Takeshi Aoyagi

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 fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys.

Background. CD40-CD154 pathway blockade by anti-CD154 monoclonal antibodies (mAbs) significantly prolongs allograft survival in nonhuman primates. However, thromboembolic complications have prevented clinical application. Thus, blockade of the counter molecule by a novel fully human anti-CD40 mAb, 4D11, is an attractive alternative. Methods. Kidney transplantations were performed between outbred cynomolgus monkeys (stimulation index >3 in a mixed lymphocyte reaction). The animals were divided into five groups: nontreatment control (Group 1, n=3), 10-week treatment with either 10 mg/kg (Group 2, n=3), 20 mg/kg (Group 3, n=3), or 40 mg/kg (Group 4, n=1), and 4-week treatment (Group 5, n=1 each) with 10 mg/kg, 20 mg/kg, or 40 mg/kg followed by monthly administration. Graft survival, biochemistry, complete blood counts, lymphocyte phenotypes, blood drug levels, antidonor and antidrug antibodies, and renal histology were examined. Results. Survival (days) was as follows: Group 1 (5, 6, 7), Group 2 (150, 108, 108), Group 3 (84, 108, 379), Group 4 (147), and Group 5 (147, 102, 112). Two animals in Group 3 with normal graft function were killed upon development of hydronephrosis and cerebral infarction. B lymphocytes fell to one-third of the preoperative value at 4 weeks after transplantation in all animals. Antidonor antibodies developed in most of the animals after stopping drug treatment or at the time of death. No animals except for one formed anti-4D11 antibody. Conclusion. 4D11 appears to be a promising agent for antirejection treatment in clinical organ transplantation.

The Novel NF-κB Inhibitor, Dehydroxymethylepoxyquinomicin, Prevents Local and Remote Organ Injury Following Intestinal Ischemia/Reperfusion in Rats

BACKGROUND Nuclear factor-kappaB regulates the expression of several genes involved in inflammation, the immune response, apoptosis, cell survival, and proliferation. Many of these same genes are activated during ischemia/reperfusion (I/R) injury. Here, we examined the anti-inflammatory efficacy of a newly developed nuclear factor-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in the intestinal I/R injury model of rats. MATERIALS AND METHODS Intestinal ischemia was induced by occluding the superior mesenteric artery for 60 min. The experimental animals were divided into two groups: untreated group, control; treated group, DHMEQ-treated (20 mg/kg). DHMEQ were administered intraperitoneally at 60 min prior to clamping and 5 min prior to reperfusion. Animal survival rates, intestinal tissue blood flow, serum levels of tumor necrosis factor-alpha, and interleukin-6, and the histopathology of both the intestine and the lung were analyzed. RESULTS The DHMEQ-treated animals exhibited higher values of intestinal tissue blood flow and suppression of tumor necrosis factor-alpha and interleukin-6 production, resulting in marked prolongation of their survival times. Histopathological findings obtained by examining tissues from control animals revealed severe intestinal mucosal damage and disruption of the lung alveolar architecture accompanied by hemorrhage and marked neutrophilic infiltration. These findings were significantly ameliorated in DHMEQ-treated animals. CONCLUSION DHMEQ effectively prevented both intestine and lung injuries in rat intestinal I/R models. This agent may possess a good potency for clinical application in various pathological settings including intestinal I/R and/or inflammatory acute lung injury.

ASKP1240, a fully human anti-CD40 monoclonal antibody, prolongs pancreatic islet allograft survival in nonhuman primates.

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti‐CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti‐CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201–12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post‐PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti‐donor cellular responses and development of anti‐donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.

Control of allograft rejection by applying a novel nuclear factor-κB inhibitor, dehydroxymethylepoxyquinomicin

Background. Nuclear factor (NF)-&kgr;B plays a crucial role in lymphocyte activation, proliferation, and survival. We examined the immunosuppressive effect of a newly developed NF-&kgr;B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in allotransplantation. Methods. Purified C57BL/6 (H-2b) T cells were used for in vitro studies examining activation, proliferation, cytokine production and nuclear NF-&kgr;B and nuclear factor of activated T cells (NFAT) protein levels. A fully major histocompatibility complex incompatible BALB/c (H-2d)-to-C57BL/6 mice cardiac transplantation model was utilized for in vivo studies. DHMEQ was given intraperitoneally to transplant recipients at a various dose starting from day 0. In some, DHMEQ was administered concomitantly with tacrolimus. Results. DHMEQ significantly suppressed &agr;CD3+&agr;CD28 monoclonal antibody–triggered T-cell proliferation, CD25/CD69 expressions, and both interleukin-2 and interferon (IFN)-&ggr; production in a dose-dependent fashion. DHMEQ blocked nuclear translocation of NF-&kgr;B but not NFAT in activated T cells. Combined treatment with DHMEQ and tacrolimus significantly suppressed T cell activation as compared to that of mono-therapy with either agent alone. Single DHMEQ treatment moderately prolonged cardiac allograft survival. Further, combination of DHMEQ plus tacrolimus markedly prolonged graft mean survival time (MST) to 59.5 days when compared to either DHMEQ (MST: 10 days) or tacrolimus (MST: 13 days) treatment alone. Such effect was associated with inhibition of mixed lymphocyte reaction against donor antigen, IFN-&ggr; producing splenocytes and graft cellular infiltration as examined at 5 and 12 days posttransplantation. Conclusion. DHMEQ inhibits nuclear translocation of NF-&kgr;B but not NFAT in activated T cells, and prolongs allograft survival. Blocking both NF-&kgr;B and NFAT by DHMEQ and tacrolimus induces potent immunosuppression, which may become a new modality in controlling allograft rejection.

Repeat endovascular treatment of recurring hepatic artery stenoses in orthotopic liver transplantation.

Hepatic artery stenosis (HAS) is a complication that impacts the results of orthotopic liver transplantation (OLT). Interventional radiological techniques are important therapeutic options for HAS. The aim of this retrospective study was to evaluate the outcome of repeated radiological treatments in recurring HAS after OLT. Of the 941 patients who underwent OLT at our center from January 1998 to September 2010, 48 (5%) were diagnosed with HAS, 37 (77%) of whom underwent transluminal interventional therapy with the placement of an endovascular stent. Success rate, complications, hepatic artery patency and follow‐up were reviewed. After stent placement, artery patency was achieved in all patients. Three patients developed complications, including arterial dissection and hematoma. HAS recurrence was observed in 9 patients (24%), and hepatic artery thrombosis (HAT) occurred in 4 (11%). Radiological interventions were repeated 10 times in 8 patients without complications. At a median follow‐up of 66 months (range 10–158), hepatic artery patency was observed in 35 cases (94.6%). The 5‐year rates for graft and patient survival were 82.3% and 87.7%, respectively. Restenosis may occur in one‐third of patients after endovascular treatment for thrombosis and HAS, but the long‐term outcomes of iterative radiological treatment for HAS indicate a high rate of success.

Liver Transplantation With Older Donors: A Comparison With Younger Donors in a Context of Organ Shortage.

Background Older liver grafts have been considered in the past decade due to organ shortage. The aim was to compare outcomes after liver transplantation with either younger or older donors. Methods Patients transplanted in our center between 2004 and 2014 with younger donors (younger than 60 years; n = 253) were compared with older donors (older than 75 years; n = 157). Multiorgan transplantations, split grafts, or non–heart-beating donors were not included. Results Donors in the older group were mostly women deceased from stroke, and only 3 patients had experienced cardiac arrest. Liver tests were significantly better in the older group than in the younger group. There was no difference regarding cold ischemia time, model for end-stage liver disease score, and steatosis. There was no significant difference regarding primary nonfunction and dysfunction, hepatic artery and biliary complications, and retransplantation rates. Graft survival was not different (65% and 64% in the older and younger groups, P = 0.692). Within the older group, hepatitis C infection, retransplantation, and emergency transplantation were associated with poor graft survival. Conclusions Provided normal liver tests and the absence of cardiac arrest in donors, older liver grafts (>75 years) may be safely attributed to non–hepatitis C-infected recipients in the setting of a first and nonurgent transplantation.

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.

Rapidly enlarging solitary nonparasitic cyst of the liver in a child presenting as acute abdomen

Symptomatic solitary nonparasitic cysts of the liver (SNCL) are rarely encountered in children, especially rapidly enlarging cysts presenting as acute abdomen. Therefore, it is difficult to establish the pre-operative diagnosis and to determine the treatment protocol of SNCL. While imaging techniques such as ultrasonography and computed tomography are modalities for diagnosis of SNCL, making a pre-operative diagnosis in the case of very large cysts remains difficult. We present a child with SNCL who initially presented with a rapidly enlarging cyst presenting as acute abdomen, and also provide a review of the literature. Moreover, we evaluate the surgical procedures and conclude that total excision of the cysts when possible is a suitable treatment procedure in children.