Masaki Kitajima

Intraportal infusion therapy as a novel approach to adult ABO-incompatible liver transplantation

Background. ABO-incompatible liver transplantation is associated with an extremely complicated postoperative course, especially when the recipients are adults. Methods. Two adult patients underwent living-donor liver transplantation from ABO-incompatible donors. The antirejection therapy included multiple perioperative plasmapheresis, splenectomy, systemic triple immunosuppressive regimen with tacrolimus, methylprednisolone, and cyclophophamide, or azathioprine. In addition to these conventional approaches, we performed intraportal infusion therapy after transplantation with methylprednisolone, prostaglandin E1, and gabexate mesilate. Results. With our protocol, antidonor blood group antibody titers in both cases remained low without any evidence of rejection or vascular complications throughout the postoperative course. Biliary complications were transient and resolved completely. The patients have now survived 30 and 12 months posttransplantation and have regained normal life activity with good liver function. Conclusions. Our experience has shown the feasibility of controlling rejection and other complications in adult ABO-incompatible liver transplantation under intraportal infusion therapy.

Overexpression of Truncated IκBα Induces TNF-α–Dependent Apoptosis in Human Vascular Smooth Muscle Cells

Dysregulation of apoptosis is one of the likely underlying mechanisms of neointimal thickening, a disorder in which proinflammatory cytokines may influence the function of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis. One of these cytokines, tumor necrosis factor-alpha (TNF-alpha), induces 2 possibly conflicting pathways, 1 leading to the activation of nuclear factor-kappaB (NF-kappaB) and the other leading to caspase-mediated apoptosis. We investigated whether specific inhibition of NF-kappaB affects TNF-alpha-dependent apoptosis in human VSMCs. To inhibit NF-kappaB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IkappaBalpha (AdexIkappaBDeltaN) that lacks the phosphorylation sites essential for activation of NF-kappaB. The IkappaBDeltaN was overexpressed by adenoviral infection and was resistant to stimulus-dependent degradation. Electromobility gel shift and luciferase assays demonstrated that overexpression of IkappaBDeltaN inhibited NF-kappaB activation induced by TNF-alpha or interleukin-1beta (IL-1beta). In cells overexpressing IkappaBDeltaN, TNF-alpha dramatically induced apoptosis, whereas IL-1beta had no effect. The induction was suppressed by treatment with a selective inhibitor of the caspase-3 family, Z-DEVD-fmk, and the overexpression of IkappaBDeltaN induced TNF-alpha-mediated caspase-3 and caspase-2 activity. These results indicate that overexpression of IkappaBDeltaN induces TNF-alpha-dependent apoptosis by efficient and specific suppression of NF-kappaB and upregulation of caspase-3 and caspase-2 activity in human VSMCs. Our findings suggest that adenovirus-mediated IkappaBDeltaN gene transfer may be useful in the treatment of disorders associated with inflammatory conditions, such as the response to vascular injury and atherosclerosis.

Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen

Background. Programmed death (PD)-1 has been implicated in peripheral tolerance. The authors investigated the roles of PD-1 and its ligands, PD-L1 and PD-L2, in the induction of regulatory cells by intratracheal delivery of alloantigen. Methods. CBA (H-2k) mice were pretreated with intratracheal delivery of C57BL/10 (H-2b) splenocytes and administration of monoclonal antibody (mAb) specific for PD-1, PD-L1, or PD-L2. Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes from the pretreated CBA mice and transplantation of C57BL/10 heart. Results. Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 7 days). Pretreatment with intratracheal delivery of C57BL/10 splenocytes prolonged graft survival significantly (MST, 65 days). Administration of control immunoglobulin (Ig) G or anti–PD-L2 mAb did not significantly affect the prolongation (MST, 72 and 68 days, respectively). In contrast, anti–PD-1 or anti–PD-L1 mAb abrogated the prolongation (MST, 18 and 17 days, respectively). Adoptive transfer from mice pretreated with intratracheal delivery of alloantigen plus control IgG or anti–PD-L2 mAb prolonged survival of C57BL/10 grafts in secondary CBA recipients (MST, 72 and 56 days, respectively). However, concurrent administration of anti–PD-1 or anti–PD-L1 mAb abrogated prolonged survival after the adoptive transfer (MST, 14 and 20 days, respectively). Conclusions. PD-1–PD-L1 interaction was essential for induction of regulatory cells by intratracheal delivery of alloantigen.

B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice.

Background. The mechanism of hyporesponsiveness induced by intratracheal (IT) delivery of alloantigen was examined and its effect on cardiac graft survival was assessed in studies in mice. Methods. In CBA (H2k) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2b) heart. To determine whether regulatory cells were involved in hyporesponsiveness, splenocytes from mice given IT delivery of alloantigen and antibodies for B7–1, B7–2, or CTLA4 were adoptively transferred to naïve secondary recipients 7 days after delivery; those recipients underwent heart transplantation the same day. Effects on cell proliferation and cytokine production of splenocytes from mice given IT delivery of alloantigen were examined in mixed leukocyte cultures (MLC). Results. Cardiac graft survival was significantly prolonged in mice given IT delivery of alloantigen (median survival time [MST], 81 days); those given syngeneic splenocytes rejected grafts acutely (MST, 7 days;P <0.05). Adoptive transfer of splenocytes also significantly prolonged survival of cardiac grafts in secondary recipients (MST, 62 days). When B7–1, B7–2, or CTLA4 antibody was combined with IT delivery of alloantigen in the first recipient, all grafts were rejected within 14 days in second recipients after adoptive transfer. In mixed leukocyte cultures, splenocytes from these mice did not respond to alloantigen and production of interleukin-4 and interleukin-10 was increased. Conclusions. Donor splenocytes delivered IT induced hyporesponsiveness and regulatory cells in our animal model, and such induction was dependent on B7–1, B7–2, and CTLA4 signals.

High dose of antithrombin III induces indefinite survival of fully allogeneic cardiac grafts and generates regulatory cells.

Background. The authors investigated whether antithrombin III (AT-III) could induce unresponsiveness to alloantigens. Methods. CBA mice were given intravenous injection of 50 or 500 U/kg AT-III or control plasma the same day as transplantation of a heart from a C57BL/6 mouse. An adoptive transfer study and mixed leukocyte culture analysis were also performed. Results. Naive CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST], 9 days). The 50-U/kg dose of AT-III induced a moderate increase in graft survival (MST, 25 days), whereas control mice rejected their graft acutely (MST, 7 days). With the 500-U/kg dose of AT-III, all grafts survived indefinitely (>100 days) and regulatory cells were generated. In vitro, AT-III suppressed proliferation of mixed leukocyte responses and generation of interleukin-2. Conclusion. AT-III can be not only an antithrombotic agent but also a strong immunomodulating agent when used at high dose.

Living Donor Liver Transplantation with Special Reference to ABO-incompatible Grafts and Small-for-size Grafts

Living donor liver transplantation (LDLT) has developed on the basis of increased safety of conventional liver surgery and the need for expanding donor sources, especially in children. Indications for LDLT were soon extended to adult patients in Japan, where cadaveric donation was limited. The right liver is now routinely transplanted to adults to avoid small-for-size graft syndrome, even though the right liver graft has the disadvantages of less remaining donor liver and the question of donor safety. Assessing the suitable size or quality of the graft, as well as of the remnant donor liver, is one of the most important problems in adult LDLT. Although several tactics have been proposed to manage the small-for-size syndrome, their efficacy remains a question. We suggest that small-for-size syndrome is preventable by engaging in careful donor selection or using effective agents for hepatic microcirculatory disturbance control. Sometimes for LDLT only ABO-incompatible grafts are available from relatives, but they must be transplanted despite the expected poor outcome in adults and older children. To overcome the problems in this situation, we developed a novel protocol including intraportal infusion therapy with methylprednisolone, prostaglandin E1, and gabexate mesylate. Two adult patients undergoing ABO- incompatible LDLT have now survived 53 and 35 months after transplantation with good liver function. However, the other two patients suffered thrombotic microangiopathy postoperatively and died owing to cerebral hemorrhage or multiple organ failure, respectively. Further investigation is needed to improve the outcome of liver transplantation across the ABO blood group barrier.

Gallbladder carcinoma with osteoclast-like giant cells

Extraskeletal tumors containing multinucleated, osteoclast-like giant cells (OGCs) are uncommon. These neoplasms are most frequently reported in the breast and pancreas. Recently, some authors have suggested that carcinomas containing OGCs may represent a distinct clinicopathological entity with a more favorable prognosis. Occurrence in the gallbladder is extremely rare, with only one previous case. We report here on an additional case of gallbladder carcinoma with an infiltrate of OGCs. A 72-year-old woman presented with postprandial abdominal pain and was found to have a mass in the body of the gallbladder with direct liver invasion. Histological examination showed an adenosquamous carcinoma with an infiltrate of benign OGCs. Immunohistochemical analysis demonstrated that the giant cells were of histiocytic origin. The patient survived for 6 years without evidence of recurrence. This case adds to a small body of literature on gallbladder carcinoma with OGCs. Further studies are required to clearly define the prognostic significance of these giant cells in gallbladder cancer and the differences between adenosquamous carcinoma with OGCs and other gallbladder carcinomas (such as adenocarcinoma and squamous cell carcinoma) with those cells.

Intratracheal delivery of a single major histocompatibility complex class I peptide induced prolonged survival of fully allogeneic cardiac grafts and generated regulatory cells

We have previously reported that intratracheal delivery of donor splenocytes in mice induces hyporesponsiveness to fully allogeneic cardiac grafts and generates regulatory cells. Here, we examined whether an allopeptide would produce the same results. A 15-mer (54-68) peptide corresponding to a hypervariable region of the K(b) molecule was given intratracheally or intravenously to CBA (H2(k)) mice 7 days before transplantation of a C57BL/10 (H2(b)) or BALB/c (H2(d)) heart and was also used in adoptive transfer experiments. Cardiac grafts in recipients given K(b) peptide intratracheally experienced a median survival time (MST) of 56 days, whereas those in recipients given the peptide intravenously were rejected acutely (MST=7.5 days). Adoptive transfer of splenocytes from mice pretreated intratracheally with K(b) peptide to naïve secondary recipients prolonged survival of cardiac grafts (MST = 35 days). Intratracheal delivery of a single major histocompatibility complex class I peptide induced hyporesponsiveness to allogeneic cardiac grafts and generated regulatory cells.

Tumor necrosis factor suppression and microcirculatory disturbance amelioration in ischemia/reperfusion injury of rat liver after ischemic preconditioning

Background: Brief periods of hepatic ischemia produce immediate tolerance for subsequent prolonged ischemia. Although the beneficial effect of this ischemic preconditioning is recognized, the mechanism itself remains poorly understood.

The role of nitric oxide after a short period of liver ischemia-reperfusion

BACKGROUNDnLiver ischemia-reperfusion injury is a serious problem during liver resection and transplantation. Nitric oxide (NO) has been suggested to have a cytoprotective effect for microcirculation, while the interaction of active oxygen species and NO produces peroxynitrite anion. The present study attempts to clarify the role of NO in liver ischemia-reperfusion injury.nnnMETHODSnWistar male rats were subjected to 30 min of hepatic ischemia followed by reperfusion. The model rats were divided into the three following groups: a control group that was not administered NO synthase inhibitors, and two experimental groups that were administered either N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine. In each group, we examined active oxygen species and nitric oxide production, and investigated liver function by measuring serum transaminase levels. In addition, we conducted histopathologic examinations and microcirculation examinations using intravital videomicroscopy.nnnRESULTSnIn the control group, NO concentrations in the plasma increased with time after reperfusion. A decrease in NO production was detected in the groups administered NO synthase inhibitors. Elevated serum transaminase levels became more prominent after L-NAME administration, while aminoguanidine administration reduced its level. The degree of microcirculation failure was found to be more prominent in the L-NAME-administered group over both the control group and the aminoguanidine-administered group. A significantly lower survival rate was observed at 6 h after reperfusion in the L-NAME-administered group over that of the other groups.nnnCONCLUSIONSnA reduction of the ischemia-reperfusion injury is important in inhibiting the production of high-output NO and peroxynitrite, and in maintaining NO levels necessary for maintenance of microcirculation.