Sunghoon Hurh

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Generation of soluble human tumor necrosis factor-α receptor 1-Fc transgenic pig.

Background. Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-&agr; (hTNF-&agr;) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs. Methods. The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested. Results. In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-&kgr;B activation in the HEK293-NF-&kgr;B-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-&agr;-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-&agr;. Conclusions. We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-&agr;-mediated activation of PECs. Because TNF-&agr; is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.

Expression Analysis of Combinatorial Genes Using a Bi-Cistronic T2A Expression System in Porcine Fibroblasts

In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient.

Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno-immune response.

With the introduction of the α1, 3‐galactosyltransferase gene‐knockout (GT‐KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long‐term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti‐inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli.

CD70–CD27 ligation between neural stem cells and CD4+ T cells induces Fas–FasL-mediated T-cell death

IntroductionNeural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. One of the remaining obstacles for NSC therapy is to overcome the alloimmune response on NSCs by the host.MethodsTo investigate the mechanisms of immune modulatory function derived from the interaction of human NSCs with allogeneic T cells, we examined the immune regulatory effects of human NSCs on allogeneic T cells in vitro.ResultsSignificantly, NSCs induced apoptosis of allogeneic T cells, in particular CD4+ T cells. Interaction of CD70 on NSCs and CD27 on CD4+ T cells mediated apoptosis of T cells. Thus, blocking CD70–CD27 interaction prevented NSC-mediated death of CD4+ T cells.ConclusionsWe present a rational explanation of NSC-induced immune escape in two consecutive stages. First, CD70 constitutively expressed on NSCs engaged CD27 on CD4+ T cells, which induced Fas ligand expression on CD4+ T cells. Second, CD4+ T-cell apoptosis was followed by Fas–Fas ligand interaction in the CD4+ T cells.

The Introduction of Human Heme Oxygenase-1 and Soluble Tumor Necrosis Factor-α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice.

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase‐1 (HO‐1) or soluble tumor necrosis factor‐α receptor type I with human IgG1 Fc (sTNF‐αR‐Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO‐1, sTNF‐αR‐Fc, sTNF‐αR‐Fc/HO‐1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood–derived CD34+ stem cells into NOD‐scid‐IL‐2Rγnull mice. Both HO‐1 and sTNF‐αR‐Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig‐to‐humanized mice transplantation. The sTNF‐αR‐Fc/HO‐1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor‐α and IL‐6 in treatment groups; however, frequency of pig‐specific interferon‐γ–producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO‐1 or sTNF‐αR‐Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO‐1 or sTNF‐αR‐Fc transgenic pigs have potential for islet xenotransplantation.

Human antibody reactivity against xenogeneic N-glycolylneuraminic acid and galactose-α-1,3-galactose antigen

Despite the development of α1,3‐galactosyl transferase‐knockout (GTKO) pigs, acute humoral xenograft rejection caused by antibodies against non‐Gal antigens, along with complement activation, are hurdles that need to be overcome. Among non‐Gal antigens, N‐glycolylneuraminic acid (Neu5Gc) is considered to play an important role in xenograft rejection in human.

Inflammatory Responses in α1,3-Galactosyltransferase Gene-Knockout (GTKO) Porcine Vascular Retransplantation to Cynomolgus Monkeys

Introduction Various pro-inflammatory factors are produced in response to a xenograft. However, it is poorly understood the differences of inflammatory responses by preformed anti-pig antibodies. In this study, we developed the animal model of xenogeneic antibody-mediated rejection of vascular xenografts in sensitized recipients and examined the systemic inflammatory responses. Methods Cynomolgus monkeys (2.5 ~ 4 kg, n=4) received femoral artery grafts from GTKO pig. Immunosuppressive therapy was based on costimulation blockade using anti-CD154 mAb (20 mg/kg). They also received cobra venom factor (0.05 mg/kg), steroid, and tacrolimus. In addition to that, two monkeys received anti-thymocyte globulin (ATG, 20 mg/kg) to minimize T-cell-mediated rejection while allowing the development of donor-specific antibodies. Immunosuppression have been stopped at the time of graft removal (n=4, day 24, 28 or 32). Same immunosuppression protocol was applied for the second vascular transplantation. Whole blood and serum samples were obtained from recipients before and serially after vascular transplantation. They were tested C-reactive protein (C-RP), pro-inflammatory cytokines and chemokines, and blood cell count. In addition, expression of tissue factor (TF) on the transplanted artery was analyzed by immunofluorescence assay. Results In vascular xenograft recipients, significantly high levels of C-RP were detected in first and second vascular recipients in comparison with before transplantation. The levels of IL-6 and TNF-&agr; were not significantly higher after first vascular transplantation. However, MCP-1 and IL-8 levels were increased after first vascular transplantation. In second xenograft recipients, the level of IL-6, MCP-1, and IL-8 were increased after transplantation, but not TNF-&agr;. Absolute numbers of monocytes and neutrophils were elevated after transplantation. Additionally, peripheral blood leukocytes were increased in the group that ATG was not given after transplantation. TF expressions were increased on the second transplanted artery compare to first transplanted artery. Conclusion We have developed a model of xenogeneic antibody-mediated rejection in recipients with preformed anti-pig antibodies. The porcine vascular transplantation to Cynomolgus model showed more severe inflammation in second transplanted recipients compare to first transplanted recipients. It means that sensitization to porcine antigens is increased in the vascular retransplanted recipients. This study was supported by the Ministry of Science, ICT & Future Planning (#2014M3A9D3034034).

Additive Expression of hTBM on hCD46 Stable Cell Line Has Synergistic Effect on Complement Regulation

Background Coagulation dysregulation and intravascular thrombosis during acute humoral xenograft rejection (AHXR) still remains the major obstacle in solid organ xenotransplantation. TBM or CD46 have been reported as an anti-coagulant or complement regulatory molecules and also potent anti-inflammatory molecules. In this study, we showed whether additional TBM expression on CD46 stable cell has synergistic effects on anti-coagulant or complement regulation. Methods & Results Fetal pig fibroblasts were transfected with hTBM-hCD46 vector and colonies expressing high amounts of the fusion protein were selected and used as nucleus donor for somatic cell nuclear transfer (SCNT) and three gilts became pregnant and one farrowed one live male piglet, which was no healthy and moved to the SPF facility but died one day after and two of the pregnant gilts, however, aborted during gestation. We isolated fibroblast from the ear of TG pig expressing hTBM-hCD46 and check the expression levels of each genes and the regulatory functions on complement and coagulation. Serum toxicity was significantly reduced in the fibroblast from hTBM-hCD46 TG pig compared with wild type pig’s fibroblast at 10% or 20% serum treatment. Thrombin generation was dramatically reduced and coagulation time was prolonged in the fibroblast from hTBM-hCD46 TG pig compared with wild type pig’s fibroblast. Conclusion The fibroblast from hTBM-hCD46 TG pig has synergistic effects on anti-coagulant or complement regulation therefore, genetically modifying pigs expressing TBM and CD46 would be a promising candidate for xenotransplantation to prolong the graft survival.

Up-regulation of fibrinogen-like protein 2 in porcine endothelial cells by xenogeneic CD40 signal

ABSTRACT Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.