Wataru Nakanishi

Attenuation of cross-talk between the complement and coagulation cascades by C5a blockade improves early outcomes after intraportal islet transplantation.

Background. Complement 5a factor (C5a) elicits a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. C5a is also linked to the coagulant activity in autoimmune diseases. Therefore, C5a most likely plays a crucial role in the instant blood-mediated inflammatory reaction. Methods. Intraportal transplantation of 2.5 islet equivalents/g of syngeneic rat islet grafts was performed in two groups of streptozotocin-induced diabetic rats: controls and C5a inhibitory peptide (C5aIP)-treated group. Results. The thrombin-antithrombin complex was significantly suppressed in the C5aIP group (P=0.003), and both the curative rate and the glucose tolerance were significantly improved in the C5aIP group (P<0.05 and P<0.005, respectively). Expression of tissue factor on granulocytes in recipient livers was up-regulated 1 h after islet infusion (P<0.0001), which was significantly suppressed by C5aIP (P<0.005). However, C5aIP was unable to regulate tissue factor expression on isolated islets. Furthermore, no differences were detected between the groups, regarding infiltration of CD11b-positive cells and deposition of C5b-9 on the islet grafts. Conclusions. These data suggest that C5aIP attenuates cross-talk between the complement and coagulation cascades through suppressing up-regulation of tissue factor expression on leukocytes in recipient livers but not on islet grafts, a process leading to improvement in islet engraftment. Therefore, C5aIP in combination with conventional anticoagulants could be a strong candidate strategy to control the instant blood-mediated inflammatory reaction induced in clinical islet transplantation.

Risk Factors for Hepatic Artery Thrombosis After Microsurgical Vascular Reconstruction in Liver Transplantation

OBJECTIVE In liver transplantation, microsurgical reconstruction of a hepatic artery is essential but requires challenging techniques. Especially in living-donor liver transplantation, the recipient artery is short and located deep in the abdominal cavity. Furthermore, hepatic artery thrombosis (HAT) can be a lethal complication. This study sought to uncover the risk factors for HAT after microsurgical vascular reconstruction. METHODS From 1991 to 2011, we performed 151 microsurgical vascular reconstructions, including 3 deceased-donor liver transplantations. We retrospectively investigated the cases, performing univariate and multivariate analyses to identify independent risk factors for HAT. The patients had undergone ultrasonographic examinations for HAT over the first 14 days after transplantation. RESULTS Upon univariate analysis, the risk factors identified to be associated with P < .20 were young age (P = .0484), low body weight (P = .0466), short height (P = .0128), high graft-to-recipient weight ratio (P = .0031), small liver graft volume (P = .0416), small amounts of gabexate mesilate infusion (P = .0516), and the conventional technique (without a back-wall support suture; P = .1326). A multiple logistic regression analysis identified low body weight to be the only independent risk factor for HAT. CONCLUSION On the univariate analysis, we found that using the back-wall support suture technique contributed to the reduction of HAT, whereas on multivariate analysis, the only independent risk factor for HAT was low body weight.

C5a-Inhibitory Peptide Combined with Gabexate Mesilate Prevents the Instant Blood-Mediated Inflammatory Reaction in a Rat Model of Islet Transplantation

BACKGROUND The instant blood-mediated inflammatory reaction (IBMIR), in which the activation of both the coagulation and the complement cascades plays a key role, is one of the main obstacles to successful islet transplantation. At present, however, no useful protocol is clinically available. Therefore the aim of this study was to examine whether complementary peptides against an active region of C5a were safe to suppress IBMIR, owing to their extremely low molecular mass, when combined with a clinically available anticoagulant. METHODS Complement receptors on pancreatic tissues and isolated islets were analyzed by immunohistochemical staining and flow cytometry. Two-and-a-half islet equivalents per gram of syngeneic rat islet grafts were transplanted intraportally into 4 groups of 10-13 animals each after streptozotocin induction of diabetes: control, gabexate (Gab), C5a-inhibitory peptide (C5aINH), and C5aINH plus Gab. Recipients injected with equivalent amounts of saline solution served as control subjects. Plasma samples were collected at 0, 0.5, 1, 3, 6, and 24 h after transplantation for analysis. We also evaluated the curative rate, intravenous glucose tolerance test, and insulin amounts in the liver of the recipients. RESULTS C3a receptor (C3aR) was scarcely expressed on the isolated islets with relatively strong expression of C5a receptor (C5aR): C3aR: 0.44 +/- 0.38%; C5aR: 7.91 +/- 2.83%). However, C5aR was not expressed on pancreatic tissues before the isolation procedures. Thrombin-antithrombin complex was significantly suppressed in the 3 treated groups (P = .0015). The curative rate was also significantly improved (0% vs 33% vs 67% vs 100%, respectively; P = .03). Glucose tolerance was significantly improved among the 3 treated groups (P < .0005). Insulin amounts in the liver were considerably higher among treated versus control hosts. Notably, the treatment did not affect the increased body weight of the recipient. CONCLUSIONS This study suggested that C5a-inhibitory peptide combined with gabexate mesilate may be a useful approach to control the IBMIR induced in clinical islet transplantation and one that is free of side effects.

Prevalence and risk factors of obesity, hypertension, dyslipidemia and diabetes mellitus before and after adult living donor liver transplantation.

The development of metabolic abnormalities after liver transplantation (LTx) contributes to cardiovascular events and mortality. We analyzed the prevalence and risk factors of obesity, hypertension, dyslipidemia and diabetes mellitus (DM) after adult living donor liver transplantation.

Expression of receptors for anaphylatoxins C3a and C5a on rat islet preparations.

BACKGROUND Complement activation has been implicated in the development of the instant blood-mediated inflammatory reaction (IBMIR). In particular, anaphylatoxins C3a and C5a elicit a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. We have previously shown that 2 types of receptors for C5a are expressed on isolated islets. In the present study, we investigated this component in detail. METHODS C3aR, C5aR, and C5L2, together with CD11b and CD31, on freshly isolated islets (fresh group) and islets cultured with (cytokine group) or without (culture group) TNF-α, IL-1β, and IFN-γ for ∼12 hours were analyzed by flow cytometry. In addition, these 3 kinds of receptors were analyzed on nonendocrine cells. RESULTS C5aR and C5L2 were expressed on the isolated islets (C5aR: 7.91 ± 2.83%; C5L2: 2.45 ± 1.34%) and the expression of both C5a receptors was markedly attenuated by culture for 12 hours (C5aR: P < .005; C5L2: P < .05). Compared with the culture group, the expression was significantly up-regulated in the cytokine group (C5aR: P < .05; C5L2: P = .05). C5aR-positive cells expressed CD11b but not CD31. In contrast to islets, nonendocrine cells expressed C5L2 predominantly. C3aR was scarcely expressed on isolated islets or nonendocrine cells. CONCLUSIONS These data suggest that C5aR and C5L2 are expressed on CD11b-positive leukocytes in islet preparations. Depletion of C5a receptors by culturing appropriately could be an attractive therapeutic strategy in clinical islet transplantation.

Edaravone, a Free Radical Scavenger, Improves the Graft Viability on Liver Transplantation From Non–heart-beating Donors in Pigs

BACKGROUND Although liver transplantation from non-heart-beating donors (NHBDs) is an effective way to overcome shortage of donors, primary graft nonfunction is often noted in these grafts. We have previously reported that edaravone, a free radical scavenger, has a cytoprotective effect on warm ischemia-reperfusion injury and improves the function of liver grafts from NHBDs in a rat model of ischemia-reperfusion. The purpose of this study was to investigate the effects of edaravone on liver transplantations from NHBDs. METHODS Pigs were divided into three groups: (1) a heart-beating (HB) group (n = 5), in which liver grafts were retrieved from HB donors; (2) a non-heart-beating (NHB) group (n = 4), in which liver grafts were retrieved under apnea-induced NHB conditions; and (3) an edaravone-treated (ED) group (n = 5), in which liver grafts were retrieved in the same manner as the NHB group and treated with edaravone at the time of perfusion (3 mg/L in University of Wisconsin [UW] solution), cold preservation (1 mg/L in UW solution), and after surgery (1 mg/kg/d). The grafts from all groups were transplanted after 4 hours of cold preservation. RESULTS In the ED group, the 7-day survival rate was significantly higher than that in the NHB group (80% versus 0%, P = .0042, Kaplan-Meier log-rank test). Furthermore, on histologic examination, the structure of sinusoids in the ED group was well preserved and similar to that in the HB group. CONCLUSIONS Edaravone may improve the viability of liver grafts from NHBDs.

Arterial and biliary complications after living donor liver transplantation: a single-center retrospective study and literature review

AimThe mortality of patients on the waiting list for deceased donor liver transplantation (DDLT) is high, especially in countries where donation rates are low. Thus, living donor liver transplantation (LDLT) is an attractive option. However, compared with DDLT, LDLT is associated with increased rates of arterial and biliary complications. We examined the rates of complications and risk factors following LDLT.MethodsWe retrospectively investigated and compared the rates of complications of DDLT and LDLT in our institute. We also performed univariate and multivariate analyses to identify the independent risk factors for these complications. The complications and specific disadvantages of LDLT were reviewed and discussed.ResultsThe incidence rate of arterial complications in LDLT was 6.0%, compared with 3.2% (13/441) in DDLT. A multivariate analysis identified low body weight (P = 0.032) as the only independent risk factor for hepatic artery thrombosis. The rate of all biliary complications in LDLT was 17.3%, compared with 18.7% in DDLT. The risk factors for biliary stricture identified by the multivariate analysis were recurrent cholangitis and the number of bile ducts. The durations of hospital stay and overall survival rates were similar between the two groups.ConclusionGiven the shortage of deceased donor organs, we believe that LDLT is acceptable in an attempt to meet demand.

Impact of the Trough Level of Calcineurin Inhibitor on the Prevalence of Donor-specific Human Leukocyte Antigen Antibodies During Long-term Follow-up After Pediatric Liver Transplantation: Antibody Strength and Complement-binding Ability

Background In pediatric patients, long-term immunosuppression after liver transplantation (LT) is typically minimal. However, posttransplant donor-specific HLA antibodies (DSAs) may be prevalent under these conditions. Here, we evaluated the effects of minimized calcineurin inhibitor (CNI) on DSA development to assess the validity of minimized/withdrawn immunosuppression. Methods We retrospectively examined 66 patients who underwent pediatric LT at our institution between July 1991 and October 2013. Patients were divided into 2 groups based on the CNI trough level. The cutoff trough levels were 3 and 30 ng/mL for tacrolimus and cyclosporine, respectively. Luminex single-antigen bead assays were performed, and the cutoff for a positive reaction was set at a mean fluorescence intensity (MFI) of at least 1000. Results The mean recipient ages at the time of LT were 29.1 and 77.2 months for the low and regular CNI groups, respectively (P = 0.0007). Univariate logistic regression analysis revealed that recipient age at LT younger than 3 years (P = 0.0099) and low CNI (P < 0.0001) were significantly associated with DSA development. In multivariate analysis, low CNI was an independent risk factor of DSA development (P = 0.0011). Of 15 high-MFI DSAs, 3 were anti-DR, and 12 were anti-DQ. Two of 3 anti-DR DSAs and 11 of 12 anti-DQ DSAs had complement-binding ability and high MFIs. Conclusions CNI minimization was an independent risk factor for posttransplant DSA during long-term follow-up after pediatric LT. Adjusting CNI to appropriate levels is a safe first step to prevent the immunological effects of DSA.

Effect of Recipient Age at Liver Transplantation on Prevalence of Post-Transplant Donor-Specific HLA Antibody

BACKGROUND Post-transplant donor-specific HLA antibodies (DSA) may have a detrimental effect on long-term outcomes of organ transplantation. The aim of this study was to specifically evaluate the effect of recipient age on the prevalence of DSA over a long-term follow-up after living donor liver transplantation (LDLT). MATERIAL AND METHODS A retrospective analysis of DSA evaluations was performed in 50 pediatric patients with HLA data available. Patients were divided into 2 groups based on their age at the time of LDLT: younger (Y) group, age <3 years; older (O) group, age ≥3 years. DSA evaluation was performed using Luminex single-antigen bead assays, with a mean fluorescence intensity ≥1000 used as a cut-off for positive results. RESULTS There were no between-group differences in terms of sex, ABO incompatibility or acute rejection. Only one of our 50 patients tested positive for class I DSA. Significantly more patients tested positive for HLA-DR DSA in group Y (40.6%) than in group O (11.1%; p=0.02). Recipients <3 years of age at the time of LDLT may be at a higher risk of testing positive for class II DSA. CONCLUSIONS These findings can inform the implementation of cost-effective screening of post-transplant DSA in pediatric LDLT recipients.

A Precise Analysis of C5a Inhibitory Peptide on Inflammatory Mediators Induced After Islet Transplantation

OBJECTIVE We recently reported that C5a inhibitory peptide (C5aIP) prevents the instant blood-mediated inflammatory reaction by attenuating cross talk between complement and coagulation cascades. C5aIP has also been shown to possess a broad range of anti-inflammatory effects. Due to methodological limitations, it is difficult to perform detailed analyses on wide range of inflammatory mediators in rat model. Therefore, we examined whether C5aIP suppressed various inflammatory cytokines induced after islet transplantation using a mouse model. METHODS Six islet equivalents per gram of syngeneic mouse islet grafts were transplanted intraportally into two groups of streptozotocin-induced diabetic mice: control group (n = 8) and C5aIP group (n = 6). The C5aIP group was treated with a bolus of 4 mg/kg just after islet infusion and a continuous infusion of 0.4 mg/kg/h, whereas the control group was injected with equivalent amounts of saline. Serum samples were collected at 0, 6, and 24 hours after transplantation. We analyzed 23 types of cytokines: interleukins-1a, -1b, -3, -4, -5, -6, -9, -10, -12, -13, and -17; eotaxin; G-CSF; GM-CSF; interferon (INF) gamma; KC; MCP-1; MIP-1 and -1b, RANTES and tumor necrosis factor-alpha. Leukocytes in the recipient liver were isolated at 6 hours after transplantation to examine IFN gamma production by fluorescence activated cell sorting (FACS). RESULTS No significant difference was detected in terms of the major inflammatory cytokines between the two groups. INF gamma production on CD11b(+)Gr-1(+) cells in the liver was not significantly inhibited by C5aIP (control 30.0% vs C5aIP 24.1%). CONCLUSIONS These data suggested that beneficial effects of C5aIP on islet engraftment are mainly due to blockade of cross talk between complement and coagulation cascades, rather than the suppression of inflammatory mediators.