Pig Islets Research Articles

Fate of alphaGal +/+ pancreatic islet grafts after transplantation into alphaGal knockout mice.

Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-alpha1,3-galactose (alphaGal) epitope on endothelial cell surfaces and therefore have preformed anti-alphaGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigated the role of anti-alphaGal antibodies in rejection of non-vascularized alphaGal+/+ grafts in alphaGal -/- mice. alphaGal +/+ and alphaGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) alphaGal -/- mice and (2) alphaGal +/+ mice. alphaGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-alphaGal antibody levels. Six of the 18 alphaGal -/- mice rejected the alphaGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with alphaGal +/+ skin grafts. Although all alphaGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of alphaGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-alphaGal antibody. The results suggest that the level of alphaGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy.

No transmission of porcine endogenous retrovirus after transplantation of adult porcine islets into diabetic nude mice and immunosuppressed rats.

The aim of this study was to investigate whether transmission of porcine endogenous retrovirus (PERV) occurs in a model of diabetes reversal by the xenotransplantation of adult porcine islets (APIs) into immunoincompetent diabetic rodents. Black-6 nu/nu mice and Lewis rats were immunosuppressed with cyclosporin A (CsA) and FTY 720, and rendered diabetic with streptozotocin. Purified APIs were transplanted into the renal subcapsular space; 5,000 islet equivalents (IEQs) were used in the nude mice (n = 4) and 40,000 IEQs in the rats (n = 4). The nude mice were sacrificed at 75 days after transplantation. In order to confirm chronic xenograft function, the graft-bearing kidney was removed prior to sacrifice. The rats were followed until xenograft rejection, at which time they were sacrificed. Immediately after sacrifice, tissue samples (liver, spleen, and small intestine) were taken for analysis. Quantitative polymerase chain reaction (PCR) was used to assess evidence of PERV transmission, and porcine cell chimerism. All animals became normoglycemic within 48 h of transplantation. The nude mice remained normoglycemic during the 75-day study period, with removal of the graft-bearing kidney resulting in prompt hyperglycemia. The rats remained normoglycemic until xenograft rejection, which occurred at 66 +/- 28 days. Despite the evidence of porcine cell microchimerism in recipients, real-time PCR detected no evidence of PERV transmission in any of the tissue specimens tested. There was no evidence of PERV transmission following transplantation of pig islets into diabetic nude mice and immunosuppressed rats.

Low–molecular weight dextran sulfate abrogates the instant blood-mediated inflammatory reaction induced by adult porcine islets both in vitro and in vivo

Background One of the main obstacles to clinical application of islet xenotransplantation is the injurious inflammatory reaction elicited by porcine islets when they are exposed to fresh human blood in vitro and in vivo. This instant blood-mediated inflammatory reaction (IBMIR) causes rapid binding of platelets to the islet surface, activation of the coagulation and complement systems, and leukocyte infiltration of the islets. As a consequence of IBMIR, morphological destruction of porcine islets occurs within the first few hours after transplantation. Materials and methods In the present study, by analyzing the plasma samples and performing immunohistochemical investigation, we assessed the effect of adding low–molecular weight dextran sulfate (LMW-DS) at 0.01–1 mg/mL to an in vitro tubing loop assay in which porcine islets were exposed to fresh human blood. The effect of LMW-DS also was investigated in an in vivo model using diabetic athymic mice, which provides an innate inflammatory milieu without influence of T cells. The possible toxicity of LMW-DS was assessed by culturing pig islets in the presence or absence of LMW-DS for 3 days. Results In the in vitro study, in the presence of LMW-DS at 0.01 mg/mL, platelet consumption, coagulation, and complement activation were reduced, and, at 0.1 mg/mL, LMW-DS totally prevented IBMIR. Immunohistochemical investigation showed that leukocyte infiltration was totally abrogated at the highest dose. A similar finding was observed in the in vivo study. No adverse effect of LMW-DS was observed on the quality of the islets. Conclusions LMW-DS appears to be an effective drug candidate that is able to control the strong innate immune response induced by pig islets in contact with human blood.

Development of a novel digestion chamber for human and porcine islet isolation

Background The current technique of human pancreas digestion for islet isolation relies on selective distribution of collagenase delivered via the pancreatic duct to produce digestion and removal of peri-acinar fibrous tissue. However, the collagenase has relatively little effect on the interlobular fibrous tissue, which must therefore be broken down by mechanical means within the digestion chamber so as to release the contained acini and islets. The current way of achieving this in the Ricordi chamber is to place five or six stainless steel balls within the chamber and shake vigorously. The shaking presumably breaks down the interlobular fibrous tissue by a combination of shear force induced by the movement of tissue through the shaking process, assisted by numerous blows from the steel balls. Intuitively, one would expect some islets would be destroyed rather than released by such a battering. Methods In an attempt to improve the efficiency of islet isolation we have designed a new digestion/filtration chamber that consists of a glass cylinder, sealed with Teflon plates holding in mesh filters at each end, secured in place by a central threaded tie-rod and external knurled nuts. A ring-shaped piston within the cylinder can be pushed up and down the travel by two rods passing out through sealed ports in the Teflon disk at one end and connected to an external handle. The handle is used to gently push the piston up and down the travel of the cylinder, which pushes the fluid and tissue through the central lumen of the ring-piston. A series of hooks attached to the central tie-rod catch the fibrous strands of the passing tissue; the shearing forces produced cause disruption by a process thought to be similar to teasing the tissue apart with fine forceps. Results A series of initial experiments with human pancreas showed the prototype to be too large, causing temperature control problems, and a redesigned smaller chamber was produced, maintaining the crucial design features. Experience processing five human pancreata has now demonstrated that in three of five pancreata the new chamber produced a good yield (>200,000 I.E.) of remarkably well separated and intact islets, the entire dispersion process being under 1 hour. However, in two isolations the collagenase digestion was poor, with few free islets. A copy of the new chamber (reserved for porcine work only) has been produced, as well as a copy of the Ricordi chamber. We have confirmed that the new chamber can isolate porcine islets in large numbers (>5000 islets/g pancreas [n = 2], but note that pig islets are small). Conclusion These preliminary studies are sufficiently encouraging to justify further direct comparison with the Ricordi chamber for the purpose of animal and human islet isolation.

A study of the xenoantigenicity of adult pig islets cells

The pig pancreas is considered to be the most suitable source of islets for xenotransplantation into patients with type I diabetes. The purpose of this study was to assess the antigenicity of pig islets, including the Galalpha1-3Galbeta1-4GlcNAc-R (the alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. The expression of alpha-Gal on islets from adult pigs was investigated by immunohistochemical staining and flowcytometric analysis. The alpha1,3 galactosyltransferase (alpha1,3GT) activity of islets was determined by high-performance liquid chromatography. Antigenicity to human natural antibodies, including the H-D antigen of pig islets was next examined by treatment of pig islets with tunicamycin, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and/or neuraminidase. In addition, complement-mediated islets lysis was examined using factor D-deficient and C1-deficient sera. Adult pig islets expressed negligible amounts of alpha-Gal epitope, and alpha1,3GT activity was also undetectable. However, human natural antibodies, immunoglobulin G and M, and the anti H-D antibody react to the adult islet. Treatment of pig islets with tunicamycin, but not PDMP, led to a drastic reduction in antigenicity to human serum, indicating the importance of N-linked sugars on the islets. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens that reacted with the human natural antibody. The complement deposition of C4, C3 and factor B on islets was demonstrated. The alternative pathway-mediated pig islet killing accounted for approximately 30% of that by the total complement pathway. The origin of antigenicity of pig islets is mainly N-linked sugars including sialic acid antigens, but not the alpha-Gal, and pig islets can be injured by both the classical and the alternative complement pathway in human serum.

Low molecular weight dextran sulfate prevents the instant blood-mediated inflammatory reaction induced by adult porcine islets.

One of the main obstacles to clinical islet xenotransplantation is the injurious instant blood-mediated inflammatory reaction (IBMIR) that causes rapid binding of platelets to the islet surface, activation of the coagulation and complement systems, and leukocyte infiltration of the islets when the islets are exposed to blood. This study assesses the effect of low molecular weight dextran sulfate (LMW-DS) on IBMIR induced by porcine islets in an in vitro tubing loop assay using human blood and in an in vivo model using diabetic athymic mice. In vitro experiments demonstrated that platelet consumption, coagulation, and complement activation were already reduced in the presence of LMW-DS at 0.01 mg/mL, and that at 0.1 mg/mL, LMW-DS prevented IBMIR. Immunohistochemical investigation showed that the leukocyte infiltration was abrogated at the highest dose. In vivo experiments showed that the transplanted pig islets survived for a significantly longer period in recipients treated with LMW-DS, and morphologic examination of transplanted islets showed a reduction in IBMIR analogous to that demonstrated by in vitro studies. Given that LMW-DS has been used in clinical studies without serious adverse reactions, it has potential as a drug candidate that can control the strong innate immune response induced by pig islets when transplanted through the portal vein.

Intraportal pig islet xenotransplantation into athymic mice as an in vivo model for the study of the instant blood‐mediated inflammatory reaction

One of the main obstacles to successful intraportal islet transplantation is the instant blood-mediated inflammatory reaction (IBMIR) elicited by the isolated islets when exposed to fresh human blood. In the present study, we investigated whether intraportal transplantation of pig islets into diabetic athymic mice could be used as a small animal model to study xenogeneic IBMIR in vivo. Adult porcine islets (APIs) or rat islets were implanted into the portal vein or under the renal subcapsular space of diabetic athymic mice. Graft survival and morphology were evaluated by measuring blood glucose levels and by performing immunohistochemical staining, respectively. Transplantation of rat islets, irrespective of implantation site, cured all diabetic athymic mice. APIs transplanted subcapsularly also cured all diabetic athymic mice, while none of the animals transplanted with an equivalent amount of APIs via the portal vein remained normoglycemic for more than 10 days after transplantation. Immunohistochemical staining on day 7 showed that most of intraportally transplanted APIs were entrapped in clots and infiltrated with CD11b+ leukocytes. Intraportal transplantation of APIs into athymic mice induced IBMIR, thus providing a small animal model for studying xenogeneic IBMIR.

T-cell-specific immunosuppression results in more than 53 days survival of porcine islets of langerhans in the monkey.

Transplantation of islets of Langerhans can restore insulin production in diabetic patients. Because of the shortage of human donor organs, transplantation of porcine islets may be an alternative solution. The present study was aimed at the characterization of rejection mechanisms of porcine islets transplanted into eight nondiabetic monkeys under the kidney capsule. Cultured adult pig islets were used, which showed no expression of the galactose(alpha1,3)galactose epitope, major histocompatibility complex class II, or CD45, and no binding of antibodies or complement after exposure to monkey serum. Immunosuppression consisted of cyclophosphamide, cyclosporine A (CsA), and steroids (group 1); or antithymocyte globulin, anti-interleukin-2 receptor antibody, CsA, and steroids (group 2). In three animals of group 2, islets were also transplanted in the portal vein. Although all monkeys had preformed anti-pig antibodies, no correlation was found between antibody titers and rejection and no deposition of antibodies or complement was observed in the grafts. Group 1 showed islets up to day 11, followed by T-cell infiltration and rejection at approximately day 14. In group 2, two monkeys showed infiltrates consisting predominantly of T cells starting at approximately day 29, whereas two monkeys showed well-preserved islets without infiltration up to day 53. In the livers of the three monkeys that also received islets intraportally and were resectioned on days 21, 33, and 49, no islets could be detected. This study demonstrates that cultured adult pig islets can survive in the monkey for more than 53 days without signs of rejection under standard immunosuppression.

Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43 degrees C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1beta, IFN-gamma, or TNF-alpha was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.

CD46 protects pig islets from antibody but not cell-mediated destruction in the mouse.

We have previously reported that islets present in cultured fetal pig pancreas (FPP) are resistant to destruction by Galalpha(1,3)Gal antibodies and compliment, but are susceptible to the 'secondary' antibody response which occurs on transplanting pig islet tissue to Galo/o murine recipients. In a model of antibody-mediated graft rejection, we tested the resistance of porcine islets to antibody. Using FPP from human CD46 transgenic pigs, we now report that the complement regulator, CD46, affords protection from antibody-mediated rejection when mouse anti-pig serum (MAPS) was administered to scid mice bearing PFF grafts from either CD46 transgenic or normal pigs. Indeed, whereas normal pig islets were destroyed by an intraperitoneal (i.p.) injection of 0.1 to 0.2 ml of MAPS antibody, destruction of CD46-expressing transgenic islets required 0.5 ml, i.e. up to five times the amount. In contrast, there was no prolongation of the survival of CD46 transgenic mouse skin or heart major histocompatibility complex-compatible or -incompatible allografts--rejected by predominantly cellular immune mechanisms, as opposed to xenograft rejection. Although complement regulators have been examined for their protective role in hyperacute rejection of vascularized xenografts, it is clear that they also have protective effects in the later, antibody-mediated responses, but are unlikely to effect the inflammatory response in cell-mediated rejection.

Heat-exposed pig islets are protected from inflammatory mediators but express proapoptotic proteins

Heat-exposed pig islets are protected from inflammatory mediators but express proapoptotic proteins

Subcutaneous transplantation of macroencapsulated porcine pancreatic endocrine cells normalizes hyperglycemia in diabetic mice.

The ultimate goal of islet transplantation is the unlimited availability of insulin-secreting cells to be transplanted in a simple procedure that requires no use of immunosuppressive drugs. Immunoisolation of xenogeneic pig islets for transplantation has great potential therapeutic benefits for treatment of diabetes. Approximately 4 x 10(6) porcine pancreatic endocrine cells (PEC) isolated from 6-month-old pigs were macroencapsulated in agarose-poly(styrene sulfonic acid) mixed gel and implanted into a prevascularized subcutaneous site in streptozotocin-induced C57BL/6 diabetic mice. Animals receiving an equal number of free porcine PEC were used as controls. After transplantation, nonfasting blood glucose, body weight, intraperitoneal glucose tolerance test, and immunohistologic evaluations were processed. All 10 animals receiving the subcutaneous xenografts of the macroencapsulated porcine PEC normalized hyperglycemia within 5 days after transplantation, maintained the duration of normoglycemia for 24 to 76 days, and gradually gained weight. The subcutaneous xenografts of free porcine PEC could not reverse hyperglycemia. The recipient became hyperglycemic again when the implanted graft was retrieved at day 45 after transplantation. The glucose clearances were significantly ameliorated at day 21 and day 45 after transplantation when compared with those in diabetic mice. The immunohistochemical results revealed an inherent intact structure of the macroencapsulated porcine PEC and positive double-immunofluorescence staining for insulin and glucagon. Subcutaneous transplantation of macroencapsulated porcine PEC normalized hyperglycemia in diabetic mice. Our results identified a potential for a favorable development of subcutaneous transplantation of porcine PEC as a cure for diabetes.

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.

Adenovirus-mediated expression of human CD55 or CD59 protects adult porcine islets from complement-mediated cell lysis by human serum.

Protection against complement activation may reduce acute islet damage in pig-to-human islet xenotransplantation. Expression of the human complement regulatory proteins decay-accelerating factor (DAF, CD55) or CD59 was induced on intact adult porcine islets (APIs) by adenoviral transduction. The functional capacity of the transgenes was examined in vitro after exposure to fresh human serum. Intact APIs were transduced with adenoviral vectors Ad.hDAF or Ad.hCD59 or a control vector. After 3 days, the islets were trypsin dissociated to a single-cell suspension. A cytotoxicity assay was performed in which the islet cells were incubated with human complement active AB serum. Flow cytometry and immunohistochemistry were used to evaluate transgene expression. APIs could be transduced to express hDAF or hCD59. Flow cytometry analysis of islet single cells revealed that only a fraction of the cells expressed the transgene; immunohistochemical staining of transduced islets demonstrated that mainly cells located in the periphery of the islets were expressing the protein. Cells from nontransduced islets or islets expressing the control protein were sensitive to lysis in human sera (66+/-4.0% and 73+/-3.7% cytotoxicity, respectively). Single cells from islets transduced with hDAF and hCD59 were partially protected from lysis. Islet cells expressing hCD59 were slightly less sensitive to lysis (33+/-3.3%) than cells expressing hDAF (45+/-3.5%). These data show that intact pig islets can be transduced to express human regulators of complement activation on the surface and that pig islet cells expressing hDAF or hCD59 are less sensitive to complement-mediated lysis.

Pig islet xenografts are resistant to autoimmune destruction by non-obese diabetic recipients after anti-CD4 treatment.

In addition to providing a large source of donor tissue, xenogeneic islet transplantation might avoid recurrent autoimmunity in patients with type 1 diabetes. To examine this possibility further, xenogeneic pig islets were transplanted into recipient mice in the presence or absence of autoimmunity. Spontaneously, non-obese diabetic (NOD) recipients rejected isografts rapidly whether or not the recipients were depleted of CD4+ T-cells. Young NOD mice made diabetic with streptozotocin accepted islet isografts without immunosuppression, indicating that destructive autoimmunity did not develop in these recipients. Pig xenografts were rejected equally quickly in the two types of NOD recipients in the absence of immunosuppression and survived for up to 9 weeks in both types of NOD recipients after CD4 depletion. BALB/c mice often accepted pig xenografts indefinitely after anti-CD4 antibody treatment. These results suggest that pig islets are resistant to recurrent autoimmunity when CD4+ T-cells are depleted. The difficulty in obtaining indefinite islet xenograft survival in NOD recipients occurs independently from the development of destructive autoimmunity.

The role of chemokines and their receptors in the rejection of pig islet tissue xenografts.

The mechanism by which inflammatory cells are recruited to pig islet tissue (proislet) xenografts was investigated by examining the intragraft mRNA expression of murine alpha- and beta-chemokines in CBA/H mice from days 3 to 10 post-transplant. Xenograft rejection was associated with early intragraft transcript expression for monocyte chemotactic protein-1 (MCP-1) (3 to 5 days), IP-10 (3 to 4 days) and macrophage inflammatory protein-1alpha (MIP-1alpha) (3 to 5 days) and subsequent expression of eotaxin (days 4 to 10), MIP-1beta (days 4 and 5) and regulated on activation, normal T cell expressed and secreted (RANTES) (days 4 to 6) mRNA. This pattern was consistent with the early recruitment of macrophages (MCP-1, MIP-1alpha), the influx of CD4 T cells (MCP-1, MIP-1alpha, MIP-1beta, IP-10 and RANTES) and the characteristic infiltrate of eosinophils (eotaxin and RANTES) associated with islet xenograft rejection. Inhibition of beta-chemokine signaling in CCR2-/- mice (which lack the major co-receptor for MCP-1) resulted in retarded macrophage and CD4 T cell recruitment, enhanced eosinophil influx and a minor delay in rejection, compared with wildtype mice; there was little effect on leukocyte infiltration in xenografts harvested from CCR5-/- mice (lacking the co-receptor for MIP-1alpha, MIP-1beta and RANTES). The impeded migration of leukocytes into xenografts in CCR2-/- hosts was associated with delayed intragraft expression of MCP-1 and RANTES mRNA; absence of MCP-1/CCR2-mediated signaling led to enhanced intragraft expression of MCP-1, MIP-1alpha and MIP-1beta mRNA. These findings suggest that MCP-1 plays an important role in regulating macrophage and CD4 T cell infiltration to xenograft sites via the CCR2 signaling pathway. Additional treatment of xenografted CCR2-/- transplant recipients with anti-interleukin-(IL)-4 and anti-IL-5 mAbs further delayed xenograft rejection demonstrating the potential for combined antirejection strategies in facilitating pig islet xenotransplantation.

Alginate microcapsules for pancreatic islet cell graft immunoprotection: struggle and progress towards the final cure for type 1 diabetes mellitus

Lights and shadows have been associated with the use of alginate/polyaminoacidic microcapsules for transplantation of pancreatic islet cells for the therapy of diabetes mellitus, with no recipient pharmacological immunosuppression. In fact, preliminary success in rodents has generally not matched the results achieved in diabetic higher mammals. The restricted availability of cadaveric human donor organs/tissue, coupled to regulatory hurdles in the use of microcapsules in patients, has significantly delayed the progress of microencapsulated islet grafts into pilot clinical trials. While the basic formulation of microcapsules from the author’s laboratory, originally comprised of an alginate gel (AG) core, a double poly-L-ornithine (PLO) coat and an outer AG coat, has virtually remained unchanged, highly purified ‘clinical grade’ AG has been introduced in order to try to surmount regulatory restrictions. In parallel, novel insulin-producing cell types have been employed to fill the capsules, with particular regard to non-human tissue, such as adult and, more recently, neonatal porcine islets. In particular, using neonatal porcine islets enveloped in AG-PLO microcapsules, hyperglycaemia has been corrected in several diabetic animal models. Should standardisation and optimisation problems associated with both AG procurement and other membrane physical–chemical fabrication parameters be surmounted, microcapsules containing either human or, possibly, pig islets, could be close to approval for Phase I human clinical trials.

Xenotransplantation of pig islets in diabetic dogs with use of a microcapsule composed of agarose and polystyrene sulfonic acid mixed gel.

The authors have designed a microcapsule composed of agarose and polystyrene sulfonic acid (PSSa) mixed gel that provides a protective barrier against complement attack. Xenografts of islets, encapsulated in an agarose-PSSa microcapsule, have been shown to normalize blood glucose in rodents with chemically induced diabetes for extended periods of time without immunosuppression. To investigate the efficacy of agarose-PSSa microencapsulated pig islets in reversing diabetes in a large animal model. Diabetes was induced in beagle recipients by total pancreatectomy. Each recipient received three to five intraperitoneal injections of either encapsulated (n = 5) or nonencapsulated pig islets (n = 2). In all dogs receiving microencapsulated islets, the graft function was achieved for 7.4 +/- 3.1 weeks (mean +/- standard error), as determined by elimination or reduction of exogenous insulin requirement. In three recipients, the fasting blood glucose levels were maintained at < or = 200 mg/dL without any exogenous insulin for a period of 6, 50, and 119 days. Circulating porcine C-peptide was detected in the sera of all dogs after transplantation of encapsulated islets. Immunohistologic examination revealed the presence of insulin-positive cells in the microcapsules. In contrast, in two dogs receiving nonencapsulated islets there was no graft function. This preliminary study demonstrates that agarose-PSSa microencapsulated pig islets can survive and function for weeks or months in totally pancreatectomized diabetic dogs without immunosuppression.

Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice.

Pig islets could transmit porcine endogenous retroviruses (PERV) to diabetic patients. Our previous work showed that pig islets expressed low levels of PERV mRNA and were not likely to transmit PERV to human cells in vitro. The real risk of infection during pig tissue xenografts can only be evaluated by in vivo experiments. Nude mice bearing tumours containing human 293 cells were grafted with specific pathogen-free pig islets or PERV-producing pig PK15 cells to determine whether pig cells could transmit PERV to mouse and human cells in vivo. Infection was monitored by PCR, long PCR, RT-PCR and long RT-PCR. As detection of PERV sequences could be due to the presence of residual pig cells, we looked for pig mitochondrial (mt) DNA. Quantitative PCR for PERV and pig mt DNA was done to compare the PERV-to-pig mt (P-to-M) ratio of each sample with the reference ratio for grafted pig cells. Among 78 mouse tissues from PK15-grafted mice, 54 and 72 were positive for gag and pig mt DNA, respectively. Human tumours developed in these mice were positive for PERV (78%) and pig mt (89%). The P-to-M ratios for mouse tissues and PERV-positive human tumours from PK15-grafted mice were higher than the ratio in PK15 cells. Among 41 tissues from pig islet cell-grafted mice, 7 were positive for PERV (3 lymph nodes, 1 kidney, 2 salivary glands, 1 ovary), and 14 were positive for pig mt DNA. Three of these samples (1 lymph node, 1 kidney and 1 salivary gland) were positive for gag DNA, but negative for pig mt DNA. One human tumour in these mice was positive for PERV DNA. P-to-M reference ratio in grafted islet cells was 0.05+/-0.03. The three PERV-positive lymph nodes contained 78 gag/3 mt copies (P-to-M: 26), 101 gag/3 mt copies (P-to-M: 34), and 4 gag/0 mt copies. The two PERV-positive salivary glands contained 14 gag/1 mt copies, and 28 gag/0 mt copies. The ovary and the kidney contained 46 gag/3 mt and 69 gag/0 mt copies, respectively. The PERV-positive human tumour contained 47 gag/3 mt copies. Microchimerism and PERV transmission were frequently observed in both mouse and human tissues during grafting of pig PK15 cells into nude mice bearing human tumours, and sometimes during pig islet xenograft in this model. This strengthens the notion that there is a risk of transmitting PERV during pig islet xenograft.

Feeding NOD mice with pig splenocytes induces transferable mechanisms that modulate cellular and humoral xenogeneic reactions against pig spleen or islet cells.

We have reported previously that oral administration of pig cells to NOD mice modified xenogeneic cellular response against pig islet cells (PICs), and hypothesized that it may have induced active suppression. This preliminary report evaluated only the effect of feeding pig cells by 'primary' proliferation, i.e. when splenocytes from fed mice are confronted with pig cells in vitro. The present study also considered 'secondary' proliferation and cytokine production after feeding and subsequent in vivo graft of pig cells. Additionally, serum IgM and IgG isotypes were quantified by ELISA using pig target cells. Induction of active mechanism by feeding was hypothetical, which led us here to transfer splenocytes from mice fed pig spleen cells (PSC) and evaluate 'primary' (after transfer) and 'secondary' (after transfer and subsequent graft of pig cells) proliferations and cytokine secretions in recipient mice. We also determined whether the effects of feeding pig cells persisted after depression of suppressor mechanisms by cyclophosphamide. Mice fed with PSC displayed increased 'primary' splenocyte proliferation to PSC or PIC (P < 0.0001), while 'secondary' responses were decreased (P < 0.03) in those fed PSC and subsequently grafted with PSC. The increased 'primary' and decreased 'secondary' proliferations were reduced (P < 0.04) by pretreatment with cyclophosphamide. The IL-10/ and IL-4/IFNgamma ratios produced in response to PSC increased (P < 0.04) in mice fed and grafted with PSC compared to those grafted only with PSC. IgM and IgG levels against pig cells were, respectively, increased (P < 0.04) and decreased (P < 0.04) in mice fed and grafted with PSC. IgG2a and IgG2b, but not IgG1, levels were lower (P < 0.01). These effects of feeding PSC on 'secondary' proliferation, cytokine and antibody productions, were not detected when mice were fed PSC only after graft with PSC. Transfer with splenocytes from mice fed PSC increased 'primary' proliferation of splenocytes from recipient mice in response to PSC (P < 0.02) or PIC (P < 0.05). After transfer with splenocytes from PSC-fed mice and graft with PSC, 'secondary' proliferation to pig cells were reduced (P < 0.04), and the IL-10/IFNgamma ratio produced in response to PSC was increased fourfold. Thus, oral administration of PSC induces active transferable mechanisms, characterized by a biphasic pattern with early increased 'primary' xenogeneic cellular reactions to both PSC and PIC, followed by decreased 'secondary' responsiveness and a concomitant shift of the Th1/Th2 balance towards greater Th2 influence. Decreased responsiveness may be due to active suppression, even though induction of anergy or deletion cannot be excluded.