Daiki Yoshii

Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury

Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl-peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic administration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neutrophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide–FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R.

New insight into reactive ductular cells of biliary atresia provided by pathological assessment of SOX9

BackgroundBiliary atresia (BA) patients may survive until adolescence after effective Kasai procedure (KP). If liver fibrosis progresses even after successful KP, liver transplantation (LTx) is inevitable. Elucidation of its cause and pathophysiology would open the possibility of treating these patients by non-invasive management. SOX9 is a transcription factor that regulates bile duct development and contributes to liver regeneration and fibrosis. To elucidate the role of SOX9 in BA liver, we investigated the SOX9 expression pattern.MethodImmunostaining with anti-SOX9 antibody was done on hepatic specimens obtained at the time of KP or LTx. We analyzed the association of SOX9 expression with clinical data.ResultsIn BA livers, SOX9 was expressed in reactive ductular cells (RDCs), mostly with a nuclear-dominant pattern. SOX9 was also ectopically expressed in hepatocytes, which was more conspicuous at the timing of KP than LTx. SOX9 expression level was significantly correlated with age (days) at which KP was performed, AST and WBC count.ConclusionsSOX9 may contribute to RDC formation in BA patients, by affecting both RDCs and hepatocytes. SOX9 could be a key molecule to understand the mechanism of RDC formation, and this understanding would provide a therapeutic strategy for effective treatment of BA.

Intravital imaging of neutrophil recruitment in intestinal ischemia-reperfusion injury

BACKGROUND Neutrophils are known to be key players in innate immunity. Activated neutrophils induce local inflammation, which results in pathophysiologic changes during intestinal ischemia-reperfusion injury (IRI). However, most studies have been based on static assessments, and few have examined real-time intravital neutrophil recruitment. We herein report a method for imaging and evaluating dynamic changes in the neutrophil recruitment in intestinal IRI using two-photon laser scanning microscopy (TPLSM). METHODS LysM-eGFP mice were subjected to 45 min of warm intestinal ischemia followed by reperfusion. Mice received an intravenous injection of tetramethylrhodamine isothiocyanate-labeled albumin to visualize the microvasculature. Using a time-lapse TPLSM technique, we directly observed the behavior of neutrophils in intestinal IRI. RESULTS We were able to image all layers of the intestine without invasive surgical stress. At low-magnification, the number of neutrophils per field of view continued to increase for 4 h after reperfusion. High-magnification images revealed the presence or absence of blood circulation. At 0-2 h after reperfusion, rolling and adhesive neutrophils increased along the vasculature. At 2-4 h after reperfusion, the irregularity of crypt architecture and transmigration of neutrophils were observed in the lamina propria. Furthermore, TPLSM imaging revealed the villus height, the diameters of the crypt, and the number of infiltrating neutrophils in the crypt. In the IRI group, the villus height 4 h after reperfusion was significantly shorter than in the control group. CONCLUSIONS TPLSM imaging revealed the real-time neutrophil recruitment in intestinal IRI. Z-stack imaging was useful for evaluating pathophysiological changes in the intestinal wall.

Outcomes of treatment with daclatasvir and asunaprevir for recurrent hepatitis C after liver transplantation

The development of direct‐acting oral agents has dramatically changed the treatment strategy of hepatitis C virus (HCV) infection. Here we aimed to reveal the efficacy and safety of daclatasvir (DCV) and asunaprevir (ASV) for recurrent HCV genotype 1 infection after liver transplantation (LT).

Effect of SOX9 on Ductular Reaction and Fibrogenesis

Background Ductular reaction (DR), which is induced when self-renewal and regeneration of the liver are damaged, aims to repair and compensate for anatomical and functional loss. DR is also thought to have crucial role in the etiology of fibrosis, and strongly associated with liver diseases, but the pathogenesis is still unknown. Sex determining region Y-box 9 (SOX9) is involved in the organogenesis of several tissues and organs. In the liver, SOX9 has an important function in bile duct generation and liver regeneration. We have studied about an association between DR and SOX9. The aim of this study was to evaluate an effect of SOX9 on DR and liver fibrogenesis and to explore the applicability of treatment of cholestatic liver disease. Methods To induce fibrotic reaction by cholestasis and inflammation, we ligated the common bile duct in mice with wild type (WT) and with a liver-specific inactivation of SOX9 (SOX9KO). On day 7 and day 14 after the procedure, liver function tests were performed concurrently with immunohistochemistry and gene expression analyses of liver specimens. Results Total bilirubin in WT mice was significantly higher than that in SOX9KO mice on day 7 and day 14, respectively (p<0.01 and p<0.05). Gene expression analysis revealed that the expression of cytokeratin 19, a marker of biliary cells, in SOX9KO mice was significantly lower than that in WT mice on day 14 (p<0.05), and the expression of osteopontin, a marker of biliary cells, in SOX9KO mice was significantly lower than that in WT mice (p<0.05). The expression of Col1A2, a marker of fibrogenesis, in SOX9KO mice showed lower increase rate than that in WT mice. Conclusion In the liver damage mice model by common bile duct ligation, SOX9 gene knockout resulted in lower expression of biliary markers and inhibition of DR and fibrogenesis. SOX9 may become a promising molecular target in the treatment of cholestatic liver disease.

Living Donor Liver Transplantation for Progressive Familial Intrahepatic Cholestasis Type 1: Two Reported Cases

BACKGROUND Progressive familial intrahepatic cholestasis type 1 (PFIC1) is an inherited disease characterized by cholestatic features. We report two patients with PFIC1 who underwent liver retransplantation. CASE REPORT One patient was a 3-year-old female who underwent liver transplantation for PFIC1. She presented with severe diarrhea and fatty liver, and went into liver failure. She therefore underwent liver retransplantation and external biliary diversion 8 years after the initial liver transplantation. The explanted liver was histologically diagnosed with chronic rejection. Her intractable diarrhea stopped after the retransplantation. She was diagnosed with a fatty liver 8 months after the retransplantation and died 4 years after retransplantation due to bleeding from an ileostomy. The other patient was a 3-year-old male. This patient underwent liver retransplantation due to liver cirrhosis caused by steatohepatitis 9 years after the initial liver transplantation. The biliary tract was not diverted. He also experienced severe diarrhea after the retransplantation and requires home parenteral nutrition due to an eating disorder. CONCLUSIONS Liver transplantation is the only treatment to resolve life-threatening issues due to PFIC1, but requires further improvement as a therapeutic modality.

Ledipasvir and sofosbuvir for recurrent hepatitis C after liver transplantation.

Management of recurrent hepatitis C following liver transplantation still remains a challenge. Here, we report five patients who achieved viral responses following combined treatment with ledipasvir and sofosbuvir. All the patients received tacrolimus for immunosuppression. No dose adjustment was made before the ledipasvir and sofosbuvir therapy. All completed the intended 12-week treatment course with the full dose of ledipasvir and sofosbuvir. There were no significant adverse events greater than grade 2. During the study period, no acute rejection episodes were detected. The trough levels of tacrolimus were maintained stably. Hepatitis C virus RNA was not detected at week 12 in any of the patients. Based on the findings from this pilot study, combined ledipasvir and sofosbuvir therapy for 12 weeks is effective and safe for living - donor liver transplantation recipients with recurrence of hepatitis C virus.