Enhanced effect of human mesenchymal stem cells expressing human TNF‐αR‐Fc and HO‐1 gene on porcine islet xenotransplantation in humanized mice

Background

Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood‐Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor‐ɑ receptor type I (sTNF‐αR) and heme oxygenase (HO)‐1 genes (HO‐1/sTNF‐αR) have been shown to improve the viability and functionality of porcine islets after transplantation.

Methods

In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO‐1/sTNF‐αR genes (HO‐1/sTNF‐αR‐MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO‐1/sTNF‐αR‐MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO‐1/sTNF‐αR‐MSCs.

Results

According to the results, the HO‐1/sTNF‐αR‐MSC‐treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO‐1/sTNF‐αR‐MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice.

Conclusions

Our results suggest that HO‐1/sTNF‐αR‐MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information for pre‐clinical animal models and future clinical trials of porcine islet xenotransplantation.     

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft‐infiltrating Th17 cells and activated B cells

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig‐to‐humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45⁺ cells comprising activated human B cells, CD4⁺CD44⁺IL‐17⁺ Th17 cells, and CD68⁺ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long‐term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI‐SP), and peri‐transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI‐SP treatment in controlling human immune cells in promoting long‐term islet xenograft survival.     

Beneficial effects of the transgenic expression of human sTNF-αR-Fc and HO-1 on pig-to-mouse islet xenograft survival

Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.     

The immunobiology of pig‐to‐nonhuman primate islet xenotransplantation: insights,innovation, and impact

Previous studies of pig‐to‐non‐human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets. Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell‐mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long‐term diabetes reversal after intraportal islet xenotransplantation in non‐human primates immunosuppressed with anti‐CD40L but not with anti‐CD40 antibody‐based protocols suggests that the therapeutic efficacy of anti‐CD40L in this transplantation setting likely involves the depletion of donor‐reactive, activated T cells besides CD40:CD40L costimulation blockade. Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4‐Ig and rapamycin was mediated largely by IL‐15‐primed, CXCR3+CD8+ memory T cells recruited by IP‐10 (CXCL10) positive pig islets and macrophages that showed staining for IL‐12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN‐γ+ and IL‐17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF‐β, Foxp3, HO‐1, and IL‐10. Anti‐pig non‐Gal IgG antibody elicitation was suppressed in both groups. We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig‐to‐mouse islet xenograft survival to >250 days when combined with peritransplant B cell‐depletion and rapamycin. This costimulation blockade‐sparing, antigen‐specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti‐CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve – as previously with anti‐CD40L antibodies – more complete, yet selective depletion of donor‐reactive, activated T‐cells for the purpose of stable xenograft acceptance.     

Transplantation of neonatal islets from LEA29Y‐transgenic pigs restores normoglycemia in streptozotocin‐diabetic NSG‐mice

Beta cell replacement therapy represents the only way towards complete restoration of the physiological glucose homeostasis in patients with Type 1 diabetes. Xenotransplantation of transgenic pig islets expressing immunomodulatory molecules might represent a promising approach to overcome the problems of lacking organ donors, adverse effects of the required systemic immunosuppression, and disappointing transplantation outcomes. LEA29Y, a second generation CTLA‐4‐Ig fusion protein exerts potent immunosuppressive effects by selective blockade of the B7 costimulatory pathway. To assess the impact of high local LEA29Y concentrations on xenogeneic islet graft rejection, a transgenic pig model with islet‐specific LEA29Y transgene expression was generated. Islet‐like clusters from neonatal pigs expressing LEA29Y under the control of the porcine insulin promoter and from wild‐type pigs were isolated by collagenase digestion and subsequent in vitro culture. After 6 days 2500 clusters/mouse were transplanted under the kidney capsule of streptozotocin‐diabetic NOD‐scidIL2Rgamma^null (NSG) mice. After an initial period of insulin dependency, mice developed stable normoglycemia within 8 weeks after transplantation. Transplanted mice of both groups (Tx, wt; Tx, LEA29Y) exhibited normal and as compared to non‐transplanted normoglycemic NSG mice even improved fasting glucose and glucose tolerance resulting from glucose‐responsive graft‐derived porcine insulin secretion. Beta cell‐specific expression of LEA29Y was detectable in neonatal porcine pancreas, in the grafts of transplanted mice, and was also measurable in the plasma of normoglycemic mice. Furthermore, mice with LEA29Y transgenic grafts exhibited a glucose‐dependent LEA29Y release during glucose tolerance testing. The present study demonstrates that LEA29Y transgenic islet‐like clusters display normal beta cell function and have the same potential as wild‐type islet clusters to restore normoglycemia in streptozotocin‐diabetic NSG mice after an in vivo maturation period towards a functional endocrine tissue. NSG mice with LEA29Y islet grafts may therefore represent a promising model to study the modulation of human‐anti‐pig xenograft rejection in detail. In ongoing experiments xenograft rejection is analyzed in “humanized” mice after the transfer of human PBMCs or isolated CD34⁺ stem cells.     

Pig islet xenograft rejection in a mouse model with an established human immune system

Abstract: Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34⁺ fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C‐peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C‐peptide in the sera, and severe intra‐graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T‐cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T‐cell‐depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T‐cell‐dependent, which is consistent with previous observations in non‐human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.     

Comparison of exendin‐4 on beta‐cell replication in mouse and human islet grafts

Exendin‐4 can stimulate β‐cell replication in mice. Whether it can stimulate β‐cell replication in human islet grafts remains unknown. Therefore, we compared the effects of exendin‐4 on β‐cell replication in mouse and human islet grafts. Islets, isolated from mouse and human donors at different ages, were transplanted into diabetic mice and/or diabetic nude mice that were given bromodeoxyuridine (BrdU) with or without exendin‐4. At 4 weeks post‐transplantation, islet grafts were removed for insulin and BrdU staining and quantification of insulin⁺/BrdU⁺ cells. Although diabetes was reversed in all mice transplanting syngeneic mouse islets from young or old donors, normoglycemia was achieved significantly faster in exendin‐4 treated mice. Mouse islet grafts in exendin‐4 treated mice had significantly more insulin⁺/BrdU⁺β cells than in untreated mice (P < 0.01). Human islet grafts from ≤22‐year‐old donors had more insulin⁺/BrdU⁺β cells in exendin‐4 treated mice than that in untreated mice (P < 0.01). However, human islet grafts from ≥35‐year‐old donors contained few insulin⁺/BrdU⁺β cells in exendin‐4 treated or untreated mice. Our data demonstrated that the capacity for β‐cell replication in mouse and human islet grafts is different with and without exendin‐4 treatment and indicated that GLP‐1 agonists can stimulate β‐cell replication in human islets from young donors.     

Pre‐clinical results in pig‐to‐non‐human primate islet xenotransplantation using anti‐CD40 antibody (2C10R4)‐based immunosuppression

Background

Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti‐CD154 monoclonal antibody (mAb) in pig‐to‐non‐human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti‐CD40 (2C10R4) mAb‐based studies have not been reported.

Methods

Nine streptozotocin (STZ)‐induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen‐free (DPF) miniature pigs. They were treated with anti‐CD40 mAb‐based immunosuppressive regimen and were divided into 3 groups: anti‐CD40 only group (n = 2), belatacept group (anti‐CD40 mAb+belatacept, n = 2), and tacrolimus group (anti‐CD40 mAb+tacrolimus, n = 5). All monkeys received anti‐thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C‐peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted.

Results

All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow‐up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C‐peptide concentration of >0.15 ng/mL, was 31, 27, and 60 days for anti‐CD40 only, belatacept, and tacrolimus groups, respectively. Anti‐αGal IgG levels in serum and the number of interferon‐γ secreting T cells in peripheral blood mononuclear cells did not increase in most animals.

Conclusion

These results showed that anti‐CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti‐CD40 mAb was not as effective as anti‐CD154 mAb in terms of preventing early islet loss.     

Potential pathological role of pro‐inflammatory cytokines (IL‐6, TNF‐α, and IL‐17) in xenotransplantation

The major limitation of organ transplantation is the shortage of available organs from deceased human donors which leads to the deaths of thousands of patients each year. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection and coagulation dysfunction are two major hurdles for the successful survival of pig xenografts in primate recipients. Pro‐inflammatory cytokines, such as IL‐6, TNF‐α, and IL‐17, play important roles in many diseases and in allotransplantation. However, the pathological roles of these pro‐inflammatory cytokines in xenotransplantation remain unclear. Here, we briefly review the signaling transduction and expression regulation of IL‐6, TNF‐α, and IL‐17 and evaluate their potential pathological roles in in vitro and in vivo models of xenotransplantation. We found that IL‐6, TNF‐α, and IL‐17 were induced in most in vitro or in vivo xenotransplantation model. Blockade of these cytokines using gene modification, antibody, or inhibitor had different effects in xenotransplantation. Inhibition of IL‐6 signaling with tocilizumab decreased CRP but did not increase xenograft survival. The one possible reason is that tocilizumab can not suppress IL‐6 signaling in porcine cells or organs. Other drugs which inhibit IL‐6 signaling need to be investigated in xenotransplantation model. Inhibition of TNF‐α was beneficial for the survival of xenografts in pig‐to‐mouse, rat, or NHP models. Blockade of IL‐17 using a neutralizing antibody also increased xenograft survival in several animal models. However, the role of IL‐17 in the pig‐to‐NHP xenotransplantation model remains unclear and needs to be further investigated. Moreover, blockade of TNF‐α and IL‐6 together has got a better effect in pig‐to‐baboon kidney xenotransplantation. Blockade two or even more cytokines together might get better effect in suppressing xenograft rejection. Better understanding the role of these cytokines in xenotransplantation will be beneficial for choosing better immunosuppressive strategy or producing genetic modification pig.     

Porcine IL‐6, IL‐1β, and TNF‐α regulate the expression of pro‐inflammatory‐related genes and tissue factor in human umbilical vein endothelial cells

Whether porcine cytokines are induced after pig‐to‐primate xenotransplantation and activate human cells remains unknown. First, we investigated the regulation of porcine IL‐6, IFN‐γ, IL‐1β, and TNF‐α in xenotransplantation using an in vitro model in which porcine aortic endothelial cells (PAECs) and porcine peripheral blood mononuclear cells (PBMCs) were stimulated with human serum. Downstream cytokines/chemokines were monitored. Pro‐inflammatory cytokines (IL‐6, IFN‐γ, and IL‐1β) and chemokines (IL‐8, MCP‐1, and CXCL2) were upregulated in the both cell types. TNF‐α was induced 10‐fold in PAECs, but not in PBMCs. Then, we assessed the role of porcine IL‐6, IFN‐γ, IL‐1β, and TNF‐α in xenotransplantation using western blotting and real‐time PCR. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells. Signaling pathways and downstream genes, such as those related to adhesion, inflammation, and coagulation, and chemokines were investigated. Porcine IL‐1β and TNF‐α significantly activated NF‐κB and P38, and STAT3 was activated by porcine IL‐6 in HUVECs. The adhesion genes (E‐selectin, VCAM‐1, and ICAM‐1), inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α), chemokines (MCP‐1 and IL‐8), and the pro‐coagulation gene (tissue factor) were upregulated by porcine IL‐1β and TNF‐α. Porcine IL‐6 increased the expression of ICAM‐1, IL‐6, MCP‐1, and tissue factor, but decreased IL‐8 expression slightly. Surprisingly, porcine IFN‐γ could not activate STAT1 or regulate the expression of any of the above genes in HUVECs. In conclusion, these findings suggest that porcine IL‐6, IL‐1β, and TNF‐α activate HUVECs and regulate downstream genes expression, which may promote inflammation and coagulation response after xenotransplantation.     

Enhanced Immunoprotective Effects by Anti‐IL‐17 Antibody Translates to Improved Skeletal Parameters Under Estrogen Deficiency Compared With Anti‐RANKL and Anti‐TNF‐α Antibodies

Activated T cell has a key role in the interaction between bone and immune system. T cells produce proinflammatory cytokines, including receptor activator of NF‐κB ligand (RANKL), tumor necrosis factor α (TNF‐α), and interleukin 17 (IL‐17), all of which augment osteoclastogenesis. RANKL and TNF‐α are targeted by inhibitors such as denosumab, a human monoclonal RANKL antibody, and infliximab, which neutralizes TNF‐α. IL‐17 is also an important mediator of bone loss, and an antibody against IL‐17 is undergoing phase II clinical trial for rheumatoid arthritis. Although there are a few studies showing suppression of Th17 cell differentiation and induction of regulatory T cells (Tregs) by infliximab, the effect of denosumab remains poorly understood. In this study, we investigated the effects of anti‐TNF‐α, anti‐RANKL, or anti‐IL‐17 antibody administration to estrogen‐deficient mice on CD4⁺ T‐cell proliferation, CD28 loss, Th17/Treg balance and B lymphopoesis, and finally, the translation of these immunomodulatory effects on skeletal parameters. Adult Balb/c mice were treated with anti‐RANKL/‐TNF‐α/‐IL‐17 subcutaneously, twice a week, postovariectomy (Ovx) for 4 weeks. Animals were then autopsied; bone marrow cells were collected for FACS and RNA analysis and serum collected for ELISA. Bones were dissected for static and dynamic histomorphometry studies. We observed that although anti‐RANKL and anti‐TNF‐α therapies had no effect on Ovx‐induced CD4⁺ T‐cell proliferation and B lymphopoesis, anti‐IL‐17 effectively suppressed both events with concomitant reversal of CD28 loss. Anti‐IL‐17 antibody reduced proinflammatory cytokine production and induced Tregs. All three antibodies restored trabecular microarchitecture with comparable efficacy; however, cortical bone parameters, bone biomechanical properties, and histomorphometry were best preserved by anti‐IL‐17 antibody, likely attributable to its inhibitory effect on osteoblast apoptosis and increased number of bone lining cells and Wnt10b expression. Based on the superior immunoprotective effects of anti‐IL‐17, which appears to translate to a better skeletal preservation, we propose beginning clinical trials using a humanized antibody against IL‐17 for treatment of postmenopausal osteoporosis. © 2014 American Society for Bone and Mineral Research.     

A review of piscine islet xenotransplantation using wild‐type tilapia donors and the production of transgenic tilapia expressing a “humanized” tilapia insulin

Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long‐term normoglycemia and provide human‐like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T‐cell‐dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co‐stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.     

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice

Abstract: Background: We have studied cellular components required for xenograft survival mediated by anti‐CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4⁺ but not CD8⁺ cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade‐induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Methods: Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jα18^null, CD1^null, or beta2 microglobulin (β2M)^null NK 1.1 depleted, or perforin^null. Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti‐CD154 mAb treatments. Additional groups received mAb or cells only. Results: We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jα18 component of the T‐cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jα18^null and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jα18^null recipients. These data imply a role for Jα18⁺ NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1^null mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1⁺ cells adversely affects rat skin graft survival. An additional assay in β2M^null mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin‐secreting NK cells. Conclusions: We conclude that MHC class I⁺ CD1⁺ Jα18⁺ NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti‐CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.     

Liraglutide,a long‐acting human glucagon‐like peptide 1 analogue,improves human islet survival in culture

The culture of human islets is associated with approximately 10–20% islet loss, occasionally preventing transplantation. Preconditioning of the islets to improve postculture yields would be of immediate benefit, with the potential to increase both the number of transplanted patients and their metabolic reserve. In this study, the effect of liraglutide, a long‐acting human glucagon‐like peptide 1 analogue, on cultured human islets was examined. Culture with liraglutide (1 μmol/l) was associated with a preservation of islet mass (significantly more islets at 24 and 48 h, compared to control; P ≤ 0.05 at 24 and 48 h) and with the presence of larger islets (P ≤ 0.05 at 48 h). These observations were supported by reduced apoptosis rates after 24 h of treatment. We also demonstrated that human islet engraftment is improved in C57Bl/6‐RAG^?/? mice treated with liraglutide 200 μg/kg sc twice daily (P ≤ 0.05), suggesting that liraglutide should be continued after transplantation. Overall, these data demonstrate the beneficial effect of liraglutide on cultured human islets, preserving islet mass. They support the design of clinical studies looking at the effect of liraglutide in clinical islet transplantation.     

In Vivo Controlling of Cellular Response to Pig Islet Xenografts by Adenovirus-Mediated Expression of Either Membrane-Bound Human FasL or Human Decoy Fas

The critical problem with clinical islet transplantation for patients with type 1 diabetes is the severe shortage of human donors. Pig islet xenotransplantation has the potential to provide a virtually unlimited source of donor pancreata. However, our previous studies demonstrated that cell-mediated rejection, especially human CD8⁺ cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a major obstacle for long-term islet xenograft survival. Moreover, we have demonstrated that the overexpression of either membrane-bound human FasL (mFasL) or human decoy Fas antigen (decoy Fas) in pig islets not only prevented CTL xenocytotoxicity in vitro, but also prolonged histological survival of pig islet xenografts in vivo. Therefore, the aim of the present study was to determine whether adenoviral transfer of these genes into pig islets ex vivo prior to transplantation had a beneficial effect on posttransplantation glycemic control of diabetic recipients. Isolated pig islets were transfected with adenovirus vector carrying complementary DNA (cDNA) of either mFasL or decoy Fas. The transfected islets were transplanted under the kidney capsule of diabetic recipient rats. Rats transplanted with either mFasL- or decoy Fas-transfected pig islet grafts showed significantly suppressed blood glucose levels from 12 hours to 18 hours posttransplantation compared with control groups transplanted with empty vector-transfected pig islets. Unfortunately, blood glucose levels of these groups were increased, with no significant difference observed at 24 hours posttransplantation. However, transgenic expression of these molecules with clinically tolerable amount of immunosuppressants may be more effective to achieve islet xenograft survival in the future.     

A CD200FC immunoadhesin prolongs rat islet xenograft survival in mice

BACKGROUND: A solubilized form of the CD200 molecule, CD200Fc, has been shown to suppress allograft rejection and development of collagen-induced arthritis in mice. We investigated whether the same molecule could prolong survival of rat islet xenografts. METHODS: Streptozocin-treated mice, receiving injections with anti-asialo-GM1 antibody, received rat islets ( approximately 400/mouse) under the kidney capsule or injected into the portal vein, along with rapamycin treatment. Thereafter mice received injections of CD200Fc (10 microg/mouse/injection) or control mouse IgG2. Blood glucose was monitored daily. Some mice received additional injections of anti-CD200/-CD200R monoclonal antibodies. RESULTS: Portal vein delivery of islets led to more extended resolution of diabetes than did transplantation under the kidney capsule. CD200Fc further prolonged survival in either case, an effect abolished by anti-CD200 or F(ab')2 anti-CD200R mAbs, but not by whole anti-CD200R (anti-CD200R Ig). Spleen cells taken from CD200Fc-treated mice showed polarization to type-2 cytokine production (interleukin-4, interleukin-10) on restimulation with rat splenocytes in culture, in comparison to cells from control mice (type-1 cytokines, interlulin-2, interferon-gamma). CONCLUSION: CD200:CD200R interactions are important in regulating rat islet xenograft survival.     

Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41–59, BMI 26–38, cold ischemic time < 10 h). Collagen IV, pan‐laminin, perlecan and laminin‐α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin‐α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan‐laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan‐laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.     

The potential of local immunomodulation and tolerance induction in porcine islet xenotransplantation

Transplantation of pancreas or isolated islet cells is currently the only option to cure type 1 diabetes. The success of islet transplantation is still limited by the requirement of large numbers of high quality islets and the shortage of organ donors. Porcine islets are a promising cell source, but the intensive immunosuppressive regimen required to suppress rejection prevents the translation into clinical practice. We aimed to develop a novel method to inhibit the human‐anti‐pig immune reaction by the expression of immunomodulatory molecules in porcine beta cells. Thus, a transgenic pig was generated expressing LEA29Y – a second generation human CTLA4‐Ig fusion protein, which inhibits activation of T cells by CD80/CD86‐CD28 costimulation – under the control of the porcine insulin promotor. Islet‐like clusters (ICC) from neonatal pigs were isolated and transplanted under the kidney capsule of diabetic NOD‐scid‐IL2γnull (NSG) mice. After an in vivo maturation period mice transplanted with wildtype (wt) as well as with LEA29Y transgenic (tg) ICCs developed normal glucose homeostasis. Within 30 days after the transfer of human PBMCs 80% of NSG mice transplanted with wt‐ICCs developed diabetes indicating xenograft rejection. By contrast, LEA‐tg ICCs were completely protected from rejection in all animals (1). Immunohistochemistry revealed a massive intra‐islet T cell infiltration, which was absent in the LEA‐tg ICCs. This proof of principle study suggests that specific expression of immunomodulatory molecules in beta cells does not disturb beta cell function and may have the potential to modulate immune response locally at the transplantation site without systemic immunosuppression. To overcome the strong xenogeneic barrier of the human and cellular immune system a combination of LEA29Y with additional immunomodulatory factors may be required. Recently, Yi and coworkers demonstrated that the treatment with in vitro expanded regulatory T cells (Treg) prevents porcine islet rejection in humanized NSG mice by the suppression of the T cell‐mediated graft destruction (2). Other potential candidates to induce a state of tolerance against porcine islets currently under investigation are molecules targeting innate immunity and factors that prevent the reoccurrence of autoimmunity. Recent advances in xenotransplantation suggest that it may be possible to start with clinical trials using porcine neonatal or adult islets within the near future. References: 1. KLYMIUK N, VAN BÜRCK L, BÄHR Aet al. Xenografted islet‐cell‐clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 2012; 61:1527–1532. 2. YI S, JI M, WU J et al. Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin‐10 in humanized mice. Diabetes 2012; 61:1180–1191.     

TNF‐α–induced NF‐κB signaling reverses age‐related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RI^null), or TNF receptor 2‐null (TNF‐RII^null) mice. VEGF induction was inhibited when we used TNF‐RI^null‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:229–235, 2009