Yoichiro Uchida

Progressive graft fibrosis and donor‐specific human leukocyte antigen antibodies in pediatric late liver allografts

The role of donor‐specific anti‐human leukocyte antigen antibodies (DSAs) that develop late after living donor liver transplantation is unknown. Seventy‐nine pediatric recipients who had good graft function and underwent protocol liver biopsy more than 5 years after transplantation (median = 11 years, range = 5‐20 years) were reviewed. DSAs were determined with the Luminex single‐antigen bead assay at the time of the last biopsy, and complement component 4d (C4d) immunostaining was assessed at the times of the last biopsy and the previous biopsy. The donor specificity of antibodies could be identified in 67 patients: DSAs were detected in 32 patients (48%), and they were usually against human leukocyte antigen class II (30 cases) but were rarely against class I (2 cases). These patients had a higher frequency of bridging fibrosis or cirrhosis (28/32 or 88%) than DSA‐negative patients (6/35 or 17%, P < 0.001). Fibrosis was likely to be centrilobular‐based. DSA‐positive patients, in comparison with DSA‐negative patients, had higher frequencies of diffuse/focal endothelial C4d staining (P < 0.001) and mild/indeterminate acute rejection [15/32 (47%) versus 5/35 (14%), P = 0.004]. Four DSA‐negative patients were off immunosuppression, whereas no patients in the DSA‐positive group were (P = 0.048). In conclusion, the high prevalence of graft fibrosis and anti–class II DSAs in late protocol biopsy samples suggests that humoral alloreactivity may contribute to the process of unexplained graft fibrosis late after liver transplantation. Liver Transpl 18:1333–1342, 2012.

T-Cell Immunoglobulin Mucin-3 Determines Severity of Liver Ischemia/Reperfusion Injury in Mice in a TLR4-Dependent Manner

BACKGROUND & AIMS T-cell immunoglobulin mucin (TIM) genes are expressed by T cells and regulate host immunity and tolerance. CD4(+) T cells mediate innate immunity-dominated liver ischemia-reperfusion injury (IRI) by unknown mechanisms. TIM-1 is involved in liver IRI, which is activated in part by the Toll-like receptor (TLR)4; we investigated the role of TIM-3 and TLR4 in IRI. METHODS Using an antibody against TIM-3 (anti-TIM-3), we studied TIM-3 signaling in mice following partial warm liver ischemia and reperfusion. RESULTS Mice given anti-TIM-3 had more liver damage than controls. Histological studies revealed that anti-TIM-3 increased hepatocellular damage and local neutrophil infiltration, facilitated local accumulation of T cells and macrophages, and promoted liver cell apoptosis. Intrahepatic neutrophil activity; induction of proinflammatory cytokines and chemokines; and expression of cleaved caspase-3, nuclear factor-κB, and TLR4 all increased in mice given anti-TIM-3. Administration of anti-TIM-3 followed by anti-galectin-9 (Gal-9 is a TIM-3 ligand) increased production of interferon-γ by concanavalin A (ConA)-stimulated spleen T cells and expression of tumor necrosis factor-α and interleukin-6 in ConA-stimulated macrophages co-cultured with T cells. Anti-TIM-3 did not affect liver IRI in TLR4-deficient mice. CONCLUSION TIM-3 blockade exacerbated local inflammation and liver damage, indicating the importance of TIM-3-Gal-9 signaling in maintaining hepatic homeostasis. TIM-3-TLR4 cross-regulation determined the severity of liver IRI in TLR4-dependent manner; these findings provide important information about the modulation of innate vs adaptive responses in patients that received liver transplants. Negative co-stimulation signaling by hepatic T-cells might be developed to minimize innate immunity-mediated liver tissue damage.

The emerging role of T cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse.

The T cell immunoglobulin and mucin domain‐containing molecules (TIM) protein family, which is expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity‐dominated organ ischemia–reperfusion injury (IRI), remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti–TIM‐1 antibody (Ab), we studied the role of TIM‐1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti–TIM‐1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared with controls. Histological examination has revealed that anti–TIM‐1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM‐1 ligand–expressing TIM‐4+ cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of proinflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in vitro studies, anti–TIM‐1 Ab suppressed interferon‐γ (IFN‐γ) production in concanavalin A (conA)–stimulated spleen T cells, and diminished tumor necrosis factor α (TNF‐α)/interleukin (IL)‐6 expression in a macrophage/spleen T cell coculture system. This is the first study to provide evidence for the novel role of TIM‐1 signaling in the mechanism of liver IRI. TIM‐1 regulates not only T for the role of cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM‐1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation. (HEPATOLOGY 2010.)

Interleukin-22: implications for liver ischemia-reperfusion injury.

Background. Ischemia-reperfusion injury (IRI) is common in general surgery and organ transplantation, and in the case of liver, it triggers proinflammatory innate immune cascade and hepatic necrosis, leading to increased incidence of early and late organ rejection. Interleukin (IL)-22, an inducible cytokine of T-cell origin and a member of the IL-10 superfamily, acts on target tissues through IL-22 receptor (IL-22R1). Methods. Partial hepatic warm ischemia was induced in C57Bl/6 wild-type (WT) and type 1 interferon receptor-deficient (KO) mice for 90 min followed by 6 to 24 hr of reperfusion. WT mice were treated at 30 min before the ischemia insult with recombinant IL-22 or anti-IL-22 neutralizing antibody; phosphate-buffered saline and IgG served as respective controls. Results. IL-22 was detected at 24 hr but not 6 hr of liver IRI. The expression of IL-22R1 was increased by 6 hr of reperfusion in WT but not type 1 interferon receptor KO mice that were protected from IRI. Treatment of WT mice with recombinant IL-22 decreased serum aspartate aminotransferase levels, ameliorated cardinal histological features of IR damage (Suzukis score) and diminished leukocyte sequestration, along with the expression of IL-22R1 and pro-inflammatory cytokines. IL-22 antibody did not appreciably affect IRI but increased IL-22R1 transcription in the liver. Administration of IL-22 protein exerted hepatoprotection by STAT3 activation. Conclusions. This is the first report investigating immune modulation by T-cell-derived IL-22 in liver injury caused by warm ischemia and reperfusion. Treatment with IL-22 protein may represent a novel therapeutic strategy to prevent liver IRI in transplant recipients.

Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury.

Programmed death‐1 (PD‐1)/B7‐H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity‐dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD‐1/B7‐H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD‐1 signaling after anti–B7‐H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7‐H1 immunoglobulin (B7‐H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well‐preserved liver architecture. The therapeutic potential of B7‐H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti‐necrotic/apoptotic Bcl‐2/Bcl‐xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)‐10. Neutralization of IL‐10 re‐created liver IRI and rendered B7‐H1Ig–treated hosts susceptible to IRI. These findings were confirmed in T cell–macrophage in vitro coculture in which B7‐H1Ig diminished tumor necrosis factor‐α/IL‐6 levels in an IL‐10–dependent manner. Our novel findings document the essential role of the PD‐1/B7‐H1 pathway in liver IRI. Conclusion: This study is the first to demonstrate that stimulating PD‐1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD‐1 upon T cell–Kupffer cell cross‐talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL‐10–dependent cytoprotection. (HEPATOLOGY 2010.)

The Inhibition of Neutrophil Elastase Ameliorates Mouse Liver Damage Due to Ischemia and Reperfusion

Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS‐D chloroacetate esterase stains, and immunohistochemistry (anti–Ly‐6G). The expression of pro‐inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C‐X‐C motif) ligand 1 (CXCL‐1), CXCL‐2, and CXCL‐10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining and caspase‐3 activity. In addition, toll‐like receptor 4 (TLR4) expression was diminished in NEI‐pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939–947, 2009.

The membrane attack complex (C5b‐9) in liver cold ischemia and reperfusion injury

Activation of the complement cascade represents an important event during ischemia/reperfusion injury (IRI). This work was designed to investigate the role of the membrane attack complex (MAC; C5b‐9) in the pathogenesis of hepatic IRI. Livers from B&W/Stahl/rC6(+) and C6(−) rats were harvested, stored for 24 hours at 4°C, and then transplanted [orthotopic liver transplantation (OLT)] to syngeneic recipients. There were 4 experimental groups: (1) C6(+)→C6(+), (2) C6(+)→C6(−), (3) C6(−)→C6(+), and (4) C6(−)→C6(−). At day +1, C6(−) OLTs showed decreased vascular congestion/necrosis, contrasting with extensive necrosis in C6(+) livers, that was independent of the recipient C6 status (Suzuki score: 7.2 ± 0.9, 7.3 ± 1.3, 4.5 ± 0.6, and 4.8 ± 0.4 for groups 1‐4, respectively, P < 0.05). The liver function improved in recipients of C6(−) grafts (serum glutamic oxaloacetic transaminase: 2573 ± 488, 1808 ± 302, 1170 ± 111, and 1188 ± 184 in groups 1‐4, respectively, P < 0.05). Intragraft macrophage infiltration (ED‐1 immunostaining) and neutrophil infiltration (myeloperoxidase activity) were reduced in C6(−) grafts versus C6(+) grafts (P = 0.001); these data were confirmed by esterase staining (naphthol). The expression of proinflammatory interferon‐γ, interleukin‐1β, and tumor necrosis factor messenger RNA/protein was also reduced in C6(−) OLTs in comparison with C6(+) OLTs. Western blot–assisted expression of proapoptotic caspase‐3 was decreased in C6(−) OLTs versus C6(+) OLTs (P = 0.006), whereas antiapoptotic Bcl‐2/Bag‐1 was enhanced in C6(−) OLTs compared with C6(+) OLTs (P = 0.001). Terminal deoxynucleotidyl transferase–mediated dUTP nick end‐labeling staining of apoptotic cells was enhanced (P < 0.05) in C6(+) OLTs compared with C6(−) OLTs. Thus, the terminal products of the complement system are essential in the mechanism of hepatic IRI. This is the first report using a clinically relevant liver cold ischemia model to show that local MAC inhibition attenuates IRI cascade in OLT recipients. Liver Transpl 14:1133–1141, 2008.

Long‐Term Outcome of Adult‐to‐Adult Living Donor Liver Transplantation for Post‐Kasai Biliary Atresia

Our objective was to analyze problems in the perioperative management and long‐term outcome of living donor liver transplantation (LDLT) for biliary atresia (BA). Many reports have described the effectiveness of liver transplantation (LT) for BA, particularly in pediatric cases, but little information is available regarding LT in adults (≥16 years old). Between June 1990 and December 2004, 464 patients with BA underwent LDLT at Kyoto University Hospital, of whom 47 (10.1%) were older than 16 years. In this study, we compared the outcomes between adult (≥16 years old) and pediatric (<16 years old) patients. The incidence of post‐transplant intestinal perforation, intra‐abdominal bleeding necessitating repeat laparotomy and biliary leakage was significantly higher (p < 0.0001, <0.001 and <0.001, respectively) in adults. Overall cumulative 1‐, 5‐ and 10‐year survival rates in pediatric patients were significantly higher (p < 0.005) than in adults. Two independent prognostic determinants of survival were identified: a MELD score over 20 and post‐transplant complications requiring repeat laparotomy. Outcome of LDLT in adult BA patients was poorer than in pediatric patients. It seems likely that LT will be the radical treatment of choice for BA and that LDLT should be considered proactively at the earliest possible stage.

The impact of meticulous management for hepatic artery thrombosis on long‐term outcome after pediatric living donor liver transplantation*

Abstract:  To analyze the risk factors in the development of hepatic artery thrombosis (HAT) and assess the impact of our perioperative management for HAT on the long‐term outcome after pediatric living donor liver transplantation (LDLT), we reviewed 382 patients under 12 yr of age who underwent 403 LDLT from January 1996 to December 2005. One‐ and 10‐yr patient survival rates were 78% and 78% in the patients with HAT (27 patients; 6.7%), and 84% and 76% in the patients without HAT, respectively (p = n.s.). Univariate analysis showed gender (female), body weight (lower), and graft‐to‐recipient weight ratio (higher) were significant risk factors in the patients with HAT (p < 0.05). Patients with Doppler ultrasound signal loss of the hepatic artery (HA) accompanied by an increase of liver enzymes underwent thrombectomy and reanastomosis (S‐group, n = 13), and patients with a weak HA signal underwent anticoagulant therapy (M‐group, n = 13). One patient underwent re‐LDLT. One‐ and five‐yr patient survival rates were 83% and 83% in the S‐group, and 77% and 77% in the M‐group (p = n.s.). The incidence of biliary complications in the S‐group (58%) was significantly higher than that of the M‐group (15%). For a successful long‐term outcome, the early detection of HAT and prompt medical and surgical intervention are crucial to minimize the insult of HAT.

The Protective Function of Neutrophil Elastase Inhibitor in Liver Ischemia/Reperfusion Injury

Background. A neutrophil elastase (NE) inhibitor, Sivelestat, has been approved for the treatment of acute lung injury associated with systemic inflammation in humans. Some reports have also shown its protective effects in liver inflammatory states. We have recently documented the importance of NE in the pathophysiology of liver ischemia/reperfusion injury, a local Ag-independent inflammation response. This study was designed to explore putative cytoprotective functions of clinically available Sivelestat in liver ischemia/reperfusion injury. Methods. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6 or 24 hr of reperfusion. The mice were given Sivelestat (100 mg/kg, subcutaneous) at 10 min before ischemia, 10 min before reperfusion, and at 1 and 3 hr of reperfusion thereafter. Results. Sivelestat treatment significantly reduced serum alanine aminotransferase levels and NE activity, when compared with controls. Histological liver examination has revealed that unlike in controls, Sivelestat ameliorated the hepatocellular damage and decreased local neutrophil activity and infiltration. The expression of proinflammatory cytokines (tumor necrosis factor-&agr; and interleukin-6), chemokines (CXCL-1, CXCL-2, and CXCL-10), and toll-like receptor 4 was significantly reduced in the treatment group, along with diminished apoptosis through caspase-3 pathway. Moreover, in vitro studies confirmed downregulation of proinflammatory cytokine and chemokine programs in mouse macrophage cell cultures, along with depression of innate toll-like receptor 4 signaling. Conclusion. Sivelestat-mediated NE inhibition may represent an effective therapeutic option in liver transplantation and other inflammation disease states.