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Islet transplantation is poised to play an important role in the treatment of type 1 diabetes mellitus (T1DM). However, there are several challenges limiting its widespread use, including the instant blood‐mediated inflammatory reaction, hypoxic/ischemic injury, and the immune response. Mesenchymal stem/stromal cells (MSCs) are known to exert regenerative, immunoregulatory, angiogenic, and metabolic properties. Here, we review recent reports on the application of MSCs in islet allo‐ and xenotransplantation. We also document the clinical trials that have been undertaken or are currently underway, relating to the co‐transplantation of islets and MSCs. Increasing evidence indicates that co‐transplantation of MSCs prolongs islet graft survival by locally secreted protective factors that reduce immune reactivity and promote vascularization, cell survival, and regeneration. MSC therapy may be a promising option for islet transplantation in patients with T1DM. [ABSTRACT FROM AUTHOR]

Abstract: Antibody‐mediated rejection is a barrier to the clinical application of xenotransplantation, and xenoantigens play an important role in this process. Early research suggested that N‐acetyl‐D‐galactosamine (GalNAc) could serve as a potential xenoantigen. GalNAc is the immunodominant glycan of the Sda antigen. Recently, knockout of β1,4‐N‐acetylgalactosaminyltransferase 2 (β1,4GalNAcT‐II) from the pig results in a decrease in binding of human serum antibodies to pig cells. It is believed that this is the result of the elimination of the GalNAc on the Sda antigen, which is catalyzed by the enzyme, β1,4GalNAcT‐II. However, research into human blood group antigens suggests that only a small percentage (1%‐2%) of people express anti‐Sda antibodies directed to Sda antigen, and yet a majority appear to have antibodies directed to the products of pig B4GALNT2. Questions can therefore be asked as to (i) whether the comprehensive structure of the Sda antigen in humans, that is, the underlying sugar structure, is identical to the Sda antigen in pigs, (ii) whether the human anti‐Sda antibody binds ubiquitously to pig cells, but not to human cells, and (iii) what role the Sda++ (also called Cad) antigen is playing in this discrepancy. We review what is known about these antigens and discuss the discrepancies that have been noted above. [ABSTRACT FROM AUTHOR]

Under normal conditions, the activity of platelets is stringently and precisely balanced between activation and quiescent state. This guarantees rapid hemostasis and avoids uncontrolled thrombosis. However, excessive platelet activation and resulting thrombotic microangiopathy are frequently observed in pig-to-primate xenotransplantation models. Endothelium-derived inhibitory mechanisms play an important role in regulation of platelet activation. These mainly include nitric oxide (NO), prostacyclin PGI2, and adenosine, which are synthesized by endothelial NO synthases (eNOS), prostacyclin synthase, and CD39/CD73, respectively. We investigated whether endothelium-derived regulatory mechanisms are affected in porcine aortic endothelial cells (PAECs) after exposure to human serum. In the present study, exposure of PAECs or porcine iliac arteries to human serum suppressed gene expression of eNOS and prostacyclin synthase, while induced gene expression of prostaglandin G/H synthase and thromboxane synthase. Simultaneously, exposure to human serum reduced NO and PGI2 production in PAEC culture supernatants. Thus, human serum altered the balance of endothelium-derived inhibitory mechanisms in PAECs, which may indicate a regulatory mechanism of excessive platelet activation in pig-to-primate xenotransplantation. [ABSTRACT FROM AUTHOR]

Background To investigate graft survival after full-thickness corneal xenotransplantation from α1,3-galactosyltransferase gene-knockout (GTKO) pigs expressing a human complement regulatory protein ( GTKO/ CD46 pigs) in rhesus monkeys. Methods Rhesus monkeys (n=10) were transplanted with full-thickness corneas from wild-type ( WT; n=4) and GTKO/ CD46 (n=4) pigs or from monkeys (n=2). All recipient monkeys received post-transplant subconjunctival injections of betamethasone. Corneal grafts were evaluated by slit-lamp. Histopathology, immunohistochemistry of the grafts, and cytokine concentrations in the aqueous humor were tested 6 months after transplantation. Anti-pig IgM/IgG and anti-galactose-α1,3-galactose (Gal) antibodies were determined by flow cytometry and ELISA, respectively. Results The longest graft survival of WT and GTKO/ CD46 xenografts was 157 and 171 days, respectively. There was no significant difference in graft survival between WT and GTKO pig corneas. Anterior synechiae occurred in two recipients of WT and all recipients of GTKO/ CD46 grafts. All xenograft recipients developed a retrocorneal membrane, inflammatory cells ( CD3+ T lymphocytes) infiltrated the corneal stroma, and in the aqueous humor, IL-6 was increased 6 months after transplantation. Induced antibody responses were documented against Gal and/or non-Gal pig antigens. In contrast, allografts survived >180 days without any rejection, with no increase in cytokines in the aqueous humor, and no elicited serum anti-pig antibodies were detected. Conclusions α1,3-galactosyltransferase gene-knockout/ CD46 pig corneas were not associated with prolonged graft survival or a reduced antibody response compared with WT pig corneas. The prevention of the development of anterior synechiae and a retrocorneal membrane after corneal xenotransplantation would appear to be important if prolonged corneal xenograft survival is to be achieved. [ABSTRACT FROM AUTHOR]

Long-term success in pig-to-primate xenotransplantation is currently hampered by acute vascular rejection ( AVR), characterized by endothelial cell ( EC) activation and injury. Klotho has anti-apoptotic, anti-inflammatory effects on EC and protects EC against reactive oxygen species, rendering klotho a promising molecule to control AVR. In this study, porcine ECs were pre-incubated with klotho and then exposed to xenoreactive antibodies and complement. Real-time PCR revealed that klotho suppressed antibody-induced pro-inflammatory gene expression of VCAM-1 and IL-1α. NF-κB activation, IκBα phosphorylation, was also attenuated by klotho administration. Furthermore, klotho induced in porcine EC resistance against complement-dependent cytotoxicity. Accompanying this change, the binding of IgG and IgM xenoreactive antibodies to porcine EC was decreased and the expression of anti-inflammatory gene HO-1 was upregulated. These findings indicated that klotho protein protected porcine EC from activation and injury caused by binding of xenoreactive antibodies and was a promising candidate molecule in a multitransgenic pig strategy for xenotransplantation. [ABSTRACT FROM AUTHOR]

There is an increasing worldwide shortage of organs and cells for transplantation in patients with end-stage organ failure or cellular dysfunction. This shortage could be resolved by the transplantation of organs or cells from pigs into humans. What competing approaches might provide support for the patient with end-stage organ or cell failure? Four main approaches are receiving increasing attention - (i) implantable mechanical devices, although these are currently limited almost entirely to devices aimed at supporting or replacing the heart, (ii) stem cell technology, at present directed mainly to replace absent or failing cells, but which is also fundamental to progress in (iii) tissue engineering and regenerative medicine, in which the ultimate aim is to replace an entire organ. A final novel potential approach is (iv) blastocyst complementation. These potential alternative approaches are briefly reviewed, and comments added on their current status and whether they are now (or will soon become) realistic alternative therapies to xenotransplantation. [ABSTRACT FROM AUTHOR]

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4+ T cells. A human dominant-negative mutant class II transactivator (CIITA-DN) transgene under a CAG promoter with an endothelium-specific Tie2 enhancer was constructed. CIITA-DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA-DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4+ T-cell response to cells from CIITA-DN and wild-type (WT) pigs was compared. Lymphocyte subset numbers and T-cell function in CIITA-DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen-presenting cells from CIITA-DN pigs was significantly reduced (40-50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4+ T-cell response to CIITA-DN pAECs was significantly weaker than to WT pAECs (60-80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4+ cells in the PBMCs from CIITA-DN (20%) than from WT (30%) pigs ( P < 0·01), T-cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA-DN pigs should be partially protected from the human cellular immune response. [ABSTRACT FROM AUTHOR]

After hyperacute rejection in pig‐to‐primate xenotransplantation had been overcome by the introduction of α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs, acute and chronic antibody‐mediated rejection became one of the major barriers to long‐term graft survival. This was associated with exposure of non‐Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC‐immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1‐regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non‐Gal antigens. [ABSTRACT FROM AUTHOR]

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

Accurate and timely diagnosis of transplant rejection is essential for the long‐term survival of solid‐organ transplant recipients. Biopsy is the "gold standard" for the diagnosis of rejection, but this approach is a time‐consuming, expensive, and invasive process. An efficient, cheap, and noninvasive tool for monitoring of immune rejection is urgently needed. Circulating miRNA and circulating DNA can be easily amplified and quantified by PCR which could be potential biomarkers for monitoring organ survival and/or immune rejection. In this review, we briefly introduce the origins and characteristics of circulating miRNA and circulating DNA. Furthermore, we review their clinical application and potential as noninvasive biomarkers for rejection in organ transplantation. Finally, we sum up their potential as biomarkers, which may help scientists and surgeons choose the proper biomarker for routine assays. [ABSTRACT FROM AUTHOR]

The article presents the cover page for the March 1, 2018 issue of the journal.