction determination. Control and HFD donor pig islets were isolated by using our standard isolation protocol to determine islet yield. Islet isolation characteristics and islet quality were assessed in both groups, and the results were compared. RESULTS: There were no significant differences in the donor characteristics (age, body weight, glucose disposal rate, acute C-peptide response to intravenous glucose, cholesterol, and aspartate aminotransferase) except fasting blood glucose level between the control and treatment groups (84 ± 6 vs 99 ± 12 mg/dL; P = .0317). The stimulated insulin and C-peptide levels between groups were similar. However, the dithizone score was slightly higher in the treatment group compared with the control group (95.4 ± 38.5 vs 62.6 ± 23.9; P = .1208). Digestion time, digested pancreas weight, pellet volume, and the fragility index were similar in both groups. However, the average islet count (islet equivalent number/g pancreas) at the digest level was significantly higher in the HFD group than in the control group (1578 ± 994 vs 738 ± 202; P = .0344). The functional viability of 2- and 7 day-cultured islets, as assessed by using oxygen consumption rate corrected for DNA, was similar in both groups. CONCLUSIONS: Pretreatment of pigs with HFD enriched with soybean oil could potentially be used to improve the islet mass in donor pigs. Further studies are needed to confirm and optimize the use of HFD for the purpose of increasing islet yield from young donor pigs.

Severely fibrotic pancreases from young patients with chronic pancreatitis: evidence for a ductal origin of islet neogenesis.

While it is known that islet cell mass increases considerably after birth, general uncertainty surrounds the source of new beta cells in humans. Chronic pancreatitis (CP) presents a natural injury model for studying postnatal beta-cell regeneration in the human pancreas. In this report, we present histological evidence from human CP pancreases to support the theory that islet neogenesis can occur from ductal precursor cells after birth. Three young patients (ages 16, 12, and 28 years) underwent total pancreatectomy for the management of CP followed by islet isolation and autologous transplantation to prevent or minimize postsurgical diabetes. In all cases, the pancreases had extensive fibrosis, a rock-like consistency, and calcifications in the ducts. During islet isolations, we observed the unusual release of islets with many ductal fragments. In histopathological evaluation of these pancreases, solid cords of cells sometimes formed islet like structures intraductally or extending from ductal structures. Immunofluorescence staining for chromogranin, insulin, proinsulin, PDX1, glucagon, and cytokeratins confirmed these structures to be composed of chromogranin-positive endocrine cells which included both ß-cells and α-cells. Labeling for Ki67 to demonstrate mitotic activity showed frequent labeling of duct epithelial cells and of some periductal cells. Using insulin and wide-spectrum cytokeratin double immunofluorescent labeling, we found insulin-positive cells to be present within the ductal lumens, among the cytokeratin-positive ductal epithelium, and extending from the ductal epithelium into surrounding connective tissues, providing evidence for a ductal origin of islet neogenesis.

Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.

BACKGROUND: Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. METHODS: Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). RESULTS: Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% +/- 9% vs 47% +/- 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% +/- 2%) and the A1AT-treated groups (99% +/- 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE). CONCLUSION: Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.

Human islet isolation for autologous transplantation: comparison of yield and function using SERVA/Nordmark versus Roche enzymes.

Islet autotransplantation (IAT) is used to preserve as much insulin-secretory capacity as possible in patients undergoing total pancreatectomy for painful chronic pancreatitis. The enzyme used to dissociate the pancreas is a critical determinant of islet yield, which is correlated with posttransplant function. Here, we present our experience with IAT procedures to compare islet product data using the new enzyme SERVA/Nordmark (SN group; n = 46) with the standard enzyme Liberase-HI (LH group; n = 40). Total islet yields (mean +/- standard deviation; 216,417 +/- 79,278 islet equivalent [IEQ] in the LH group; 227,958 +/- 58,544 IEQ in the SN group; p = 0.67) were similar. However, the percentage of embedded islets is higher in the SN group compared to the LH group. Significant differences were found in pancreas digestion time, dilution time, and digested pancreas weight between the two groups. Multivariate linear regression analysis showed the two groups differed in portal venous pressure changes. The incidence of graft function and insulin independence was not different between the two groups. The SN and LH enzymes are associated with similar outcomes for IAT. Further optimization of the collagenase/neutral protease ratio is necessary to reduce the number of embedded islets obtained when using the SN enzyme.

Acute fatty liver of pregnancy: a report of two cases.

Acute fatty liver of pregnancy is an uncommon, potentially fatal disorder. Between 1998 and 2000, two patients with acute fatty liver of pregnancy presented at the Christian Medical College Hospital, Vellore. Both patients were in the thirty-sixth week of pregnancy. jaundice and encephalopathy were the predominant symptoms. Both the mothers died after they delivered a stillborn Infant each. The maternal deaths were due to multiorgan failure and/or postpartum haemorrhage and sepsis. The route of delivery was vaginal in both the patients. Extrahepatic and metabolic complications in both cases Included renal failure, sepsis, hypoglycaemia, disseminated intravascular coagulation and gastrointestinal bleeding. Liver biopsy done in both patients was consistent with the diagnosis of acute fatty liver of pregnancy. To the best of our knowledge, this is the first report from India on acute fatty liver of pregnancy.

Epithelioid haemangioendothelioma of the liver.

Epithelioid haemangioendothelioma is a well-recognized vascular neoplasm but can be mistaken for metastatic adenocarcinoma or other primary liver tumors. We present a case of epithelioid haemangioendothelioma of the liver in a 62 year old woman, in whom the diagnosis was missed by various pathologists. This patient presented with loss of appetite and weight, and fever. She rapidly deteriorated and died presumably of liver cell failure. The characteristic histological features and the positive immunostaining for endothelial markers (CD31 & Ulex europaeus) confirmed the diagnosis.

Vesical varix in cirrhosis of liver.

Vesical varices in portal hypertension are rare. We report a patient with portal hypertension who developed recurrent painless hematuria. Cystoscopy was normal. Doppler ultrasound and MR angiography showed a dilated paraumbilical vein within the falciform ligament coursing down to the urinary bladder wall and draining into the right internal iliac vein. He underwent liver transplantation for decompensated chronic liver disease. He is in good health and has not had further episodes of hematuria.

Clinical manifestations and survival pattern of Wilson's disease.

BACKGROUND: Wilson's disease usually presents in childhood. Early recognition and treatment is crucial to retard the progression of this disease, which can be debilitating, if not fatal. We analysed the clinical manifestations and survival pattern of patients admitted at our centre from 1993 to 1996. METHODS: Hospital records of patients were reviewed to obtain the clinical manifestations. The survival status of patients was determined by a prospective follow up. The Kaplan-Meier survival curve and univariate Cox proportional hazards model were used to determine the survival pattern and risk for death in Wilson's disease. RESULTS: A total of 60 patients were studied. The median age at onset of symptoms was 12 years (range 5-52 years). The log rank test showed a significant difference in the mean (SD) survival between patients who presented with hepatic [87.36 (15.26) months] and neurological symptoms [114.33 (9.14) months]. Cox proportional hazards analysis showed a hazard ratio of 4.9 for patients with a hepatic presentation compared to those with neurological presentation. CONCLUSION: The presentation of Wilson's disease is not limited to the paediatric age group. Patients with a hepatic presentation had a five-fold higher risk of mortality when compared to those with a neurological presentation.

Classification of children as slow or rapid acetylators based on concentrations of isoniazid in saliva following oral administration of body-weight and surface-area-related dosages of the drug.

The acetylator phenotype of 180 children aged 3-11 years was determined on the basis of isoniazid concentrations in saliva collected at 5 hours after oral administration of body-weight and surface-area-related dosages of the drug in a syrup form. Isoniazid 2.5 mg/kg was administered on one occasion and 75 mg/m2 surface-area on another, with an interval of 3 days between the occasions. A cross-over design was employed and the sequence was determined by random allocation. The distribution of the concentrations was bimodal with both procedures, indicating the presence of two groups namely, the slow and rapid acetylators. The criterion for a rapid acetylator was a concentration of 0.3 micrograms/ml or less by body-weight-related dosage and 0.4 micrograms/ml or less by that based on surface-area. Based on these criteria, 62% of the children were classified as slow acetylators and 38% as rapid acetylators by body-weight, and 59 and 41%, respectively by surface-area, and the findings were similar in children in the different age-groups. The agreement between the two procedures was 98%.