Yukisato Kitamura

Tissue inhibitor of metalloproteinase-1 in the liver of patients with chronic liver disease.

BACKGROUND/AIMS Tissue inhibitor of metalloproteinase (TIMP)-1 is an important regulator of matrix metalloproteinase activity. To clarify the changes in TIMP-1 in diseased livers, we measured TIMP-1 concentrations in liver tissue samples from patients with chronic liver disease. The relationship between serum and liver levels of TIMP-1 was also examined in some patients. METHODS The subjects were 68 patients who underwent liver biopsy. The liver TIMP-1 concentration was measured using an enzyme immunoassay after the extraction of TIMP-1 with 2 M guanidine. RESULTS As compared with the controls (n=10), the liver TIMP-1 level was increased 2.2-fold in the 24 chronic active hepatitis 2A patients, 2.9-fold in the 10 chronic active hepatitis 2B patients and 4.1-fold in the six liver cirrhosis patients, but no significant increase was observed among the 18 chronic persistent hepatitis patients. The liver TIMP-1 levels were closely correlated with the histological degrees of periportal necrosis, portal inflammation, and liver fibrosis. When the localization of TIMP-1 was examined immunohistochemically, TIMP-1 was stained mainly in hepatocytes, and the intensity was stronger in the livers of chronic active hepatitis and liver cirrhosis patients than in those of the chronic persistent hepatitis patients. The serum TIMP-1 and liver TIMP-1 levels were significantly correlated, indicating that serum TIMP-1 could reflect the change of liver TIMP-1 in patients with chronic liver disease. CONCLUSION Liver TIMP-1 concentration increases with progression of the liver disease, when the degradation of extracellular matrix proteins is decreased, resulting in the development of liver fibrosis.

Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan‐1

Bafilomycin A1, a specific inhibitor of vacuolar type H+‐ATPase, can inhibit the growth of a variety of cultured cells in a dose‐dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan‐1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitroand in vivowas examined using an MTT assay and an in vivotumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan‐1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan‐1 cell line tumour received 4 weeks of bafilomycin A1 (1·0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P<0·05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan‐1 human pancreatic cancer cells through apoptosis.

c-erbB-2 protein is expressed in hepatolithiasis and cholangiocarcinoma.

The c‐erbB‐2 proto‐oncogene encodes a transmembrane protein which is highly homologous to epidermal growth factor receptor. Overexpression of this c‐erbB‐2 protein has been reported in many human carcinomas, including breast carcinoma. However, there have been few studies of the expression of c‐erbB‐2 in cholangiocarcinoma and hepatolithiasis, a condition occasionally associated with cholangiocarcinoma.

Effects of chitin and chitosan particles on BALB/c mice by oral and parenteral administration

Chitin and chitosan were administered orally and parenterally into mice and their toxicity was investigated. When 5 mg of chitin were injected intraperitoneally every 2 weeks over a 12-week period, the mice were apparently normal, but histologically, many macrophages with hyperplasia were observed in the mesenterium and foreign-body giant-cell-type polykaryocytes were observed in the spleen. The polykaryocytes were also observed in the spleen of the mice injected subcutaneously with 5 mg of chitin, but no other changes were observed. When 5 mg of chitosan were injected intraperitoneally, the body weights of the mice decreased significantly and inactivity was observed in the fifth week. Histologically, many macrophages with hyperplasia were observed in the mesenterium. Subcutaneous injection of 5 mg of chitosan did not evoke the general and cellular abnormalities. Oral administration of 5% chitosan via a casein diet caused mouse body weights to decrease and also decreased the number of Bifidobacterium and Lactobacillus in normal flora of the intestinal tract. These results indicate that special care should be taken in the clinical use of chitin and chitosan over a long time period.

Expression of epithelial-cadherin, alpha-catenin and beta-catenin during human intrahepatic bile duct development: a possible role in bile duct morphogenesis

BACKGROUND/AIMS Cell adhesion molecules play an important role in the morphogenesis of developing organs. However, little is known about their expression during intrahepatic bile duct development. METHODS We immunohistochemically investigated the expression of E-cadherin (E-Cad), alpha-catenin (A-Cat) and beta-catenin (B-Cat) during human intrahepatic bile duct development, using 31 fetal livers of various gestational ages. The developmental stages of bile ducts were classified into the ductal plate, migrating biliary cells (remodeling stage), and immature bile ducts (remodeled stage). RESULTS E-Cad was broadly and strongly expressed in the ductal plate with a cytoplasmic pattern, heterogeneously and weakly expressed in the migrating biliary cells with a cytoplasmic pattern, and broadly and strongly expressed in immature bile ducts with a membranous pattern. A-Cat was broadly and strongly expressed in the ductal plate with a membrane pattern, broadly and moderately expressed in the migrating biliary cells with a membranous pattern, and broadly and strongly expressed in immature bile ducts with a membranous pattern. In contrast, expression of B-Cat was weak or slight in the ductal plate, but B-Cat was expressed broadly and strongly with a membranous pattern in migrating biliary cells and in immature bile ducts. Immature hepatocytes rarely expressed E-Cad and A-Cat, but expressed B-Cat with a membranous pattern throughout development. CONCLUSIONS These results suggest that E-Cad, A-Cat and B-Cat are involved in the normal developmental morphogenesis of human intrahepatic bile ducts.

Endosomal Accumulation of Toll-Like Receptor 4 Causes Constitutive Secretion of Cytokines and Activation of Signal Transducers and Activators of Transcription in Niemann–Pick Disease Type C (NPC) Fibroblasts: A Potential Basis for Glial Cell Activation in the NPC Brain

Niemann–Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-β, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1−/− mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1−/− mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1−/− mouse brain.

Plasma transforming growth factor‐β1 concentrations in patients with chronic viral hepatitis

Transforming growth factor (TGF)‐β1 is an important cytokine involved in the pathobiology of tissue fibrosis through its stimulation of the production of, and inhibition of the degradation of, extracellular matrix proteins. We examined the clinical usefulness of plasma TGF‐β1 concentration as a marker of fibrogenesis in patients with chronic viral hepatitis. Thirty‐five patients, 11 with minimal chronic hepatitis, 14 with mild chronic hepatitis and 10 with moderate chronic hepatitis and 20 healthy subjects were studied. Transforming growth factor‐β1 concentrations in platelet‐poor plasma were measured with a TGF‐β1 enzyme‐linked immunosorbent assay system kit after acid‐ethanol extraction. Plasma TGF‐β1 levels were significantly elevated in patients with mild and moderate chronic hepatitis, but not in those with minimal chronic hepatitis, compared with the levels in the controls. Plasma TGF‐β1 levels were increased in parallel with the histological degree of necroinflammation and of liver fibrosis. Plasma TGF‐β1 levels were positively correlated with blood levels of procollagen type III N‐peptide, and 7S fragment and central triple‐helix of type IV collagen. These results suggest that plasma TGF‐β1 level is a useful marker in assessing the situation of liver active fibrogenesis in patients with chronic viral hepatitis.

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2‐40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF‐C), VEGF‐D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF‐D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF‐C or VEGF‐D expression. High LVD, induced by VEGF‐C or VEGF‐D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.

Collagen typing of granulation tissue induced by chitin and chitosan

Granulation tissue induced by chitin- or chitosan-coated polyester nonwoven fabric (chitin-NWF, chitosan-NWF) and uncoated NWF (control) implanted in the feline abdominal wall was evaluated by macroscopic and microscopic observation. Tissue growth around the implant was most marked with chitosan-NWF and least with chitin-NWF. The inflammatory reaction was much stronger with chitosan-NWF. In the tissue surrounding the implant, many giant cells and prominent angiogenesis were induced by chitin-NWF compared to the other NWFs. Types I, III and IV collagen were synthesized at higher levels in the chitin-NWF implants, especially type IV collagen, compared with the other implants. At 14 days after implantation, the levels of types III and IV collagen were also greater in the tissue surrounding the chitin-NWF. The pattern of synthesis of each type of collagen induced by chitosan-NWF in the surrounding tissue closely resembled that of the control.

Endocrine cells in hepatobiliary cystadenomas and cystadenocarcinomas

We investigated the distribution of endocrine cells in hepatobiliary cystadenoma (n=5, two associated with mesenchymal stroma) and cystadenocarcinoma (n=3) immunohistochemically. In normal livers (n=20) and livers affected by hepatolithiasis (n=15) used as controls, endocrine cells revealed by chromogranin immunostaining were located exclusively in normal or proliferating intrahepatic peribiliary glands. In the eight cases of hepatobiliary cystadenoma and cystadenocarcinoma, endocrine cells were present in four cases (50%) (1 cystadenoma, 1 cystadenoma with mesenchymal stroma, and 2 cystadenocarcinomas). Endocrine cells tended to be located beneath and among the columnar epithelial cells. Intrahepatic peribiliary glands were located in the vicinity of cystadenoma or cystadenocarcinoma in six (75%) of the eight cases, and they frequently showed cystic dilatation and contained endocrine cells. Intrahepatic peribiliary glands were located in the vicinity of the endocrine cells in all cystadenomas and cystadenocarcinomas that were positive for endocrine cells. These data show that about 50% of hepatobiliary cystadenomas and cystadenocarcinomas contain endocrine cells and suggest that hepatobiliary cystadenoma and cystadenocarcinoma may originate from intrahepatic peribiliary glands.