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.

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.

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.

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.

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.

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.

Positive contrast visualization of SPIO-labeled pancreatic islets using echo-dephased steady-state free precession.

OBJECTIVE: MRI has recently been introduced as a promising method of monitoring the transplanted pancreatic islets labelled with superparamagnetic iron oxide (SPIO). However, the traditional [Formula: see text]-weighted approach frequently yields ambiguous results because of the negative contrast of the SPIO particles on the background of other body components. This obstacle could be overcome with the use of a novel method known as echo-dephased steady state free precession (SSFP), generating positive contrast in the presence of paramagnetic material. METHODS: In phantoms, we achieved exact localisation and clear positive contrast visualisation of human SPIO labelled islets. Using the proposed method we demonstrated the ability to detect even a single pancreatic islet against a homogeneous background. RESULTS: In vivo experiments in rats confirmed reliable and accurate localisation of transplanted SPIO labelled islets. CONCLUSION: The echo-dephased SSFP technique could successfully visualise SPIO-labelled human and rat pancreatic islets yielding a positive contrast.

[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.

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.

Perfluorocarbon improves post-transplant survival and early kidney function following prolonged cold ischemia.

BACKGROUND: The two-layer organ preservation method (TLM) based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution has been successfully used in clinical islet and experimental heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve kidney function in a model of syngeneic kidney transplantation with prolonged ischemia time. METHODS: Kidneys were stored for 24 h either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation. RESULTS: One-month survival rates in the UW, TLM and control groups were 12.5, 62.5 and 100%, respectively (UW vs. TLM, p < 0.01). Median creatinine levels 24 h after transplantation were 381, 299 and 121 microM, respectively (UW vs. TLM, p < 0.02). Histological scoring showed more severe tissue damage in the UW group than in the TLM group (p < 0.05). Apoptosis was more frequent in the UW group than in the TLM group (p < 0.05). CONCLUSION: We demonstrated for the first time that conservation with TLM significantly improves the outcome of kidney transplantation in a rat model and should therefore be further studied in larger animals.

In vivo differentiation of human umbilical cord blood-derived cells into insulin-producing beta cells.

In our study we confirmed the potential of human umbilical cord blood cells to differentiate into insulin-producing cells following transplantation into immunocompromised mice. The average number of C-peptide-positive human cells per animal was 18 +/- 13 as assessed by immunofluorescence staining and fluorescence in situ hybridization specific for human ALU sequence. Differentiation into insulin-producing cells was further confirmed by reverse transcription-polymerase chain reaction specific for human insulin mRNA. Successful differentiation required sublethal irradiation of xenogeneic recipient at least at a dose of 3 Gy. However, transplantation of human umbilical cord blood cells did not improve hyperglycaemia in diabetic animals. The results of our study show that human umbilical cord blood may be considered as a potential source of stem cells for treatment of diabetes mellitus.

Clinical magnetic resonance imaging of pancreatic islet grafts after iron nanoparticle labeling.

There is a crucial need for noninvasive assessment tools after cell transplantation. This study investigates whether a magnetic resonance imaging (MRI) strategy could be clinically applied to islet transplantation. The purest fractions of seven human islet preparations were labeled with superparamagnetic iron oxide particles (SPIO, 280 microg/mL) and transplanted into four patients with type 1 diabetes. MRI studies (T2*) were performed prior to and at various time points after transplantation. Viability and in vitro and in vivo functions of labeled islets were similar to those of control islets. All patients could stop insulin after transplantation. The first patient had diffuse hypointense images on her baseline liver MRI, typical for spontaneous high iron content, and transplant-related modifications could not be observed. The other three patients had normal intensity on pretransplant images, and iron-loaded islets could be identified after transplantation as hypointense spots within the liver. In one of them, i.v. iron therapy prevented subsequent visualization of the spots because of diffuse hypointense liver background. Altogether, this study demonstrates the feasibility and safety of MRI-based islet graft monitoring in clinical practice. Iron overload (spontaneous or induced) represents the major obstacle to the technique.

Differentiation of CD133-positive pancreatic cells into insulin-producing islet-like cell clusters.

Adult pancreatic stem and progenitor cells could represent an alternative source of insulin-producing tissue for diabetes treatment. In order to identify these cells, we have focused on the human pancreatic cells expressing cell surface molecule CD133, a marker of adult stem cells. We found that population of human CD133-positive pancreatic cells contains endocrine progenitors expressing neurogenin-3 and cells expressing human telomerase, ABCG2, Oct-3/4, Nanog, and Rex-1, markers of pluripotent stem cells. These cells were able to differentiate into insulin-producing cells in vitro and secreted C-peptide in a glucose-dependent manner. Based on our results, we suppose that the CD133 molecule represents another cell surface marker suitable for identification and isolation of pancreatic endocrine progenitors.

Immunosuppression in pancreas transplantation: the Euro SPK trials and beyond.

The Immunosuppression in Pancreas Transplantation was historically based on the fact that the pancreas is an extremely immunogenic organ. Quadruple drug therapy with polyclonal or monoclonal antibodies induction was the mainstay therapy since the introduction of Cyclosporine A. In the modern era of Immunosuppression, Mycophenolate Mofetil replaced Azathioprine while Tacrolimus-another potent calcineurin inhibitor-had-and still has-a difficult challenge to replaced Cyclosporine A, due to its potential diabetogenic effect. Thanks to the first two EuroSPK studies which prospectively tried to answer several questions in that field. But, the future challenge will be in understanding the impact of innate immunity and ischemic reperfusion injuries on the long-term graft function. Hopefully, new drugs will be available and tested to block unspecific deleterious reactions to attenuate the proinflammatory response. It will be the aim of the third Euro SPK Study.

Isolation and characterization of human CXCR4-positive pancreatic cells.

The existence of an adult PSC that may be used in the treatment of diabetes is still a matter of scientific debate as conclusive evidence of such a stem cell in the adult pancreas has not yet been presented. The main reason why putative PSC has not yet been identified is the lack of specific markers that may be used to isolate and purify them. In order to increase the list of potential PSC markers we have focused on the human pancreatic cells that express cell surface receptor CXCR4, a marker of stem cells derived from different adult tissues. Here we report that CXCR4-positive pancreatic cells express markers of pancreatic endocrine progenitors (neurogenin-3, nestin) and markers of pluripotent stem cells (Oct-4, Nanog, ABCG2, CD133, CD117). Upon in vitro differentiation, these cells form ILCC and produce key islet hormones including insulin. Based on our results, we assume that CXCR4 marks pancreatic endocrine progenitors and in combination with other cell surface markers may be used in the attempt to identify and isolate PSC.

[Transplantation in the treatment of diabetes]. / Transplantace v lécbe diabetu.

Diabetes mellitus continues to be the most common cause of chronic kidney failure, blindness acquired in adulthood, non-traumatic amputations and severe forms of neuropathy. Therefore it is necessary to look for new forms of therapy capable of achieving long-term normalisation of blood sugar levels. The only standard method so far is pancreas transplantation. Most often, it is performed in combination with kidney transplantation and only exceptionally as an isolated procedure. A new and considerably less invasive option is transplantation of isolated Langerhans islets. While the number of pancreas transplantations in IKEM has exceeded 300, the program of islet transplantation is in its formative phase, with 10 clinical surgeries having been performed since May 2007. However, the number of suitable patients who could benefit from this method of treatment largely exceeds the availability of organs suitable for transplantation. Therefore, new possibilities of acquiring insulin producing cell lines are searched for, both from animal tissue and, primarily, from embryonic or adult stem cells. Also the possibility of in vivo regeneration of endogenous or transplanted beta cells of the pancreas has now became an object of study. Combined transplantation of the kidney and pancreas is still the best available method in the treatment of uremic type 1 diabetes patients and its long-term results have shown to be very good, even though their further improvement has been but of a lesser degree. Isolated transplantation of the pancreas is still reserved for a limited group of patients with very labile diabetes. The transplantation of isolated Langerhans isletes is an alternative option which is far safer for the patient, but the long-term results of which still leave much to be desired. The method currently used in the Institute of Clinical and Experimental Medicine (IKEM) is organ transplantation of the pancreas while a program of transplantation of isolated islets has been launched, and also studied are the possibilities of insulin producing cell lines propagation.

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).

Insulin-pump treatment of pancreas-graft rejection.

Continuous subcutaneous insulin infusion (CSII) was combined with antirejection therapy in four diabetic recipients of pancreas and kidney grafts with persisting hyperglycemia due to pancreas rejection. In three of the patients, full function of the pancreas was restored after as many as 40, 86, and 139 days of CSII. In another patient, the dose of insulin was halved, and his graft function was classified as partial. Pancreas rejection treated without CSII was reversible only in one of four other recipients. We conclude that restoration of the function of a pancreas graft damaged by rejection can be achieved even after a long period with the help of CSII therapy.

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.