Diabetic rats transplanted with adult porcine islets and immunosuppressed with cyclosporine A, mycophenolate mofetil, and leflunomide remain normoglycemic for up to 100 days

BACKGROUND: Transplantation of adult porcine islets (APIs) offers a possible means of treating diabetes. However, isolating APIs has been notoriously difficult. Furthermore, islet xenograft rejection must be prevented. MATERIALS AND METHODS: APIs were isolated by a modified automated method. API quality was assessed by static glucose stimulation (SGS), by transplantation to diabetic nude mice and by intraperitoneal glucose tolerance tests (IPGTTs). The morphologic characteristics of API xenograft rejection in rats were studied immunohistochemically. Furthermore, APIs were transplanted to diabetic rats that were either left untreated or immunosuppressed with cyclosporine A (CsA), mycophenolate mofetil (MMF) and leflunomide (LEF). B-glucose and porcine C-peptide levels were monitored and grafts were studied morphologically. RESULTS: Large numbers of APIs were isolated. At SGS, insulin release increased significantly. All nude mice transplanted with APIs were normoglycemic within 24 hr and remained so for up to 1 year. During IPGTTs, B-glucose levels were rapidly regulated to porcine levels. In untreated rats, API xenografts were destroyed within 6 days by a cellular infiltrate consisting mainly of macrophages. In untreated diabetic rats normoglycemia was sustained for 5.5+/-0.3 days. Rats immunosuppressed with CsA+MMF+LEF remained normoglycemic for 59.6+/-11.3 days. In 3 of 11 rats, normoglycemia was sustained for up to 101 days. Porcine C-peptide was detected in serum. At recurrence of hyperglycemia, many mononuclear cells were found close to the xenografts. However, only occasional cells infiltrated the grafts and many APIs were intact. CONCLUSIONS: Well-functioning APIs can be isolated in large numbers. API xenografts can be protected from rejection and can maintain an adequate function for up to 100 days, in rats immunosuppressed with CsA+MMF+LEF.     

Immunosuppression with FTY720 and cyclosporine A inhibits rejection of adult porcine islet xenografts in rats

BACKGROUND: Our aim was to evaluate the effect of FTY720 in discordant islet xenotransplantation. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into normoglycemic rats that were either left untreated or treated with FTY720 only, with FTY720 plus cyclosporine A (CsA) or with CsA only. Twelve or 24 days after transplantation, graft morphology was evaluated immunohistochemically. Furthermore, adult porcine islets (APIs) were transplanted into diabetic rats immunosuppressed with FTY720 plus CsA. Blood glucose and porcine C-peptide levels were monitored. RESULTS: In untreated rats, the ICC xenografts were completely rejected after 12 days. Treatment with CsA had only a marginal effect on the rejection. In animals given FTY720, only the number of infiltrating cells was somewhat reduced. However, at 12 days, no intact ICCs remained. Immunosuppression with FTY720 plus CsA had a marked inhibitory effect on islet xenograft rejection and plentiful morphologically intact ICCs remained. Twelve days after transplantation, only occasional macrophages and T cells could be detected. At 24 days after transplantation, the findings were similar. Furthermore, diabetic rats transplanted with APIs and immunosuppressed with FTY720 plus CsA remained normoglycemic for 53.0+/-15.8 days. In fact, one animal remained normoglycemic for more than 100 days. Serum levels of porcine C-peptide remained at levels similar to those for human C-peptide in healthy individuals. CONCLUSIONS: Immunosuppression with FTY720 plus CsA inhibited almost all morphological signs of pig-to-rat islet xenograft rejection for up to 24 days after transplantation. Diabetic rats transplanted with APIs and immunosuppressed with FTY720 plus CsA remained normoglycemic for 53.0+/-15.8 days.     

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

Abstract:  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.     

No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts

Transplantation of pig tissues into humans has the potential for cotransferring pig infections. Knowledge of the epidemiology of pig infections transmissible to humans allows the development of risk limitation strategies at the source herd level, but potentially infectious pig endogenous retrovirus (PERV) is ubiquitous in all domestic pigs and therefore is not avoidable. Using a specific and sensitive RT-PCR and nested PCR for PERV nucleic acids with primers, the screening of pigs from New Zealand herds for the presence and expression of the PERV was conducted. The presence of PERV proviral DNA (pol and env region) and viral RNA was demonstrated in all tested pig tissues including pancreas, liver, spleen, brain, heart, and PBMC. Using the same assays it was established that different tissues (liver, spleen, and heart) of nude and nonobese diabetic (NOD) mice previously transplanted with nonencapsulated pig islets were PERV DNA and RNA negative. Alginate polylysine capsules prepared with encapsulated pig islets were tested for possible leakage of viral particles or viral nucleic acids. RNA was extracted from the supernatant of viable encapsulated pig islet cells grown in culture for 2 months. No evidence of PERV RNA or of cellular nucleic acids could be found. Two adult type I diabetic subjects were transplanted with 1 x 10(6) neonatal pig islets encased in alginate capsules into the peritoneal cavity. One patient was immunosuppressed. Both showed evidence of graft function (up to 34% reduction in insulin dose, corresponding increase in serum pig C-peptide) for up to 2 years. DNA and RNA were extracted from PBMC and blood plasma of both patients at 19 months posttransplant. No evidence of PERV proviral DNA or RNA could be detected. Piglet islets contain PERV DNA and RNA, but this does not traverse the capsules used or produce any evidence of infection in nude and nonobese diabetic (NOD) mice or humans.     

Transplantation of porcine islets in nude mice: Implications for islet replacement therapy in humans

Abstract: Porcine islets obtained from adult donors were transplanted under the kidney capsule of streptozotocin diabetic nude mice. Over a period of 30 days or more, blood glucose levels fell to values lower than those of normal mice but comparable to those of normal pigs; this change in the mice was probably being driven by a lower set point for glucose-induced insulin secretion of porcine islets. Oral and intraperitoneal glucose tolerance tests had lower glucose profiles than those carried out in control mice. The mass of beta cell tissue in the porcine islet graft that cured the diabetes was about 1 mg, close to the normal beta cell mass of a mouse pancreas. When graft-bearing kidneys were perfused in situ, there was a marked increase of insulin secretion to challenges with glucose and arginine. These results suggest that porcine islets could be a good source of tissue for human islet replacement therapy. Some of the ramifications of this possibility are discussed.     

Xenotransplantation of neonatal porcine islets and Sertoli cells into nonimmunosuppressed streptozotocin-induced diabetic rats

The testis has been shown to be a privileged site for transplantation of allogenic islets in rodents, and the testicular cell aggregates are thought to confer this immunologic privilege. Recently, a group in Mexico reported transplantation of cocultured neonatal porcine islets and Sertoli cells resulting in insulin independence in nonimmunosuppressed type 1 diabetes patients. We have transplanted similar islets alone (naked islets) or cocultured islets with Sertoli cells (islet/Sertoli cells) into an omental site and other locations of nonimmunosuppressed, streptozotocin-induced diabetic male Sprague Dawley (SD) rats. Histologic examination showed viable neonatal porcine islets survived in xenografted rodents for at least 2 days, and some glucagon and inhibin stained cells appear to have survived for 4 days posttransplantation. However, histological examination did not demonstrate any difference in xenograft survival in the islets/Sertoli cells mixture compared to naked islets when transplanted into these nonimmunosuppressed diabetic rats.     

Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation

BACKGROUND: The long-term viability and function of transplanted encapsulated neonatal porcine islets was examined in a diabetic patient. METHODS AND RESULTS: A 41-yr-old Caucasian male with type 1 diabetes for 18 yr was given an intraperitoneal transplant of alginate-encapsulated porcine islets at the dose of 15,000 islet equivalents (IEQs)/kg bodyweight (total dose 1,305,000 IEQs) via laparoscopy. By 12 weeks following the transplant, his insulin dose was significantly reduced by 30% (P = 0.0001 by multiple regression tests) from 53 units daily prior to transplant. The insulin dose returned to the pre-transplant level at week 49. Improvement in glycaemic control continued as reflected by total glycated haemoglobin of 7.8% at 14 months from a pre-transplant level of 9.3%. Urinary porcine C-peptide peaked at 4 months (9.5 ng/ml) and remained detectable for 11 months (0.6 ng/ml). The patient was followed as part of a long-term microbiologic monitoring programme which subsequently showed no evidence of porcine viral or retroviral infection. At laparoscopy 9.5 yr after transplantation, abundant nodules were seen throughout the peritoneum. Biopsies of the nodules showed opacified capsules containing cell clusters that stained as live cells under fluorescence microscopy. Immunohistology noted sparse insulin and moderate glucagon staining cells. The retrieved capsules produced a small amount of insulin when placed in high glucose concentrations in vitro. An oral glucose tolerance test induced a small rise in serum of immuno-reactive insulin, identified as porcine by reversed phase high pressure liquid chromatography. CONCLUSION: This form of xenotransplantation treatment has the potential for sustained benefit in human type 1 diabetics.     

Natural antibodies prevent in vivo transmission of porcine islet-derived endogenous retrovirus to human cells

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.     

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver

Di Nicuolo G, D’Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RAFM. Long‐term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell–based Academic Medical Center bioartificial liver. Xenotransplantation 2010; 17: 431–439. © 2010 John Wiley & Sons A/S. Abstract: Background: Clinical use of porcine cell–based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow‐up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. Methods: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center‐BAL (AMC‐BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real‐time polymerase chain reaction assay has been used. Results: Eight patients who received a liver transplant after AMC‐BAL treatment are still alive under long‐term pharmacological immunosuppression. The current clinical follow‐up ranges from 5.6 to 8.7 yr after BAL treatment. A new q‐real‐time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥5 copies per 1 × 10⁵ PBMCs. The linear dynamic range was from 5 × 10⁰ to 5 × 10⁶ copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. Conclusion: Up to 8.7 yr after exposure to treatment with porcine liver cell–based BAL, no PERV infection has been found in long‐term immunosuppressed patients and in HCWs by a new highly sensitive and specific q‐real‐time PCR assay.     

Experimental transplantation with principal islets of teleost fish (Brockmann bodies). Long-term function of tilapia islet tissue in diabetic nude mice.

Certain teleost fish have macroscopically visible islets called BBs that are anatomically discrete. BBs were harvested from Oreochromis nilotica (tilapia) with microscissors, divided, and cultured overnight at 37 degrees C before transplantation into STZ-induced diabetic nude mice. Each mouse received BB fragments from 3-5 fish weighing in aggregate approximately 1.7 kg. Non-FPGs were monitored 5 days/wk. Recipients remained normoglycemic (plasma glucose < 11.1 mM) for 50 days posttransplantation. Mice bearing 50-day-old grafts had essentially normal GTTs. Left nephrectomies then were performed to remove the grafts, and plasma glucose levels in recipient mice rose to > 22.2 mM. Histological examination of graft-bearing kidneys showed viable, vascularized islet tissue containing numerous well-granulated beta-cells; examination of recipient native pancreases revealed small islets composed predominantely of non-beta-cells.     

Resistance of established porcine islet xenografts to humoral rejection by hyperimmune sera.

BACKGROUND: Although preformed natural antibodies cause hyperacute rejection of primarily vascularized xenografts, tissue grafts such as skin or islets are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term porcine islet xenografts in diabetic nude mice. METHODS: Porcine islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic BALB/c athymic mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of BALB/c mice with porcine lymph node cells. Splenic T cells were enriched by nylon wool column separation. Tissues were examined by immunohistology using murine- and porcine-specific monoclonal antibodies. RESULTS: Porcine islets survived in nude mice for > 100 days with high levels of circulating porcine C-peptide and maintenance of normoglycemia. Injection of the hyperimmune sera (IgG) into normoglycemic nude mice bearing porcine islets for > 70 days failed to induce rejection despite the continued presence of circulating anti-porcine cytotoxic antibody. Injection of sensitized T cells caused acute rejection of long-term (>140 days) porcine islets, whereas injection of naive T cells had no effect. Histologically, porcine islets removed from mice treated with hyperimmune serum showed no staining for IgG. Long-surviving porcine islet grafts showed strong staining for interleukin (IL)-10 and a lesser amount of IL-4 but no staining for IL-2 or interferon-gamma. Although fresh porcine islets were positive for swine leukocyte antigen class 1 antigen and intercellular adhesion molecule (ICAM)-1 but negative for mouse platelet endothelial cell adhesion molecule and ICAM-2, long-surviving porcine islets showed positive endothelial staining for mouse platelet endothelial cell adhesion molecule and ICAM-2. CONCLUSIONS: Established islet xenografts are resistant to hyperimmune serum as a result of a lack of target endothelial antigens, whereas they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate injected naive T cells.     

成人胰岛大容量分离技术的改进及胰岛功能检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能,为利用同种异体胰岛移植治疗1型和部分2型糖尿病提供理论依据和技术基础。方法 采用改良的自动分离技术连续分离28例人胰岛,然后用连续性密度样度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量(islet equivalent,IEQ)表示。胰岛功能的测定分别为体外测定胰岛的胰岛素／DNA比率;静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内胰岛功能鉴定并随后进行腹腔糖耐量试验,连续测定移植鼠血糖水平及其体内C肽浓度。结果 28例成人胰腺分离的胰岛收获量为5000～1030000IEQ／胰腺,平均为291635IEQ／胰腺,前13例平均每个胰腺收获49123IEQ,平均每克组织收获846IEQ。平均纯度为87％,随着技术的改进后15例的分离结果则分别为：平均每个胰腺501813IEQ,平均每克组织7003IEQ,平均纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常鼠。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的实验基础。     

Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.     

Delayed functional maturation of neonatal porcine islets in recipients under strict glycemic control

BACKGROUND: The aim of this study was to compare the functional maturation of neonatal porcine islet (NPI) grafts exposed to long-term hyperglycemia with those implanted under euglycemic conditions. METHODS: mice Neonatal porcine islets were transplanted under the left renal capsule of diabetic SCID mice (group H), or in diabetic SCID mice who were also implanted with 500 BALB/c islets under the right renal capsule (group N). On day 42, the right kidneys were removed in both groups. RESULTS: No animals in group H achieved euglycemia within 3 weeks after transplantation. Thus, these mice were exposed to long-term hyperglycemia. Mice in group N became euglycemic immediately after transplantation, however after removal of BALB/c grafts on day 42 they exhibited significantly higher blood glucose levels than in group H and showed glucose intolerance after glucose administration. Cellular insulin content of NPI grafts harvested on day 58 or 72 was significantly lower in group N mice compared to group H. CONCLUSIONS: These results suggest that tight control of glycemia reduces the functional maturation of NPI grafts.     

大量人胰岛分离技术的改进及相应胰岛功能的检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能。方法采用经典及改良技术分离、纯化28例人胰岛。胰岛收获量以胰岛当量(IEQ)表示。胰岛功能测定分别为体外测定胰岛素/DNA比率、静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内功能鉴定并行糖耐量试验,连续测血糖水平及其体内C肽浓度。结果 前13例采用经典方法,平均每个胰腺收获49123 IEQs,平均846 IEQs/g胰腺组织,平均纯度为87％。改进技术后15例的分离结果则分别为:501813 IEQs/胰腺,7003 IEQ/g胰腺组织及纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的基础。