Masaaki Awane

Suppression of VEGFR-3 signaling inhibits lymph node metastasis in gastric cancer.

In gastric cancer, lymph node metastasis is one of the major prognostic factors and forms the basis for surgical removal of local lymph nodes. Recently, several studies have demonstrated that overexpression of lymphangiogenic growth factor VEGF‐C or VEGF‐D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models. We examined whether these processes could be inhibited in naturally metastatic tumors by blocking of their cognate receptor VEGFR‐3 signaling pathway. Using a mouse orthotopic gastric cancer model which has a high frequency of lymph node metastasis, we estimated lymphatic vessels in gastric cancers by immunostaining for VEGFR‐3 and other specific lymphatic markers, LYVE‐1 and prox‐1. Then we systemically administered anti‐VEGFR‐3 blocking antibodies. This treatment resulted in the inhibition of regional lymph node metastasis and reduction of lymphatic vessel density in the primary tumors. In addition, increased density of LYVE‐1‐positive lymphatic vessels of primary tumors was closely correlated with lymph node metastasis in human samples of gastric cancer. Antilymphangiogenesis by inhibiting VEGFR‐3 signaling could provide a potential strategy for the prevention of lymph node metastasis in gastric cancer.

Living related liver transplantation in children

We reviewed 37 living related liver transplantations (LRLT) performed by our department during the last 27 months on children with end-stage liver disease. The patients were 15 boys and 22 girls aged 7 months to 15 years with biliary atresia (27), cryptogenic cirrhosis (3), Budd-Chiari syndrome (2), progressive intrahepatic cholestasis (2), protoporphyria (1), Wilsons disease (1), and fulminant hepatitis (1). The donors were 14 fathers and 23 mothers. Grafts were made from the left lateral segment (19), left lateral segment with partial S4 (11), left lobe (6), and right lobe (1). After graft harvesting all donors resumed normal liver function and normal life. The recipient underwent total hepatectomy with preservation of the inferior vena cava. FK506 and low-dose steroids were used for immunosuppression. The survival rate was 90% (27/30) in elective cases and 57% (4/7) in emergency cases. Six recipients had functioning grafts but died of extrahepatic complications. Hepatic vein stenosis occurred in 3 cases at 3 months after LRLT and was successfully treated by balloon dilatation. Portal vein stenosis occurred in 1 case at 8 months after LRLT and was also safely dilated. We incurred no hepatic artery thrombosis after introducing microsurgery techniques. Among 12 viral, 5 bacterial, and 3 fungal postoperative infections, 1 Candida pneumonia and 1 EBV-associated lymphoma were lethal. Three patients with ABO-blood group compatible grafts and one with an incompatible graft developed acute rejection, which was controlled in evey case by steroid bolus and/or increasing the dose of FK506. There were no definite episodes of rejection in ABO-identical cases. Children with moderate growth retardation (> or = -1.5 SD of normal growth) caught up in growth soon after LRLT, but those with severe retardation (<-1.5 SD) were slow to attain age-normal height. Appropriate timing, meticulous surgical procedures, and comprehensive management of complications are crucial for successful outcome with LRLT. LRLT is a promising option for alleviating the shortage of livers for pediatric transplantation and may be regarded as an independent modality to supplement cadaver donation.

Compartmentalization of Peyer’s Patch Anlagen Before Lymphocyte Entry

We have shown that Peyer’s patch (PP) first develops as a simple and even cell aggregation during embryogenesis. To investigate when and how such a simple cell aggregation forms the complex PP architecture, we analyzed the distribution of cells expressing IL-7Rα (PP inducer cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature lymphocytes by whole-mount immunostaining of 17.5 days postcoitus to 2 days postpartum mouse gut. Our results show that compartmentalization of PP anlagen commences at day 18.5 of gestation by clustering and subsequent follicle formation of IL-7Rα+, VCAM-1+, and CD11c+ cells. This process adds the primitive architecture of PP anlage with several follicles in which IL-7Rα+ cells localize in the center, while VCAM-1+ and CD11c+ cells localize at the fringe. This follicle formation is accompanied by the establishment of PP-specific vascular network expressing mucosal addressin cellular adhesion molecule-1. Mature B and T lymphocytes entering in the PP anlage are distributed promptly to their own target zones; B cells to the follicle and T cells to nonfollicular zones. Our analysis of scid/scid mouse indicate that the initial processes including formation of PP-specific vascular network occur in the absence of lymphocytes. These observations indicate that the basic architecture of PP is formed by a set of cell lineages assembled during the initial phase of induction of PP anlagen before entry of mature lymphocytes.

A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases.

OBJECTIVES To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. DESIGN AND METHODS We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. RESULTS This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. CONCLUSIONS The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.

Analyses of the risk and operative stress for donors in living-related partial liver transplantation

Operative risk and stress were analyzed in 28 parent-donors whose children received partial liver transplantations at the Second Department of Surgery, Kyoto University Hospital between June 1990 and December 1991. Graft-harvesting operations were classified into three types: left lobectomy (group L, n=12), left lateral segmentectomy (group S, n=15), and right lobectomy (n=l). Since donor safety is a primary concern, great care was taken to minimize potential damage to the remaining lobes of the donor liver as well as the graft liver by avoiding stressful maneuvers such as hepatic vascular clamping during the transection of the hepatic parenchyma. In all cases the arterial ketone body ratio, which reflects the hepatic mitochondrial redox potential, was maintained at over 0.7 throughout the donor operation as well as postoperatively. There was no significant difference in the postoperative RBC, WBC, serum GOT, and total bilirubin between groups L and S, although the abnormalities exhibited by the single right lobectomy case tended to be larger and more prolonged. All cases were within the range acceptable for immediate discharge, and all donors of groups L and S were subsequently discharged within 14 days without any postoperative complications. The single right lobectomy donor was discharged on POD 17 because of transient slight icterus, which is the only postoperative complication encountered in this series thus far. The present analyses would indicate that the risk and operative stress to the donor in living-related partial liver transplantation can be minimal when the left lobe or left lateral segment of the liver is used for the graft.

The ratio of reduced glutathione/oxidized glutathione is maintained in the liver during short-term hepatic hypoxia

Controversy persists as to whether reperfusion-induced injuries actually occur in the hepatocyte. The liver is the major source of glutathione, a scavenger of hydrogen peroxide. The aim of this study was to evaluate the sensitivity of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) [GSH: GSSG] as an index of hepatic metabolic stress. A total of 121 rats were studied. The superior mesenteric vein (SMV) was occluded for 30 min, and this was followed by 0, 10, or 120 min of reperfusion. Total glutathione and GSSG levels in the liver, bile, and plasma were quantified, using glutathione reductase-coupled enzymatic assays. Results indicated that the hepatic GSH/GSSG ratio was maintained after an occulusion of the SMV, despite a decrease in adenosine triphosphate (ATP) level and energy charge potential. However, plasma levels of total glutathione and GSSG in the inferior vena cava increased after SMV occlusion and continued to increase after reperfusion. Biliary GSSG efflux decreased during 30-min occlusion of the SMV, and remained low even after reperfusion. The liver maintains homeostasis despite a decrease in biliary GSSG efflux, probably by secreting excess GSSG into the hepatic vein when the SMV is occluded. We conclude that the total amount of glutathione and GSSG in the plasma is directly correlated with oxidative stress in the liver.

Reducing environment protects sinusoidal lymphocytes isolated from normal human liver from apoptosis

BACKGROUND/AIMS We previously reported that the populations of lymphocytes and the expression of activated antigens in human sinusoidal mononuclear cells were different from those in peripheral blood mononuclear cells. Attempts to culture these cells for further study failed because they died rapidly under standard culture conditions in vitro after isolation from the liver. In this study, we evaluated the characteristics of cell death and the effects of various culture conditions on the viability of these cells. METHODS Sinusoidal mononuclear cells were isolated from University of Wisconsin solution that had been perfused through the portal veins of normal healthy human livers harvested for transplantation into living related recipients. RESULTS 70% of sinusoidal mononuclear cells cultured in vitro were nonviable within 48 h after isolation, while only 10% of peripheral blood mononuclear cells died under the same conditions. Sinusoidal mononuclear cells showed DNA ladder formation of DNA on electrophoresis and characteristic morphological pattern on electron microscopic examination that suggested they had died in an apoptotic manner. The addition of human liver extracts or 2-mercaptoethanol and reduced glutathione to the cultures rescued the sinusoidal mononuclear cells from apoptosis. Furthermore, diamide, a sulfhydryl group specific oxidant, negated the effect of the liver extract. CONCLUSION In comparison with peripheral blood mononuclear cells, human sinusoidal mononuclear cells were more subject to death by apoptosis ex vivo, which was reversed by exogenous agents producing reducing conditions. These results suggested that hepatic sinusoidal mononuclear cells might express a different sensitivity to redox environment than peripheral blood mononuclear cells.

CHANGES IN GLUCOSE TRANSPORTER 2 AND CARBOHYDRATE-METABOLIZING ENZYMES IN THE LIVER DURING COLD PRESERVATION AND WARM ISCHEMIA: The Possibility of Evaluating the Viability of the Liver Graft before Transplantation

In order to examine glucose metabolism in liver grafts during cold preservation (24 and 48 hr), warm ischemia (60 and 120 min), a combination of the two and reperfusion, the amount of protein and mRNA of glucose transporter 2 and the activities of enzymes in glycolysis (glucokinase, phosphofructokinase, pyruvatekinase), gluconeogenesis (glucose 6-phosphatase, fructose 1,6-bisphosphatase), and the pentose phosphate pathway (glucose 6-phosphate dehydrogenase) were measured. It appeared that glucose transport, the pentose phosphate pathway, and gluconeogenesis were maintained during cold preservation and warm ischemia. The activity of glucokinase significantly decreased from the control value of 1.33 +/- 0.23 IU/g protein to 0.70 +/- 0.17 (24 hr, P<0.05) and 0.57 +/- 0.12 (48 hr, P<0.01) only during cold preservation. However, the activity of phosphofructokinase significantly decreased from the control value of 4.37 +/- 0.06 IU/g protein to 2.67 +/- 0.15 (60 min, P<0.0001) and 1.53 +/- 0.06 (120 min, P<0.0001) only during warm ischemia. This indicates that glycolysis deteriorates during both cold preservation and warm ischemia and demonstrates further that the balance between glycolysis and gluconeogenesis shifts to gluconeogenesis. Even when cold preservation was combined with warm ischemia, the activity of glucokinase decreased only during cold preservation and the activity of phosphofructokinase decreased only during warm ischemia. Furthermore, these changes were time-dependent. It is suggested that they can be used as a clock to measure the durations of cold preservation and warm ischemia separately and that the magnitude of an ischemic injury to a liver and a liver grafts viability can be indirectly estimated before transplantation.

Evaluating the significance of HLA class II genotype matching in living-related liver transplantation

To evaluate the clinical significance of HLA class II matching in living-related liver transplantation, the genotypes of HLA class II including DPB1 were determined by the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method and their matching was compared to the postoperative course. Conventional serotypes included 15.6% and 13.6% errors in DR and DQ, respectively. Among 42 consecutive cases that were followed up over 1 year after transplant, rejection occurred in seven cases. There was no correlation between the matching for each locus and the frequency of rejection episodes. In this study, rejection-free cases were investigated in terms of the potency of immunosuppressive therapy and graft function during 1 year after operation. The doses of tacrolimus in unmatched cases tended to be larger than those in matched cases for every locus except for DQA1. Its trough levels in matched cases were lower than those in unmatched cases, especially at 9-12 months after operation for DRB1 (p < 0.05). The termination of steroid administration tended to be postponed in unmatched cases for every locus. Serum levels of asparatan transaminase, alanine transaminase gamma-glutamyltranspeptidase (gamma-GTP) and total bilirubin were generally lower in matched cases than in unmatched ones for DRB1, DQB1 and DPB1, while in DQA1 the tendency was the opposite, especially total bilirubin and gamma-GTP, which were p < 0.02 and p < 0.05, respectively, at 6 months after operation. Investigation of subclinical immune responses other than rejection episodes showed that DRB1, DQB1 and DPB1 matching had a beneficial effect on graft function, while DQA1 matching seemed to have a varied effect.

Analysis of Deep Posterior Anal Fistulas by Magnetic Resonance Imaging: Site of Primary Abscess and Extension Patterns According to the Primary Abscess Depth

Objectives: The aim of this study was to use magnetic resonance imaging (MRI) to elucidate the site and depth of the primary abscesses associated with deep posterior anal fistulas and their extension patterns. Methods: We analyzed 176 consecutive patients with deep posterior anal fistulas and classified the fistulas according to whether the MRI-detected site of the primary abscess was at a superficial or a deep external anal sphincter (EAS) level. Results: The distance between the anal center and the primary abscess center was significantly shorter than the length of the EAS and radius at an angle of 45°. In addition, deep posterior anal fistulas with primary abscesses located at the deep EAS level penetrated the EAS significantly more laterally and made external openings at a significantly more lateral site than when the primary abscess was located at a superficial EAS level. Conclusions: Primary abscesses associated with deep posterior anal fistulas are located in the posterior intersphincteric space or in the EAS muscle itself, not in Courtneys space, as had previously been claimed.