Susumu Shibasaki

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.

Risk factors for portal vein complications in pediatric living donor liver transplantation.

Shibasaki S, Taniguchi M, Shimamura T, Suzuki T, Yamashita K, Wakayama K, Hirokata G, Ohta M, Kamiyama T, Matsushita M, Furukawa H, Todo S. Risk factors for portal vein complications in pediatric living donor liver transplantation.
Clin Transplant 2010: 24: 550–556.
© 2009 John Wiley & Sons A/S.

IL-6 down-regulates HLA class II expression and IL-12 production of human dendritic cells to impair activation of antigen-specific CD4(+) T cells.

Immunosuppression in tumor microenvironments critically affects the success of cancer immunotherapy. Here, we focused on the role of interleukin (IL)-6/signal transducer and activator of transcription (STAT3) signaling cascade in immune regulation by human dendritic cells (DCs). IL-6-conditioned monocyte-derived DCs (MoDCs) impaired the presenting ability of cancer-related antigens. Interferon (IFN)-γ production attenuated by CD4+ T cells co-cultured with IL-6-conditioned MoDCs corresponded with decreased DC IL-12p70 production. Human leukocyte antigen (HLA)-DR and CD86 expression was significantly reduced in CD11b+CD11c+ cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors by IL-6 treatment and was STAT3 dependent. Arginase-1 (ARG1), lysosomal protease, cathepsin L (CTSL), and cyclooxygenase-2 (COX2) were involved in the reduction of surface HLA-DR expression. Gene expressions of ARG1, CTSL, COX2, and IL6 were higher in tumor-infiltrating CD11b+CD11c+ cells compared with PBMCs isolated from colorectal cancer patients. Expression of surface HLA-DR and CD86 on CD11b+CD11c+ cells was down-regulated, and T cell-stimulating ability was attenuated compared with PBMCs, suggesting that an immunosuppressive phenotype might be induced by IL-6, ARG1, CTSL, and COX2 in tumor sites of colorectal cancer patients. There was a relationship between HLA-DR expression levels in tumor tissues and the size of CD4+ T and CD8+ T cell compartments. Our findings indicate that IL-6 causes a dysfunction in human DCs that activates cancer antigen-specific Th cells, suggesting that blocking the IL-6/STAT3 signaling pathway might be a promising strategy to improve cancer immunotherapy.

Efficacy of DHMEQ, a NF-κB inhibitor, in islet transplantation: II. Induction DHMEQ treatment ameliorates subsequent alloimmune responses and permits long-term islet allograft acceptance.

Background Long-term graft deterioration remains a major obstacle in the success of pancreatic islet transplantation (PITx). Antigen-independent inflammatory and innate immune responses strengthen subsequent antigen-dependent immunity; further, activation of nuclear factor (NF)-&kgr;B plays a key role during these responses. In this study, we tested our hypothesis that, by the inhibition of NF-&kgr;B activation, the suppression of these early responses after PITx could facilitate graft acceptance. Methods Full major histocompatibility complex (MHC)–mismatched BALB/c (H-2d) mice islets were transplanted into streptozotocin-induced diabetic C57BL/6 (B6: H-2b) mice. The NF-&kgr;B inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) was administered for either 3 or 14 days after PITx. To some PITx recipients, tacrolimus was also administered. Islet allograft survival, alloimmune responses, and in vitro effects of DHMEQ on dendritic cells (DCs) were assessed. Results With a vehicle treatment, 600 islet allografts were promptly rejected after PITx. In contrast, 3-day treatment with DHMEQ, followed by 2-week treatment with tacrolimus, allowed permanent acceptance of islet allografts. The endogenous danger-signaling molecule high mobility group complex 1 (HMGB1) was elevated in sera shortly after PITx, whereas DHMEQ administration abolished this elevation. DHMEQ suppressed HMGB1-driven cellular activation and proinflammatory cytokine secretion in mouse bone marrow–derived DCs and significantly reduced the capacity of DCs to prime allogeneic T-cell proliferation in vitro. Finally, the DHMEQ plus tacrolimus regimen reverted the diabetic state with only 300 islet allografts. Conclusions Inhibition of NF-&kgr;B activation by DHMEQ shortly after PITx suppresses HMGB1, which activates DCs and strengthens the magnitude of alloimmune responses; this permits long-term islet allograft acceptance, even in case of fewer islet allografts.

Percutaneous transhepatic gallbladder drainage followed by elective laparoscopic cholecystectomy in patients with moderate acute cholecystitis under antithrombotic therapy.

Standard treatment for acute cholecystitis (AC) in patients receiving antithrombotic drugs has not been established. We evaluated the safety of percutaneous transhepatic gallbladder drainage (PTGBD) followed by elective laparoscopic cholecystectomy (LC) in patients with moderate AC who were receiving antithrombotics.

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.

Successful transplantation of rat hearts subjected to extended cold preservation with a novel preservation solution

Since prolonged cold preservation of the heart deteriorates the outcome of heart transplantation, a more protective preservation solution is required. We therefore developed a new solution, named Dsol, and examined whether Dsol, in comparison to UW, could better inhibit myocardial injury resulting from prolonged cold preservation. Syngeneic heterotopic heart transplantation in Lewis rats was performed after cold preservation with UW or Dsol for 24 or 36 h. In addition to graft survival, myocardial injury, ATP content, and Ca2+ ‐dependent proteases activity were assessed in the 24‐h preservation group. The cytosolic Ca2+ concentration of H9c2 cardiomyocytes after 24‐h cold preservation was assessed. Dsol significantly improved 7‐day graft survival after 36‐h preservation. After 24‐h preservation, Dsol was associated with significantly faster recovery of ATP content and less activation of calpain and caspase‐3 after reperfusion. Dsol diminished graft injury significantly, as revealed by the lower levels of infarction, apoptosis, serum LDH and AST release, and graft fibrosis at 7‐day. Dsol significantly inhibited Ca2+ overload during cold preservation. Dsol inhibited myocardial injury and improved graft survival by suppressing Ca2+ overload during the preservation and the activation of Ca2+ ‐dependent proteases. Dsol is therefore considered a better alternative to UW to ameliorate the outcome of heart transplantation.

Does reduced-port laparoscopic surgery for medically uncontrolled ulcerative colitis do more harm than good?

Reduced‐port laparoscopic surgery is a novel minimally invasive surgery. However, reduced‐port surgery for ulcerative colitis (UC) remains controversial. Here, we describe the clinical outcomes of single‐incision plus one port laparoscopic surgery (SILS + 1) for medically uncontrolled UC.

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.