Aki Yamamoto

Complement regulation in the GalT KO era

Miyagawa S, Yamamoto A, Matsunami K, Wang D, Takama Y, Ueno T, Okabe M, Nagashima H, Fukuzawa M. Complement regulation in the GalT KO era.
Xenotransplantation 2010; 17: 11–25.

Survey of glycoantigens in cells from α1‐3galactosyltransferase knockout pig using a lectin microarray

Miyagawa S, Takeishi S, Yamamoto A, Ikeda K, Matsunari H, Yamada M, Okabe M, Miyoshi E, Fukuzawa M, Nagashima H. Survey of glycoantigens in cells from α1‐3galactosyltransferase knockout pig using a lectin microarray.
Xenotransplantation 2010; 17: 61–70.

Effects of Blocking the Chemokine Receptors, CCR5 and CXCR3, with TAK-779 in a Rat Small Intestinal Transplantation Model

Background. The effect of blocking lymphocyte chemokine receptors with TAK-779 was investigated using a rat intestinal transplantation model. Methods. Dark Agouti rat small intestines were heterotopically transplanted into Lewis rats. The recipients were treated with TAK-779 (10 mg/kg per day) by subcutaneous injection. Graft survival, histologic changes, mixed lymphocyte reaction, and antibody production were examined. Furthermore, expression of the chemokine receptors on the graft-infiltrating lymphocytes in the mesenteric lymph node (MLN) and Peyer’s patches (PP) were measured using fluorescence-activated cell sorter analysis. Results. Average duration of survival was greater for group T (with TAK-779: 9.8±1.4) than group A (without TAK-779: 7.0±0.6). Histologic findings and immunohistochemistry of the graft were consistent with the prolonged survival in group T. In the fluorescence-activated cell sorter analysis, several CD4+ and CD8+ cells were significantly suppressed in the blood, spleen, and MLN of the recipient and in the PP of the graft on postoperative day (POD) 6, but recovered in recipient spleen and MLN by POD 9. However, double-staining of graft-infiltrating lymphocytes in MLN and PP showed a significant reduction in the numbers of T cells expressing CCR5 and CXCR3 by POD 9. On the other hand, mixed lymphocyte reaction and cytokine production, and the antidonor antibody titer were suppressed on POD 6 but not on POD 9. Conclusion. TAK779 diminished not only the number of the graft-infiltrating cells and their expression of CCR5 and CXCR3, but also the total number of recipient T cells that play a role in graft rejection. Further exploration of these effects will provide the novel treatment of the intestinal transplantation.

A novel strategy for preventing PERV transmission to human cells by remodeling the viral envelope glycoprotein

Miyagawa S, Nakatsu S, Hazama K, Nakagawa T, Kondo A, Matsunami K, Yamamoto A, Yamada J, Miyazawa T, Shirakura R. A novel strategy for preventing PERV transmission to human cells by remodeling the viral envelope glycoprotein. Xenotransplantation 2006; 13: 258–263.

Trial using pig cells with the H–D antigen knocked down

PurposeThis report describes an attempt to reduce the expression level of Hanganutziu–Deicher (H–D) antigens by small interfering RNA (siRNA) for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (pCMAH).MethodsA pig endothelial cell (PEC) line, and PEC and fibroblasts from an α1,3galactosyltransferase knockout (GalT-KO) piglet were used. Real-time PCR was used to evaluate the degradation of mRNA by siRNA. The H–D antigen was stained, and then the cells were incubated with human serum for the FACS analysis. The extent of lysis in human serum was next calculated using an LDH assay.ResultsSuppression of the mRNA of pCMAH by each siRNA was first determined. The mixture of siRNAs for pCMAH reduced the expressions of the H–D antigen on the PEC and fibroblasts to a considerable extent. The further reduction in the xenoantigenicity for human serum of the GalT-KO cells was then confirmed. In addition, the PEC line showed a significant downregulation in complement-dependent cytotoxicity by the siRNAs, thus indicating that the anti-H–D antigen in human serum is capable of causing lysis of the pig cells.ConclusionpCMAH silencing by siRNA reduced the expression of the H–D antigen and its antigenicity, thus confirming that the H–D antigen is one of the major non-Gal antigens in this situation.

Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model

The effects of FK506, and TAK-779, antagonists of CCR5 and CXCR3, were investigated using a rat intestinal transplantation model. Small intestines from DA rats were heterotopically transplanted into LEW rats. The recipients were treated with FK506 (1mg/kg/day, day 0-5) and TAK-779 (10mg/kg/day, day 0-10). Graft survival and immunological responses to these materials were estimated by mixed lymphocyte reactions and IFN-γ production. The expression of chemokine receptors on lymphocytes was also examined. The average duration of survival was 7.0±0.3, 12.0±1.0, 9.8±0.5 and 18.0±1.5days in the allogeneic, FK506, TAK-779 and the two-drug combined groups, respectively. Cell proliferative responses and IFN-γ production were suppressed to a significant extent in the FK506 group compared with the TAK-779 group. In addition, the two-drug combination showed a tendency for stronger suppression than FK506 alone, correlated with in vivo and histopathological data. The numbers of both CD4(+) and CD8(+) cells were significantly suppressed in the blood of the recipients of both the FK506 and the TAK-779 groups, and in Peyers patches of the graft of the TAK-779 group, but the FK506 group was not, as evidenced by FACS analysis. In addition, double-staining of graft-infiltrating lymphocytes showed a significant reduction in lymphocyte numbers, expressing CCR5 and CXCR3 in the TAK-779 group, but not evident in the FK506 group, compared to the allogeneic group. While FK506 suppresses cell proliferation and effecter function, it has less effect on the expression of CCR5 and CXCR3 in lymphocytes. Further exploration of the effects of a combined therapy with TAK-779 could represent a novel treatment for intestinal transplantation.

A newly cloned pig dolichyl-phosphate mannosyl-transferase for preventing the transmission of porcine endogenous retrovirus to human cells.

Porcine endogenous retrovirus (PERV) is a major problem associated with successful clinical xenotransplantation. In our previous study, reducing the high mannose type of N‐glycan content proved to be very effective in downregulating PERV infectivity. In this study, dolichyl‐phosphate mannosyltransferase (D‐P‐M), an enzyme related to the early stages of N‐linked sugar synthesis was studied. The pig cDNA of the encoding D‐P‐M was newly isolated. The RNA interference (siRNA) for the D‐P‐M was applied and transfected to PEC(Z)/PB cells, a pig endothelial cell line with the Lac Z gene and PERV‐B, to reduce the levels of high mannose type N‐glycans. Compared with the mock line, the temporary PEC(Z)/PB lines showed a decreased mRNA expression for pig D‐P‐M, and each line then showed a clear destruction of PERV infectivity to human cells in the Lac Z pseudotype assay. The PEC(Z)/PB was next transfected with pSXGH‐siRNA, H1‐RNA gene promoter. The established PEC(Z)/PB clones with pSXGH‐siRNA clearly led to the downregulation of PERV infectivity, as evidenced by the decreased levels of the mRNA for pig D‐P‐M. Reducing D‐P‐M enzyme activity represents a potentially useful approach to address the problem of PERV infections in clinical xenotransplantations.

Differential human serum-mediated neutralization of PERV released from pig cells transfected with variants of hDAF.

Abstract:  Background:  Expression of complement regulatory proteins (CRP) on pig endothelial cells (PEC) is an effective means of avoiding induction of hyperacute rejection by human sera. However, pig endogenous retrovirus (PERV) from PEC transfected with CRP may acquire resistance to human sera. This study investigated a form of transfected CRP that is easily expressed on PERV particles.

Expression of complement regulatory protein on porcine endogenous retrovirus (PERV) depends on molecular size.

Expression of complement regulatory proteins (CRP) on pig cells is an effective means to avoid hyperacute rejection. However, pig endogenous retrovirus (PERV) from pig cells transfected with CRP may acquire resistance to human serum (HS). The present study investigated the size limitations of the transfected CRP that can be easily expressed and function on PERV particles. cDNAs of various sized DAF(CD55)s, including single-, double-, triple-, tetra-, as well as 2.1- and 2.2-DAF, were prepared. Pig endothelial cells (PEC) were transduced with the LacZ gene, and were then infected with PERV-B to produce PEC(Z)/PB. The extent of complement-mediated lysis by the transfectant molecules on PEC(Z)/PB was then determined. HEK293 cells were incubated with PEC(Z)/PB culture supernatants in the presence of HS and the LacZ pseudo-type assay was then carried out. Amelioration of complement-mediated lysis by the hybrid molecules was verified in each PEC(Z)/PB clone. All molecules appeared to effectively protect xenogeneic cells against complement-mediated lysis. While PERVs from the PEC(Z)/PB with both the single-DAF and double-DAF were resistant to HS, PERVs from the triple-DAF and tetra-DAF showed no significant increase in resistance. In addition, the PERVs from PEC(Z)/PB with 2.1-DAF and 2.2-DAF were less resistant than PEC with double-DAF. Resistance to HS was steadily attenuated with increasing size of the DAF molecule. The resistance to HS was disappeared by the anti-DAF blocking mAb, indicating that PERVs from the transfectants express DAF molecules on the surface of the PERV. The data clearly indicate that, to avoid the induction of resistance to HS in PERV particles, relatively large CRPs, such as triple-DAF and tetra-DAF or DAF with other large molecules, should be employed in the production of transgenic pigs.

AN ANALYSIS OF PIG CYTIDINE MONOPHOSPHO-N-ACETYLNEURAMINIC ACID (CMP-NEUAC) HYDROXYLASE.: 1537

K. Ikeda1, A. Yamamoto2, A. Kondo3, Y. Takama3, M. Fukuzawa3, H. Matsunari4, H. Nagashima4, S. Miyagawa5 1Organ Transplantation, Osaka University Graduate School of Medicine, suita/JAPAN, 2Division Of Organ Transplantation, Osaka UniversityGraduate School of Medicine, Suita/JAPAN, 3Transplantation, Osaka University Graduate School of Medicine, Suita/JAPAN, 4, Osaka University Graduate School of Medicine, Suita/JAPAN, 5Transplantation, Osaka University Graduate School of Medicine, Suita/Osaka/JAPAN