Ryota Shirakura

The mechanism of discordant xenograft rejection.

The mechanism of discordant xenograft rejection using the guinea pig-to-rat heart graft model was studied. In this model, we found that (A) Rejection occurred rapidly, in 17.5 +/- 8.3 min (mean +/- SD) (n = 8). (B) The graft survived longer when the recipient rat was pretreated with cobra venom facter (CVF)

In vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion injury in rat.

Heat shock protein 70 (HSP70) has been reported to be involved in the myocardial self-preservation system. To obtain the evidence that HSP70 plays a direct role in the protection from myocardial ischemia-reperfusion injury, rat hearts were transfected with human HSP70 gene by intracoronary infusion of hemagglutinating virus of Japan (HVJ)-liposome containing human HSP70 gene. The control hearts were infused with HVJ-liposome without the HSP70 gene. The hearts from whole-body heat-stressed or nontreated rats were also examined. Western blot and immunohistochemical analysis showed that apparent overexpression of HSP70 occurred in the gene transfected hearts and that gene transfection might be more effective for HSP70 induction than heat stress. In Langendorff perfusion, better functional recovery as well as less creatine phosphokinase leakage after ischemia were obtained in the gene transfected hearts with HSP70 than in the control or nontreated hearts. Furthermore, the gene transfected hearts showed better functional recovery than the heat-stressed hearts. These results indicated that overexpressed HSP70 plays a protective role in myocardial injury, suggesting the possibility that gene transfection with HSP70 may become a novel method for myocardial protection through enforcing the self-preservation systems.

Report of the xenotransplantation advisory committee of the international society for heart and lung transplantation : The present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases

An urgent and steadily increasing need exists world-wide for a greater supply of donor thoracic organs. Xenotransplantation offers the possibility of an unlimited supply of hearts and lungs that could be available electively when required. However, anti-body- mediated mechanisms cause the rejection of pig organs transplanted into non-human primates, and these mechanisms provide major immunologic barriers that have not yet been overcome. Having reviewed the literature on xenotransplantation, we present a number of conclusions on its present status with regard to thoracic organs, and we make a number of recommendations relating to eventual clinical trials. Although pig hearts have functioned in heterotopic sites in non-human primates for periods of several weeks, median survival of orthotopically transplanted hearts is currently ,1 month. No transplanted pig lung has functioned for even 24 hours. Current experimental results indicate that a clinical trial would be premature. A potential risk exists, hitherto undetermined, of transferring infectious organisms along with the donor pig organ to the recipient, and possibly to other members of the community. A clinical trial of xeno-transplantation should not be undertaken until experts in microbiology and the relevant regulatory authorities consider this risk to be minimal. A clinical trial should be considered when approximately 60% survival of life-supporting pig organs in non-human primates has been achieved for a minimum of 3 months, with at least 10 animals surviving for this minimum period. Furthermore, evidence should suggest that longer survival (.6 months) can be achieved. These results should be achieved in the absence of life-threatening complications caused by the immunosuppressive regimen used. The relationship between the presence of anti-HLA antibody and anti-pig antibody and their cross-reactivity, and the outcome of pig-organ xenotransplantation in recipients previously sensitized to HLA antigens require further investigation. We recommend that the patients who initially enter into a clinical trial of cardiac xenotransplantation be unacceptable for allotransplantation, or acceptable for allotransplantation but unlikely to survive until a human cadaveric organ becomes available, and in whom mechanical assist-device bridging is not possible. National bodies that have wide-reaching government-backed control over all aspects of the trials should regulate the initial clinical trial and all subsequent clinical xenotransplantation procedures for the foreseeable future. We recommend coordination and monitoring of these trials through an international body, such as the International Society for Heart and Lung Transplantation, and setting up a registry to record and widely disperse the results of these trials. Xenotransplantation has the potential to solve the problem of donor-organ supply, and therefore research in this field should be actively encouraged and supported.

EFFECTS OF TRANSFECTED COMPLEMENT REGULATORY PROTEINS, MCP, DAF, AND MCP/DAF HYBRID, ON COMPLEMENT-MEDIATED SWINE ENDOTHELIAL CELL LYSIS

We established several swine endothelial cell (SEC) lines, expressing human MCP (CD46), DAF (CD55), and MCP/DAF hybrid by transfection of cDNA, and assessed the function of these transfectant molecules on complement-mediated cell lysis as an in vitro hyper-acute rejection model of swine to human discordant xenograft. Discordant organ xenografts are hyper-acutely rejected by complement activation. Amelioration of complement-mediated lysis by these transfectant molecules was tested in each SEC line by lactate dehydrogenase assay. Naive swine endothelial cells were markedly damaged by human complement mainly via the classical pathway, activating only minimally the alternative pathway of human complement. Both MCP and DAF protected SEC from human complement attack in parallel with the expression density, with DAF being more effective than MCP. The MCP/DAF hybrid was more effective than MCP alone, and as effective as DAF in this system. The results suggest that the transfection of DAF or the MCP/DAF hybrid cDNA into organs to be transplanted could protect against hyperacute rejection.

Remodeling of the Major Pig Xenoantigen by N-Acetylglucosaminyltransferase III in Transgenic Pig

We have been successful in generating several lines of transgenic mice and pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

Efficiency of In Vivo Gene Transfection Into Transplanted Rat Heart by Coronary Infusion of HVJ Liposome

BACKGROUND Current methods of in vivo gene transfer into myocardium are limited by low efficiency. To improve in vivo gene transfer, a gene transfer method using hemagglutinating virus of Japan (HVJ) as a viral vector can be an alternative. METHODS AND RESULTS In vivo gene transfection of FITC-labeled oligonucleotide (F-ODN) and cDNA of beta-galactosidase (beta-gal) was examined with use of the HVJ liposome (H group) or without it (C group). In the H group, F-ODN or cDNA of beta-gal were complexed with liposomes, DNA binding nuclear protein (HMG1), and the viral protein coat of HVJ. After the harvest of donor rat hearts arrested by cardioplegia, the coronary artery was infused with the liposome gene complex. The hearts were transplanted into the abdomens of recipient rats and harvested 3 days after transplantation. Regarding F-ODN, the H group clearly showed FITC staining in the nuclei of the myocytes and endothelial cells in almost all layers of the myocardium as compared with the C group. Regarding the expression of beta-gal, the H group showed a clear expression of beta-gal on myocytes, whereas very low expression of beta-gal was seen in the C group. CONCLUSIONS The donor hearts were transfected with F-ODN and beta-gal gene in almost all layers of the myocardium as a result of coronary infusion of the HVJ liposome during cardioplegic arrest. Our method is seen as a novel in vivo gene transfer technique for the heart and may provide a new tool for both research and therapy of heart transplantation.

A study of the xenoantigenicity of adult pig islets cells.

Abstract:  Background:  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 Galα1‐3Galβ1‐4GlcNAc‐R (the α‐Gal) and Hanganutziu‐Deicher (H‐D) antigens, and the pathway involved in human complement activation.

Ambulatory electrocardiographic study of the frequency and cause of ventricular arrhythmia after correction of tetralogy of fallot

Ambulatory 24-hour electrocardiographic monitoring with a Holter recording system was performed in 100 patients after repair of tetralogy of Fallot. The incidence and severity of ventricular arrhythmia (VA) were studied relative to operative age, follow-up period after corrective surgery, hemodynamic data, ventricular function and operative method. Significant VA (Lown grade 2 to 4) was detected in 41 patients. Patients with significant VA (group I) were older (11.7 +/- 10.0 years old) at operation than those without VA (group II) (5.5 +/- 5.8 years, p less than 0.001). The follow-up period after operation in group I (9.5 +/- 4.8 years) was significantly (p less than 0.001) longer than that in group II (5.5 +/- 4.3 years). The incidence of elevated right ventricular (RV) systolic pressure was significantly (p less than 0.005) higher in group I (43%) than in group II (7%). RV ejection fraction in group I (48 +/- 6%) was significantly (p less than 0.001) lower than that in group II (56 +/- 5%). The new operative method for tetralogy of Fallot without or with minimal right ventriculotomy was more frequently performed in group II (49%) than in group I (15%, p less than 0.005). Pulmonary regurgitation, RV and left ventricular size, and left ventricular ejection fraction were not related to severity of VA. Thus, serious VA was related to higher age at operation, longer interval after surgery, elevated RV systolic pressure, depressed RV ejection fraction and RV scar.

Survival of adult islet grafts from transgenic pigs with N-acetylglucosaminyltransferase-III (GnT-III) in cynomolgus monkeys.

Abstract:  Background:  Because of a severe shortage of human donor pancreases, pig islets are considered to be an attractive donor source. Our previous in vitro study revealed that adult pig islets have strong non‐Galα1‐3Galβ1‐4GlcNAc‐R (α‐Gal) antigenicity, including the Hanganutziu‐Deicher (H‐D) antigen, especially in N‐linked sugars. In this study, the issue of whether islets from N‐acetylglucosaminyltransferase‐III (GnT‐III) transgenic pigs can prolong their survival in cynomolgus monkeys was examined.

Selective chemokine and receptor gene expressions in allografts that develop transplant vasculopathy

BACKGROUND Chemokine systems probably play a role in transplant vasculopathy; however, a comprehensive study of the expression of chemokines and their receptors in this disease has not been performed. METHODS The expression of all the rat chemokines and chemokine receptor genes for which the nucleotide sequences are known were quantitatively monitored using the fluorescence-based real-time reverse-transcriptase polymerase chain reaction technique, and selected cytokine-receptor pairs were determined using immunohistochemical staining. The analysis covered the whole time course of transplant vasculopathy in 2 different graft models (cardiac and aortic grafts) with 4 different strain combinations of rats. RESULTS Among the 13 receptor genes examined, the CXCR3, CCR5, and CCR2 genes and those of their corresponding ligands were selectively and strongly induced in grafts that develop transplant vasculopathy. The expression patterns of the receptors were similar in both cardiac and aortic allografts, although their induction and their absolute levels of expression were amplified several fold in the grafted aorta compared with heart grafts. The genes were induced before morphologic changes became apparent and expression was sustained during the whole period of neointimal formation. Interestingly, immunohistochemical staining for CXCR3 showed a unique pattern of expression: we found weak expression on cells in the outer layer of the neointima and adventitia and found the strongest staining in the innermost layer of the neointima. CONCLUSIONS This study suggested diagnostic as well as potential functional roles of the chemokine-receptor pairs IP10-CXCR3, RANTES-CCR5, and MCP1-CCR2 in rat models of transplant vasculopathy.