The influence of porcine pancreas digestion parameters and islet histomorphology on islet isolation outcome.

Transplantation of the pig islets of Langerhans is considered as the future treatment for patients suffering from type I diabetes mellitus. Despite the adaptation of modified Ricordi method and highly purified collagenase, the results of pancreas digestions are precarious. Selection of proper donor and optimal digestion procedure are fundamental. The aim of this study was to assess the impact of pancreas procuring parameters on pig islets yield. The pancreata were harvested from 69 market sows weighting over 150 kg. After intraductal injection of cold collagenase solution pancreata were transported in UW solution or under conditions of two layer method (TLM). In laboratory pancreata were digested at 37 degrees C according to Ricordi isolation method or stationary in the bottle. The particular parameters of isolation procedure were considered as substantial. Pig weight, volume of infused collagenase solution, TLM application and pancreas dividing before digestion positively affected islet yield. Additionally, the influence of pancreatic islet tissue histomorphology on isolation outcome was studied. Proper donor selection as well as adequate digestion parameters could improve pig islet recovery during islet isolation.

Comparison of two methods of pancreas islets immunoisolation.

The efficacy of two methods of Langerhans islets immunoisolation was compared. For this purpose the function of islets encapsulated with alginate/polyethylenimine/protamine/heparin (APPH) or with alginate/poly-L-lisine/alginate (APA) membranes was assessed: in vitro according to their survival and response to glucose challenges, and in vivo according to their capability to provide sufficient insulin delivery to maintain normal fasting blood glucose following xenotransplantation to streptozotocin diabetic mice. In vitro insulin secretion and the response to glucose challenge of APPH and APA encapsulated islets were comparable to free islets. In vivo intraperitoneal concordant xenotransplantation of APA encapsulated rat islets reversed the diabetic state of streptozotocin diabetic mice for a longer period, than APPH islet grafts. This study clearly demonstrated the inadequacy of in vitro methods in the prediction of in vivo results of islets transplantation.

Reversal of hyperglycemia in streptozotocin diabetic mice by xenotransplantation of microencapsulated rat islets.

Rat pancreatic islets were immunoisolated within alginate capsules with additional polyethyleneimine-protamine-heparin highly biocompatible membrane. Perifusion study in vitro demonstrated satisfactory similarities between the insulin release profiles of encapsulated and free islets. Concordant xenotransplantation of microencapsulated rat islets significantly prolonged mean time of restored normoglycemia (46 +/- 15 days) in streptozotocin-diabetic BALB/c mice recipients comparing to uncoated grafts (7 +/- 2 days).

Experience with pancreas islets separation, immunoisolation and cryopreservation.

Experience of Warsaw Pancreas Laboratory is presented. Some improvement in the methods of rat, human and pig pancreases digestion, and in identification of Langerhans islets by means of intravenous injection of I-DTZ was achieved. For immunoisolation of islets, 2 methods were elaborated: capsules containing alginate/polyethyleneimine/protamine/heparin membrane prepared by modified Sun method, and microcapsules based on Zekorn method. Biocompability of hollow fibers, prepared with polypropylene (PP), surface modified PP (PPS) and polysulphone (PS) was assessed in vitro. Only PS fibers were fully compatible. It was shown, that the mixture of exocrine tissue did not influence in vitro insulin secretion, providing that alginate in which islets are embedded remain gelled. The efficacy of 3 methods of islets cryopreservation was compared: freezing in "semicontrolable" conditions, in programmable Kriomedpol machine, and vitrification. The highest percentage of frozen/thawed living cells, and the most reliable results were obtained with Kriomedpol method.

Recipient uridine 5'-diphospho-glucuronosyltransferase UGT1A9 c.98T>C variant determines transplanted kidney filtration rate.

Mycophenolic acid preparations are commonly used in prophylaxis of kidney allograft acute rejection. The medication is metabolized by uridine diphosphate glucuronosyltransferases, mainly UGT1A9 present in liver, kidney, and intestine. The effect of UGT1A9 allelic variants on drug metabolism in healthy volunteers and transplant recipients has been previously evaluated; these studies included the UGT1A9 c.98T>C polymorphism (rs72551330, p.Met33Thr) causing methionine-to-threonine substitution in the polypeptide chain. The study objective was to evaluate the relationship between UGT1A9 c.98T>C polymorphism and kidney graft function and survival and the risk of acute allograft rejection. Kidney recipients who underwent transplantation between 2000 and 2007 at the Medical University of Warsaw were included. Clinical data originated from standard medical records, UGT1A9 c.98T>C was genotyped using a polymerase chain reaction-restriction fragment length polymorphism method. A group of 243 kidney transplant recipients was enrolled in the study. The frequency of the c.98C allele was 2.4% (12 of 486). Most of the carriers of the allelic variant (10 of 12) received cyclosporine A at transplantation. In the c.98C allele carriers, worse function of the renal allograft, manifested by a significantly lower glomerular filtration rate, was found in the first posttransplantation month and persisted at a lower level for 8 years after the procedure (for comparison of the UGT1A9 c.98 TT vs the UGT1A9 c.98 TC groups, P = .03). No association was found between the presence of the UGT1A9 c.98C allele and the risk of delayed renal graft function, acute rejection, dysfunction of the renal graft, or dialysis treatment reintroduction. Significantly lower estimated glomerular filtration rate of the renal allograft in UGT1A9 c.98C carriers did not translate into decreased allograft survival.

Uridine diphosphate glucuronosyltransferase 2B7 variant p.His268Tyr as a predictor of kidney allograft early acute rejection.

BACKGROUND: Uridine diphosphate glucuronosyltransferase (UGT2B7) is responsible for conversion of mycophenolic acid to mycophenolic acyl-glucuronide (acylMPAG). Conflicting data exist regarding the role of UGT2B7 p.His268Tyr (802C>T, rs7439366) variant in the clinical course following organ transplantation. STUDY AIM: The aim of this study was to reveal an association between UGT2B7 p.His268Tyr (802C>T, rs7439366) polymorphism and kidney transplantation outcome. STUDY DESIGN, PATIENTS, AND METHOD: Genomic DNA of 235 kidney transplant recipients was genotyped for UGT2B7 802C>T using TagMan single nucleotide polymorphism (SNP) genotyping assay. Maintenance immunosuppression used mycophenolate mofetil (MMF) and cyclosporine A (n = 137) or tacrolimus (n = 98). Primary end-point was biopsy-confirmed acute rejection within 3 and 12 post-transplantation months. Secondary end-points included gastrointestinal side effects, leukopenia, lymphopenia, neutropenia, and infections. Statistical analysis was performed with the aid of SAS System using kernel-smoothed estimates of acute graft rejection hazard function. The log-rank test and hazard ratio were used to reflect association between UGT2B7 802C>T variant and risk of acute graft rejection. RESULTS: Within 3 postimplantation months 38 (16.2%) patients experienced acute rejection; 33 were allele C carriers in UGT2B7 802C>T SNP and 5 were TT homozygotes (P < .0457). Allele C-associated risk of rejection was 2.50 and remained between 2.19 and 3.02 after adjustment for clinical confounders, ie, HLA mismatch, panel-reactive antibodies, donor age, repeated transplantation, induction therapy, donor type, delayed graft function, applied calcineurin inhibitor, or MMF dosing. We found no association between the polymorphism and gastrointestinal side effects, leukopenia, lymphopenia, neutropenia, and infections. CONCLUSION: UGT2B7 802C>T genotyping may help identify patients with excessive early acute rejection risk.

Lymphocyte counts in kidney allograft recipients are associated with IMPDH2 3757T>C gene polymorphism.

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme for de novo synthesis of guanine nucleotides, is required for lymphocyte proliferation. Inhibition of IMPDH by mycophenolic acid (MPA) constitutes part of an immunosuppressive therapy in kidney allograft recipients. The 3757T>C polymorphic variant (rs11706052) of the IMPDH2 gene, which encodes 1 of 2 IMPDH isoenzymes, has been associated with increased IMPDH activity and reduced ability of MPA to exert antiproliferative effects on lymphocytes. The association of IMPDH2 3757T>C SNP with posttransplant courses of kidney allograft recipients remains unclear. Therefore, the aim of the present study was to evaluate associations between this single nucleotide polymorphism and common posttransplant complications among Polish kidney allotransplant recipients. We observed that the frequency of IMPDH2 3757C allele in this group (n=177) did not differ significantly from a control cohort representing the background population of Poland (n=550). There were no significant differences between patients carrying the IMPDH2 3757CT and TT genotypes with respect to acute rejection risk, neutropenia, or incidences of serious infections or gastrointestinal side effects. However, we noted that the 3757C allele was associated with higher lymphocyte counts and a reduced incidence of lymphopenia among kidney allograft recipients. Our findings may be of practical significance to tailor immunosuppressive regimens in kidney transplant recipients.

Multidrug resistance-associated protein 2 gene (ABCC2) variant in kidney allograft recipients.

Multidrug resistance-associated proteins may play crucial roles in the protection of internal organs, particularly the kidneys and liver, from various toxic agents. Little is known about their significance in transplanted organ function and survival. The aim of the present study was to evaluate the frequency of functional ABCC2 4544A>G polymorphism in 165 renal transplant recipients. DNA was isolated from peripheral blood and ABCC2 4544A>G polymorphism was assessed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Analysis of allele and genotype frequencies revealed that the presence of ABCC2 4544 A allele was associated with prolonged observation time and may have an impact on a greater frequency of proteinuria. This observation strongly suggested that particular alleles of the ABCC2 gene may contribute to transplantation outcomes.

In vitro and in vivo evaluation of protamine-heparin membrane for microencapsulation of rat Langerhans islets.

Rat pancreatic islets were microencapsulated with multilayer protamine-heparin (PH) membrane. Basal and stimulatory insulin secretion of microencapsulated islets was similar to the controlled free islets in vitro. During the long-term culture (up to 2 weeks) mean insulin release of encapsulated islets did not significantly differ from the mean of free ones (the ratio of mentioned means was 54-167%). Empty PH microcapsules transplanted into Wistar rats intraperitoneally and under the kidney capsule were generally harmless up to 4 months. In only a few cases traces of fibrotic tissue around capsules entrapped in the omentum were found. No damage of microcapsules structure was observed. The worst results were obtained in the instance of retroperitoneal transplantation. We conclude, therefore, that PH membrane was proved to be highly biocompatible, nontoxic for islets, and did not impair viability and glucose-dependent insulin secretion of Langerhans islets in in vitro culture.

[Experiments with pancreas islet xenotransplantation]. / Doswiadczenia w ksenotransplantacji wysp trzustkowych.

Different methods of human, porcine and rat pancreata digestion Langerhans islets purification, immuno-isolation and cryopreservation were compared. The results obtained were assessed in vitro and in vivo. The longest concordial xenograft survival was observed after transplantation of rats islets immunoisolated by Sun's method to streptozotocin diabetic mice. Due to its simplicity and lesser time consumption using Kriomedpol machine was recommended.