Calcium : A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas

Background: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. Methods: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. Results: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. Conclusions: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.Peer reviewe

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells

The effect of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibition on the cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in primary insulin-releasing pancreatic β-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca2+-deficient medium the individual primary β-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca2+]i followed by an explosive transient elevation. The [Ca2+]i transients were preferentially observed at low intracellular concentrations of the Ca2+ indicator fura-2 and were unaffected by pre-treatment with 100 μM ryanodine. Whereas 20 mM caffeine had no effect on basal [Ca2+]i or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca2+]i transients as well as inositol 1,4,5-trisphosphate-mediated [Ca2+]i transients in response to carbachol. In striking contrast to the primary β-cells, caffeine readily mobilized intracellular Ca2+ in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca2+ from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca2+-induced Ca2+ release (CICR). In primary pancreatic β-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal β-cells

Single dose anti-CD4 monoclonal antibody for induction of tolerance to cardiac allograft in high- and low-responder rat strain combinations

Repeated administration of monoclonal antibodies (mAb) directed against the CD4 lymphocyte receptor may induce specific, long-lasting unresponsiveness to fully MHC-mismatched cardiac allografts in rats without additional immunosuppression. We assessed the effect of a single dose of murine anti-rat depleting anti-CD4 mAb (OX-38) on allograft survival in high- and low-responder rat strain combinations. Isogenic strains of DA (RT1(av1)), PVG (RT1(c)), AUG (RT1(c)), and WF (RT1(u)) rats were used. Recipients in antibody treated groups were given one dose of 5 mg/kg OX-38 mAb on the day of transplant, a dose which was shown to effectively deplete (or block) circulating CD4+ T cells. Other groups were treated for 10 days with cyclosporin A (CsA) and/or Linomide, a novel immunomodulator, which is the first compound able to fully eliminate the effect of CsA in the rat cardiac allograft model. The DA strain was identified as a low-responder to the allogeneic haplotype RT1(c) (PVG or AUG), but not to RT1(u) (WF), and developed true tolerance following RT1(c) grafting and OX-38 or low-dose CsA (5 mg/kg) induction, as verified by the response to retransplantation of a graft from the same donor strain or a third-party challenge. PVG recipients of DA grafts were characterized by high response and only modest (OX-38; median 9.5 days) or moderate (CsA; 23.5 days) prolongation of graft survival. Contrasting graft survival results were obtained in the low-responder combination, either very early rejection (at 10 days) or permanent graft survival (> 100 days). Linomide challenge affected CsA treatment in the high-responder combination but not tolerance induction in the low-responder combination, or the effect of OX-38. It was concluded that in rat heart transplantation a single-dose anti-CD4 mAb therapy may induce permanent donor-specific unresponsiveness in a low-responder strain combination, and that anti-CD4 mAb seems to be unique among immunosuppressive agents while being resistent to challenge by Linomide

Donor-derived urologic cancers after renal transplantation : A retrospective non-randomized scientific analysis

BACKGROUND: Malignancies in the urinary tract and the kidney graft are quite common after kidney transplantation. In some selected cases tumours develop from donor-derived tissue. OBJECTIVES: We hypothesised that there is a clinical value to investigate donor/recipient origin in urologic malignancies in renal transplant recipients. METHODS: In this retrospective study, including patients transplanted between the years 1969 and 2014 at Uppsala University Hospital, Sweden, 11 patients with malignancies in urinary tract and 4 patients with malignancies in kidney transplants were investigated. Donor/recipient origin of tumour tissue was analysed by polymerase chain reaction (PCR) of human leucocyte antigen (HLA) genotypes or by fluorescence in situ hybridization (FISH analysis) of sex chromosomes. HLA genotype and sex chromosomes of the tumour were compared to the known HLA genotype and sex chromosomes of recipient and donor. RESULTS: Three of ten cancers in the urinary tract and three of four cancers in the kidney transplants were donor-derived. CONCLUSIONS: We suggest that urologic malignancies in renal transplant recipients can be investigated for transplant origin. In addition to conventional therapy the allograft immune response against these tumours can be valuable to treat donor-derived cancers

A Randomized, Doubleblind, Placebo-Controlled, Study of Single-Dose Rituximab as Induction in Renal Transplantation

We performed a prospective, double blind, randomized, placebo-controlled multicenter study on the efficacy and safety of rituximab as induction therapy, together with tacrolimus, mycophenolate mofetil, and steroids. The primary endpoint was defined as acute rejection, graft loss, or death during the first 6 months. Secondary endpoints were creatinine clearance, incidence of infections, and incidence of rituximab-related adverse event. Results. We enrolled 140 patients (44 living donor and 96 deceased donor), and of those, 68 rituximab and 68 placebo patients fulfilled the study. In all the patients receiving rituximab, there was a complete depiction of CD 19/CD20 cells, whereas there was no change in the number of CD19/CD20 cells in the placebo group. There were 10 treatment failures in the rituximab group versus 14 in the placebo group (P=0.348). There were eight rejection episodes in the rituximab group versus 12 in the placebo group (P=0.317) Creatinine clearance was 66 +/- 22 mL/min in the study group and 67 +/- 23 mL/min in the placebo group. There was no difference in the number of bacterial infections, cytomegalovirus infections, and BK virus infections or fungal infections. Conclusion. We performed a placebo-controlled study of rituximab induction in renal transplantation. There was a tendency toward fewer and milder rejections during the first 6 months in the rituximab group. Although induction with one dose of rituximab induced a complete depletion B cells, there was no increase in the incidence of infectious complications or leukopenia and it seems safe, therefore, to conduct further studies on the use of rituximab in transplantation

Prior antithymocyte globulin therapy and survival in post-transplant lymphoproliferative disorders

Background: Treatment with antithymocyte globulin (ATG) is a well-recognized risk factor for the development of post-transplant lymphoproliferative disorders (PTLD) after solid organ transplantation, but it is unknown how its use affects overall survival after PTLD. Methods: A total of 114 patients with PTLD and available data on immunosuppressive regimen were included from a nation-wide case series of solid organ transplant recipients in Sweden. Prior use of ATG was correlated to clinical features, PTLD subtype, and survival. Results: A total of 47 (41%) patients had received ATG prior to the diagnosis of PTLD. The ATG-treated patients were more likely to be recipients of hearts or lungs, and less likely of kidneys (p < 0.01). They had experienced more acute rejections (p = 0.02). The PTLDs arose earlier, median 2.0 vs. 6.6 years post-transplant (p = 0.002) and were more often situated in the allograft (32% vs. 7%, p < 0.001) in patients with prior ATG vs. no ATG treatment. The PTLDs in the ATG group were more often Epstein-Barr virus-positive (80% vs. 40%, p < 0.001). There were more polymorphic PTLDs (17% vs. 1.5%, p = 0.004) and less T-cell PTLDs (4% vs. 19%, p = 0.02) in the ATG group than in the no ATG group. Diffuse large B-cell lymphoma was equally common in patients with and without prior ATG therapy, but the non-germinal center subtype was more frequent in the ATG group (p = 0.001). In an adjusted Cox proportional hazards regression model, prior ATG treatment and better performance status were associated with superior overall survival, whereas older age, T-cell subtype of PTLD, presence of B symptoms, and elevated lactate dehydrogenase were associated with inferior overall survival. Patients receiving ATG solely as rejection therapy had superior overall survival compared with those receiving ATG as induction therapy or both (p = 0.03). Conclusions: ATG therapy, especially rejection therapy, prior to PTLD development is an independent prognostic factor for superior overall survival after PTLD diagnosis

Characteristics of recipeint derived cancers in the urinary tract.

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