Toshiyuki Hata

Splenectomy does not offer immunological benefits in ABO-incompatible liver transplantation with a preoperative rituximab.

Background. Preformed anti-ABO antibodies are primarily responsible for antibody-mediated rejection (AMR) after ABO-incompatible (ABO-I) liver transplantation (LT) resulting in lethal hepatic necrosis and biliary complications. Splenectomy, an integral part of protocol for ABO-I LT, decreases anti-ABO antibodies. With the preoperative rituximab prophylaxis, role of the splenectomy for ABO-I LT is now under debate. We investigated the necessity of splenectomy by retrospective analyses of the short-term anti-ABO antibody response and long-term outcomes of ABO-I LT. Methods. Thirty-seven ABO-I LTs performed from May 2006 through July 2009, at Kyoto University Hospital, Kyoto, Japan, were retrospectively analyzed. Twenty-seven patients who underwent splenectomy (splenectomy group) received 329.6±35.8 mg rituximab 17.7±11.9 days before living donor LT. Ten patients without splenectomy (nonsplenectomy group) received 320.0±10.3 mg rituximab 26.6±21.3 days before transplantation. All patients received a posttransplant hepatic artery infusion with prostaglandin E1 and methylprednisolone. Perioperative anti-ABO immunoglobulin M and immunoglobulin G antibody titers, rejections, biliary complications, infections, and survival results were compared. Results. Preoperative rituximab with plasma exchange effectively reduced anti-ABO antibodies in both patient groups at the time of LT. There was no statistically significant difference observed in anti-ABO immunoglobulin M and immunoglobulin G antibody titers between the “splenectomy” and “nonsplenectomy” groups during the initial 8 weeks. The clinical outcomes, including AMR, biliary complications, infections, and survival, were similar in both the groups. Conclusions. Preoperative rituximab effectively decreased the anti-ABO antibodies sufficiently to prevent the AMR irrespective of splenectomy. Splenectomy does not offer any immunological benefit in ABO-I LT with preoperative rituximab.

TIMP-1 attenuates blood-brain barrier permeability in mice with acute liver failure

Blood-brain barrier (BBB) dysfunction in acute liver failure (ALF) results in increased BBB permeability that often precludes the patients from obtaining a life-saving liver transplantation. It remains controversial whether matrix metalloproteinase-9 (MMP-9) from the injured liver contributes to the deregulation of BBB function in ALF. We selectively upregulated a physiologic inhibitor of MMP-9 (TIMP-1) with a single intracerebroventricular injection of TIMP-1 cDNA plasmids at 48 and 72 hours, or with pegylated-TIMP-1 protein. Acute liver failure was induced with tumor necrosis factor-α and D-(+)-galactosamine in mice. Permeability of BBB was assessed with sodium fluorescein (NaF) extravasation. We found a significant increase in TIMP-1 within the central nervous system (CNS) after the administration of TIMP-1 cDNA plasmids and that increased TIMP-1 within the CNS resulted in an attenuation of BBB permeability, a reduction in activation of epidermal growth factor receptor and p38 mitogen-activated protein kinase signals, and a restoration of the tight junction protein occludin in mice with experimental ALF. Pegylated TIMP-1 provided similar protection against BBB permeability in mice with ALF. Our results provided a proof of principle that MMP-9 contributes to the BBB dysfunction in ALF and suggests a potential therapeutic role of TIMP-1 in ALF.

Transplantation of engineered chimeric liver with autologous hepatocytes and xenobiotic scaffold

Objective: Generation of human livers in pigs might improve the serious shortage of grafts for human liver transplantation, and enable liver transplantation without the need for deceased or living donors. We developed a chimeric liver (CL) by repopulation of rat hepatocytes in a mouse and successfully transplanted it into a rat recipient with vessel reconstruction. This study was designed to investigate the feasibility of CL for supporting the recipient after auxiliary liver grafting. Methods: Hepatocytes from luciferase transgenic or luciferase/LacZ double-transgenic rats were transplanted into 20- to 30-day-old urokinase-type plasminogen activator/severe-combined immunodeficiency (uPA/SCID) mice (n = 40) to create CLs with rat-origin hepatocytes. After replacement of mouse hepatocytes with those from rats, the CLs were transplanted into wild-type Lewis (n = 30) and analbuminemia (n = 10) rats, followed by immunosuppression using tacrolimus (TAC) with/without cyclophosphamide (CPA) or no immunosuppression. Organ viability was traced by in vivo bioimaging and Doppler ultrasonography in the recipient rats for 4 to 6 months. Rat albumin production was also evaluated in the analbuminemia rats for 4 months. In addition, histological analyses including Ki67 proliferation staining were performed in some recipients. Results: Both immunosuppressive protocols significantly improved graft survival and histological rejection of CLs as compared to the nonimmunosuppressed group. Although rat albumin production was maintained in the recipients for 4 months after transplantation, ultrasonography revealed patent circulation in the grafts for 6 months. Ki67 staining analysis also revealed the regenerative potential of CLs after a hepatectomy of the host native liver, whereas immune reactions still remained in the mouse-origin structures. Conclusions: This is the first report showing that engineered CLs have potential as alternative grafts to replace the use of grafts from human donors.

Simple and reproducible hepatectomy in the mouse using the clip technique.

AIM To investigate the reliability of massive hepatectomy models by using clip techniques. METHODS We analyzed anatomical findings in 100 mice following massive hepatectomy induced by liver reduction > 70%. The impact of various factors in the different models was also analyzed, including learning curves, operative time, survival curves, and histopathological findings. RESULTS According to anatomical results, models with 75%, 80%, and 90% hepatectomy produced massive hepatectomy. Learning curves and operative times were most optimal with the clip technique. Each hepatectomy performed using the clip technique produced a reasonable survival curve, and there were no differences in histopathological findings between the suture and clip techniques. CONCLUSION Massive hepatectomy by the clip technique is simple and can provide reliable and relevant data.

Oxidative stress and extracellular matrices after hepatectomy and liver transplantation in rats

AIM To investigate oxidative stress (OS)-mediated damage and the behavior of extracellular matrices in various rat models because shear stress with portal hypertension and cold ischemia/warm reperfusion injury trigger the liver regeneration cascade after surgery. These injuries also cause fatal liver damage. METHODS Rats were divided into four groups according to the surgery performed: control; hepatectomy with 40% liver remnant (60% hepatectomy); orthotopic liver transplantation (OLT) with whole liver graft (100% OLT); and split OLT (SOLT) with 40% graft (40% SOLT). Survival was evaluated. Blood and liver samples were collected at 6 h after surgery. Biochemical and histopathological examinations were performed. OS-induced damage, 4-hydroxynonenal, ataxia-telangiectasia mutated kinase, histone H2AX, phosphatidylinositol 3-kinase (PI3K) and Akt were evaluated by western blotting. Behavior of extracellular matrices, matrix metalloproteinase (MMP)-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were also evaluated by western blotting and zymography. RESULTS Although 100% OLT survived, 60% hepatectomy and 40% SOLT showed poor survival. Histopathological, immunohistological, biochemical and protein assays revealed that 60% hepatectomy, 100% OLT and 40% SOLT showed liver damage. PI3K and Akt were decreased in 60% hepatectomy and 40% SOLT. For protein expression, 40% SOLT showed differences in MMP-9, MMP-2 and TIMP-2. TIMP-1 showed differences in 60% hepatectomy and 40% SOLT. For protein activity, MMP-9 demonstrated significant differences in 60% hepatectomy, 100% OLT and 40% SOLT. CONCLUSION Under conditions with an insufficient liver remnant, prevention of OS-induced damage via the Akt/PI3K pathway may be key to improve the postoperative course. MMP-9 may be also a therapeutic target after surgery.

Pretreatment of liver grafts in vivo by γ-aminobutyric acid receptor regulation reduces cold ischemia/warm reperfusion injury in rat.

BACKGROUND Gamma-aminobutyric acid (GABA) is found throughout the body. The regulation of GABA receptor (GABAR) reduces oxidative stress (OS). Ischemia/reperfusion injury after orthotopic liver transplantation (OLT) causes OS-induced graft damage. The effects of GABAR regulation in donors in vivo were investigated. MATERIAL AND METHODS Donor rats received saline, a GABAR agonist or GABAR antagonist 4 h before surgery. Recipient rats were divided into four groups according to the donor treatments: laparotomy, OLT with saline, OLT with GABAR agonist and OLT with GABAR antagonist. Histopathological, biochemical and immunohistological examinations were performed at 6, 12 and 24 h after OLT. Protein assays were performed at 6 h after OLT. The 4-hydroxynonenal (4-HNE), ataxia-telangiectasia mutated kinase (ATM), phosphorylated histone H2AX (gammaH2AX), phosphatidylinositol-3 kinase (PI3K), Akt and superoxide dismutase (SOD) were assessed by western blot analysis. RESULTS In the univariate analysis, histopathological and biochemical profiles verified that the GABAR agonist reduced graft damage. Immunohistology revealed that the GABAR agonist prevented the induction of apoptosis. Measurement of 4-4-HNE levels confirmed OS-induced damage after OLT, and the GABAR agonist improved this damage. In the gammaH2AX, PI3K, Akt and antioxidant enzymes (SODs), ATM and H2AX were greatly increased after OLT, and were reduced by the GABAR agonist. In the multivariate analyses between multiple groups, histopathological assessment, aspartate aminotransferase level, immunohistological examinations for apoptotic induction and gammaH2AX showed statistical differences. CONCLUSIONS A specific agonist demonstrated regulation of GABAR in vivo in the liver. This activation in vivo reduced OS after OLT via the ATM/H2AX pathway.

Effect of specific activation of γ‐aminobutyric acid receptor in vivo on oxidative stress‐induced damage after extended hepatectomy

Aim:  γ‐Aminobutyric acid (GABA) is a multifunctional molecule with various physiological effects throughout the body. The regulation of GABA receptor (GABAR) plays a key role in reducing the damage mediated by oxidative stress (OS). Extended hepatectomy causes fatal OS‐induced injury in the liver remnant. We aimed to investigate the effect of a GABAR agonist in extended hepatectomy.

Transplantable liver production plan: "Yamaton"-liver project, Japan

Organ grafts developed in the xenogeneic pig scaffold are expected to resolve most issues of donor safety and ethical concerns about living-donor liver transplantation in Japan. We have been working on so-called “Yamaton” projects to develop transplantable organs using genetically engineered pigs. Our goal is to produce chimeric livers with human parenchyma in such pigs. The Yamaton-Liver project demonstrated the proof of concept by showing that rat–mouse chimeric livers could develop in mice and be successfully transplanted into syngeneic or allogeneic rats. Under conventional immunosuppression, the transplanted livers showed long-term function and protection against rejection. Because chimeric liver grafts have xenogeneic components, additional strategies, such as humanization of pig genes, induction of hematopoietic chimeras in donors, and replacement of pig endothelial cells with human ones, might be required in clinical use. Our projects still need to overcome various hurdles but can bring huge benefits to patients in the future.

Pretreatment of Small-for-Size Grafts In Vivo by γ-Aminobutyric Acid Receptor Regulation against Oxidative Stress-Induced Injury in Rat Split Orthotopic Liver Transplantation

Background. Graft pretreatment to limit postoperative damage has the advantage of overcoming a current issue in liver transplantation (LT). The strategic potential of graft pretreatment in vivo by a specific agonist for γ-aminobutyric acid receptor (GABAR) was investigated in the rat LT model with a small-for-size graft (SFSG). Methods. Recipient rats were divided into three groups according to donor treatments and recipient surgeries: (i) saline and laparotomy, (ii) saline and split orthotopic liver transplantation (SOLT) with 40%-SFSG, and (iii) GABAR agonist and SOLT with 40%-SFSG. Survival was evaluated. Blood and liver samples were collected 6 h after surgery. Immunohistological assessment for apoptotic induction and western blotting for 4-hydroxynonenal, ataxia-telangiectasia mutated kinase (ATM), histone H2AX, phosphatidylinositol-3 kinase (PI3K), Akt, and free radical scavenging enzymes were performed. Results. Pretreatment by GABAR showed improvement in survival, histopathological assessment, and biochemical tests. Apoptotic induction and oxidative stress were observed after SOLT with an SFSG, and this damage was limited by GABAR regulation. GABAR regulation appeared to reduce DNA damage via the ATM/H2AX pathway and to promote cell survival via the PI3K/Akt pathway. Conclusions. Pretreatment in vivo by GABAR regulation improves graft damage after SOLT with an SFSG. This strategy may be advantageous in LT.

Matrix metalloproteinase-9 as a therapeutic target for the progression of fulminant liver failure with hepatic encephalopathy: A pilot study in mice

If progressive liver injury and subsequent hepatic encephalopathy can be prohibited in fulminant liver failure (FLF), it would be ideal for intensive care of FLF and provide an expanded opportunity for liver transplantation (LT). We hypothesized that matrix metalloproteinase (MMP)‐9 plays an important role in FLF progression, and investigated MMP‐9 behaviors in a murine FLF model, especially at the coma stage.