Ryoichi Goto

A Human Anti‐CD40 Monoclonal Antibody, 4D11, for Kidney Transplantation in Cynomolgus Monkeys: Induction and Maintenance Therapy

Blockade of CD40–CD154 signaling pathway is an attractive strategy to induce potent immunosuppression and tolerance in organ transplantation. Due to its strong immunosuppressive effect shown in nonhuman primate experiments, anti‐CD154 monoclonal antibodies (mAbs) have been tried in clinical settings, but it was interrupted by unexpected thromboembolic complications. Thus, inhibition of the counter molecule, CD40, has remained an alternative approach. In the previous preliminary study, we have shown that 4D11, a novel fully human anti‐CD40 mAb, has a fairly potent immunosuppressive effect on kidney allograft in nonhuman primates. In this study, we aimed to confirm the efficacy and untoward events of the 2‐week induction and 180‐day maintenance 4D11 treatments. In both, 4D11 significantly suppressed T‐cell‐mediated alloimmune responses and prolonged allograft survival. Addition of weekly 4D11 administration after the induction treatment further enhanced graft survival. Complete inhibition of both donor‐specific Ab and anti‐4D11 Ab productions was obtained only with higher‐dose maintenance therapy. No serious side effect including thromboembolic complications was noted except for a transient reduction of hematocrit in one animal, and decrease of peripheral B‐cell counts in all. These results indicate that the 4D11 appears to be a promising candidate for immunosuppression in clinical organ transplantation.

A pilot study of operational tolerance with a regulatory T‐cell‐based cell therapy in living donor liver transplantation

Potent immunosuppressive drugs have significantly improved early patient survival after liver transplantation (LT). However, long‐term results remain unsatisfactory because of adverse events that are largely associated with lifelong immunosuppression. To solve this problem, different strategies have been undertaken to induce operational tolerance, for example, maintenance of normal graft function and histology without immunosuppressive therapy, but have achieved limited success. In this pilot study, we aimed to induce tolerance using a novel regulatory T‐cell‐based cell therapy in living donor LT. Adoptive transfer of an ex vivo‐generated regulatory T‐cell‐enriched cell product was conducted in 10 consecutive adult patients early post‐LT. Cells were generated using a 2‐week coculture of recipient lymphocytes with irradiated donor cells in the presence of anti‐CD80/86 monoclonal antibodies. Immunosuppressive agents were tapered from 6 months, reduced every 3 months, and completely discontinued by 18 months. After the culture, the generated cells displayed cell‐number‐dependent donor‐specific inhibition in the mixed lymphocyte reaction. Infusion of these cells caused no significant adverse events. Currently, all patients are well with normal graft function and histology. Seven patients have completed successful weaning and cessation of immunosuppressive agents. At present, they have been drug free for 16‐33 months; 4 patients have been drug free for more than 24 months. The other 3 recipients with autoimmune liver diseases developed mild rejection during weaning and then resumed conventional low‐dose immunotherapy. Conclusions: A cell therapy using an ex vivo‐generated regulatory T‐cell‐enriched cell product is safe and effective for drug minimization and operational tolerance induction in living donor liver recipients with nonimmunological liver diseases. (Hepatology 2016;64:632‐643)

A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice

BACKGROUND In inflammatory bowel disease (IBD), gut inflammation is associated with the activation of nuclear factor kappa B (NF-κB), a key pro-inflammatory transcription factor. AIM To investigate the therapeutic potential of a novel, specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), we examined its effect on IBD using murine experimental colitis models. METHODS The in vitro effect of DHMEQ was evaluated by inflammatory cytokine production and p65 immunostaining using HT-29 and RAW264.7 cells. The in vivo therapeutic effect of DHMEQ was studied in colitis induced by dextran sulphate sodium (DSS) and trinitrobenzenesulphonic acid (TNBS). In these, progression and severity of colitis was mainly assessed by the disease activity index (DAI), histopathology, cellular infiltration, and mRNA expression levels of pro-inflammatory cytokines in the colonic tissues. RESULTS In RAW264.7 cells, DHMEQ significantly inhibited tumour necrosis factor (TNF)-α and interleukin (IL)-6 production induced by LPS in a dose-dependent manner by blocking the nuclear translocation of NF-κB. In addition, DHMEQ inhibited IL-8 production induced by LPS in HT-29 cells. DHMEQ significantly ameliorated DSS colitis as assessed by DAI scores, colonic oedema, and histological scores. Immunohistochemistry revealed that DHMEQ inhibited colonic infiltration of nuclear p65(+) cells, CD4(+) lymphocytes, and F4/80(+) macrophages. mRNA expression levels of the pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-12p40, IL-17, and MCP-1 were also suppressed by DHMEQ administration. Furthermore, DHMEQ significantly ameliorated TNBS colitis as assessed by body-weight changes and histological scores. CONCLUSION DHMEQ ameliorated experimental colitis in mice. These results indicate that DHMEQ appears to be an attractive therapeutic agent for IBD.

Does the Permanent Portacaval Shunt for a Small-for-Size Graft in a Living Donor Liver Transplantation Do More Harm Than Good?

In order to obviate a small‐for‐size graft syndrome (SFSGS), a portacaval (PC) shunt had been considered in a case of adult‐to‐adult living donor liver transplantation (AA‐LDLT). In a recent AA‐LDLT case, we adopted the PC shunt to resolve SFSGS; however, graft atrophy was observed in the late period of LDLT, thereby resulting in liver dysfunction. Due to the surgical closure of the PC shunt at 11 months post‐LDLT, the graft regenerated gradually and resulted in the recovery of the liver function. This experience indicates that the portacaval shunt would overcome SFSGS in the early period of LDLT, while it would cause the graft atrophy and the graft dysfunction in the late period of LDLT.

Inhibition of macrophage activation and suppression of graft rejection by DTCM-glutarimide, a novel piperidine derived from the antibiotic 9-methylstreptimidone

ObjectiveWe have previously synthesized a novel piperidine compound, 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide), that inhibits LPS-induced NO production, and in the present research we studied further the anti-inflammatory activity of DTCM-glutarimide in a macrophage cell line and in mice bearing transplanted hearts.Materials and methodsMouse macrophage-like RAW264.7 cells were employed for the evaluation of cellular inflammatory activity. DTCM-glutarimide was synthesized in our laboratory. The AP-1 activity was measured by nuclear translocation and phosphorylation. For the heart transplantation experiment, male C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as donor and recipient, respectively. DTCM-glutarimide was administered intraperitoneally.ResultsDTCM-glutarimide inhibited the LPS-induced expression of iNOS and COX-2 in macrophages; but, unexpectedly, it did not inhibit LPS-induced NF-κB activation. Instead, it inhibited the nuclear translocation of both c-Jun and c-Fos. It also inhibited LPS-induced c-Jun phosphorylation. Moreover, it inhibited the mixed lymphocyte reaction in primary cultures of mouse spleen cells; and furthermore, in mice it prolonged the graft survival in heart transplantation experiments.ConclusionThe novel piperidine compound, DTCM-glutarimide, was found to be a new inhibitor of macrophage activation, inhibiting AP-1 activity. It also inhibited graft rejection in mice, and thus may be a candidate for an anti-inflammatory agent.

Immunosuppressive effects of DTCM-G, a novel inhibitor of the mTOR downstream signaling pathway.

Background A newly developed compound, 3-[(dodecylthiocarbonyl)methyl]-glutarimide (DTCM-G), has been shown to inhibit nuclear translocation of c-Fos/c-Jun in a murine macrophage cell line. Herein, we studied the immunosuppressive properties and potency of DTCM-G. Methods Using purified mouse T cells, the in vitro effects of DTCM-G on activation, cytokine production, proliferation, and cell cycle progression were assessed, and a possible molecular target of DTCM-G was investigated. In a BALB/c (H-2d) to C57BL/6 (H-2b) mouse heart transplantation model, transplant recipients were administered DTCM-G, a calcineurin inhibitor (tacrolimus), and a nuclear factor-&kgr;B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). Treatment drugs were administered daily for 14 days after transplantation. Alloimmune responses were assessed in addition to graft survival time. Results After anti-CD3+anti-CD28 monoclonal antibody stimulation, DTCM-G significantly suppressed proliferation, interferon-&ggr; production, and cell cycle progression of activated T cells but not CD25 expression or interleukin-2 production. These effects were accompanied by inhibition of 70-kDa S6 protein kinase phosphorylation, a downstream kinase of the mammalian target of rapamycin. The addition of tacrolimus and DHMEQ to DTCM-G resulted in a robust inhibition of T-cell proliferation. In vivo combination therapy of DTCM-G plus either tacrolimus or DHMEQ significantly suppressed alloreactive interferon-&ggr;–producing precursors and markedly prolonged cardiac allograft survival. Furthermore, combination of all three agents markedly inhibited alloimmune responses and permitted long-term cardiac allograft survival. Conclusions DTCM-G inhibits T cells by suppressing the downstream signal of mammalian target of rapamycin. DTCM-G in combination with tacrolimus and DHMEQ induces a strong immunosuppressive effect in vivo.

Dendritic cells conditioned with NK026680 prolong cardiac allograft survival in mice.

Background Pharmacologically modulated dendritic cells (DCs) can potentially regulate alloimmune responses. We examined the characteristics of immunoregulatory DCs induced by a novel triazolopyrimidine derivative, NK026680, which has been previously shown to inhibit DC maturation. Methods DCs were generated from bone marrow progenitor cells from C57BL/6 (B6, H-2b haplotype) mice with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. DCs were cultured with allogeneic BALB/c (H-2d) splenocyte lysates with or without NK026680. DC functions were examined in vitro after stimulation of tumor necrosis factor &agr; and in vivo by the intravenous injection of C3He/J (C3H, H-2k) DCs cultured with B6 cell lysates and NK026680 into C3H mice. Seven days later, DC-treated mice received B6 heart allografts, and graft survival and alloimmune responses were assessed. Results In NK026680-treated DCs (NK-DCs), significant inhibition of the up-regulation of surface activation markers (CD40, CD80, CD86, and major histocompatibility complex class II) and IL-12 p40 production was observed after stimulation of tumor necrosis factor &agr; compared with that of control DCs. Furthermore, NK-DCs suppressed alloreactive T-cell proliferation. The modulation of NK-DCs was likely associated with the inhibition of phosphorylation of p38 mitogen-activated protein kinase and the up-regulation of indolamine 2,3-dioxygenase expression. Compared with both noninjected and control DC–injected mice, mice that received a single in vivo infusion of NK-DCs showed significant increases in splenocyte IL-10 production and the splenic CD4+ IL-10high T-cell population 7 days after injection, a significantly increased splenic CD4+CD25+FoxP3+ T-cell population 14 days after injection, and markedly prolonged cardiac allograft survival. Conclusions Ex vivo NK026680 conditioning allows DCs to acquire immunoregulatory properties that suppress alloimmune responses and prolong cardiac allograft survival.

Immunomodulatory effect of nuclear factor-κB inhibition by dehydroxymethylepoxyquinomicin in combination with donor-specific blood transfusion.

Background. Nuclear factor-&kgr;B (NF-&kgr;B) is a key molecule in alloimmune responses, however, its role in tolerance induction is not clear. We have previously reported that dehydroxymethylepoxyquinomycin (DHMEQ), a novel NF-&kgr;B inhibitor, prolongs cardiac allograft survival. In this study, we evaluated the immunomodulatory effects of DHMEQ when combined with a donor-specific blood transfusion (DST), and assessed whether the treatment induces tolerance in a mouse heart transplantation model. Methods. DST (20×106 splenocytes) was given intravenously at day −7. DHMEQ (30 mg/kg/day) was administered intraperitoneally for 14 days after DST. Graft survival and histology were evaluated. The underlying mechanisms of immunomodulation by DST and DHMEQ treatments were investigated by assessing alloimmune responses after transplantation. Results. In fully mismatched H2d-to-H2b heart transplants, DST alone prolonged allograft median survival time to 15 days, whereas when DST was combined with DHMEQ treatment, the graft median survival time was prolonged to 39.5 days. When the donor-recipient strain combination was reversed, that is, H2b-to-H2d, heart transplants were accepted (>150 days survival) in more than 60% of recipients treated with a DST and DHMEQ, whereas control allografts were all rejected within 8 days. The combined therapy markedly inhibited immune responses by both the direct and indirect allorecognition pathways mainly attributed to promotion of activation-induced cell death and Treg generation. Conclusions. Our results demonstrate the distinctive ability of NF-&kgr;B inhibition in combination with donor alloantigen to promote transplantation tolerance through multiple cellular mechanisms.

Asunaprevir and daclatasvir for recurrent hepatitis C after liver transplantation; a Japanese multicenter experience

The safety and efficacy of an IFN‐free regimen using asunaprevir (ASV) and daclatasvir (DCV) for recurrent hepatitis C virus (HCV) infection after liver transplantation (LT) have not been evaluated in Japan. A multicenter study of LT recipients (n = 74) with recurrent HCV genotype 1b infection treated with ASV‐DCV for 24 weeks was performed. Medical history was positive for pegylated interferon and ribavirin (Peg‐IFN/RBV) in 40 (54.1%) patients, and for simeprevir (SMV) with Peg‐IFN/RBV in 12 (16.2%) patients. Resistance‐associated variants (RAVs) were positive at D168 (n = 1) in the NS3, and at L31 (n = 4), Y93 (n = 4), and L31/Y93 (n = 1) in the NS5A region of the HCV genome. Sixty‐one (82.4%) patients completed the 24‐week treatment protocol. Although sustained viral response (SVR) was achieved in 49 (80.3%) patients, it was achieved in only two (16.7%) patients among those with histories of receiving SMV (n = 12). Univariate analysis showed that a history of SMV (P < .01) and the presence of mutations in NS5A (P = .02) were the significant factors for no‐SVR. By excluding the patients with either a history of SMV‐based treatment or RAVs in NS3/NS5A, the SVR rate was 96.4%. By excluding the patients with a history of SMV and those with RAVs in NS3/NS5A, viral clearance of ASV‐DCV was favorable, with a high SVR rate.

NK026680 inhibits T-cell function in an IL-2-dependent manner and prolongs cardiac allograft survival in rats

NK026680 is a triazolopyrimidine derivative that has been shown to inhibit dendritic cell maturation and activation. Here, we examined the immunosuppressive properties of NK026680 on T-cell function and assessed its immunosuppressive efficacy in an ACI (RT1(av1) haplotype) to Lewis (RT1(l)) rat heart transplantation model. The effects of NK026680 on T-cell proliferation, activation, and cytokine production were investigated in vitro. Heart transplant recipient rats were administered NK026680 daily for 14 days post-transplantation. In addition to graft survival time, alloimmune responses and graft histology at 4-10 days post-transplantation were assessed. NK026680 was found to inhibit proliferation, CD25 upregulation, IL-2 production, and cell cycle progression in αCD3/αCD28-stimulated murine T cells. These effects were likely due to suppression of the p38 mitogen-activated protein kinase pathway and the subsequent inhibition of p65, c-Fos, and to a lesser extent, c-Jun. Daily NK026680 treatment suppressed alloimmune responses, prevented cellular infiltration into allografts, and prolonged graft survival. The anti-rejection effects of NK026680 were enhanced by tacrolimus. In conclusion, NK026680 inhibits the activation of T cells and prolongs cardiac allograft survival in rats. These features make it a potential candidate immunosuppressant for the treatment of organ transplant patients in the future.