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Abstract: Background: Pig islet xenotransplantation is a promising alternative to allogeneic transplantation. However, the wide immunologic barrier between pigs and primates limits the long‐term survival of the graft. MD‐3, a novel monoclonal antibody (mAb) that recognizes a particular epitope of human ICAM‐1, can render T cells tolerant to a xenograft by arresting dendritic cell maturation. We report the long‐term survival of adult wild‐type pig islets and successful retransplantation in nonhuman primates using a protocol comprising induction with MD‐3 mAb and maintenance with anti‐CD154 mAb and sirolimus. Methods: Eleven rhesus monkeys were assigned to three groups. Group 1 (n = 4) involved treatment with MD‐3 induction, short‐term (<4 months) administration of anti‐CD154 mAb, and maintenance therapy with sirolimus. Group 2 (n = 4) involved treatment with MD‐3 induction and long‐term maintenance therapy with anti‐CD154 mAb and sirolimus. Group 3 (n = 3) involved only maintenance therapy with anti‐CD154 mAb and sirolimus. Diabetes was induced in monkeys by streptozotocin, and pig islets (61 000‐112 000 IEQ/kg for each transplant; up to 280 000 IEQ/kg per recipient) were infused through the portal vein. The in vivo functional potency of the isolated islets was tested by minimal model transplant in streptozotocin‐induced diabetic NOD/SCID mice, and the mean AUC of blood glucose level divided by the number of follow‐up days was calculated. Results: The islet grafts survived more than 6 months (between 225 and 727 days) in nine of 12 transplants of MD‐3‐treated groups 1 and 2, whereas in the absence of MD‐3 mAb, survival was <40 days. In three transplants of the MD‐3‐treated Group 2, functional graft survival was only for 104, 125, and 154 days. In these cases, a retrospective analysis suggested that the relatively short survival duration was associated with the relatively high AUC value in the NOD/SCID bioassay. Notably, when retransplantation was performed in Group 3, blood glucose control was extended up to 956 days, which was supported by MD‐3 mAb‐based suppression of adaptive immunity. No replication of cytomegalovirus genes was observed. Conclusions: Long‐term survival of pig islet xenografts and successful retransplantation were achieved with MD‐3 mAb‐based immunosuppression regimen in this pig‐to‐monkey transplantation model. It should be emphasized that these encouraging results were achieved following the transplantation of islets from pigs that had not been genetically modified. Considering that it is possible to further substantially reduce the destruction of grafted islet using genetically modified pig islet, the islet requirement could be reduced and much longer graft survival can be achieved. [ABSTRACT FROM AUTHOR]

Abstract: Background: Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti‐CD154 monoclonal antibody (mAb) in pig‐to‐non‐human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti‐CD40 (2C10R4) mAb‐based studies have not been reported. Methods: Nine streptozotocin (STZ)‐induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen‐free (DPF) miniature pigs. They were treated with anti‐CD40 mAb‐based immunosuppressive regimen and were divided into 3 groups: anti‐CD40 only group (n = 2), belatacept group (anti‐CD40 mAb+belatacept, n = 2), and tacrolimus group (anti‐CD40 mAb+tacrolimus, n = 5). All monkeys received anti‐thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C‐peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted. Results: All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow‐up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C‐peptide concentration of >0.15 ng/mL, was 31, 27, and 60 days for anti‐CD40 only, belatacept, and tacrolimus groups, respectively. Anti‐αGal IgG levels in serum and the number of interferon‐γ secreting T cells in peripheral blood mononuclear cells did not increase in most animals. Conclusion: These results showed that anti‐CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti‐CD40 mAb was not as effective as anti‐CD154 mAb in terms of preventing early islet loss. [ABSTRACT FROM AUTHOR]

Background Islet allotransplantation is a promising way to treat some type 1 diabetic (T1D) patients with frequent hypoglycemic unawareness, and islet xenotransplantation is emerging to overcome the problem of donor organ shortage. Our recent study showing reproducible long-term survival of porcine islets in non-human primates ( NHPs) allows us to examine whether autologous regulatory T-cell (Treg) infusion at peri-transplantation period would induce transplantation tolerance in xenotransplantation setting. Methods Two diabetic rhesus monkeys were transplanted with porcine islets from wild-type adult Seoul National University ( SNU) miniature pigs with immunosuppression by anti-thymoglobulin ( ATG), cobra venom factor, anti- CD154 monoclonal antibody ( mAb), and sirolimus. CD4+ CD25high CD127low autologous regulatory T cells from the recipients were isolated, ex vivo expanded, and infused at the peri-transplantation period. Blood glucose and porcine C-peptide from the recipients were measured up to 1000 days. Maintenance immunosuppressants including a CD40- CD154 blockade were deliberately discontinued to confirm whether transplantation tolerance was induced by adoptively transferred Tregs. Results After pig islet transplantation via portal vein, blood glucose levels of diabetic recipients became normalized and maintained over 6 months while in immunosuppressive maintenance with a CD40- CD154 blockade and sirolimus. However, the engrafted pig islets in the long-term period were fully rejected by activated immune cells, particularly T cells, when immunosuppressants were stopped, showing a failure of transplantation tolerance induction by autologous Tregs. Conclusions Taken together, autologous Tregs infused at the peri-transplantation period failed to induce transplantation tolerance in pig-to- NHP islet xenotransplantation setting. [ABSTRACT FROM AUTHOR]

Background To understand humoral responses elicited after xenotransplantation, we compared the induction of anti-non-Gal antibodies vs. anti-Gal antibodies in non-human primates ( NHPs) after intraportal porcine islet transplantation ( PITX). Methods Anti-Gal and anti-non-Gal IgGs were analyzed in serial plasma samples of NHP recipients after PITX by enzyme-linked immunosorbent assay ( ELISA) using synthetic Gal and by flow cytometry using α-1,3-galactosyltransferase gene knockout ( GTKO) porcine endothelial cells, respectively. Anti-non-Gal IgG was detected in some recipients after PITX. The specificity of anti-non-Gal IgG was investigated by two-dimensional electrophoresis of the protein extract from GTKO porcine endothelial cells, Western blot analysis of recipient pre- and post- PITX plasma, and MALDI- TOF/ TOF mass spectrometry, revealing albumin, a non-glycosylated protein in the serum supplement of the islets solution, as a putative antigen for anti-non-Gal IgG. The binding of IgG antibodies to human albumin ( HA), bovine albumin ( BA), porcine albumin ( PA), and Gal was compared by ELISA in pre- and post- PITX plasma samples of 30 NHP recipients subjected to intraportal PITX, which were grouped according to the use of CD40- CD154 blockade and sirolimus. Results One of the immunoblot-matched spots was identified as BA by mass spectrometry. By ELISA, the plasma used in the immunoblot analysis revealed strong IgG binding to BA and PA, but not to HA. Anti- PA, anti- BA, and anti-Gal antibodies in NHP recipients 1 month after PITX were detected in 5 (100%), 3 (60%), and 5 (100%), respectively, of the 5 recipients receiving various immunosuppression ( IS) without CD40- CD154 blockade (group I) and in 0 (0%), 0 (0%), and 4 (16%), respectively, of the 25 recipients receiving IS with CD40- CD154 blockade and sirolimus (group II). This finding revealed significant differences between the groups (P < 0.0001, P = 0.0011 and P = 0.0013, respectively). Interestingly, among 15 recipients achieving graft survival longer than 1 month in group II, anti- PA IgG was detected in only 1 recipient (6.7%) 180 days after PITX. However, an increase in anti-Gal IgG was detected in 7 recipients (46.7%) despite maintenance IS with anti- CD154 and sirolimus. This result indicates that anti-Gal IgG is more frequently induced than anti- PA IgG (P = 0.0352). Moreover, induction IS with anti- CD154 and sirolimus suppressed anti-Gal IgG, but not anti- PA and anti- BA IgG, responses in sensitized recipients given a repeat transplantation. Conclusions In NHP recipients of PITX, anti- PA and anti- BA IgG antibodies are elicited by porcine serum included as a supplement in porcine islet preparation. IS including CD40- CD154 blockade and sirolimus suppresses these antibody responses in naïve recipients, but not in sensitized recipients. The elicitation of anti-xenogenic albumin antibodies, a humoral response to a model protein antigen, is distinct from that of anti-Gal antibodies, a response to carbohydrate antigen. [ABSTRACT FROM AUTHOR]

To develop an international consensus regarding the appropriate conditions for undertaking clinical trials in xenocorneal transplantation, here we review specific ethical, logistical, scientific, and regulatory issues regarding xenocorneal transplantation, and propose guidelines for conduct of clinical xenocorneal transplantation trials. These proposed guidelines are modeled on the published consensus statement of the International Xenotransplantation Association regarding recommended guidelines for conduct of clinical islet xenotransplantation. It is expected that this initial consensus statement will be revised over time in response to scientific advances in the field, and changes in the regulatory framework based on accumulating clinical experience. [ABSTRACT FROM AUTHOR]

Background To establish the consensus about the conditions for undertaking clinical trials in xenocorneal transplantation in Korea, specific issues regarding the xenocorneal transplantation on ethical and regulatory aspects are addressed, and the guidelines to conduct clinical trial of the xenocorneal transplantation are proposed. Method and Results Chapter 1 reviews the key ethical requirements and progress of a Korean regulatory framework for clinical trials of xenocorneal transplantation. Chapters 2-7 provide recommendations on source pigs, quality control of porcine corneal procurement, preclinical efficacy required to justify a clinical trial, strategies to prevent transmission of porcine endogenous retrovirus ( PERV), patient selection for clinical trials, and informed consent in xenocorneal transplantation using either cellularized or decellularized porcine graft, which are essentially based on the International Xenotransplantation Association ( IXA) islet xenotransplantation consensus statement. The consensus statement of the inclusion criteria for the patients' selection has been made by the executive board members in Korean External Eye Disease Society. Conclusions This consensus statement will be a good initiative for Korean Food and Drug Administration to discuss final regulatory guidelines in conducting clinical trials of xenocorneal transplantation in Korea and for International Xenotransplantation Association to develop International Consensus Standards of Xenocorneal Transplantation. [ABSTRACT FROM AUTHOR]

Lee J-I, Shin J-S, Lee J-E, Jung W-Y, Lee G, Kim M-S, Park C-G, Kim S-J. Reference values of hematology, chemistry, electrolytes, blood gas, coagulation time, and urinalysis in the Chinese rhesus macaques ( Macaca mulatta). Xenotransplantation 2012; 19: 244-248.. © 2012 John Wiley & Sons A/S. Abstract: Background: Due to increasing preclinical study using the rhesus monkeys, the physiological data of these monkeys are very important for the objective evaluation of experimental results. Here, we report the physiological values of the Chinese rhesus monkeys acclimated under a well-controlled laboratory environment. Methods: Seventy healthy rhesus monkeys of both genders (29 males and 41 females) were used in this study. Available data on hematology, serum biochemistry, electrolytes, blood gas, coagulation time, urinalysis, water consumption, urine volume, and body weight were examined. Results: The lymphocyte values were 1.5-2.5 times higher than the neutrophil values in both genders. In serum biochemistry, there was no significant difference between the two genders. Interestingly, the values of alkaline phosphatase from the monkeys were very high. According to isoenzyme analysis, the percentage of alkaline phosphatase that originated from the bone was 60%, and this value was significantly higher than that from the liver (39%, P = 0.020). Conclusion: Physiological data are very important parameters that can be directly and indirectly related to organ function (e.g. islet, kidney, liver, heart, etc.) in the transplantation model. In this respect, our data constitute valuable references for preclinical study using the Chinese rhesus monkeys. [ABSTRACT FROM AUTHOR]

Lee KG, Lee H, Ha JM, Lee YK, Kang HJ, Park C-G, Kim SJ. Increased human tumor necrosis factor-α levels induce procoagulant change in porcine endothelial cells in vitro. Xenotransplantation 2012; 19: 186-195. © 2012 John Wiley & Sons A/S. Abstract: Background: Intravascular thrombosis and systemic coagulation abnormalities are major hurdles to successful xenotransplantation and are signs of acute humoral rejection. Increased expression of tissue factor (TF) is associated with the development of microvascular thrombosis in xenografts. To develop an effective strategy to prevent accelerated coagulation in xenografts, we investigated the mechanism by which porcine endothelial cells (PECs) become procoagulant after contact with human blood. Methods: The changes in TF mRNA levels and activity in PECs after incubation with 20% human serum or human bioactive molecules, including C5a, tumor necrosis factor-α (TNFα) and interleukin (IL)-1α, were evaluated using real-time PCR and the factor Xa chromogenic assay, respectively. The procoagulant changes in PECs by these agonists were evaluated by measuring the coagulation time of human citrated plasma suspended with PECs pretreated with each agonist. TF expression and coagulation times were also assessed in PECs transfected with short interfering RNA (siRNA) designed to knock down porcine TF. We also examined the production of proinflammatory cytokines in human whole-blood or plasma after contact with PECs, which were screened using the cytometric bead array system. TNFα levels were measured using ELISA in whole-blood after contact with PECs, with or without the addition of xenoreactive antibodies or C1 esterase inhibitor. Results: Porcine TF mRNA and activity in PECs were up-regulated in response to human TNFα and IL-1α but were not affected by C5a or 20% human serum. Up-regulation of TF expression by human TNFα or IL-1α shortened PEC-induced coagulation time, while siRNA-mediated knockdown of TF expression prolonged coagulation time. The incubation of PECs with human whole-blood led to a significant increase in human TNFα levels in the blood, which was promoted by the addition of xenoreactive antibodies and prevented by C1 esterase inhibitor. Conclusions: Human TNFα level increases in human blood after contact with PECs, which is attributed to xenoreactive antibody binding and subsequent complement activation. Human TNFα induces procoagulant changes in PECs with increased TF expression. This study suggests that human TNFα may be one of the mediators linking complement activation with procoagulant changes in the xenoendothelium. [ABSTRACT FROM AUTHOR]

Jin S-M, Shin JS, Kim KS, Gong C-H, Park SK, Kim J-S, Yeom S-C, Hwang ES, Lee CT, Kim S-J, Park C-G. Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation 2011; 18: 369-379. © 2011 John Wiley & Sons A/S. Abstract: Background: In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen-free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs. Methods: Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP-grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics. Results: Proportion of islets larger than 200 μm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 μm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue-free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal. Conclusions: Use of the newer bovine nervous tissue-free enzymes including a GMP-grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high-quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered. [ABSTRACT FROM AUTHOR]