Masahito Kuroda

Interleukin-6 induces proliferation of rat hepatocytes in vivo

Abstract Background/Aims: The aim of this study was to assess the effect of interleukin-6 (IL-6) on the proliferation of hepatocytes and to study the interaction between IL-6 and hepatocyte growth factor (HGF) in vivo . Methods: IL-6 was injected at a dose of 200 μg/mg subcutaneously into rats every day for 14 days. Liver and blood samples were obtained at 1, 3, 7 and 14 days during IL-6 administration. Hepatocyte proliferative activity of sera was measured using 3 H-thymidine incorporation into cultured rat hepatocytes. To evaluate the proliferative activity of the hepatocytes in tissue sections, hepatic DNA content and immunostaining of the liver tissue sections for proliferating cell nuclear antigen (PCNA) were performed. Plasma HGF levels were measured using specific EIA. In addition, total RNA was extracted from the liver and expression of HGF mRNA was detected by RT-PCR. Results: The DNA contents of liver taken from IL-6-treated rats were increased during IL-6 administration compared with untreated rats. Sera taken from IL-6-treated rats at various intervals during administration also significantly increased 3 H-thymidine incorporation by cultured rat hepatocytes compared with sera from untreated rats, suppressing 3 H-thymidine incorporation at day 1 and 3 by anti-HGF antibody. IL-6 itself did not increase 3 H-thymidine incorporation. Increased expression of PCNA in these hepatocytes was noted from 1 day after IL-6 administration, and at 14 days, the number of PCNA-positive cells was sevenfold greater than in the livers of untreated rats. However, plasma HGF levels showed a peak at day 1, decreased gradually from day 3, and became undetectable by day 14. HGF mRNA expression in livers of IL-6-treated rats was suppressed from day 3 to day 14 of IL-6 administration. Conclusions: These data show that IL-6 induces an early phase of liver cell growth in vivo and suggest that an increase level of HGF mediates this effect.

Clinicolaboratory characteristics of patients with primary biliary cirrhosis associated with CREST symptoms.

Aims: Patients with primary biliary cirrhosis (PBC) occasionally suffer complications from other autoimmune diseases. When PBC was associated with calcinosis, Raynauds phenomenon, esophageal dysmotility, sclerodactyly and telangiectasias (CREST) symptoms, it has been proposed that it is a distinct clinical entity. This study aimed to investigate whether PBC associated with CREST symptoms is a distinct disease complex. Method: Clinicolaboratory data, HLA type of leukocytes and disease prognosis were compared between 31 patients with PBC associated with CREST symptoms and 68 patients with PBC alone. Results: The characteristic findings and significant differences observed in patients with PBC associated with CREST symptoms compared with PBC alone are as follows: all women with older age with milder clinical features of both PBC (asymptomatic PBC in 84%) and CREST syndrome (incomplete CREST in 81%), more frequent occurrence of esophageal varices (28.6 vs. 9.3%), better prognosis (87.5 vs. 45.5% in 10 years survival), lower serum levels of AST (39.8 vs. 63.6 IU/l) and IgM (460 vs. 676 mg/dl), higher prevalence of discrete speckled pattern of antinuclear antibodies (93.5 vs. 12.3%), higher median titers of anti-CENP-B antibodies (1.22 vs. 0.31), lower median titers of antimitochondrial antibody (1:80 vs. 1:160), and a higher prevalence of HLA-DR9 (54.5 vs. 24.3%). Conclusion: These findings support the presence of a subgroup in PBC as PBC associated with CREST symptoms.

Primary sclerosing cholangitis with marked eosinophilic infiltration in the liver

A 16-year-old boy was diagnosed as having primary sclerosing cholangitis (PSC), based on retrograde cholangiography showing mixed features of narrowing and dilatation of the common hepatic and intrahepatic bile ducts. However, periductal fibrosis was not observed in the needle biopsy liver specimen. The liver biopsy specimen obtained 11 years previously, at the onset of the disease had disclosed a marked infiltration of eosinophils in the portal tract with eosinophilic catinonic protein immunostaining, with marked eosinophilia (54%) being noted. In Japanese reports, eosinophilia of more than 7% was reported in 13 of 32 (40.6%) PSC patients. However, the early stage of PSC, with marked eosinophilia and eosinophilic infiltration in the liver, such as in the present case, has rarely been reported. The findings in this case suggest that eosinocytes are related to the pathogenesis of PSC.

Analysis of peripheral blood mononuclear cell stimulated with pyruvate dehydrogenase complex, T-cell receptors from patients with primary biliary cirrhosis

Abstract: Progressive destruction of the intrahepatic bile ducts in patients with primary biliary cirrhosis (PBC) is thought to be mediated by cytotoxic T cells which recognize certain epitopes, such as the pyruvate dehydrogenase complex (PDC). To clarify the T-cell repertoire in PBC, we analyzed T-cell receptor (TCR) Vβ-chain messages expressed in peripheral blood mononuclear cells (PBMCs) stimulated with PDC and in liver biopsy specimens. PBMCs from 12 PBC patients and 6 healthy controls were examined. The TCR Vβ repertoires of unstimulated PBMCs and PBMCs stimulated with PDC purified from bovine heart were analyzed, using the reverse transcriptase-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism (SSCP). Liver biopsy specimens from 5 PBC patients were also analyzed. In the PBC patients, several different T-cell clones, some of which showed the same mobility, were evident in both the PDC-stimulated and unstimulated PBMCs, as demonstrated by SSCP analysis. In addition, TCR clonality of infiltrating lymphocytes in the liver was also observed in PBC patients, showing common clonal T-cell accumulation with that seen in PBMCs stimulated with PDC. These data indicate that common clonal T-cell accumulation specific for PDC may be present in both peripheral PBMCs and the liver of patients with PBC.

Antibodies to E1 and E2/Protein X components of pyruvate dehydrogenase complex in sera of patients with primary biliary cirrhosis

AIMS/METHODS Using purified E1 component of pyruvate dehydrogenase complex (PDC) from bovine heart, we measured the levels of anti-E1 antibodies in PBC sera using ELISA and determined the degree of inhibition that these antibodies exerted on E1 enzyme activity. We also estimated levels of anti-E2/Protein X (Pro-X) antibodies in PBC sera using purified E2 and Pro-X of PDC which were copurified with E1. RESULTS/CONCLUSIONS Anti-E1 antibodies were detected in 87.5% (35/40) of PBC sera. Some of these sera inhibited E1 enzyme activity but inhibition did not correlate with levels of anti-E1 antibodies. A high positive correlation (r = 0.918) was found between levels of anti-E1 and anti-E2/Pro-X antibodies, suggesting that anti-PDC antibody production was stimulated by PDC itself. Levels of IgG class anti-E2/Pro-X antibodies were significantly higher in sera of symptomatic PBC patients than in those of asymptomatic PBC patients. It was also found that patients who were positive for only IgM class anti-E2/Pro-X antibodies had early-stage PBC.

Cyst-like extension of hepatic subcapsular bleeding caused by ruptured hepatocellular carcinoma into the bursa omentalis.

An 81‐year‐old man, who experienced upper abdominal pain after shoveling snow, was admitted to a local hospital where a computed tomography (CT) showed a cystic lesion adjoining the pancreas. He was transferred to our department for detailed investigations and treatment. On ultrasonography, a tumor of the caudate lobe of the liver, with which the cystic lesion was continuous, was seen. The tumor of the caudate lobe of the liver was enhanced in the early phase of the CT but was washed out in the delayed phase. Subsequently, T1‐weighted and T2‐weighted magnetic resonance imaging (MRI) images showed a low intensity and a high intensity, respectively. Because the cystic lesion was continuous with the tumor of the caudate lobe of the liver, its CT value was higher than that of water, and both the T1‐weighted and T2‐weighted MRI images showed a high intensity, which was attributed to a hematoma. Examination of the image suggested that rupture of a hepatocellular carcinoma (HCC) might have caused intracavitary hemorrhage. After the HCC was treated by transcatheter arterial embolization therapy, the patient was discharged. Subsequently, tumor enlargement was confirmed, and surgical removal of the tumor was conducted at the hospital where the patient had originally presented. On histology, moderately differentiated HCC was diagnosed, but the cyst‐like lesion was confirmed to be a hepatic subcapsular hematoma extending into the bursa omentalis. Although ruptured HCC often causes intraperitoneal bleeding, this rare case showed a cyst‐like imaging finding in the form of a subcapsular hematoma within the bursa omentalis.

Characterization of Autoantibodies against the E1α Subunit of Branched-Chain 2-Oxoacid Dehydrogenase in Patients with Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMAs) that react with the lipoyl-containing E2 subunits of 2-oxoacid dehydrogenase complexes such as BCOADC and PDC. The lipoyl domains of E2 contain the major epitopes essential for immunopathology. However, the non-lipoyl-containing E1 subunits are also frequently targeted. Since anti-E1 antibodies always appear in combination with anti-E2 antibodies, the mechanisms underlying the autoimmunity against E1 may be linked to, but distinct from, those against E2. Here, we demonstrate that intermolecular and intramolecular determinant spreading underlies the autoimmunity against E1. We performed characterizations and epitope mapping for anti-BCOADC-E1α antibodies from both the intermolecular and intramolecular points of view. The antibody reactivities form a cluster against the BCOADC complex that is distinct from that against the PDC complex, and the anti-BCOADC-E1α antibodies arise as part of the cluster against the BCOADC complex. Multiple epitopes are present on the surface of the BCOADC-E1α molecule, and the major epitope overlaps with the active center. Sera with anti-BCOADC-E1α antibodies strongly inhibited the enzyme activity. These findings suggest that the E1α subunit as part of the native BCOADC complex is an immunogen, and that determinant spreading is involved in the pathogenesis of AMA production.

High serum levels of vascular endothelial growth factor in patients with human hepatocellular carcinoma

Vascular endothelial growth factor (VEGF) is intimately involved in neovascularization. In addition, it is know that in human hepatocellular carcinoma (HCC), angiogenesis is indispensable for tumor growth. In this study, we measured the serum VEGF levels of patients with HCC and studies the relationship between the serum VEGF level and maximum tumor diameter as well as that between the serum VEGF level and the serum α-fetoprotein (AFP) level. Mean serum VEGF level were 5.33 ± 0.77, 3.97 ± 0.68, 2.64 ± 0.78, and 2.57 ± 0.97 ng/ml for patients with HCC, chronic hepatitis (CH), or liver cirrhosis (LC) and normal controls (NC), respectively, with that of the HCC patients being significantly (P < 0.05) higher than that of the LC patient or NC. In addition, the serum VEGF level was significantly (r = 0.53, P < 0.05) correlated with the maximum tumor diameter in the HCC patients, and the sera of the patients with hypervascular HCC showed a significantly (P < 0.01) higher VEGF titer than the sera of the patients with isovascular or hypovascular HCC. However, there was no significant correlation between serum VEGF level and serum AFP level. These findings suggest that VEGF may play an important role, apart from that in AFP production, in the development of HCC.

Effect of pyruvate dehydrogenase on production of interleukin-6 in peripheral blood mononuclear cells from patients with primary biliary cirrhosis

Abstract We investigated the effect of pyruvate dehydrogenase (PDH), a mitochondrial autoantigen, on the production of interleukin-6 (IL-6) by peripheral blood mononuclear cells (PBMC) from nine patients with primary biliary cirrhosis (PBC) and nine patients with other chronic liver disease (CLD) as a control. IL-6 activity was measured by the bioassay using MH-60 BSF2 cells. The mean level of spontaneous secretion of IL-6 from PBMC of PBC patients was significantly (P

A case of gastric lipoma resected by endoscopic submucosa dissection with difficulty in preoperative diagnosis

A 66-year-old man was referred to our hospital with an increasing subepithelial lesion in the gastric antrum. Using esophagogastroduodenoscopy, a tumor with a steep, 20-mm-high rise protruding in the lumen was observed. The mucosal surface of the tumor was reddish, with ulcers forming at the base. Moreover, the tumor was mobile and soft. A biopsy specimen was taken from the ulcer, but tumor tissue was not collected from the submucosa. Endoscopic ultrasonography (EUS) showed a high echoic mass in the submucosa. However, because the mucosal surface of the ulceration was red, the mesenchymal tumor with internal bleeding was inferred to be lipoma. Additionally, because the tumor was small, flexible, and soft, collecting tumor tissue under EUS-guided fine-needle aspiration was inferred as difficult. We were unable to make a final diagnosis because the lesion showed a small tumor with atypical macroscopic morphology. Therefore, endoscopic submucosa dissection (ESD) was chosen for the diagnostic treatment. Sodium hyaluronate sufficient for separation from the muscular layer was injected into the submucosa. Then submucosal dissection was performed just above the muscle layer. Results demonstrate the possibility of removing the tumor reliably without perforation. Pathological evaluation of the ESD specimen indicated a diagnosis of gastric lipoma.