Warfarin or Low-Molecular-Weight Heparin Therapy does not Prolong Pig-To-Primate Cardiac Xenograft Function

Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low-molecular-weight heparin (LMWH) anti-coagulation on xenograft function using a heterotopic pig-to-primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti-CD20 and TPC, an alpha-galactosyl-polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti-coagulant drugs. The median duration of xenograft function was 20 days (range 3-62 days), 18 days (range 5-109 days) and 15 days (range 4-53 days) in Groups 1 to 3 respectively. Anti-coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti-factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti-coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti-coagulant strategies to achieve long-term xenograft function cannot be excluded.     

A multicenter study of total pancreatectomy with islet autotransplantation (TPIAT): POST (Prospective Observational Study of TPIAT)

Background/objectives

Total pancreatectomy with islet autotransplantation (TPIAT) is considered for managing chronic pancreatitis in selected patients when medical and endoscopic interventions have not provided adequate relief from debilitating pain. Although more centers are performing TPIAT, we lack large, multi-center studies to guide decisions about selecting candidates for and timing of TPIAT.

Improvement of porcine islet isolation by inhibition of trypsin activity during pancreas preservation and digestion using α1-antitrypsin

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast?), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.     

Islet purification method using large bottles effectively achieves high islet yield from pig pancreas

Porcine islets are a promising resource for xenotransplantation. However, low efficacy of islet isolation because of their marked fragility remains a problem. Recently we found that the standard purification method using COBE 2991 cell processor (COBE) with Ficoll density gradient solution damaged islets mechanically by high shearing force. In this study, we evaluated our new purification method using large plastic bottles for the efficacy of islet purification. Ten porcine pancreata were used. The average warm ischemic time was over 40 min; therefore, these pancreata were considered to be in a marginal condition. After digestion, the digested tissue was divided into three groups. Each group was purified using either top loading method with bottle (top group) or bottom loading method with bottle (bottom group) or standard COBE method (COBE group). Islet yield per pancreas weight (IEQ/g) and the rate of postpurification recovery in the top group were significantly higher than the COBE group (top: 8060 ± 1652 IEQ/g, bottom: 4572 ± 614 IE/g, COBE: 3900 ± 734 IE/g. p < 0.02 in top vs. COBE; top percentage of recovery: 99.3 ± 12.3%, bottom: 62.6 ± 8.8%, COBE: 49.5 ± 6.7%, p < 0.02 in top vs. bottom and COBE). The average sizes of purified islets in the top and bottom groups were significantly larger than COBE group (Average diameter top: 156 ± 8 μm, bottom: 147 ± 6 μm, COBE: 119 ± 6 μm, p < 0.01 in top vs. COBE and in bottom vs. COBE), which indicated that bottle method can reduce shear force during purification. Our new purification using top loading bottle method enabled us to obtain a high yield of porcine islets from marginal pancreata.     

Islet cell transplantation for the treatment of type 1 diabetes in the USA

Islet cell transplantation (ICTx) is one of the most effective treatments for type 1 diabetes and is less invasive compared to whole organ transplantation. The US has been the leader in the research and clinical applications of ICTx for the last 40 years. ICTx requires complex procedures, including pancreas procurement and preservation; pancreas digestion; islet purification; and transplantation. Even with the dramatic progresses in each of the procedures listed above, there are still challenges to make ICTx the standard therapy. These challenges are: (1) obtaining enough islets from a single donor and (2) preventing graft loss due to allogenic rejection and recurrence of autoimmune islet destruction. A new preservation strategy for pancreata and pancreatic ducts using ET-Kyoto solution as well as a new islet purification method using iodixanol has substantially improved islet yields. Continuous research to improve the efficacy of islet isolation will solve the issue of obtaining enough islets from a single donor. Immunological tolerance is an ideal solution for the issue of rejection and autoimmune recurrence and a regulatory T cell strategy seems promising. Moreover, the SUITO index is a simple and powerful tool to assess engrafted islet mass and is, therefore, useful for evaluating the efficacy of new immunosuppressant strategies. Once ICTx becomes a standard treatment, the donor shortage will become the next challenge. Marginal or living donor islet transplantations could help alleviate this issue; however, bio-artificial islet transplantation with animal islets could be the ultimate solution.     

Comparison of ulinastatin, gabexate mesilate, and nafamostat mesilate in preservation solution for islet isolation

For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.     

Blood groups and transfusions in pigs

The blood groups of pigs are important to transplantation research because some are also important transplantation antigens and because pigs undergoing organ or hematopoietic transplantation may require transfusion support. There is considerable literature on the subject but much of it is not in transplant related journals. We will review this literature and also give some practical advice on transfusion support.     

Inhibition of glucose-stimulated activation of extracellular signal-regulated protein kinases 1 and 2 by epinephrine in pancreatic beta-cells

Glucose sensing is essential for the ability of pancreatic beta-cells to produce insulin in sufficient quantities to maintain blood glucose within the normal range. Stress causes the release of adrenergic hormones that increase circulating glucose by promoting glucose production and inhibiting insulin release. We have shown that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are responsive to glucose in pancreatic beta-cells and that glucose activates ERK1/2 by mechanisms independent of insulin. Here we show that glucose-induced activation of ERK1/2 is inhibited by epinephrine through the alpha2-adrenergic receptor. Epinephrine and the selective alpha2-adrenergic agonist UK14304 reduced insulin secretion and glucose-stimulated ERK1/2 activation in a pertussis toxin-sensitive manner, implicating the alpha subunit of a Gi family member. Alpha2-adrenergic agonists also reduced stimulation of ERK1/2 by glucagon-like peptide 1 and KCl, but not by phorbol ester or nerve growth factor. Our findings suggest that alpha2-adrenergic agonists act via a Gi family member on early steps in ERK1/2 activation, supporting the idea that ERK1/2 are regulated in a manner that reflects insulin demand.