Finding Eden - alternative transplantation sites for pancreatic islets.

Pancreatic islets transplantation is an established treatment method for type 1 diabetic patients with the hypoglycemia unawareness syndrome in whom a therapy with modern technologies fails. Islet transplantation is most commonly done using an interventional radiology method: a tissue suspension of pancreatic islets is applied into a branch of the portal vein through a percutaneously installed catheter. Although being minimally invasive unlike pancreas organ transplant, this method is associated with many technical difficulties. Possible complications of the procedure include hemorrhage and portal vein thrombosis. Unlike their natural dispersed localization in exocrine pancreas, isolated pancreatic islets are exposed to hypoxia, toxins and immunosuppressive drugs in the liver parenchyma. Direct contact with the recipients blood causes an instant blood mediated inflammatory reaction (IBMIR) resulting in the death of more than half of the pancreatic islets shortly after their application. Therefore the size of the islet graft is often insufficient and a number of transplanted patients require administration of exogenous insulin. All of these are reasons for seeking an alternative transplantation site with more hospitable conditions for long-term islet survival. Various transplantation sites have been tested in experimental and clinical research. The advantages and disadvantages of some of them are summarized in this paper. Currently, transplantation into the greater omentum seems most promising, which has already been used in clinical practice at several institutions.

Incidence and Risk Factors of Diabetic Foot Syndrome in Patients Early After Pancreas or Kidney/Pancreas Transplantation and its Association with Preventive Measures.

Diabetic foot (DF) can develop in diabetic patients after organ transplantation (Tx) due to several factors including peripheral arterial disease (PAD), diabetic neuropathy and inappropriate DF prevention. Aim: To assess the occurrence of DF and associated risk factors in transplant patients. Methods: Fifty-seven diabetic patients were enrolled as part of this prospective study. All patients underwent organ Tx (01/2013-12/2015) and were followed up for minimum of 12 months up to a maximum of 50 months. Over the study period we evaluated DF incidence and identified a number of factors likely to influence DF development, including organ function, presence of late complications, PAD, history of DF, levels of physical activity before and after Tx, patient education and standards of DF prevention. Results: Active DF developed in 31.6% (18/57) of patients after organ Tx within 11 months on average (10.7 ± 8 months). The following factors significantly correlated with DF development: diabetes control (p = .0065), PAD (p<0.0001), transcutaneous oxygen pressure (TcPO2;p = .01), history of DF (p = .0031), deformities (p = .0021) and increased leisure-time physical activity (LTPA) before Tx (p = .037). However, based on logistic stepwise regression analysis, the only factors significantly associated with DF during the post-transplant period were: PAD, deformities and increased LTPA. Education was provided to patients periodically (2.6 ± 2.5 times) during the observation period. Although 94.7% of patients regularly inspected their feet (4.5 ± 2.9 times/week), only 26.3% of transplant patients used appropriate footwear. Conclusions: Incidence of DF was relatively high, affecting almost 1/3 of pancreas and kidney/pancreas recipients. The predominant risk factors were: presence of PAD, foot deformities and higher LTPA before Tx. Therefore, we recommend a programme involving more detailed vascular and physical examinations and more intensive education focusing on physical activity and DF prevention in at-risk patients before transplantation.

A Preliminary Characterization of a Novel Recombinant Clostridial Collagenase Blend.

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.

Reprogramming of Human Pancreatic Organoid Cells into Insulin-Producing ß-Like Cells by Small Molecules and in Vitro Transcribed Modified mRNA Encoding Neurogenin 3 Transcription Factor.

Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.6 ± 1.2 % of the total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) needed for the proper function of pancreatic ß-cells. Additionally, we have revealed a positive effect of ALK5 inhibitor RepSox on the overall reprogramming efficiency. However, the reprogrammed IPCs possessed only a partial insulin-secretory capacity, as they were not able to respond to the changes in the extracellular glucose concentration by increasing insulin secretion. Based on the achieved results we conclude that due to the incomplete reprogramming, the IPCs have immature character and only partial properties of native human ß-cells.

Lipasin/betatrophin is differentially expressed in liver and white adipose tissue without association with insulin resistance in Wistar and Goto-Kakizaki rats.

Lipasin is a recently identified lipokine expressed predominantly in liver and in adipose tissue. It was linked to insulin resistance in mice and to type 1 and type 2 diabetes (T1D, T2D) in humans. No metabolic studies concerning lipasin were performed yet in rats. Therefore, we used rat model of T2D and insulin resistance, Goto-Kakizaki (GK) rats, to determine changes of lipasin expression in liver and in white adipose tissue (WAT) over 52 weeks in the relation to glucose tolerance, peripheral tissue insulin sensitivity and adiposity. GK rats were grossly glucose intolerant since the age of 6 weeks and developed peripheral insulin resistance at the age of 20 weeks. Expression of lipasin in the liver did not differ between GK and Wistar rats, declining with age, and it was not related to hepatic triacylglycerol content. In WAT, the lipasin expression was significantly higher in Wistar rats where it correlated positively with adiposity. No such correlation was found in GK rats. In conclusion, lipasin expression was associated neither with a mild age-related insulin resistance (Wistar), nor with severe genetically-based insulin resistance (GK).

Severe Epidermal Nerve Fiber Loss in Diabetic Neuropathy Is Not Reversed by Long-Term Normoglycemia After Simultaneous Pancreas and Kidney Transplantation.

Whether nerve fiber loss, a prominent feature of advanced diabetic neuropathy, can be reversed by reestablishment of normal glucose control remains questionable. We present 8-year follow-up data on epidermal nerve fiber (ENF) density and neurological function in patients with type 1 diabetes after simultaneous pancreas and kidney transplantation (SPK) with long-term normoglycemia. Distal thigh skin biopsies with ENF counts, vibration perception thresholds (VPTs), autonomic function testing (AFT) and electrophysiological examinations were performed at time of SPK and 2.5 and 8 years after SPK in 12 patients with type 1 diabetes. In comparison to controls, baseline ENF density, VPT and AFT results of patients indicated severe neuropathy. At follow-up, all SPK recipients were insulin independent with excellent glycemic control and kidney graft function; however, the severe ENF depletion present at baseline had not improved, with total ENF absence in 11 patients at 8-year follow-up. Similarly, no amelioration occurred in the VPT and AFT results. Numerical improvement was seen in some electrophysiological parameters; however, statistical significance was achieved only in median motor nerve conduction velocity. ENF loss and functional deficits in advanced diabetic peripheral neuropathy are rarely reversible, even by long-term normoglycemia, which underscores the importance of neuropathy prevention by early optimal glycemic control.

Formation of Cholangiogenic Cysts Following Intrahepatic Islet Transplantation in Streptozotocin Diabetic Rats.

Permanent hyperinsulinemia and the resulting overstimulation of the insulin receptor signaling pathway is suspected as a trigger of cancer genesis in the livers of type 2 diabetic patients. Liver tissue (LT) surrounding transplanted pancreatic islets (PI) can be permanently exposed to insulin in even higher concentrations than in type 2 diabetic patients. Therefore, this study examines the effect of PI transplantation (Tx) on LT in animals with streptozotocin (STZ)-induced diabetes mellitus. The suboptimal mass (400 or 1000) of isogeneic PI was transplanted into either the portal vein or under the kidney capsule of diabetic Brown Norway (BN) rats. Healthy BN rats treated with 400 isogeneic PI transplanted in the portal vein served as a control group. During the first 6 months after PI Tx, small and infrequent cystic lesions developed in animals with STZ diabetes, irrespective of the Tx site. In 10 months, frequent and complex cystic lesions appeared in these animals. In the control group, several small lesions were detected but not until 10 months after the PI Tx. In summary, STZ is the likely main inductor of hepatic cystic lesions, but the contribution of PI was not confirmed.

Portal versus systemic venous drainage of the pancreatic graft: the effect on glucose metabolism in pancreas and kidney transplant recipients.

Two different methods of graft venous drainage are used in pancreas transplantation: portal (PVD) and systemic (SVD). PVD is considered to be more physiologic due to its similarity to venous outflow of the native pancreas. The aim of our study was to compare glucose metabolism in Type 1 diabetic recipients of kidney and pancreatic grafts with PVD versus SVD by intravenous glucose tolerance test (IVGTT). We examined 28 insulin-independent patients after simultaneous pancreas and kidney transplantation: 14 recipients with PVD of the pancreatic graft and 14 with SVD after a mean post-transplant period of 1 year. All recipients had stable good function of the kidney graft. Fasting glycemia, insulin levels, glycosylated hemoglobin (HbA1c), and standard IVGTT with coefficient of glucose assimilation (KG) calculation were assessed. Insulin sensitivity and production were evaluated using the homeostasis model assessment (homeostasis model assessment of insulin resistance [HOMA-IR], homeostasis model assessment of B-cell function [HOMA-B]). Total C-peptide and insulin secretions were calculated as areas under the curves (AUCs) from the serum levels during the IVGTT. PVD and SVD groups did not differ in age, body mass index (BMI) and duration of post-transplantation period (P ≥ .05). We did not find any significant difference in fasting glycemia, HbA1c, KG, HOMA-IR, parameters of C-peptide level, fasting insulin level, and response during IVGTT. HOMA-B and AUC of insulin level were higher in the SVD group (45.1 ± 35.1 versus 19.8 ± 15.5, P =.03 and 1075 ± 612 versus 1799 ± 954 mIU/L/60 minutes, P < .03, respectively). In the PVD group, 1 patient had an abnormal response to the glucose stimulus, 8 patients had an impaired glucose tolerance, and 5 patients had a normal glucose tolerance. In the SVD group, an abnormal response was present in none, impaired glucose tolerance in 4, and normal glucose tolerance in 10 recipients. Athough this was not a prospectively randomized trial, we conclude that the change of surgical technique from SVD to PVD did not lead to any substantial change in terms of glucose tolerance.

Effect of mesenchymal stem cells on the vascularization of the artificial site for islet transplantation in rats.

An adequate vascularization of the artificially created cavity is crucial for subsequent transplantation of isolated pancreatic islets. In a reported study, dynamic contrast-enhanced magnetic resonance imaging was used to assess the effect of mesenchymal stem cells on neoangiogenesis within connective tissue surrounding an implantable biocompatible device. The signal increase detected after injection of magnetic resonance contrast agent in each target region was considered to be an effect of contrast agent, which was related to the blood supply. To minimize the influence of variability in contrast agent application, all outcomes measured in the implanted devices were normalized to the signal intensity of kidney tissue. When supported by mesenchymal stem cells, the mean signal increase intra-abdominally was 42%, 41%, and 64% and within subcutaneously implanted devices was 23%, 54%, and 52% of that measured in kidney.

Pancreatic islet autotransplantation after completion pancreatectomy for pancreatic fistula after hemipancreatoduodenectomy for carcinoma.

OBJECTIVE: Pancreatic islet autotransplantation (IAT) has a potential to prevent brittle diabetes in patients after total pancreatectomy. Because of the fear of tumor spread, IAT has rarely been used in case of malignancy. We report our experience with patients who underwent hemipancreatoduodenectomy for carcinoma and later completion pancreatectomy for pancreatic fistula with islet autotransplantation at our institution. METHODS: From August 2007 to December 2012, 5 patients underwent IAT after completion pancreatectomy for pancreatic fistula after hemipancreatoduodenectomy for carcinoma. Islets were isolated from the pancreatic tail with the use of digestion with collagenase. Nonpurified islet suspension was infused into the portal vein during surgery. RESULTS: The median number of islets transplanted was 175,000 islet equivalents (range, 70,000-365,000). One patient died after surgery for reasons unrelated to IAT. Another 3 patients had stable diabetes with partial graft function (fasting C-peptide levels 0.23, 0.41, and 0.61 nmol/L and HbA1c 4.8%, 4.6%, and 6.9% at 24, 24 and 9 months after IAT, respectively). The 1st patient, with pancreatic head carcinoma, was alive 28 months after IAT with lymph node and liver recurrence since 18 months after IAT. The 2nd patient, with gall bladder and distal bile duct carcinoma, died 47 months after IAT with tumor recurrence. The 3rd patient, with ampullary carcinoma, died 12 months after IAT with local recurrence and solitary liver metastasis. The last patient had been off insulin 9 months after IAT without tumor recurrence (fasting C-peptide, 0.89 nmol/L; HbA1c, 4.2%). CONCLUSIONS: Autotransplantation of pancreatic islets isolated from the residual pancreatic tissue in patients who previously underwent hemipancreatoduodenectomy for cancer may provide stable glucose control and thus improve quality of life. In this small series we did not observe early development of multiple liver metastases caused by islet suspension contamination with malignant cells. Oncologic outcome of the patients was not worse than what would be expected without IAT.

The effects of DPP-IV inhibition in NOD mice with overt diabetes.

Sitagliptin is a dipeptidyl peptidase IV (DPP-IV) inhibitor that exerts an anti-hyperglycaemic effect by preventing degradation of glucagon-like peptide 1 with subsequent ß-cell stimulation and potential regeneration. We tested whether sitagliptin therapy in symptomatic non-obese diabetic (NOD) mice would lead to changes in the immune cell profile, improve ß-cell survival and induce diabetes remission. Flow cytometry analysis of immune cells in the spleen and peripheral lymph nodes, immunohistology of the pancreas and DPP-IV activity were investigated in diabetic NOD mice, either treated or non-treated with sitagliptin, at 0, 7, 14 and 28 days after hyperglycaemia onset, and in non-diabetic NOD controls. While compared to diabetic controls sitagliptin prevented increase of the CD8+/CD4+ ratio in pancreatic nodes after four weeks (0.443 ± 0.067 vs. 0.544 ± 0.131; P < 0.05), the population of Tregs in lymph nodes increased from day 0 to 28 in both treated and non-treated diabetic groups (8 ± 1.76 vs. 13.45 ± 5.07 % and 8 ± 1.76 vs. 13.19 ± 5.58 %, respectively). The severity of islet infiltration was similar in both diabetic groups and decreased in parallel with ß-cell loss. Surprisingly, sitagliptin blocked the DPP-IV activity only temporarily (on day 7, 277.68 ± 89.2 vs. 547.40 ± 94.04 ng/ml in the diabetic control group) with no apparent effect later on. In conclusion, sitagliptin administered after the onset of overt hyperglycaemia in NOD mice had only a marginal immunological effect and did not lead to diabetes remission. Failure to block DPP-IV over time represents an important finding that requires further explanation.

Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor stimulates trans-differentiation of human non-endocrine pancreatic cells into insulin-producing cells.

Differentiation of pancreatic ß-cells is regulated by a wide range of signalling pathways. The aim of our current work was to evaluate the effect of the Jak/Stat signalling pathway on the differentiation of human non-endocrine pancreatic cells into insulin-producing cells. Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor (LIF) stimulated differentiation of C-peptide-negative human non-endocrine pancreatic cells into insulin-producing cells in 6.3 ± 2.0 % cells (N = 5) and induced expression of pro-endocrine transcription factor neurogenin 3, Notch signalling pathway suppressor HES6 and stimulator of ß-cell neogenesis REG3A. The expression of the REG3A gene and increased rate of differentiation into insulin-producing cells (10.2 ± 2.1 %) were further stimulated by a combination of LIF with nicotinamide and dexamethasone. Glucose-stimulated (5 vs. 20 mM) C-peptide secretion confirmed proper insulin secretory function of trans-differentiated insulin-producing cells (0.51 vs. 2.03 pmol C-peptide/µg DNA, P < 0.05). Our results indicate that Jak/Stat signalling critically contributes to trans-differentiation of non-endocrine pancreatic cells into functional insulin-producing cells. The positive effect of the Jak/Stat signalling pathway on trans-differentiation is mediated by the key genes that activate differentiation of pancreatic ß-cells.

Detection of pancreatic islet allograft impairment in advance of functional failure using magnetic resonance imaging.

This study evaluated the ability of magnetic resonance imaging (MRI) to predict failure of pancreatic islets (PI) transplanted into the hepatic portal vein. Brown-Norway (n = 18) and Lewis (n = 6) rats received islets isolated from Lewis donors. The rejection process in Brown-Norway recipients was mitigated by two different immunosuppressive regimens [tacrolimus + hydrocortisone for 3 months (n = 6) or tacrolimus for 12 days (n = 12)]. Longitudinal MRI monitoring of recipients at post-transplantation weeks 1, 2, 3, 4, 6, 8, 10, and 12 confirmed the ability to detect SPIO labeled PI after transplantation into the liver. The relative number of MRI signals related to PI isografts remained stable up to study completion. Recipients of PI allografts were normoglycemic until the end of study; signals declined gradually to 44 ± 17% in these animals. In animals with islets failure during post-transplant week 12, the number of signals decreased to 25 ± 10% of initial values. The difference between groups (islet function/failed) became significant post-transplant week 3. Our data demonstrate that the MRI changes attributable to rejection become apparent within 3 weeks after transplantation, i.e. at least 8 weeks before functional allograft failure.

The effect of epigenetic factors on differentiation of pancreatic progenitor cells into insulin-producing cells.

Differentiation of pancreatic progenitors into insulin-producing ß cells is regulated by various transcription factors. To be expressed the genes coding these transcription factors need to be in accessible DNA. Whether a particular gene is present in a form of active euchromatin structure with accessible DNA or in an inactive heterochromatin structure with inaccessible DNA is determined by various epigenetic modifications. We studied the effect of epigenetic modifiers on differentiation of human nonendocrine cells into insulin-producing cells with the aim to evaluate the effect of epigenetic modifications in that process. Within 3 days of cultivation nonendocrine cells form isletlike cell clusters (ILCCs) containing mainly cytokeratin-19-positive cells. After cultivation with epigenetic modifiers and further differentiation, the highest number of C-peptide-positive cells (10.3% ± 2.9%) as well as glucagon-positive cells (7.2% ± 2.8%) was observed in a sample supplemented with a combination of 5-Aza-2'-deoxycytidine modifiers, BIX01294 and MC1568. In response to glucose stimulation (5 vs 20 mmol/L) these ILCCs secreted increased amounts of C-peptide (0.45 vs 1.05 pmol C-peptide/µg DNA). Control samples treated without any epigenetic modifiers showed significantly lower numbers of C-peptide-positive cells (3.5% ± 1.6%). These results showed that a combination of epigenetic modifiers 5-Aza-2'-deoxycytidine (BIX01294 and MC1568) significantly improved reproducible differentiation of nonendocrine pancreatic cells into insulin-producing cells.

Dynamic contrast-enhanced magnetic resonance imaging as a tool to monitor the blood supply to an artificial cavity used as a site for islet transplantation in rats.

BACKGROUND: The transplantation of islets of Langerhans isolated from one donor pancreas can rarely release a diabetic recipient from insulin injections. The major reason is the destruction of 50%-60% of the transplanted tissue, which proceeds typically within a few hours after the insertion of the islets into the portal vein. Therefore, several groups have focused on development of an artificial site for islet transplantation. The main aim of the present study was to test the efficacy of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to evaluate the blood supply feeding the artificially created cavities for islet transplantation. METHODS: Two rounded devices were implanted: one device subcutaneously and the second one into the greater omentum of each animal. On the day of implantation as well as 1, 3, and 4 weeks later, we quickly injected the vascular specific MR contrast agent Vasovist (0.05 mL/100 g) intravenously. Penetration of the contrast agent was monitored by DCE-MRI. The influence of the contrast agent on the signal intensity observed within selected target areas was calculated with the use of ImageJ software. RESULTS: The penetration of the contrast agent was detected by the increase in signal intensity within implanted devices. The signal increase caused by the contrast compound was normalized to kidney tissue. On day of implantation of the device, no signal due to the contrast agent was detected in all devices. However, over the following weeks, there was an increase in signal detection within the omental device to 34%, 21%, and 14% of that of the kidney. Within the subcutaneously implanted devices there was an increase in signal detection up to 11%, 10%, and 7% of that detected in the kidney. CONCLUSIONS: The optimal time for transplantation of pancreatic islets into our omental device was 1 week after implantation of the scaffold. Also, the blood supply feeding the subcutaneous devices was regarded to be inadequate.

Effect of tacrolimus versus cyclosporine on glucose metabolism of pancreas and kidney recipients in the late (> 8 years) posttransplant period.

Diabetogenic effects of immunosuppressive agents are of great importance in pancreas or islet transplantation. The aim of our study was to compare the glucose metabolism in type 1 diabetic kidney and pancreas recipients on tacrolimus (Tacro) versus cyclosporine-based (Cyclo) immunosuppression in the late posttransplant period. We examined 26 insulin-independent patients with stabile good renal function. They were at least 7 years after simultaneous pancreas and kidney transplantation and with unchanged immunosuppressive therapy for at least 6 years. The mean follow-up in Tacro (n = 13) and Cyclo (n = 13) groups were 9.7 ± 1.9 and 10.9 ± 1.3 years, respectively (P = .08). Fasting glycemia, insulin levels, glycosylated hemoglobin (HbA(1c)), a standard intravenous glucose tolerance test (IVGTT) with coefficient of glucose assimilation (K(G)) calculation and trough Tacro/Cyclo levels were assessed. Insulin sensitivity and insulin secretion were evaluated using the homeostasis model assessment (HOMA-IR, HOMA-B). Total C-peptide and insulin secretions were calculated as areas under the curves (AUC) from the serum levels during the IVGTT. Tacro and Cyclo groups did not differ in age and body mass index. We did not find any significant difference in any examined parameters of glucose metabolism (fasting glycemia, insulin and C-peptide levels, HbA(1c,) IVGTT with K(G), HOMA-IR, HOMA-B, AUC of C-peptide and AUC of insulin; P > .05). Two patients in the Tacro group and none in the Cyclo group had K(G) <0.8%/min. Seven recipients in the Tacro group and eight in the Cyclo group had the normal glucose tolerance with K(G) ≥ 1.2%/min. Trough Tacro or Cyclo levels did not correlate with any of examined parameters. The use of different types of calcineurin inhibitors in type 1 diabetic pancreas and kidney recipients had no effect on glucose metabolism in the late posttransplant period.

Assessment of mitochondrial DNA as an indicator of islet quality: an example in Goto Kakizaki rats.

BACKGROUND: Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting ß-cells. Among the remaining ß-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. METHODS: ß-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn(2+) signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. RESULTS: The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining ß-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, respectively. Versus age-paired Wistar rats, 6- and 4-month-old GK rats showed reductions to 60 ± 15% and 50 ± 20%, respectively. CONCLUSIONS: OXPHOS of remnant ß-cells in diabetic GK was drastically impaired due to the lack of sufficient mtDNA levels. We suggest the use of mtDNA quantification to quickly assess PI quality before transplantation.

Immunoregulatory effect of anti-thymocyte globulin monotherapy on peripheral lymphoid tissues of non-obese diabetic mice.

OBJECTIVE: Experimental and clinical studies have shown that autoimmunity-causing diabetes may be abrogated by immune intervention. Several anti-T-lymphocyte antibodies focus on distinct T-cell targets. We tested the effect of murine anti-thymocyte globulin (ATG; Genzyme, Framingham, MA) in peripheral lymphoid organs of non-obese diabetic (NOD) mice after the onset of hyperglycemia. METHODS: Diabetic NOD mice were treated with two doses of ATG (1 mg totally) or maintained without treatment as controls. Blood glucose levels were monitored twice a week. The mice were terminated at day 0, 7, 14, or 28 after the initiation of the study. Subpopulations of T-lymphocytes and FoxP3+ (forkhead box P3 positive) regulatory T-cells were analyzed among elements isolated from the spleen and pancreatic lymph nodes. RESULTS: Mice with blood glucose levels greater than 13 mmol/L were included in the study. Diabetes remission occurred in 16% (3/19) of mice treated with ATG. Only one case of remission was observed in the control group (6%; 1/16). ATG therapy a significantly decreased the CD8+/CD4+ T-lymphocyte ratio. Among splenocytes, a significant difference was detected only on day 7 (0.069 versus 0.198 T-lymphocyte ratio); in lymph nodes, a decrease was observed on day 28 (0.21 versus 0.51 T-lymphocytes ratio). The regulatory T-cells population increased after ATG administration compared with the control group at day 7 (16.2% versus 10.8% in CD4+ splenocytes; 20.7% versus 10.3% in CD4+ lymph node cells). However, the increased FoxP3+ cell population was not durable. CONCLUSIONS: ATG treatment of diabetic NOD mice showed an immunoregulatory effect in peripheral lymphoid tissue with a significantly deceased CD8+/CD4+ ratio, which, however, did not normalize the metabolic parameters in a short period after the onset of overt diabetes.

Neoplasm incidence in simultaneous pancreas and kidney transplantation: a single-center analysis.

BACKGROUND: Long-term immunosuppression is associated with an increased rate of cancer. The aim of this study was to analyze the incidence of newly diagnosed tumors in simultaneous kidney and pancreas transplantation (SPKT). METHODS: We retrospectively analyzed the incidence of a neoplasm among 360 diabetic subjects who consecutively underwent SPKT from 1985 to August 2010 in a single institution. Data were retrieved from the institutional registry. We evaluated the nature of all newly diagnosed malignant tumors, including posttransplantation lymphoproliferative disease (PTLD), to compare Kaplan-Meier survival rates with those of patients free of a neoplasm. RESULTS: The median follow-up was 8 years; the overall 5-year patient survival was 84%. In 25 patients the tumors were malignant. Almost one-fourth of the cancers represented skin tumors (3 squamous cell and 4 basal cell carcinomas). PTLD was diagnosed in 5 recipients. The cumulative survival of patients with malignancies was significantly lower than that in recipients without cancer (8-year survival by 38% vs 70%; P < .001). The mean (±SD) time to diagnosis was 6 ± 3 years. Since 2004, the 12 recipients with malignancy who were switched to sirolimus at the time of diagnosis showed survivals that were not apparently better than those who remained on the established immunosuppression (46% vs 55%; P = .71). CONCLUSIONS: The risk of neoplasm development was similar to that reported by other centers. Recipients of SPKT show higher incidence of cancer, though their overall survival is still significantly better than in those usually remaining on dialysis.

[Simultaneous transplantation of the kidney and the islets of Langerhans]. / Simultánní transplantace ledviny a Langerhansových ostruvku.

Simultaneous kidney and islet transplantation is recent therapeutic alternative for diabetics with end-stage kidney disease, who are not acceptable for simultaneous pancreas-kidney transplantation. Islet transplantation has less complications but still worse long-term function compared to whole pancreas transplantation.