NK-cell-dependent acute xenograft rejection in the mouse heart-to-rat model

BACKGROUND: Acute humoral xenograft rejection is characterized by widespread intravascular thrombosis with a significant NK-cell and macrophage infiltrate. Although in vitro and ex vivo data have shown that NK cells are capable of killing xenogeneic tissue, the precise role they play in vivo is still not certain. Consequently, there are few tested strategies for dealing with NK-cell-mediated rejection, should this prove to be a problem. One reason for this has been the lack of a relevant rodent model in which rejection by these cells can be easily studied. METHODS: Prior to transplantation of mouse hearts, we depleted rat recipients of fibrinogen using a snake venom, ANCROD, from the Malayan pit viper. Graft survival was examined by manual palpation and the rejected hearts were examined by histology. Levels of circulating interferon gamma (IFN-gamma), used as a surrogate marker for NK-cell activation, were determined by an enzyme-linked immunosorbent assay. RESULTS: Depletion of fibrinogen to approximately 5% of normal allowed surgery without a significant increase in the technical failure rates and prolonged graft survival compared with that seen in unmanipulated rats. Rejected hearts showed no evidence of intravascular thrombosis but did show significant antibody and complement deposition. There was little T-cell infiltration and cyclosporin had no influence on survival. Instead, hearts were infiltrated with NK cells and macrophages and rejection was associated with significant IFN-gamma production. Depletion of NK cells with anti-asialo-GM-1 from ANCROD-treated recipients led to a further significant prolongation of graft survival. CONCLUSIONS: Inhibition of intravascular thrombosis by fibrinogen depletion, in the absence of any other manipulation, unmasks NK-cell-dependent acute xenograft rejection in the mouse-to-rat heart transplantation model. This relatively simple model is expected to be useful to investigate the mechanisms of NK-cell-mediated rejection and to provide insight into the types of graft manipulation that could modify this process.     

Short Communication: Pig islet xenotransplantation acceptance in a Latin‐American diabetic population

Abalovich A, Wechsler C, Lara S, Bervottini M. Pig islet xenotransplantation acceptance in a Latin‐American diabetic population. Xenotransplantation 2010; 17: 263–266. © 2010 John Wiley & Sons A/S. Abstract: Progress in porcine islet xenotransplantation has been accompanied by studies on acceptance of this new procedure by patients, health professionals or the general public. Such studies have not been done in the Latin‐American population. We conducted a questionnaire in 108 diabetes patients (insulin‐dependent, n = 53; insulin‐independent, n = 55) in a public hospital in Argentina. The questions addressed the general perception of the xenotransplant procedure and specific items related to the outcome (achieving insulin independence, improvement in metabolic control, delay in emergence of diabetic complications, need for repeat procedures, potential of transfer of infectious viruses, association with psychological problems, and anticipated success in relation to achieving a cure). Eighty‐six (79%) of the patients accepted islet xenotransplantation; this incidence was not different for insulin‐dependent or insulin‐independent patients, patients with or without complications, or patients with good or poor metabolic control. Also, over 75% of patients accepted the procedure if this is only associated with a reduction in insulin requirement, if the procedure just delays but not prevents the onset of complications, or if the procedure needs to be performed every 6 months. Fifty‐seven percent of patients indicated acceptance even if the potential transmission of a virus infection cannot be completely ruled out: this outcome was not affected by the outbreak of the H1N1 flu epidemic during the conduct of this study. Forty percent of patients indicated that living with porcine cells in their body could give psychological problems. We conclude that this population of Latin‐American diabetic patients shows a high acceptance rate of a porcine islet xenotransplantation product.     

Rapid failure of pig islet transplantation in non human primates

Abstract: We have previously demonstrated that adult pig islets of Langerhans are not destroyed in vitro by primate sera. Whether these islets can function when placed into the liver of non-human primates is not known. We now report on the outcome of pig islet xenotransplantation into five non diabetic primates (four baboons and one macacus fascicularis) receiving intraportally purified adult pig islets. The average number of islet-equivalent per graft was 110000 (60–180000). All animals received associations of ATG, cyclosporine or LF 195 (a deoxyspergualin analog), mycophenolate mofetil and corticosteroids. A specific porcine C-peptide (C-pep) RIA test was used to monitor insulin secretion. Two hours after grafting, porcine C-peptide was positive (from 0.37 to 4.25 ng/ml) in all monkeys except one. Primate C-pep was normal in all cases. Only two monkeys had detectable levels of porcine C-pep on day 1 or 2 with undetectable levels thereafter, even after glucagon challenge between days 6 and 10. Several normal islets with moderate inflammatory infiltration were observed in one animal liver on day 2 (the time of necropsy) as well as islets with IgM and complement deposition. Among animals sacrificed on days 14, 16 and 38, some residual islet cells could be identified only in livers collected on day 14. Partial glycaemic control was achieved in some rats receiving islets from the same preparations. In conclusion, adult pig islets are not able to maintain insulin secretion for more than 24 h when injected intraportally into non diabetic immunosuppressed monkeys, suggesting immediate islet xenograft destruction.     

Pig islet xenograft rejection is markedly delayed in macrophage-depleted mice: a study in streptozotocin diabetic animals

The present study aimed to evaluate the effect of depletion of macrophages and/or natural killer (NK) cells on islet xenograft rejection in the pig-to-mouse model. Five microliters (4,000 to 5,000 IEQ, islet equivalents) of adult pig islets were transplanted under the renal capsule of C57BL/6 mice with streptozotocin-induced diabetes. Macrophages were depleted by injection of liposome-encapsulated dichloromethylene diphosphonate (Lip-Cl2MDP) intraperitoneally (i.p.) at a dose of 100 microl/ 10 g body weight (BW) 2 days before transplantation, and 50 microl/10 g BW weekly thereafter. NK cells were depleted by injection of the monoclonal antibody NK 1.1 (anti-NK 1.1 mAb) i.p. at a dose of 100 microg/mouse 1 day before transplantation, and then 25 microg per week thereafter. Islet graft survival was monitored by daily measurements of blood glucose. Graft survival was 8 +/- 1.2 days in untreated controls, 9 +/- 1.0 days with anti-NK 1.1 mAb alone, 22 +/- 4.9 days with Lip-Cl2MDP alone (P<0.01 vs. controls), and 26 +/- 3.8 days with Lip-Cl2MDP plus anti-NK 1.1 mAb (P<0.01 vs. controls). In the last group, two of six animals were killed with functioning grafts 30 days after transplantation. In untreated controls, rejected xenografts were heavily infiltrated by F4/80+ macrophages and CD3+T cells. In Lip-Cl2MDP-treated groups, the number of F4/80+ macrophages was markedly reduced. On the periphery of xenografts, a small number of CD3+T cells were observed. In conclusion, our results suggest that strategies targeting macrophages may facilitate islet xenograft survival. A role for NK cells cannot be excluded, but appears to be of minor importance.     

A health‐economic analysis of porcine islet xenotransplantation

Beckwith J, Nyman JA, Flanagan B, Schrover R, Schuurman H‐J. A health‐economic analysis of porcine islet xenotransplantation. Xenotransplantation 2010; 17: 233–242. © 2010 John Wiley & Sons A/S. Abstract: Background: Islet cell transplantation is a promising treatment for type 1 diabetes. To overcome the shortage of deceased human pancreas donors, porcine islet cell xenotransplantation is being developed as an alternative to allotransplantation. The objective of this study was to perform a cost‐effectiveness analysis of porcine islet transplantation in comparison with standard insulin therapy. The patient population for this study was young adults, ages 20 to 40, for whom standard medical care is inadequate in controlling blood glucose levels (hypoglycemia unawareness). Since trial data were lacking, estimates used extrapolations from data found in the literature and ongoing trials in clinical allotransplantation. Cost estimates were based on the data available in the USA. Methods: Markov modeling and Monte Carlo simulations using software specifically developed for health‐economic evaluations were used. Outcomes data for ongoing clinical islet allotransplantation from the University of Minnesota were used, along with probabilities of complications from the Diabetes Control and Complications Trial. Quality‐adjusted life years (QALYs) were the effectiveness measure. The upper limit of being cost‐effective is $100 000 per QALY. Cost data from the literature were used and adjusted to 2007 US dollars using the medical care portion of the Consumer Price Index. Results: In both Markov modeling and Monte Carlo simulations, porcine islet xenotransplantation was both more effective and less costly over the course of the 20‐yr model. For standard insulin therapy, cumulative cost per patient was $661 000, while cumulative effectiveness was 9.4 QALYs, for a cost of $71 100 per QALY. Transplantation had a cumulative cost of $659 000 per patient, a cumulative effectiveness of 10.9 QALYs, and a cost per QALY of $60 700. Islet transplantation became cost‐effective at 4 yr after transplantation, and was more cost‐effective than standard insulin treatment at 14 yr. These findings are related to relative high costs in the transplantation arm of the evaluation during the first years while those in the insulin arm became higher later in follow‐up. Throughout the follow‐up period, effectiveness of transplantation was higher than that of insulin treatment. In sensitivity analysis, duplication or triplication of one‐time initial costs such as costs of donor animal, islet manufacturing and transplantation had no effect on long‐term outcome in terms of cost‐saving or cost‐effectiveness, but the outcome of transplantation in terms of diabetes complications in cases with partial graft function could affect cost‐saving and cost‐effectiveness conclusions. Conclusion: Despite limitations in the model and lack of trial data, and under the assumption that islet transplantation outcomes for young adult type 1 diabetes patients are not dependent on the source of islet cells, this health‐economic evaluation suggests that porcine islet cell xenotransplantation may prove to be a cost‐effective and possibly cost‐saving procedure for type 1 diabetes compared to standard management.     

Tacrolimus inhibits discordant islet xenograft rejection: a study in the pig-to-rat model

Abstract: The aim of the present study was to evaluate the immunosuppressive effect of tacrolimus (TAC) in discordant islet xenotransplantation. Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in normoglycemic rats treated with TAC monotherapy, TAC plus other immunosuppressive drugs or cyclosporin A (CsA) monotherapy. Twelve or 24 days after transplantation, the extent of a cellular infiltration in the xenografts was evaluated using immunohistochemistry. In some animals, the grafts were examined for antibody and complement deposition and the levels of xenoreactive antibodies in serum were determined. In untreated rats, the xenografts were completely rejected after 12 days and no intact ICCs remained. TAC monotherapy (at 0.5 and 1.0 mg/kg b.w.) almost completely inhibited rejection for up to 12 days. In animals treated with TAC monotherapy (at 0.5 mg/kg b.w.), rejection was markedly inhibited for up to 24 days. However, the effect after 24 days was not consistent and in some grafts there were signs of rejection. The protective effect of TAC observed in this study is in contrast to the findings in rats given CsA monotherapy in which no or only a marginal effect on islet xenograft rejection was observed. Only when CsA was given at 20 mg/kg b.w., an inhibitory effect could be observed. Immunosuppression with TAC at a suboptimal dose (0.3 mg/kg b.w.) plus 15-deoxyspergualin or brequinar also had an inhibitory effect on the rejection. In animals given TAC plus mycophenolate mofetil, a protective effect was observed as well; however, this effect was not consistent.     

Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model

The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.     

Beta-5 score to evaluate pig islet graft function in a primate pre-clinical model

Igarashi Y, D’hoore W, Goebbels R‐Marie, Gianello P, Dufrane D. Beta‐5 Score to evaluate pig islet graft function in a primate pre‐clinical model. Xenotransplantation 2010; 17: 449–459. © 2010 John Wiley & Sons A/S. Abstract: Background: We developed a composite scoring system to accurately assess pig islet function in pre‐clinical primate studies. Methods: Two scoring methods that have been clinically validated in human islet allotransplantation were tested in six non‐diabetic and nine streptozotocin (STZ)‐induced diabetic primates: (i) SUITO index = [1500 × fasting C‐peptide (ng/ml)]/[fasting blood glucose (FBG, mg/dl) ? 63] and (ii) CP/G ratio = [fasting C‐peptide (ng/ml) × 100]/FBG (mg/dl). Both scores were analysed as a function of the β‐cell mass of the native primate pancreas. Next, a proposed β5 score based on FBG values, daily glycosuria, post‐prandial glycosuria, polydipsia, and polyuria was validated on the same primates. Ranges of normal and pathologic values for each parameter were assessed during 5 months in non‐diabetic and diabetic primates, respectively. Finally, scores were tested on the nine STZ‐induced diabetic primates, four of which were transplanted with microencapsulated pig islets and five with macroencapsulated pig islets. All parameters required for each score were measured prior to transplantation and up to 12 weeks post‐transplantation. For the CP/G ratio after transplantation, primate C‐peptide was replaced by porcine C‐peptide. Results: The Suito index was not correlated with the pancreatic β‐cell mass in contrast to the CP/G ratio (R² = 0.17, P = 0.645 vs. R² = 0.76, P = 0.003; respectively). The internal consistency of the parameters implied by the β5 score was confirmed by a Cronbach’s alpha test of 0.97. Diabetes was confirmed by a significant decrease in the CP/G ratio and the β5 score before and after diabetes induction, respectively. After transplantation, a significant correlation was found between the CP/G ratio and the β5 score, which reflected the functionality of pig islet xenografts and diabetes control. In addition, the CP/G ratio and β5 score were correlated with the glycosylated hemoglobin course after transplantation and diabetes correction with macroencapsulated pig islets. Conclusion: The proposed β5 score provides a valid tool to accurately assess islet transplantation in a primate pre‐clinical model.     

The usefulness and limitations of the diabetic macaque model in evaluating long‐term porcine islet xenograft survival

Abstract: Background: Various groups have reported prolonged diabetes reversal and graft function after porcine islet transplantation into diabetic macaques using different experimental designs (macaque source, islet source, type of immunosuppression): subsequently, the International Xenotransplantation Association has published recommendations for entering a clinical trial. Our experiments showed limitations that affected consistent achievement of long‐term survival. We aimed to identify these limitations and underlying causes to emphasize the translational value of this highly relevant type 1 diabetic macaque model. Methods: We reviewed data from our institution and literature data on long‐term porcine islet xenograft survival in the diabetic macaque model, especially focusing on aspects of incomplete diabetes reversal relative to macaque normal values. This phenomenon was compared with diabetes reversal in an allo‐islet transplant model in macaques and with chronic insulin treatment of diabetic macaques, all with 180‐day follow‐up. This comparison enabled to identify potential model limitations and underlying causative factors. Results: Especially in the xenograft model, the achievement of long‐term graft survival revealed limitations including chronic, mild hyperglycemia and absence of body weight (BW) gain or even progressive BW loss. Metabolic incompatibilities in glycemic control (i.e., insulin kinetics) between the pig and macaque species underlie chronic, mild hyperglycemia. This phenomenon might not bear relevance for the pig‐to‐human species combination because the glycemic control in pigs and humans is similar and differs from that in nonhuman primates (NHP). Weight loss could be related to changes in the gastrointestinal tract related with local high exposure to orally administered immunosuppressants; these must be given at higher dose levels because of low bioavailability in macaques to achieve systemic exposure at therapeutic levels. This is aggravated by insufficient graft insulin production in proportion to the needs of macaques: this model limitation has no translational value to the pig‐to‐human setting. Nutritional deficits can result in incorrect interpretation of blood glucose levels and C‐peptide levels regarding graft function. Likewise, nutritional status alters physiologic responses, influencing susceptibility to infectious and noninfectious complications. Conclusion: The model‐induced confounding described interferes with accurate interpretation of safety and efficacy studies, which affects the translational value of pig‐to‐NHP islet cell transplant studies to the pig‐to‐human transplant condition.     

Induction of Human T-Cell Tolerance to Pig Xenoantigens via Thymus Transplantation in Mice with an Established Human Immune System

Thymus xenotransplantation has been shown to induce tolerance to porcine xenografts in mice and to permit survival of α1,3Gal-transferase knockout porcine kidney xenografts for months in nonhuman primates. We evaluated the ability of porcine thymus xenotransplantation to induce human T-cell tolerance using a humanized mouse (hu-mouse) model, where a human immune system is preestablished by implantation of fetal human thymus tissue under the kidney capsule and intravenous injection of CD34⁺ hematopoietic stem/progenitor cells. Human T-cell depletion with an anti-CD2 mAb following surgical removal of human thymic grafts prevented the initial rejection of porcine thymic xenografts in hu-mice. In these hu-mice, porcine thymic grafts were capable of supporting human thymopoiesis and T-cell development, and inducing human T-cell tolerance to porcine xenoantigens. Human T cells from these mice responded strongly to third-party pig, but not to the thymic donor swine leukocyte antigen (SLA)-matched pig stimulators in a mixed lymphocyte reaction (MLR) assay. Anti-pig xenoreactive antibodies declined in these hu-mice, whereas antibody levels increased in nontolerant animals that rejected porcine thymus grafts. These data show that porcine thymic xenotransplantation can induce donor-specific tolerance in immunocompetent hu-mice, supporting this approach for tolerance induction in clinical xenotransplantation .     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

Interleukin-6 in islet xenograft rejection

Earlier work on primate cardiac xenotransplantation has demonstrated a correlation between interleukin (IL)-6 levels and severity of vascular rejection. IL-6 was originally identified as a lymphokine inducing final maturation of B lymphocytes into antibody-secreting cells. The present study aimed to evaluate the role of IL-6 in fetal porcine islet-like cell cluster (ICC) xenograft rejection. Moreover, other authors have reported that eosinophils dominate the cellular response following discordant islet xenograft transplantation. Here, a technique for specific detection of eosinophils was applied. IL-6-deficient mice and wild-type controls were implanted with fetal porcine ICCs under the kidney capsule and killed 4-, 7-, and 10 days after transplantation. Xenografts were histologically evaluated, and serum samples were analyzed for IgM and IgG antibodies against ICC membrane antigens. IL-6-deficient mice and wild-type controls readily rejected the xenograft. On day 7 after transplantation, abundant numbers of F4/80⁺ and Mac-1⁺ cells were found distributed throughout the collapsing graft accompanied by small amounts of eosinophils and peripherally accumulated CD3⁺ T cells (predominantly CD4⁺). Significantly lower serum levels of IgM and IgG antibodies against ICC membrane antigens were observed in IL-6-deficient mice on day 4 or 7 after transplantation when compared to wild-type controls. No significant differences were seen on day 10 after transplantation. In both experimental groups, specific IgM and IgG antibody levels remained stable over time. In the pig-to-mouse model, IL-6 seems to be of minor importance to fetal porcine ICC xenograft rejection. Macrophages, and not eosinophils, dominate the cellular response associated with this process. Received: 10 August 1999 Revised: 21 August 2000 Accepted: 27 November 2000     

The effect of immunoglobulin immunadsorptions on delayed xenograft rejection of human CD55 transgenic pig kidneys in baboons

Delayed xenograft rejection (DXR) remains a major obstacle in discordant xenotransplantation. As strategies of complement inhibition and xenogeneic natural antibody (Ab) removal have been shown to give prolonged xenograft survival, we endeavored to determine whether combining these two strategies would lead to an additive effect in terms of graft survival. The study was initiated with two groups, A and B, where group A received normal kidneys and group B received hCD55 transgenic kidneys. Both groups underwent pre-transplant (day-1) total immunoglobulin (Ig) immunoadsorption (IA) and received an immunosuppression of cyclophosphamide, cyclosporine A, mycophenolate mofetil and corticosteroids. Two subsequent groups (C and D) receiving hCD55 transgenic pig kidneys were then performed with an 'optimized' immunosuppression (Cyclophosphamide starting 1 day earlier) but only group D recipients were immunoadsorbed. Biopsies taken during the post-transplantation period were analyzed for Ab deposition, compliment activation and cellular infiltration. No hyperacute rejection was observed. In the initial immunoadsorbed groups A and B, all baboons underwent DXR, which started surprisingly early (day 5 in most cases. In the subsequent two groups, the immunoadsorbed group D baboons also underwent DXR, again as early as day 5. In contrast, group C baboons did not show any signs of DXR on their day 6 biopsy or at their time of death. Analysis of graft biopsies from the kidneys undergoing rejection or with stable function showed strong deposition of anti-Gal IgM in all cases whereas strong complement C5b-9 deposits were only observed in biopsies at rejection. Cellular infiltration consisted mostly of monocytes/macrophages, was more pronounced in biopsies taken at rejection and was associated with a pro-inflammatory environment involving interleukins 1alpha, 6 and 8. Our findings suggest non-specific Ig (anti-Gal and non-Gal Ig of all isotypes) IA or even incomplete IA in immunosuppressed baboon recipients of transgenic pig kidneys is detrimental to graft survival by being associated with an Ab and compliment driven rejection. We speculate that the IA were insufficient in terms of Ig depletion or frequency inducing an Ab rebound or that this total Ig depletion also removed components facilitating graft survival.     

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models

Dufrane D, D'hoore W, Goebbels R‐M, Saliez A, Guiot Y, Gianello P. Parameters favouring successful adult pig islet isolations for xenotransplantation in pig‐to‐primate models.
Xenotransplantation 2006; 13: 204–214. © Blackwell Munksgaard, 2006 Abstract: Background: In the near future, adult porcine islets of Langerhans appear as an unlimited source of insulin‐producing cells which could play a major role for treating diabetes mellitus. There is, however, an obvious lack of pre‐clinical results and data in the pig‐to‐primate model. One of the main hurdles of this model is certainly related to the difficulty of reproducing regularly successful porcine islet isolation. This experimental work was designed to provide guidelines applicable in pig pancreas procurement and islet isolation for successful islet xenotransplantation into primates. Methods: Pancreases were harvested from adult Belgium Landrace pigs (n = 79) in a single centre. The impact on islet yield of (1) pancreas procurement (blood exsanguination and warm ischaemia time (WIT)), (2) cold storage solutions (classic UW and modified UW (without hydroxyethyl starch and inverse K⁺/Na⁺ concentration)), (3) a dynamic or static method of pancreas digestion, and (4) the endotoxin content and enzymatic activity from five different batches of Liberase PI was studied. In addition, pancreatic biopsies (n = 18), performed before isolation, were retrospectively analyzed to study the impact of histomorphometry on porcine islet yield. Finally, two diabetic cynomolgus monkeys were transplanted without immunosuppression with 15 000 pig islet equivalents/kg body weight of recipient to assess in vivo the function of freshly isolated islets. Univariate and multivariate analyses were performed. Results: By multiple linear regression, the most significant variables that significantly improved islet yield were, firstly, the presence of <30 EU (endotoxin units) of endotoxin in Liberase batches, followed by a WIT under 10 min and the use of blood exsanguination before pancreas harvesting (P<0.005). In contrast, isolation method (dynamic vs. static) and the solution used for storage (short‐term) (UW vs. modified UW) did not significantly influence islet yield. The correlation of retrospective histomorphometry analysis of native pancreas and extemporaneous biopsy before isolation clearly determined a positive relationship between isolated islet number and the number of islets/cm² (r = 0.708, P<0.01) or with the percentage of large islets (r = 0.680, P<0.01) found in pancreas biopsies. Pig pancreases containing more than 82 islets/cm² and more than 42% of large islets (>100 μm) thus enabled more than 120 000 islet equivalents to be obtained in 90% of the cases, which is an ideal amount of islets to transplant into a primate of 4 to 5 kg. In vivo, a reduction of blood glucose (<200 mg/dl), associated with porcine C‐peptide production, was observed in two primates after transplantation with adult pig islets. At day 7 post‐transplantation, however, loss of islet function was associated with graft destruction and immune reaction. Conclusions: Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.     

Three-yr follow-up of a type 1 diabetes mellitus patient with an islet xenotransplant

In order to alleviate the shortage of human donors, the use of porcine islets of Langerhans for xenotransplantation in diabetic patients has been proposed as a solution. To overcome rejection, we have developed a procedure for protecting the islets by combining them with Sertoli cells and placing them in a novel subcutaneous device, that generates an autologous collagen covering. A type 1 diabetic woman was closely monitored for 10 months, and then transplanted in two devices with two months of difference and a third time after 22 months. Here we present a three-yr follow-up. The close monitoring induced a rapid decrease in exogenous insulin requirements, which stabilized between 19 and 28 IU/d for nine months. After transplantation, the requirements reduced further to below 6 IU/d and for some weeks she was insulin free. Glycosylated hemoglobin levels decreased concomitantly. Porcine insulin could be detected in the serum after a glucose challenge and insulin positive cells inside a removed device after two yr. No complications have arisen and no porcine endogenous retrovirus infection has been detected through PCR and RT-PCR. This case demonstrates the feasibility of using the xenotransplantation of porcine cells to alleviate metabolic complications and insulin requirements in type 1 diabetic patients.     

Complement depletion with cobra venom factor delays acute cell‐mediated rejection in pig‐to‐mouse corneal xenotransplantation

Oh JY, Kim MK, Lee HJ, Ko JH, Kim Y, Park CS, Kang HJ, Park CG, Kim SJ, Lee JH, Wee WR. Complement depletion with cobra venom factor delays acute cell‐mediated rejection in pig‐to‐mouse corneal xenotransplantation. Xenotransplantation 2010; 17: 140–146. © 2010 John Wiley & Sons A/S. Abstract: Background: We have demonstrated earlier that porcine corneal xenografts underwent an acute cell‐mediated rejection in mice despite the absence of T cells. In the present study, we investigated the effect of complement depletion by cobra venom factor (CVF) on the corneal xenograft rejection in a pig‐to‐mouse model. Methods: Porcine corneas were orthotopically transplanted into C57BL/6 (B6) and severe combined immunodeficiency (SCID) mice. For complement depletion, 25 μg of CVF (1 g/kg bodyweight) was injected intraperitoneally on the day before and 1, 3, 5, and 7 days after transplantation. Graft survival was clinically assessed by slit lamp biomicroscopy and the median survival time (MST) was calculated. The grafts were histologically evaluated serially after transplantation using antibodies against CD4, CD8, NK1.1, and F4/80. Results: The CVF treatment significantly prolonged the porcine corneal xenograft survival in both B6 (MST 9.4 vs. 15.5 days; P = 0.0011) and SCID mice (MST 16.4 vs. 20.5 days; P = 0.0474). Histologically, whereas macrophages and CD4⁺ T cells were progressively infiltrated into porcine corneal grafts in CVF‐untreated B6 mice, the infiltration by both cells was markedly delayed and decreased in the xenografts in CVF‐treated B6 mice. Likewise, macrophage infiltration, which was prominent in rejected porcine xenografts in SCID mice, was also reduced in CVF‐treated SCID mice. Conclusions: Our results suggest that complement depletion by CVF delayed, although did not prevent, an acute cell‐mediated rejection in a pig‐to‐mouse corneal xenotransplantation.     

Paramagnetic microparticles do not elicit islet cytotoxicity with co-culture or host immune reactivity after implantation

Suszynski TM, Rizzari MD, Kidder LS, Mueller K, Chapman CS, Kitzmann JP, Pongratz RL, Cline GW, Todd PW, Kennedy DJ, O’Brien TD, Avgoustiniatos ES, Schuurman H‐J, Papas KK. Paramagnetic microparticles do not elicit islet cytotoxicity with co‐culture or host immune reactivity after implantation. Xenotransplantation 2011; 18: 239–244. © 2011 John Wiley & Sons A/S. Abstract: Background: Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular purification of porcine islets as a potential xenotransplantation product. We assessed whether MPs affect islet function or induce an adverse effect following implantation. Methods: Porcine islets were co‐cultured with 0, 500, and 1500 MPs per islet equivalent (IE) for 1 day and with 0 and 1500 MPs/IE for 7 days. Fractional viability was assessed using oxygen consumption rate normalized to DNA content (OCR/DNA) and after 7‐day co‐culture by perifusion glucose‐stimulated insulin secretion (GSIS) and by transplantation under the renal capsule of diabetic nude mice. To assess an inflammatory response or immune reaction, MPs (～10⁷) were implanted under the renal capsule of C57BL/6 mice. Results: No statistically significant differences were measured in OCR/DNA (mean ± SE) following 1‐day co‐culture with 0, 500, or 1500 MPs/IE (243.3 ± 4.5, 211.3 ± 8.1, or 230.6 ± 11.3 nmol/min·mgDNA, respectively) or following 7‐day co‐culture with 0 or 1500 MPs/IE (248.5 ± 1.4 or 252.9 ± 4.7 nmol/min·mgDNA, respectively). GSIS was not affected by the presence of MPs; first‐ and second‐phase insulin area‐under‐the‐curve (mean ± SE) reflected no statistically significant differences after 7‐day co‐culture between 0 and 1500 MPs/IE (8.36 ± 0.29 and 8.45 ± 0.70 pg/ml·min·ngDNA for first‐phase; 69.73 ± 2.18 and 65.70 ± 4.34 pg/ml·min·ngDNA for second‐phase, respectively). Islets co‐cultured with MPs normalized hyperglycemia in diabetic nude mice, suggesting no adverse effects on in vivo islet function. Implantation of MPs did not elicit tissue injury, inflammatory change or immune reactivity. Conclusion: MPs do not adversely affect islet viability or function during co‐culture, and MPs are not immune reactive following implantation.     

Circulating pig‐specific DNA as a novel biomarker for monitoring xenograft rejection

Monitoring for immune rejection is crucial for long‐term survival of pig xenografts. Circulating DNA is a promising non‐invasive biomarker for either organ injury or response to therapy. In this study, circulating pig‐specific DNA (cpsDNA) was monitored during xenograft rejection. Potential targets of cpsDNA were selected by in silico analysis, and species specificity of selected primers was confirmed by PCR. Subsequently, cpsDNA as a biomarker was evaluated using a complement‐dependent cytotoxicity (CDC) assay in vitro. Then, early diagnosis and response to rapamycin were assessed by an in vivo imaging model of pig‐to‐mouse cell transplantation. Finally, cpsDNA was monitored in a pig‐to‐monkey artery patch transplantation model. The results showed that (a) a method of cpsDNA quantitation was established for application in mouse and nonhuman primate models; (b) cpsDNA reflected CDC in vitro; (c) cpsDNA in vivo mirrored xenograft rejection, and correlated with xenograft loss in pig‐to‐mouse cell transplantation; (d) cpsDNA was significantly reduced when rapamycin was administered; and (e) dynamic cpsDNA was detectable in pig‐to‐monkey artery patch transplantation. In conclusion, measurement of cpsDNA could prove to be a less invasive, but more specific and sensitive low‐cost biomarker enabling monitoring of xenograft rejection and the response to immunosuppressive therapy.     

Species incompatibilities in the pig-to-macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation

Graham ML, Bellin MD, Papas KK, Hering BJ, Schuurman H‐J. Species incompatibilities in the pig‐to‐macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation. Xenotransplantation 2011; 18: 328–342. © 2011 John Wiley & Sons A/S. Abstract: Background: Porcine islet transplantation into diabetic non‐human primates is considered most relevant in translational research supporting a clinical application. Most studies have focused on immunosuppressive protocols, while metabolic aspects have mainly been utilized in graft monitoring. We evaluated data from our group regarding human and non‐human primate (NHP) allotransplantation and pig‐to‐NHP xenotransplantation to identify incompatibilities in metabolic factors and their consequences for transplant outcomes. Methods: Basic gluco‐metabolic parameters (fasting blood glucose, C‐peptide, and response to stimulation with arginine or glucose) were derived from literature (humans), 72 macaques, and 47 adult Landrace pigs. Islet preparations from 15 human deceased donors, 61 macaques, and 23 adult pigs were compared with respect to yield, fractional viability assessed by oxygen consumption normalized for DNA, and in vitro glucose‐induced insulin release. Metabolic parameters at day 75 after a single islet transplantation in the liver were compared for 19 patients and 9 macaques receiving an allotransplant and 11 macaques receiving a porcine xenotransplant: recipients received chronic immunosuppression. Results: Pigs differ from NHPs and humans by a much lower C‐peptide level (0.42 vs. 1.3 to 2.0 ng/ml, respectively) and a 2‐ to 7‐fold lower C‐peptide response to arginine stimulation. In contrast, NHPs have the highest metabolic demand as evidenced by a high C‐peptide and high C‐peptide response to arginine stimulation; values are about twice higher than in humans. For manufactured islet preparations, these differences are reflected by glucose‐stimulated insulin release (the stimulation index for pigs is 1.5, for humans 3.8, and for macaques 7.7), but not by fractional viability, which was in the same range. The day 75 outcome after transplantation assessed by C‐peptide was similar for allotransplanted humans and NHPs (80 to 90% good graft function) and lower in xenografted NHPs (36% good graft function); gluco‐metabolic parameters were in accordance with graft function, albeit different between species because normoglycemia under exogenous insulin is maintained more aggressively in patients than in NHPs. In xenografted NHPs, the shift in glycemic control with respect to normal values, combined with low values of circulating porcine C‐peptide, resembled more the normal condition in a pig than that in a macaque. Conclusions: The substantially lower glucose‐induced insulin response in adult porcine islet preparations as opposed to islets manufactured from humans or macaques combined with the much higher need for insulin in macaques than in humans creates an imbalance between the metabolic demand and the engrafted islet mass in the pig‐to‐NHP xenograft recipient. Engrafted islet mass is affected by dose, suggesting that a much higher dose level of islets is necessary in the xenogeneic setting than in human or NHP allotransplantation, and pig islets need to be given at a higher dose in macaques than the anticipated effective dose in humans. To cope with differences in metabolic demand and presumably also metabolic dynamics, a liberal regime in supportive exogenous insulin might be essential to achieve long‐term survival. These intrinsic characteristics of the NHP model deserve consideration to optimally design experimental studies with the perspective of translational value of results.