Takuya Kitada

In situ detection of oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease

BACKGROUND/AIMS 8-Hydroxydeoxyguanosine (8-OHdG) is a promutagenic DNA lesion produced by oxygen radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. METHODS Hepatic expression of 8-OHdG was immunohistochemically investigated in control and diseased human livers. RESULTS While no positive immunolabeling for 8-OHdG was observed in control livers, 8-OHdG was widely evident in diseased livers. Nuclear expression of 8-OHdG in the hepatocytes and bile duct cells were found in various forms of chronic hepatitis. 8-OHdG-positive hepatocytes were especially abundant in the periportal area with piecemeal necrosis and prominent cell infiltration. The number of positive hepatocytes significantly increased with the progression of severity of chronic hepatitis activity (r(s)=0.68, P<0.05). In alcoholic liver disease, nuclear expression of 8-OHdG was detected in the hepatocytes in the area of alcoholic hepatitis. Regarding primary biliary cirrhosis, 8-OHdG was preferentially detected in the nuclei of injured bile ducts (11 of 12 cases, 91.7%) and occasionally (2 of 12 cases, 16.7%) in the nuclei of hepatocytes around the bile duct lesions. CONCLUSIONS These results indicate that oxidative DNA damage is common in various forms of chronic liver disease suggesting a possible link between chronic inflammation and hepatocarcinogenesis.

Expression of antigens related to apoptosis and cell proliferation in chronic nonsuppurative destructive cholangitis in primary biliary cirrhosis

The initial injury in primary biliary cirrhosis (PBC) is the destruction of portal bile ducts. Little information is available on apoptosis and cell proliferation in such bile ducts, so we used immunohistochemical techniques to locate molecules related to apoptosis [Fas antigen, Lewis Y antigen (BM1/JIMRO), and bcl-2 protein] and to cell proliferation (proliferating cell nuclear antigen, PCNA) in 21 patients with PBC. In addition, nick-end labelling was done to locate DNA fragmentation. The expression of these molecules in chronic nonsuppurative destructive cholangitis (CNSDC) was examined. Cell death and PCNA expression were both found in portal bile ducts affected by CNSDC in 7 of the 13 CNSDC patients examined. Fas antigen was found on the plasma membrane and rough endoplasmic reticulum of bile-duct cells with CNSDC in the frozen sections of all 6 patients with CNSDC out of the 9 patients inspected, and this antigen was found also in bile-duct cells without CNSDC in 2 of these 9 patients. It was not found in anatomically normal liver (from 2 patients with Gilberts disease). The Lewis Y antigen was found in bile ducts with CNSDC and in proliferated ductules in all 16 patients examined. No bcl-2 protein was found in any bile-duct or ductule cells, but it was found in the cytoplasm of lymphocytes surrounding or invading CNSDC. DNA fragmentation was found in the nuclei of bile-duct cells with CNSDC by nick-end labelling. Our study indicated that Fas-mediated apoptosis might be involved in CNSDC, but that bcl-2 protein seems to participate less than Fas. Although the Lewis Y antigen was found in many bile ducts, the relationship between the antigen and apoptosis remains unknown because there was no evidence that this antigen mediates apoptosis.

Clinicopathological significance of hypoxia‐inducible factor‐1α expression in human pancreatic carcinoma

Aims:  The transcription factor hypoxia‐inducible factor‐1α (HIF‐1α) plays a key role in the cellular adaptation to hypoxia and the activation of several genes that have been implicated in tumour growth. The aim of this study was to investigate the clinicopathological significance of HIF‐1α expression in pancreatic carcinoma.

Pathological significance of oxidative cellular damage in human alcoholic liver disease

Aims: To investigate the pathological significance of oxidative stress‐induced lipid peroxidation and oxidative DNA damage in alcoholic liver disease.

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

Abstract. Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries.

Immunohistochemical detection of 8‐hydroxydeoxyguanosine, a marker of oxidative DNA damage, in human chronic cholecystitis

Immunohistochemical detection of 8‐hydroxydeoxyguanosine, a marker of oxidative DNA damage, in human chronic cholecystitis

Clinicopathological characterization of prion: a novel marker of activated human hepatic stellate cells

BACKGROUND/AIMS We recently demonstrated prion as a new marker for hepatic stellate cell activation in rats. Here, we have examined prion expression in normal and diseased human livers. METHODS Prion expression was examined at protein level by immunohistochemistry and at mRNA level by in situ hybridization. RESULTS While normal livers were negative for prion, all liver specimens but one from patients with chronic liver disease were positively stained. In chronic hepatitis, prion protein expression was found not only in the sinusoidal lining cells within the lobules but also in mesenchymal cells in expanded portal tracts. In alcoholic liver disease, prion-positive cells were found mainly in the areas of alcoholic hepatitis. Immunoelectronmicroscopy revealed that prion-positive cells were activated stellate cells. In situ hybridization demonstrated that the distribution of prion mRNA is similar to that of prion protein. In chronic hepatitis, the number of prion-positive cells correlated with the grade of activity but not with the stage of fibrosis. In alcoholic liver disease, levels of prion protein expression were significantly increased in the presence of alcoholic hepatitis. CONCLUSION Prion as a novel marker of activated stellate cells correlates well with disease activity in human chronic liver diseases.

Granular cell tumor of the duodenum : A case report

Granular cell tumor (GCT) in the duodenum is an extremely rare disease: only one case has been listed in a review, to date. We reported a 47-yr-old Japanese male case with GCT of the duodenum. Clinically, melena caused by bleeding from the tumor was the only symptom. The tumor cells showed abundant, granular eosinophilic cytoplasm. Although this tumor was clinically and histologically benign, highly developed tumor microvessels were demonstrated both angiographically and histologically, suggesting malignant potential of the tumor.

Expression of progenitor cell markers in livers with fulminant massive necrosis.

Studies of stem cells in various organs have greatly progressed, and progenitor cells have been confirmed even in liver by recognition of cytokeratin 14 (CK14), c-kit, flt-3, and CD34. We, therefore, immunohistochemically examined the expression of these progenitor cell markers in patients with confluent or massive necrosis, in addition to CK19, albumin, vimentin, and Ki-67. Our subjects were six survivors and 14 deceased patients. Expression of CK14 and c-kit was found in a small number of cells lining biliary ductule-like structures, and that of flt-3 was found in many lining cells in two deceased patients with multi-lobular necrosis. CK14 positive cells were positive for c-kit, flt-3, and CK19 in semi-serial sections, but were negative for albumin, Ki-67, and CD34. In conclusion, expression of CK14 and c-kit was found in a small number of cells lining biliary ductule-like structures, and that of flt-3 was found in many cells lining biliary ductule-like structures. CK14-positive cells were positive for c-kit, but negative for CD34. Since c-kit is a hematopoietic marker, our study suggests that CK14- and c-kit-positive cells may be derived from bone marrow in liver with fulminant hepatitis.

Induction of apoptosis in a human hepatocellular carcinoma cell line by a neutralizing antibody to transforming growth factor-α

A cell line derived from a Japanese man with hepatocellular carcinoma was established in culture and designated OCUH-16. The cell line has the morphological and chromosomal features of hepatocellular carcinoma cells and has a short doubling time (≈33 h). OCUH-16 cells were shown to express transforming growth factor-α, (TGF-α) in addition to albumin, DNA polymerase-α, c-JUN, and the retinoblastoma gene product. Electron microscopy revealed TGF-α immunoreactivity associated with the cell membrane, but TGF-α was not detected in medium conditioned by OCUH-16 cells by enzyme-linked immunosorbent assay. Reverse transcription and polymerase chain reaction analysis revealed the presence of TGF-α messenger RNA in these cells. Culture of OCUH-16 cells in the presence of a neutralizing antibody to TGF-α inhibited cell proliferation and induced many cells to undergo apoptosis (programmed cell death). These observations suggest that endogenous TGF-α is necessary for OCUH-16 cell growth.