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Purpose of the study The purpose of this study is to explore donor relatives' experiences of the medical interventions enabling organ donation, as well as to examine the donor relatives' attitudes towards donating their own organs, and whether or not their experiences have influenced their own inclination to donate. Methods The experiences of donor relatives were explored via in-depth interviews. The interviews covered every step from the deceased family member being struck by a severe bleeding in the brain till after the organ recovery, including the medical interventions enabling organ donation. The interviews were analysed through qualitative and quantitative content analysis. Results Brain death and organ donation proved to be hard to understand for many donor relatives. The prolonged interventions provided after death in order to enable organ donation misled some relatives to believe that their family member still was alive. In general, the understanding for what treatment aimed at saving the family member and what interventions aimed at maintaining organ viability was low. However, most donor relatives were either inspired to, or reinforced in their willingness to, donate their own organs after having experienced the loss of a family member who donated organs. Conclusions There is a need for greater transparency regarding the whole chain of events during the donation process. Yet, having experienced the donation process closely did not discourage the donor relatives from donating their own organs-but rather inspired a willingness to donate. This indicates an acceptance of the medical procedures necessary in order to enable organ donation after death.

By 2005, human organ trafficking, commercialization, and transplant tourism had become a prominent and pervasive influence on transplantation therapy. The most common source of organs was impoverished people in India, Pakistan, Egypt, and the Philippines, deceased organ donors in Colombia, and executed prisoners in China. In response, in May 2008, The Transplantation Society and the International Society of Nephrology developed the Declaration of Istanbul on Organ Trafficking and Transplant Tourism consisting of a preamble, a set of principles, and a series of proposals. Promulgation of the Declaration of Istanbul and the formation of the Declaration of Istanbul Custodian Group to promote and uphold its principles have demonstrated that concerted, strategic, collaborative, and persistent actions by professionals can deliver tangible changes. Over the past 5 years, the Declaration of Istanbul Custodian Group organized and encouraged cooperation among professional bodies and relevant international, regional, and national governmental organizations, which has produced significant progress in combating organ trafficking and transplant tourism around the world. At a fifth anniversary meeting in Qatar in April 2013, the DICG took note of this progress and set forth in a Communiqué a number of specific activities and resolved to further engage groups from many sectors in working toward the Declaration's objectives.

AIMS/HYPOTHESIS: The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. METHODS: In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. RESULTS: Human KATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7+/-0.1 micromol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 micro mol/l LC-CoA ester. The presence of 1 micro mol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5+/-0.2 nS to 2.5+/-1.3 nS. CONCLUSION/INTERPRETATION: Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate KATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (>10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

Background. Histidine-tryptophan-ketoglutarate (HTK) has been established as an alternative to University-of-Wisconsin solution (UWS) for abdominal organ preservation, but data about HTK efficiency to preserve pancreata during prolonged cold ischemia time (CIT) are conflicting. In human islet transplantation, HTK provided similar isolation outcomes after short CIT. The present study aimed to investigate whether islets can be successfully isolated from HTK-preserved pancreata after prolonged CIT compared with UWS. Materials and Methods. Sixty-four human pancreata retrieved from donors meeting criteria for kidney donation were perfused utilizing either HTK or UWS and preserved for more or less than 10 h prior to islet isolation. Along with parameters related to isolation and islet quality assessment, the dry-to-wet weight ratio was evaluated. Results. Donor-and procurement-related factors did not vary between HTK- and UWS-perfused pancreata. The dry-to-wet weight ratio was lower in HTK-preserved pancreata indicated tissue edema (21.0% +/- 3.5% versus 24.8% +/- 2.0%, P = 0.007). Isolation-related variables differed between experimental groups after prolonged CIT with respect to purified packed tissue volume (9.1 +/- 5.0 versus 17.2 +/- 8.1 mu L/g, P = 0.004) and islet yield (1910 +/- 980 versus 3150 +/- 1420 IE/g, P = 0.012). Islet purity and survival after culture were similar after HTK or UWS perfusion. The preservation solution did not affect in vitro function and transplantability of isolated islets. Conclusions. Compared with UWS, HTK has similar efficiency to preserve human pancreata for subsequent islet isolation during

In this study, we evaluated the effects of exendin-4 on free and encapsulated islet grafts in a rodent model. We also investigated the role of a transcription factor, hypoxia-inducible factor-1 (HIF-1), in mediating the beneficial effects of exendin-4. Diabetic athymic mice were transplanted with free rat islets under the kidney capsule or with macroencapsulated rat islets SC with or without exendin-4, islet preculture (exendin-4 0.1 nM for 20 h), and/or recipient treatment (IP 100 ng/day, day 0–7). The mice were followed for 4 weeks and the graft function and β-cell volume were evaluated. The effects of exendin-4 on islet HIF-1α mRNA and protein expression and on ATP content in a rat insulinoma cell line (INS-1E) were also examined. Preculture with exendin-4 followed by recipient treatment improved the outcome of both free (73% graft function vs. 26% in controls, p = 0.03) and macroencapsulated islet grafts (100% vs. 25% in controls, p = 0.02). In macroencapsulated grafts, the exendin-4-treated group had significantly larger endocrine volume, less graft necrosis, and more blood vessels around the capsule. In rat islets cultured with exendin-4, HIF-1α mRNA and protein expression were significantly enhanced. ATP content was increased in exendin-4-treated INS-1E cells under hypoxic conditions. The improved functional outcome after transplantation of a marginal islet mass with a brief initial treatment with exendin-4 is related to a larger surviving endocrine cell volume. Exendin-4 may improve islet graft resistance to hypoxia during the peritransplant period by increasing the expression of HIF-1α.

Background. The ability to predict clinical function of a specific islet batch released for clinical transplantation using standardized variables remains an elusive goal. Methods. Analysis of 10 donor, 7 islet isolation, 3 quality control, and 6 recipient variables was undertaken in 110 islet-after-kidney transplants and correlated to the pre- to 28-day posttransplant change in C-peptide to glucose and creatinine ratio (Delta CP/GCr). Results. Univariate analysis yielded islet volume transplanted (Spearman r = 0.360, P < 0.001) and increment of insulin secretion (r = 0.377, P < 0.001) as variables positively associated to Delta CP/GCr. A negative association to Delta CP/GCr was cold ischemia time (r = -0.330, P < 0.001). A linear, backward-selection multiple regression was used to obtain a model for the transplanted functional islet mass (TFIM). The TFIM model, composed of islet volume transplanted, increment of insulin secretion, cold ischemia time, and exocrine tissue volume transplanted, accounted for 43% of the variance of the clinical outcome in the islet-after-kidney data set. Conclusion. The TFIM provides a straightforward and potent tool to guide the decision to use a specific islet preparation for clinical transplantation.

The critical pathway of deceased donation provides a systematic approach to the organ donation process, considering both donation after cardiac death than donation after brain death. The pathway provides a tool for assessing the potential of deceased donation and for the prospective identification and referral of possible deceased donors.

Much remains to be done to facilitate the transplantation process for patients with end-stage renal disease. The aim here was to explore these patients' experiences of the donation process and factors related to whether the actual donors of the recipients were living or deceased and describe which issues needed attention in a quality development project.A specially constructed questionnaire was sent to 246 recipients of living and deceased kidney transplants who had been transplanted at the Karolinska University Hospital in Stockholm, Sweden. The response rate was 87%.Six conditions were identified as problematic: --Most living-donor kidney recipients perceived the evaluation period for the donors as too long. --Although a living donor was available, most living-donor kidney recipients had to undergo dialysis for a relatively long period. --A majority of the patients perceived it difficult to ask for a donation. Deceased-donor kidney recipients were least satisfied with the offered support in finding a living donor. --Patients perceived fear as the main reason for potential living donors to refuse donation. --About one-fourth of living-donor kidney recipients thought that the donors were abandoned by healthcare after nephrectomy. --Older patients and singles were least likely to receive a living-donor kidney.The problem issues outlined above should be scrutinized and improved. Checking these issues can be used in quality control when analysing living kidney donation at local and national levels.

BACKGROUND: Pancreas oxygenation during cold storage has been established in islet isolation and transplantation to prevent ischemic tissue damage using perfluorodecalin (PFD) as hyperoxygen carrier. However, studies in humans and pigs provided conflicting results about the efficiency of PFD for pancreas oxygenation. The aim of this study was to compare PFD with a newly developed oxygen carrier composed of perfluorohexyloctane and polydimethylsiloxane 5 (F6H8S5) for long-term storage of human pancreata. METHODS: After 24-hr storage in preoxygenated PFD or F6H8S5, pancreata were processed using Liberase HI for pancreas dissociation and a Ficoll gradient for islet purification. Islet quality assessment was performed measuring glucose-stimulated insulin release, viability, islet ATP content, and posttransplant function in diabetic nude mice. RESULTS: Compared with PFD, F6H8S5 significantly increased the intrapancreatic partial oxygen pressure and islet ATP content. This corresponded to an increase of islet yield, recovery after culture, glucose stimulation index, viability, and improved graft function in diabetic nude mice. CONCLUSIONS: The present findings indicate clearly that F6H8S5 improves isolation outcome after prolonged ischemia compared with PFD. This observation seems to be related to the significant lipophilicity and almost pancreas-specific density of F6H8S5. Moreover, these characteristics facilitate pancreas shipment without using custom-made transport vessels as required for PFD.

The fate of islets in clinical transplantation is unclear. To elude on this positron emission tomography combined with computed tomography (PET/CT) was performed for 60 min during islet transplantation in five patients receiving six transplants. A fraction of the islets (23%) were labeled with 18F-fluorodeoxyglucose ([(18)F]FDG) and carefully mixed with unlabeled islets just prior to intraportal transplantation. The peak radioactivity concentration in the liver was found at 19 min after start of islet infusion and corresponded to only 75% of what was expected, indicating that islets are lost during the transplantation procedure. No accumulation of radioactivity was found in the lungs. A nonphysiological peak of C-peptide was found in plasma during and immediately after transplantation in all subjects. Distribution in the liver was heterogeneous with wide variations in location and concentration. Islets found in areas with concentrations of >400 IEQ/cc liver tissue varied between 1% and 32% of the graft in different subjects. No side effects attributed to the PET/CT procedure were found. Clinical outcome in all patients was comparable to that previously observed indicating that the [(18)F]FDG labeling procedure did not harm the islets. The technique has potential to be used to assess approaches to enhance islet survival and engraftment in clinical transplantation.

BACKGROUND: The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. METHODS: Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. RESULTS: Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. CONCLUSIONS: We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.

Side effects associated with current immunosuppressive therapy complicate the use of islet transplantation as a treatment for type 1 diabetes. Multipotent mesenchymal stromal cells (MSCs) have demonstrated immunomodulatory activity. The purpose of this study was to investigate whether a murine stromal cell line affects graft rejection in a fully major histocompatibility complex (MHC)-mismatched islet transplant model. We show that stromal cells have an inhibitory effect on T cell proliferation in vitro, and that they slow down the rejection of allogeneic islets. These findings indicate a possibility to use MSCs as a treatment to prolong the survival of islet grafts.

A 7-year-old girl with severe hereditary pancreatitis underwent total pancreatectomy. A total of 160,000 islet equivalents (6400 islet/kg) were transplanted to the brachioradialis muscle of the right forearm. Her plasma C-peptide level was undetectable after pancreatectomy but increased to 1.37 ng/mL after 17 days; at this time point, her insulin requirement was 0.75 units of insulin/kg/day. At 5- and 27-months, her hemoglobin A1c (HbA1c) and insulin requirements were 4.5 and 5.3% and 0.3 and 0.18 units/kg/day, respectively. Basal and stimulated C-peptide levels were 0.67 +/- 0.07 and 3.36 +/- 1.37 ng/mL, respectively. Stimulated insulin levels were 30% higher in the islet-bearing arm compared to the contralateral arm after glucagon stimulation. After surgery and islet transplantation, the quality of life improved dramatically and she gained 8 kg of weight. In summary, a normal HbA1c, a low insulin requirement and the absence of recurrent hypoglycemia and the gradient of insulin between the arms indicate that the intramuscularly transplanted islets contribute to a long-term clinically significant metabolic control.

OBJECTIVE—In clinical islet transplantation, the instant blood-mediated inflammatory reaction (IBMIR) is a major factor contributing to the poor initial engraftment of the islets. This reaction is triggered by tissue factor and monocyte chemoattractant protein (MCP)-1, expressed by the transplanted pancreatic islets when the islets come in contact with blood in the portal vein. All currently identified systemic inhibitors of the IBMIR are associated with a significantly increased risk of bleeding or other side effects. To avoid systemic treatment, the aim of the present study was to render the islet graft blood biocompatible by applying a continuous heparin coating to the islet surface. RESEARCH DESIGN AND METHODS—A biotin/avidin technique was used to conjugate preformed heparin complexes to the surface of pancreatic islets. This endothelial-like coating was achieved by conjugating barely 40 IU heparin per full-size clinical islet transplant. RESULTS—Both in an in vitro loop model and in an allogeneic porcine model of clinical islet transplantation, this heparin coating provided protection against the IBMIR. Culturing heparinized islets for 24 h did not affect insulin release after glucose challenge, and heparin-coated islets cured diabetic mice in a manner similar to untreated islets. CONCLUSIONS—This novel pretreatment procedure prevents intraportal thrombosis and efficiently inhibits the IBMIR without increasing the bleeding risk and, unlike other pretreatment procedures (e.g., gene therapy), without inducing acute or chronic toxicity in the islets.

Background. Many nations are able to prosecute transplant-related crimes committed in their territory, but transplant recipients, organ sellers and brokers, and transplant professionals may escape prosecution by engaging in these practices in foreign locations where they judge the risk of criminal investigation and prosecution to be remote. Methods.The Declaration of Istanbul Custodian Group convened an international working group to evaluate the possible role of extraterritorial jurisdiction in strengtheningtheenforcementof existinglawsgoverningtransplant-relatedcrimesacrossnationalboundaries.Potentialpracticalandethical concerns about the use of extraterritorial jurisdiction were examined, and possible responses were explored. Results. Extraterritorialjurisdictionisa legitimate tooltocombattransplant-relatedcrimes. Further,developmentofaglobalregistryoftransnationaltransplantactivitiesinconjunctionwitha standardizedinternationalreferralsystemforlegitimate travelfortransplantationis proposed as a mechanism to support enforcement of national and international legal tools. Conclusions. States are encouraged to include provisions on extraterritorial jurisdiction in their laws on transplant-related crimes and to collaborate with professionals and international authorities in the development of a global registry of transnational transplant activities. These actions would assist in the identification and evaluation of illicit activities and provide information that would help in developing strategies to deter and prevent them. (Transplantation 2015;00: 00–00)

There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low–molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15–60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.

Islet transplantation is currently being explored as a treatment for patients with type 1 diabetes. At present, the number of patients becoming insulin-independent is rapidly increasing world-wide applying the transplantation protocol originally described by the group in Edmonton. A hallmark in this procedure is repeated infusions of islets obtained from 2 to 4 donors until normoglycemia is achieved. In order to establish islet transplantation as a widely accepted treatment modality, and make tolerance induction regimes applicable, it is essential that the donor:recipient ratio is brought down to 1:1. A conceivable strategy to achieve this goal in clinical islet transplantation is discussed.

Background: The IgG antibody response in type I diabetic patients, transplanted with fetal pig islet-like cell-clusters, was investigated using purified immunoglobulin G (IgG) fractions from sera collected 7 to 9 yr after transplantation. From our earlier studies, we knew that the immunological specificities of xenoreactive IgG1 and IgG2 antibodies are different, and that IgG1 antibodies, in contrast to the IgG2 population, are mainly directed against non-Galα1,3Gal epitopes. In this study our goal was to establish whether xenoreactive IgG1 and IgG2 antibodies react with pig islet cells and, if so, to identify the target cell type, the biological function as well as the specificity of such antibodies for islet cell antigens. Sera from xenotransplanted patients were compared with those of patients with diabetes, selected for high titres of islet-cell specific autoantibodies. Methods: IgG antibody fractions from patient sera were purified on a protein G column. Surface expression of target antigens was studied using flow cytometry as well as immunofluorescence microscopy. The biological function of islet-cell reactive sera was tested using antibody dependent cellular cytotoxicity with both xenogeneic adult pig islet cells and allogeneic human islet cells as targets. Antibody specificity was assessed using 2D Western blots with both fetal and adult pig islet as well as human islet cell antigenic preparations. Results: Some of the diabetic patients, who have been transplanted with xenogeneic fetal pig islet cells, continue to produce xenospecific IgG1 and IgG2 antibodies for 7 to 9 yr post-transplantation. A separate analysis of IgG1 and IgG2 antibodies showed that IgG1 antibodies react with pig islet β cells, whereas IgG2 antibodies mainly react with non-endocrine pig cells. Such antibodies are xenospecific, as they were found to mediate antibody-dependent cellular cytotoxicity of adult pig, but not human islet target cells. The reverse was true for antibodies from non-transplanted diabetic patients with high titres of autoantibodies against β cells. Fluorescence analysis as well as 2D gel Western blots revealed that the reactivity was variable between patient samples, indicating that target antigens for non-Galα1,3Gal-specific antibodies are heterogeneous. Conclusion: Thus, xenotransplantation of diabetic patients induces islet-β cell reactive xenospecific IgG1 antibodies, which are biologically active and can mediate antibody-dependent cellular cytotoxicity of pig islet cells.

Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets : Implications for clinical islet transplantation

0.01 vs. controls). In untreated control rats, xenoreactive antibody (IgM, IgG) levels decreased immediately after PE, but their levels rapidly returned to normal. In rats treated with DSG or DSG + Lip-Cl2MDP, the IgM levels remained low during the observation period. Immunohistochemistry showed that macrophage infiltration into the graft was suppressed in Lip-Cl2MDP-treated groups at the time of rejection. Our results demonstrate that sustained suppression of antibody levels can be achieved by PE in combination with DSG and xenograft survival is further prolonged in macrophage-depleted C6- rats. These findings suggest that strategies targeting antibody and macrophages may be useful in prolonging xenograft survival.

Intraportal transplantation of pancreatic islets offers improved glycaemic control and insulin independence in type 1 diabetes mellitus, but intraportal thrombosis remains a possible complication. The thrombotic reaction may explain why graft loss occurs and islets from more than one donor are needed, since contact between human islets and ABO-compatible blood in vitro triggers a thrombotic reaction that damages the islets. We investigated the possible mechanism and treatment of such thrombotic reactions.Coagulation activation and islet damage were monitored in four patients undergoing clinical islet transplantation according to a modified Edmonton protocol. Expression of tissue factor (TF) in the islet preparations was investigated by immunohistochemistry, immunoprecipitation, electron microscopy, and RT-PCR. To assess TF activity in purified islets, human islets were mixed with non-anticoagulated ABO-compatible blood in tubing loops coated with heparin.Coagulation activation and subsequent release of insulin were found consistently after clinical islet transplantation, even in the absence of signs of intraportal thrombosis. The endocrine, but not the exocrine, cells of the pancreas were found to synthesise and secrete active TF. The clotting reaction triggered by pancreatic islets in vitro could be abrogated by blocking the active site of TF with specific antibodies or site-inactivated factor VIIa, a candidate drug for inhibition of TF activity in vivo.Blockade of TF represents a new therapeutic approach that might increase the success of islet transplantation in patients with type 1 diabetes, in terms of both the risk of intraportal thrombosis and the need for islets from more than one donor.

THROUGHOUT THE WORLD xenotransplantation researchers are confronted not only with medical and technological difficulties but also with cultural and ethical ambiguities. Within the research program “Treatment of Diabetes by Transplantation of Porcine Islets” at Karolinska Institute/Huddinge University Hospital in Stockholm, Sweden, an interview study has been performed concerning the cultural and ethical issues related to xenotransplantation research and development. The purpose of the study is to evaluate, from cultural and ethical viewpoints, the way in which the xenotransplantation approach is perceived among individuals who either professionally or by personal plight are engaged in the scientific development of treating diabetes by transplantation of porcine islets.

Suppression of T cells results in long-term survival of mouse heart xenograft in C6-deficient rats.

We have monitored the humoral immune responses of 10 type I diabetic patients, xenotransplanted with fetal porcine islet-like cell clusters for up to 8 years after xenotransplantation. We investigated the immunoglobulin subclass distribution as well as specificity differences of xenoreactive antibodies. Hemagglutintion tests, using pig erythrocytes, showed that some patients maintained higher titers of xenoreactive IgM antibodies during the entire follow up period, compared with pretransplant levels. In microcytotoxicity tests all but one patient tested showed higher than pretransplant levels of cytotoxic antibodies against pig peripheral blood mononuclear cells (PBMC) 6-8 years after transplantation. Levels of Gal alpha 1,3Gal specific antibodies, were also high. Antibody dependent cellular cytotoxicity (ADCC) activity against a Gal alpha 1,3Gal expressing human B cell line was detected in four patients while ADCC reactivity against adult pig islet cells was detected in only two patients, 6-8 years after transplantation. Immune sera collected 30 days and 1 year after transplantation showed positive staining of adult pig islet cells in fluoromicroscopy whereas sera from later time points did not. Western blot experiments showed that some patients had IgG1 antibodies reactive against epitopes on pig cells other than Gal alpha 1,3Gal, while xenoreactive IgM and IgG2 antibodies mainly reacted with Gal alpha 1,3Gal-containing epitopes as shown by absorption experiments. These results show that patients continue to produce higher than pretransplant levels of IgM and IgG2 xenospecific antibodies against Gal alpha 1,3Gal for extended time periods following xenotransplantation. Some patients also produce xenoreactive IgG1 antibodies directed against non-Gal alpha 1,3Gal epitopes.

Background. Islet allograft rejection in sensitized recipients is difficult to control by costimulation blockade using anti-CD154 and cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig). Because leukocyte function antigen (LFA) 1 is highly expressed on memory T cells, adding an LFA-1 blockade may inhibit memory T-cell activities. We examined the effects on islet allograft survival of triple costimulation blockade in presensitized recipient mice. Methods. C57BL/6 mice were sensitized by transplantation under the kidney capsule or intraperitoneal injection of Balb/c islets. Four weeks after transplantation, sensitization was confirmed by flow-cytometric detection of alloreactive antibodies. Diabetes was induced by a single intravenous injection of streptozotocin. Recipients were transplanted with 200 Balb/c islets under the right kidney capsule. Graft function was assessed by daily blood glucose and body weight records. Transplanted animals were divided into 3 treatment groups: group 1, control antibody; group 2, anti-CD154 and CTLA-4 Ig double therapy; group 3, anti-CD154, CTLA4Ig, and anti LFA-1 triple therapy. Injections were administered every second day from day -2 to day 8. Results. Naive mice rejected islet allografts between days 7 and 29 (mean 16 +/- 6 d; n = 5), sensitized mice in group 1 between days 0 and 14 (mean 7 +/- 5 d; n = 8), in group 2 between days 4 and 16 (mean 8 +/- 4 d; n = 7), and in group 3 between days 4 and 26 (mean 11 +/- 7 d; n = 10). Conclusion. Triple costimulation blockade with anti-CD154, CTLA4Ig, and anti LFA-1 was not sufficient to improve islet allograft survival in sensitized recipients. (Less)

In 1990-1993, eight diabetic renal transplant patients had porcine fetal islets injected intraportally at Huddinge Hospital in Stockholm. Four of the patients had evidence of xenograft function reflected in the excretion of small amounts of porcine C-peptide. Two patients had the porcine fetal islets placed under the capsule of a simultaneously transplanted kidney. In one of these patients, a graft biopsy specimen taken 3 weeks after transplantation revealed morphologically intact epithelial cells staining positively for insulin and glucagon. The insulin production was in all instances insufficient to affect the patient's insulin requirements. All patients formed specific xenoantibodies (mostly anti-Gal); presumably, most of the xenoislets were destroyed by rejection. On follow-up studies carried out 6-8 years after xenotransplantation, most patients still had higher-than-pretransplant levels of xenoantibodies. There was no evidence of transmission of porcine endogeneous retroviruses to the patients. All patients expressed a positive attitude toward the use of animal tissue for treatment of disease, and none of the patients regretted participating in the trial. Cell transplantation is leading the way at present for clinical xenotransplantation. The finding that complement inhibition protects intraportally injected porcine islets from an injurious incompatibility reaction holds promise for future clinical application. A similar protective effect might be achievable with the use of islets from transgenic pigs expressing human complement receptors.

Lack of antibody production against Hanganutziu-Deicher (H-D) antigenswith N-glycolylneuraminic acid in patients with porcine exposure history[In Process Citation]

Pig islet xenograft rejection is markedly delayed in macrophage-depletedmice: a study in streptozotocin diabetic animals [In Process Citation]

Damage to porcine islets of Langerhans after exposure to human blood invitro, or after intraportal transplantation to cynomologus monkeys:protective effects of sCR1 and heparin [see comments]

Encapsulation of cellular grafts in an immunoisolation membrane device may make it possible to perform transplantation without having to give immunosuppressive drugs. A common problem is the development of an avascular fibrotic zone around the implants, leading to impaired graft survival. The TheraCyte™ macroencapsulation device has therefore been designed to facilitate neovascularization of the device's surface. In this study, we evaluated the microcirculation around empty TheraCyte™ devices implanted SC in rats at various times after implantation, using a laser Doppler probe introduced via the device port. Studies were performed on day 1 or at 1, 2, and 4 weeks or at 2, 3, and 12 months after implantation. The mean flow was 158 ± 42, 148 ± 50, 133 ± 28, 72 ± 17, 138 ± 41, 165 ± 43, and 160 ± 29 perfusion units (PU), respectively. Thus, the microcirculation around the device was significantly reduced at 4 weeks after implantation (p < 0.01) while, from 2 months onwards the circulation had improved and did not differ significantly from that on day 1. The present study shows time-related changes in the microcirculatory flow around TheraCyte™ macroencapsulation devices that agree with our previous microdialysis studies on in vivo exchange of insulin and glucose between the device and the circulation. Laser Doppler flowmetry seems to provide a reliable technique for screening blood perfusion around macroencapsulation devices.

The purpose of this study was to investigate the effect of macrophage depletion, using liposome-encapsulated dichloromethylene diphosphonate (Lip-Cl2MDP), on delayed xenograft rejection (DXR) in the guinea pig-to-C6-deficient rat heart transplantation model. In this model, hyperacute rejection does not occur, but, in untreated recipients, xenografts are still destroyed by DXR within 1-2 days. Graft survival was 68 +/- 8.4 h in splenectomized control rats, 77 +/- 16.3 h with Lip-Cl2MDP alone, 99 +/- 10.4 h with deoxysperguarlin (DSG; P < 0.01 vs. controls), and 127 +/- 19.4 h with Lip-Cl2MDP plus DSG (P < 0.01 vs. DSG alone). Treatment with DSG alone or in combination with Lip-Cl2MDP resulted in significant reductions in serum IgM levels at rejection. Immunohistological studies showed that Lip-Cl2MDP alone or in combination with DSG reduced infiltration of grafts by both EDI + and ED2 + macrophages. These experiments support the concept that macrophages play an important role in DXR and suggest that strategies targeting macrophages may be useful in controlling DXR.

Background The purpose of pancreatic transplantation in insulin-dependent diabetic patients is to restore normoglycemia and thereby prevent the secondary complications of diabetes. However, uncertainty remains as to whether the mortality rate in diabetic patients can be affected by this procedure. Method We followed 14 patients with insulin-dependent diabetes mellitus (IDDM) and end-stage diabetic nephropathy for 10 years after successful combined kidney and pancreas transplantation. Fifteen diabetic patients subjected to kidney transplantation alone have served as controls. The glycemic control has been studied annually for 10 years and diabetic polyneuropathy has been assessed in both groups after 2, 4, and 8 years. Results In recipients of pancreas-kidney grafts, metabolic control was maintained throughout the observation period, with values of glycated hemoglobin in the normal range. In contrast, glucose metabolism was impaired in the control group, with glycated hemoglobin values around 10%. Nerve conduction and parasympathetic autonomic dysfunction improved in both groups after 2 years; there was no difference between the groups. After 4 years, we found a significant difference between the study group and the control group, and after 8 years it had widened. At the 4-year evaluation, there was no difference in mortality between the groups. At 8 years, however, a significant difference was noted, which was further substantiated at 10 years with a 20% mortality rate in the pancreas-kidney group versus an 80% mortality in the kidney alone group. Conclusions We found a substantial reduction in mortality in IDDM patients 10 years after successful combined pancreas and kidney transplantation. We speculate that the decrease in mortality was due to the beneficial effect of long-term normoglycemia on diabetic late complications and suggest therefore that combined pancreas and kidney transplantation, rather than kidney transplantation alone, should be offered to IDDM patients with end-stage diabetic nephropathy.

In this study, insulin was injected into TheracyteTM immunoisolation devices to analyze changes in the permeability of the device over time after implantation. The recovery of insulin was studied after subcutaneous implantation of the devices in rats, using the microdialysis technique. The area under the insulin cocnetration vs. time curves (AUC) after insulin injection in devices implanted 1 day previously did not differ significantly from the AUC after subcutaneous injection. At 1, 2 and 4 weeks after implantation, the recovery of insulin was significantly reduced, but at 3 months, the AUC was not significantly different from that in the control group. Histological examination showed that the number of vascular profiles within 15 μm of the device were significantly higher at 2, 4 weeks and 3 months after transplantation when compared to numbers at 1 week. The design of the device allows transplantation of cells at a chosen time point after its implantation. Delayed filling of the device would allow neovascularization of the device surface before graft implanatation and we suggest that such a schedule might improve function of the encapsulated graft.

Insulin resistance and increased demand for insulin secretion occur after successful pancreas transplantation. To investigate the potential effects of immunosuppression and pancreas transplantation on fasting beta-cell function, we studied fasting proinsulin and 32,33 split proinsulin secretion cross-sectionally and longitudinally in segmental pancreatic graft recipients (SPx, n = 18); in whole-pancreas graft recipients (WPx, n = 13); in nondiabetic kidney transplant recipients (Kx, n = 14) and in normal subjects (Ns, n = 14). Basal insulin secretion rates were significantly increased in SPx 15.8 (1.7), WPx 24.4 (4.5) and Kx 22.1 (2.1) vs Ns 9.7 (1.6) pmol min(-1) l(-1), p < 0.05, mean (SEM). Total proinsulin, intact proinsulin and 32,33 split proinsulin concentrations were significantly higher in all the transplanted groups than in normal subjects (p < 0.05), whereas the total proinsulin to C-peptide ratio and the 32,33 split proinsulin ratio were higher in SPx than in WPx, Kx and Ns (< 0.05). In the longitudinal study, beta-cell function in terms of proinsulin secretion remained stable for 1 year. In conclusion, fasting glucose homeostasis in pancreas-kidney transplant recipients is obtained at the expense of increased proinsulin secretion and increased insulin secretion rates, primarily induced by immunosuppression. In segmental pancreas graft recipients, increased fasting proinsulin and 32,33 split proinsulin relative to the number of beta-cells transplanted indicate more stress on the residual beta-cell and therefore higher secretory demand than in whole pancreas transplant recipients.

Porcine fetal endocrine pancreatic tissue was placed under the kidney capsule in a diabetic renal transplant patient. In a core-needle kidney biopsy specimen obtained 3 weeks after transplantation, clusters of epithelial cells were identified in the subcapsular space. The ultrastructural and immunocytochemical features of these cells were typical of pancreatic islet cells. Some cells stained positively for insulin; others stained positively for glucagon, somatostatin or chromagranin A. There were well-defined cytoplastic storage and transport granulae that indicated hormone synthesis. The ultrastructural findings provide further evidence that porcine cells can survive after transplantation to humans.

Summary Background The study of whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for evaluating the safety of pig-to-man xenotransplantation. PERV is carried in the pig germline, and all recipients of porcine tissues or organs will be exposed to the virus. Methods We studied 10 diabetic patients who had received porcine fetal islets between 1990 and 1993, looking for evidence of PERV infection by using PCR serology, PCR, and reverse transcriptase assays. Prolonged xenograft survival (up to a year) was confirmed in five patients by porcine C-peptide excretion and detection of pig mitochondrial DNA (mtDNA) in serum. Findings Despite the evidence for extended exposure to pig cells and despite concomitant immunosuppressive therapy, we were unable to detect markers of PERV infection in any patient. Screening for two PERV sequences in peripheral blood lymphocytes collected 4–7 years after the xenotransplantation was negative. Markers of PERV expression, including viral RNA and reverse transcriptase, were undetectable in sera from both early (day 3 to day 180) and late (4–7 years) time points. Western blot analysis for antibodies was consistently negative. Interpretation These results suggested the absence of PERV infection in these patients. Also this study establishes a minimum standard for post-transplant surveillance of patients given porcine xenografts.

After pancreas-kidney transplantation, it is difficult to obtain an accurate estimate of the insulin secretion of the pancreas graft, since several pitfalls are involved using peripheral C-peptide and/or insulin measurements in this determination. In this study, the individual kinetic parameters of C-peptide and then the rates of insulin secretion were estimated by two mathematical methods, the deconvolution method and the “combined model” during slow (oral glucose) and fast (intravenous glucagon) changes in insulin secretion in six successful pancreas-kidney transplant recipients with systemic delivery of insulin (Px), six nondiabetic kidney-transplant recipients with portal insulin secretion (Kx), six nondiabetic controls (NS), and six C-peptide-negative insulin-dependent diabetes mellitus patients (IDDM). Decreased C-peptide clearance and basal and poststimulatory hyperinsulinemia were found in both Px and Kx compared with NS ( P < 0.05). Similar glucose responses were observed after intravenous glucagon in all groups, whereas the responses after oral glucose were 30% higher in Px and Kx than in NS ( P < 0.05). During oral glucose and after intravenous glucagon, both mathematical methods resulted in significantly lower maximal and incremental insulin secretion rates (ISR) in Px than in Kx ( P < 0.05). In contrast, calculations of incremental ISR in NS and Px induced by the two β-cell stimuli were about the same but significantly higher in Kx than in NS ( P < 0.05). These results differed markedly from those obtained using peripheral measurements of insulin and C-peptide alone. In conclusion, when C-peptide clearance and insulin metabolism change, such as in pancreas-kidney transplant recipients, accurate evaluation of insulin secretion from the graft can be obtained only by using individual kinetics of the peptides before calculating the ISR. This study also clearly demonstrates that insulin secretion after pancreas transplantation is still defective.

To gain insight into the pathophysiology of impaired glucose tolerance in pancreas transplantation, glucose kinetics and insulin secretion were assessed after an oral glucose load in four combined pancreas-kidney recipients with impaired glucose tolerance (IPx), in five combined pancreas-kidney recipients with normal glucose tolerance, in six nondiabetic kidney transplant recipients, and in eight normal subjects employing a dual isotope technique. β-Cell function was evaluated by calculating prehepatic insulin secretion rates, which subsequently were correlated to the ambient glucose concentrations to obtain an index of β-cell responsiveness. Oxidative and nonoxidative glucose metabolism were assessed by indirect calorimetry. Basal insulin secretion rates, the glucose-stimulated early insulin secretion rates, as well as β-cell responsiveness were markedly reduced in IPx than in the glucose-tolerant transplant subjects. Total systemic glucose appearance was similar in the groups with apparently comparable inhibition of systemic glucose release and increase in exogenous glucose appearance. The hyperglycemic response in IPx was due to a significant reduction in the glucose disappearance rates during the first 2 h after glucose ingestion. Nonoxidative glucose metabolism increased significantly less in IPx than in glucose-tolerant groups. Glucagon secretion was less suppressed in the early part of the study in IPx, which may have contributed to the excessive hyperglycemia. In conclusion, IPx after pancreas transplantation was characterized by 1) impaired early insulin secretion, 2) reduced β-cell responsiveness, 3) reduced glucose uptake, 4) impaired nonoxidative glucose metabolism, and 5) impaired early inhibition of glucagon secretion.

Alport syndrome, also termed hereditary nephritis, was initially described in 1927 by A.C. Alport [1] as an inherited kidney disease characterized by hematuria and sensorineural deafness. Later, ocular lesions were also associated with the syndrome and, with the introduction of the electron microscope, irregularities and disruptions in the glomerular basement membrane (GBM) were shown to be typical for this disorder as well [2, 31. The disease is highly progressive and usually leads to renal failure during adolescence or before middle age. The disease is primarily inherited as an X-chromosome linked dominant trait with an estimated gene frequency of 1:5,000 to 1:10,000, but autosomal forms also exist [2, 3]. The defective gene in X-linked Alport syndrome was located in 1988 and 1989 to the long arm of the X chromosome [4—61. In 1990, a gene encoding a novel basement membrane (type IV) collagen a5 chain was discovered and localized to the Alport gene region on chromosome X, and this was soon followed by identification of mutations in this gene in Alport patients [7, 8]. More recently, mutations have also been reported in the genes for the a3 and a4 type IV collagen chains in the rarer autosomal forms of Alport syndrome

Background Costimulation blockade can prevent rejection of islet xenografts in naive but not sensitized recipients. Donor-specific antibodies (DSA) may partly explain this observation. The effect of DSA on rat islet xenograft survival in mice receiving costimulation blockade was investigated. Methods Naive C57BL/6 mice with alloxan-induced diabetes were transplanted under the left kidney capsule with 100 Lewis rat islets. Recipients were divided into three groups receiving: (i) isotype control antibodies (Abs); (ii) anti-CD154 and CTLA4Ig; or (iii) anti-CD154, CTLA4Ig, and anti-LFA-1 every second day, day 0–8. At the time of transplantation (Tx), half of the animals in each group received naive mouse serum and half xenoimmune serum derived from mice previously transplanted with rat islets. Non-fasting blood glucose levels and body weight were followed daily. Cured mice were examined by intraperitoneal glucose tolerance (IPGT) tests at 1 and 4 months after transplantation. Results Donor-specific antibodies were detected in immune serum-injected recipients up to at least 96 h post-Tx. Short term (≤96 h), there was no significant difference with regard to graft mass, infiltrating and apoptotic cells between groups of mice receiving naive and immune sera. A moderate infiltration of polymorphonuclear and mononuclear cells was seen 96 h post-Tx in mice given control Abs, whether or not they received immune or naive mouse serum. Mice given costimulation blockade had well-maintained endocrine tissue and very little cell infiltration. There was no significant difference in islet xenograft function and survival long term between groups receiving naive and immune sera in combination with costimulation blockade. About half of the mice receiving costimulation blockade lost graft function within 110 days. Conclusion The presence at Tx of DSA does not appear to negatively influence early and late islet xenograft survival in mice receiving costimulation blockade.

Most current knowledge of pancreatic islet pathophysiology in diabetes mellitus has come from animal models. Even though islets from humans are readily available, only a few come from diabetic donors. We had the uncommon opportunity to acquire islets from humans with type 2 diabetes and used it to perform a study not previously done with human or animal islets.Oxidative stress has been proposed as a mechanism for impaired β-cell function in type 2 diabetes. Lipid peroxides caused by reactive oxygen species are damaging to body tissues. The objective was to determine whether lipid peroxide-protein adducts occur in pancreatic islets of humans with type 2 diabetes.Immunoblots with two antibodies to hydroxynonenal and 2 other antibodies we generated against reactive small aliphatic compounds were used to detect lipid peroxide-protein adducts in islets of patients with type 2 diabetes and controls.The antibodies reacted strongly to ≥5 islet proteins. The major hydroxynonenal adduct in the islets of type 2 diabetes patients was a 52-kDa protein seen with all 4 antibodies that was also seen in islets of nondiabetic humans, rat islets, and insulinoma cells and in mitochondria of various rat tissues. Nano-LC-MS/MS (liquid chromatography-tandem mass spectrometry) and MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) analysis identified the protein as the β-chain of the mitochondrial F-ATP synthase, an enzyme responsible for 95% of ATP formed in tissues.Lipid peroxide-protein adducts occur in β-cells in the nondiabetic state and in diabetes. Lipid peroxidation is thought to be damaging to tissues. Analogous to various other unhealthy characteristics, the presence in nondiabetic individuals of lipid peroxide-protein adducts does not necessarily indicate they are not detrimental.

Insulin resistance is a characteristic feature in recipients of a pancreas transplant, but the relative contribution of the liver and peripheral tissues to this abnormality within a spanning range of insulin concentrations is unknown. To assess the impact of insulin action on glucose metabolism after pancreas transplantation, a euglycemic-hyperinsulinemic clamp with sequential insulin infusions (5, 40, and 200 mU m−2·min−1 for 120 min each), combined with isotopic determinations of the rates of hepatic glucose production and extrahepatic glucose uptake, indirect calorimetry, and measurements of glycogen synthase and hexokinase activities in vastus lateralis muscle, were performed in six pancreas-kidney transplant recipients (Px group) and compared with those performed in six nondiabetic kidney transplant recipients with similar immunosuppression (Kx group) and six nondiabetic control subjects. The overall effects of insulin on whole-body glucose metabolism, determined as the glucose infusion rates versus the corresponding steady-state serum insulin concentrations, demonstrated a rightward shift in the dose-response curves of the transplanted groups compared with those of normal subjects. The dose-response curve for glucose disposal rates (Rd) was shifted to the right in the Px and Kx groups, and the maximal glucose disposal rate was reduced by 40% in the Px group (11.7 ±1.1 mg·kg−1 fat-free mass·min−1) and 30% in the Kx group (13.9 ± 1.2 mg·kg−1 fat-free mass·min−1) compared with that in control subjects (19.1 ±2.2 mg·kg−1 fat-free mass·min−1) (P < 0.05). The dose-response curve for suppression of hepatic glucose output rates was similar at increasing hepatic sinusoidal insulin concentrations.Glucose oxidation rates were similar in all groups, whereas nonoxidative glucose rates were reduced by 50% in the Px group and by 30% in the Kx group compared with those in the control group (P < 0.05). In the Px group, an impaired activation of the fractional velocity and absent decrease in the half-maximal stimulation of muscle glycogen synthase occurred during the insulin infusion. However, this finding could only explain in part the degree of impairment in nonoxidative glucose metabolism. No differences were found in total hexokinase activity in muscle between normal subjects and the transplant groups at basal insulinemia or after insulin stimulation. During hyperinsulinemia, glucagon and nonesterified fatty acids were not suppressed as much in the transplanted groups as they were in normal control subjects (P < 0.05). In conclusion, pancreas transplantation causes impaired peripheral action of insulin as compared with that in normal subjects and kidney transplant recipients. The main course of insulin resistance in the two transplant groups is explained by the immunosuppressive treatment, but the augmented insulin resistance in pancreas transplant recipients could partly be explained by the chronic peripheral hyperinsulinemia. The principal site of insulin resistance was a reduced insulin-stimulated nonoxidative glucose metabolism of peripheral tissues, which resulted in decreased capacity to store glucose as glycogen. The impaired peripheral insulin action could only partly be explained by a reduced activation of the glycogen synthase enzyme in skeletal muscle.