Toshiko Yamada

AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase

The novel immunosuppressant AS1387392 has been isolated from Acremonium sp. No. 27082. This compound showed a strong inhibitory effect against mammalian histone deacetylase and T-cell proliferation. Further, AS1387392 showed a good oral absorption, and its plasma concentration was higher than that of FR235222, an analog of AS1387392 that inhibited histone deacetylase previously reported. Given these findings, AS1387392 may represent an important new lead in developing immunosuppressant.

Bioconversion of AS1387392: bioconversion studies involving Amycolatopsis azurea JCM 3275

We screened actinomycetes capable of converting AS1387392 to AS1429716 and identified those strains capable of hydroxylation. Amycolatopsis azurea JCM 3275 was found to be a particularly efficient strain, capable of converting AS1387392 to AS1429716, with a yield of 44% after 9 h. This strain can metabolize not only the hydroxylation of phenylalanine at the meta and para positions but also the reduction of hydroxyketones, as shown by the isolation of bioconversion products. Examination of more suitable conversion conditions showed that pH 7.8 and 25 °C were the optimum pH and temperature for bioconversion, respectively. We also demonstrated the effect of carbon and nitrogen sources in the culture media on hydroxylation. Using this strain, we were able to efficiently produce AS1429716 as a chemical template. Further derivatization studies may provide more effective, safer immunosuppressants than those that are currently on-market.

Microarray gene expression profiling of chronic allograft nephropathy in the rat kidney transplant model

Whole genome gene expression profiles were correlated with renal function and histology in a well-established animal model of chronic allograft nephropathy (CAN). Kidneys of F344 rats were transplanted into LEW recipients treated with a brief dose of FK506 (BFK). Blood and urine samples were collected weekly. Kidney grafts were harvested at an early (day 6) or late (days 30-90) phase after transplantation. BFK kidney grafts showed remarkable changes in function, histology, and gene expression profiles when compared to the isograft controls. In the early phase, renal function and histology were barely affected, yet the expression levels of 225 genes were significantly changed, reflecting both immune and non-immune pathways. In the late phase, however, 826 genes were affected in the BFK kidney grafts, including genes in the pathways of extracellular matrix and cell adhesion. Of these genes, 214 appear to be key factors for development of CAN, since they were affected at both early and late phases, including genes involved in the immune response, the inflammatory response, apoptosis, and metabolism. Kinetic studies with gene expression profiling can identify genes involved in the progressive development of chronic allograft rejection, leading to more detailed therapeutic approaches or useful biomarkers in clinical transplantation.

Mechanism analysis of long-term graft survival by monocarboxylate transporter-1 inhibition.

Background. Monocarboxylate transporter (MCT)-1, a member of a family of molecules, transports monocarboxylates such as lactate. Inhibiting MCT-1 leads to long-term graft survival in rodent heart transplantation and induces tolerance. We evaluated an MCT-1 inhibitor, AS2495674, in a rat heart transplant model and analyzed its underlying mechanism. Methods. AS2495674 was tested on rat lymphocytes to determine its effect on lactate accumulation, proliferation, and immunoglobulin production. The effect of AS2495674 on graft survival was tested on the Brown Norway to Lewis rat strain combination with a second heart transplantation to test donor-specific suppression. Histology and ex vivo analyses were done to examine the AS2495674 effects on the immune response. Results. In vitro, AS2495674 resulted in lactate accumulation, inhibited lymphocyte proliferation, and prevented immunoglobulin production. AS2495674 induced long-term allograft survival with little evidence of chronic rejection and induced donor-specific suppression. Evaluation of the allograft and peripheral T lymphocytes from the AS2495674 group compared with that of vehicle showed (1) decreased donor-specific T lymphocyte response, (2) more forkhead box P3+ (Foxp3+) and CD45RA+ cells in the allograft, (3) higher gene expression of chemokines and chemokine receptors in the allograft, and (4) preferential inhibition of Foxp3− cells with little or no effect on Foxp3+ cells. Conclusions. AS2495674 prevents acute rejection, reduces features of chronic rejection, and induces tolerance. Our data suggest that the mechanism of AS2495674 involves generating a tolerogenic graft environment by preferentially targeting T effector cells while sparing the generation of T regulatory cells.

Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

Background Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. Methods Splenocytes from C57BL/6 J (B6) AID−/− mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID−/− mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. Results The AID−/− splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID−/− recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID−/− recipients with further reduction of intimal hyperplasia and fibrosis scores. Conclusions The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

A Histone Deacetylase Inhibitor, FR276457, Altered Characteristics of Infiltrating Cells into Allograft in a Rat Cardiac Transplant Model

Background: A recent study indicated that FR276457, a pan-histone deacetylase inhibitor, prevented allograft rejection in an ACI-to-Lewis rat heart transplant model, seemingly without affecting cellular infiltration into the transplanted heart. In this report, we investigated alterations in infiltrating cells in allografts by treatment of FR276457. Materials and Methods: Heterotopic cardiac allografts which were removed from August Copenhagen Irish rats were transplanted into the necks of Lewis rats. FR276457 40 mg/kg or vehicle was subsequently administered orally for 5 consecutive days beginning on the operation day after recovery from anesthesia (Day 0). The allograft recipients were sacrificed under anesthesia on Day 5 after transplantation. At first, we identified infiltrating cells into the allografts by the histopathological analysis. In addition, we isolated the infiltrating cells from allografts, sorted CD8 positive (CD8 + ) T cells, and then investigated cytotoxicity of CD8 + T cells against spleen cells from an August Copenhagen Irish rat. Results: Histopathological analysis of allografts on Day 5 after the heart transplant showed that infiltration of T cells was not suppressed in FR276457-treated allograft recipients, compared with those in vehicle-treated allograft recipients, although the infiltration of ED1 positive cells was tended to decrease. Ex vivo analysis revealed that alloantigen-specific cytotoxicity of CD8 + T cells, isolated from allografts in FR276457-treated allograft recipients, against ACI rat splenocytes was much lower than that in vehicle-treated allograft recipients. Conclusion: FR276457 treatment suppressed the alloantigen-specific cytotoxicity of CD8 + T cells infiltrated into