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An application of disrelation mapping to Fourier transform infrared (FT-IR) spectroscopic imaging datasets is provided to reveal different H-bonded water species within a mammalian cell. 2D correlation analysis revealed a disrelation peak at (3420 cm-1, 3220 cm-1), showing the existence of a specific water band at around 3220 cm-1 whose variation of absorbance did not follow the trend of water molecules with a well-coordinated H-bonding network. Disrelation maps constructed with disrelation intensities between (3420 cm-1, 3220 cm-1) and (3420 cm-1, 1540 cm-1) reveal that the disruption of the water network occurs around the interfacial regions between the cell and media, indicating the disintegration of the H-bonding network of bulk water due to the entrapment of water by the protein inside the cell. This hydration effect also becomes apparent around the area adjacent to the cellular nucleus, reflecting the fact that protein synthesis mainly occurs in this region. These results clearly show the presence of different molecular states of water inside living cells, which are not readily identified by conventional analysis methods.

We previously demonstrated that human TNF-alpha induces rapid apoptosis of human neutrophils. To understand better the in vivo significance of neutrophil apoptosis, we examined spontaneous, recombinant human and mouse TNF-alpha- or cycloheximide-induced apoptosis of normal peripheral blood neutrophils (PBN), PBN from rats injected i.p. with proteose peptone or a streptococcus preparation, OK-432 (inflammatory PBN), peritoneally exudated neutrophils (PEN) obtained after a proteose peptone injection, and normal bone marrow neutrophils. The following observations were made. 1) Normal PBN responded to TNF-alpha, but PEN, normal bone marrow neutrophils, and inflammatory PBN at 12 h after stimulation did not. 2) The sensitivity to TNF-alpha of the inflammatory PBN started to decrease at 3 h, was lowest at 12 h, and was almost restored at 52 h after stimulation. 3) Spontaneous apoptosis of normal and inflammatory PBN reached 25% at 12 h after in vitro incubation, but that of PEN and normal bone marrow neutrophils was very low over this period. 4) The sensitivity to cycloheximide (6 h incubation) was high for normal PBN and bone marrow neutrophils, but low for PEN and inflammatory PBN after 12 h. 5) 125I-rhTNF-alpha binding of bone marrow neutrophils was significantly lower than that of normal and inflammatory PBN and PEN. 6) TNF-alpha-induced apoptosis of normal or inflammatory PBN and bone marrow neutrophils was enhanced by treatment with low doses of cycloheximide that alone were barely able to induce neutrophil apoptosis; however, apoptosis of PEN was not. The mechanisms and in vivo significance of these phenomena are discussed.

Squamous metaplasia of the stomach is a rare clinical entity that occurs during healing of gastric ulcers or chronic inflammation. We have treated two patients with this condition, which has only occasionally been observed endoscopically. The first was a 60-year-old woman who initially presented with multiple gastric ulcers. Two months after treatment, a white patch about 4 cm in diameter was found in the lesser curvature of the cardiac region of stomach. The second patient was a 65-year-old woman, who also developed a white patch in the same region. Two months later, a small ulcer and inflamed mucosa were seen near the lesion. The white areas in both patients were stained with Lugol's iodine solution, and biopsy specimens confirmed squamous epithelium. The squamous metaplasia was observed as a white mucosal area in the stomach, and the metaplastic area stained positively with Lugol's iodine solution.

We examined whether superoxide is a factor responsible for paraquat-induced liver injury in terms of superoxide dismutase using cultured rat liver slices exposed to various concentrations of paraquat. The degree of liver injury was assessed by measurement of percentage of lactate dehydrogenase leakage into the medium and lipid hydroperoxides in the liver slices and by direct histopathological observation. Paraquat produced concentration- and time-related liver injury in the cultured rat liver slices. Notably, after exposure to 5 mmol/L paraquat, a significant increase of the percentage of lactate dehydrogenase leakage occurred from 4 hr (p less than 0.05 vs. control group), and this gradually increased up to 8 hr (p less than 0.01 and p less than 0.001 vs. control group at 6 and 8 hr, respectively). Changes in lipid hydroperoxides in the liver slices were similar to those in percentage of lactate dehydrogenase leakage (p less than 0.05 and p less than 0.01 vs. control group at 6 and 8 hr, respectively). Liver injury was located around the central vein at 6 hr and gradually spread at 8 hr. Paraquat-induced liver injury was aggravated both biochemically and histopathologically by pretreatment with 5 mmol/L diethyldithiocarbamate, an inhibitor of copper- and zinc-containing superoxide dismutase activity (percentage of lactate dehydrogenase leakage: p less than 0.01 and p less than 0.001 vs. paraquat group at 6 and 8 hr, respectively; lipid hydroperoxides: p less than 0.01 vs. paraquat group at 8 hr). Incubation of liver slices with liposome-encapsulated superoxide dismutase increased the augmentation of superoxide dismutase in the liver slices.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and H2O2, on hepatitis B virus (HBV) replication was analyzed in the HBV DNA transfected human hepatoblastoma-derived cell line HB 611. Secretion of HBV DNA from HB611 cells was inhibited by IFN-gamma, TNF-alpha, IL-1beta, and H2O2 in a dose-dependent manner. These cytokines and H2O2 also decreased HBV mRNA expression in HB611 cells, meaning that these reagents decreased the synthesis of all virally encoded components of the HBV virion. The level of manganese-SOD mRNA, indicative of occurrence of oxidative stress, increased immediately after treatment with IFN-gamma, TNF-alpha, IL-1beta, and H2O2. Moreover, the antioxidant N-acetyl-L-cysteine substantially inhibited the antiviral effect. Our findings suggest that oxidative stress may be a common factor in the antiviral effects of IFN-gamma, TNF-alpha, and IL-1beta on HBV.

The aim of this study was to investigate whether increased hepatic oxidative stress could be visualised in living animals before the onset of obvious liver injury. Acute hepatic injury was induced in mice by priming with heat-killed Corynebacterium parvum followed by injection of a low dose of lipopolysaccharide (LPS). Low frequency band electron spin resonance-computed tomography (ESR-CT) with 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was used to visualize hepatic oxidative stress. Biochemical and histological investigations performed 3 h after injection of LPS revealed no obvious injury to the liver. Conversely, significant hepatic oxidative stress could be detected at this time. Nitroxides such as carbamoyl-PROXYL are rapidly reduced to the corresponding hydroxylamine in vivo. resulting in the disappearance of their ESR signals. The kinetic clearance of carbamoyl-PROXYL after intravenous administration was delayed significantly in mice that had received LPS, due to impairment of the reduction system by hepatic oxidative stress. ESR-CT of the murine abdomen revealed a high intensity area of carbamoyl-PROXYL which consisted mainly of the liver and enlarged spleen. Time-course observations with ESR-CT using carbamoyl-PROXYL showed that the high intensity area in the liver disappeared rapidly due to reduction of carbamoyl-PROXYL. Three hours after LPS injection into the same mouse, ESR-CT images were obtained again by intravenous injection of carbamoyl-PROXYL. The ESR-CT images of the mouse with hepatic oxidative stress clearly showed that the high intensity area of carbamoyl-PROXYL in the liver persisted for a long period of time. This study is the first report to describe the use of in vivo ESR-CT for visualizing the state of increased oxidative stress in the liver before the onset of obvious hepatic injury.

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5' non-coding region (5'NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5'NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23-0.84% nucleotide position and at 0.42-0.61% amino acid position.

Hepatic lipid hydroperoxides, which are produced when reactive oxygen intermediates react with polyunsaturated lipids, were measured in patients with chronic hepatitis C. Eight patients with chronic persistent hepatitis (chronic persistent hepatitis group), 16 patients with chronic active hepatitis (chronic active hepatitis group), and 11 patients with cirrhosis (cirrhotic group) were included in the study. Measurements from ten patients without liver disease were used as controls. The lipid hydroperoxide levels in the chronic persistent hepatitis, chronic active hepatitis, and cirrhotic groups were significantly higher than that in the controls (p0.05, p0.005, and p0.005, respectively). Among the patients with chronic hepatitis C, the lipid hydroperoxide levels in the chronic active hepatitis and cirrhotic groups were significantly higher than that in the chronic persistent hepatitis group (p0.05 in both groups). The activity and progression of liver disease, represented by the Histology Activity Index, were significantly correlated with the hepatic lipid hydroperoxide level (r = 0.654, p0.01). Our findings suggest that an increased hepatic lipid hydroperoxide level is closely related to the activity and progression of chronic hepatitis C.

We report successful treatment of acute severe Budd-Chiari syndrome with portal venous thrombosis. The prognosis of patients with this condition is poor, because the therapeutic options are limited. A 38-year-old woman with polycythemia vera was admitted in a critical condition, and Budd-Chiari syndrome complicated by portal venous thrombosis was diagnosed. Tissue plasminogen activator and urokinase were infused systemically and were partially effective. Transjugular intrahepatic portosystemic shunting to reduce the high portal venous pressure was performed successfully and, eventually, her general condition improved. Our experience indicates that emergency transjugular intrahepatic portosystemic shunting is an effective therapeutic modality for controlling portal hypertension in patients with severe Budd-Chiari syndrome with portal venous thrombosis.

We evaluated the measurements of serum alpha-fetoprotein (AFP) and the protein induced by vitamin K absence (PIVKA-II) in 734 patients with chronic hepatitis (CH) and liver cirrhosis (LC) who had been followed-up for the development of hepatocellular carcinoma (HCC).Serum AFP and PIVKA-II were measured every month and abdominal ultrasonography was performed every 3 months. Youden's index (sensitivity + specificity -1) was calculated.On an average follow-up period of 374.5 days, HCC was detected in three HBsAg-positive LC patients (10.0%/yr), four anti-HCV-positive CH patients (1.35%/yr), 21 anti-HCV-positive LC patients (7.8%/yr), and one patient with both HBsAg- and anti-HCV-positive LC (22.7%/yr). At the time of HCC detection, the size of HCC was 4.7+/-0.6 (mean +/- SD) cm in HBsAg-positive patients and 2.4+/-1.3 cm in anti-HCV-positive patents. Cut-off values of 20 ng/ml for AFP (Youden's index = 0.422) and 60 mAU/ml for PIVKA-II (Youden's index = 0.316) gave the highest index for each marker. When these two markers were combined, cut-off values of 40 ng/ml for AFP and 80 mAU/ml for PIVKA-II gave the highest index (Youden's index = 0.500, sensitivity = 65.5%, specificity = 85.5%, positive predictable value = 14.8%, negative predictable value = 98.3%). The levels of AFP or PIVKA-II increased within three months before the detection of HCC.Simultaneous measurements of serum AFP and PIVKA-II levels that are performed every 3 months are useful for detecting a developing HCC. The optimal cut-off values for AFP and PIVKA-II may be 40 ng/ml and 80 mAU/ml, respectively.

A controlled trial was conducted to compare the efficacy of interferon (IFN) between two groups of patients with type C liver. Thirty-five patients were randomly assigned to group A (17 patients) or group B (18 patients). The former received 3 megaunits (MU) of human lymphoblastoid IFN six days per week for two weeks, followed by three days per week for 50 weeks; the latter group received 6 MU six days per week for two weeks followed by three days per week for 24 weeks. The percentages of biological sustained responders (B-SR) and virological sustained responders (V-SR) were 29.4 and 23.5%, respectively, in group B, and 17.6% for both in group A. The therapeutic effects were not different between two groups. HCV genotype 2 accounted for significantly higher percentage of B-SR and V-SR (both 57.1%, respectively). These findings indicate that IFN is effective in type C cirrhosis with genotype 2.

Detecting plasma protein loss from the stomach is quite difficult by measuring fecal alpha1-AT clearance because alpha1-AT is rapidly destroyed in the gastric juice at values below pH 3. We examined protein loss from the stomach using fecal alpha1-AT clearance with lansoprazole, a proton pump inhibitor. A 38-year-old Japanese male presented with hypoproteinemia (total serum protein: 4.4 g/dl). Abdominal scintigraphy using technetium-99m-labeled albumin revealed distinct radioactivity accumulation in the small intestine. It strongly suggested excessive protein loss to the gastrointestinal tract. Although, regular fecal alpha1-AT clearance was within the normal range (13 ml/day), the fecal alpha1-AT clearance with the administration of lansoprazole was 80.5 ml/day. The results indicated that this method using lansoprazole is simple and useful for detecting protein-losing gastropathy.

The aim of this work was to study the prevalence, potential risk factors, clinical and laboratory features of GB virus C (GBV-C) infection in general population from an area endemic for hepatitis C. A reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of GBV-C RNA was used to examine the prevalence of GBV-C RNA in both hepatitis C virus (HCV) endemic (R town) and nonendemic areas (M town) in Yamagata prefecture, Japan. In R town, GBV-C RNA was detected in 23 (2.9%) out of the 800 residents, whereas anti-HCV and HCV-RNA were found in 226 (28.3%) and 163 (20.4%), respectively. The prevalence of GBV-C RNA in R town (2.9%) was higher than that in M town (1.0%), although the difference was not statistically significant. The individuals with anti-HCV had significantly higher frequency of active GBV-C-infection than those without anti-HCV in both towns. No evidence indicating that GBV-C infection affected the severity of hepatitis C was obtained. The multivariate analysis revealed that the young anti-HCV positive individuals with a history of blood transfusion had higher incidence of active GBV-C infection. The phylogenetic analysis showed that the GBV-C isolates from both R and M towns were divided into two separate branch groups designated HG and Asia GB groups.

We examined the prevalence on HGV infection in a hepatitis C epidemic area where 27.8% of inhabitants 40 years or over were anti-HCV positive. Of the 200 HCV RNA positive inhabitants 17(8.5%) were HGV RNA positive, although one of the 100 anti-HCV negative inhabitants in the same epidemic area, showed HGV RNA positive (1.0%). It might suggest that HGV infections in this HCV infection epidemic area have occurred in relation to HCV infection. The rate of abnormal transaminase in inhabitants HGV RNA and HCV RNA both positive was not different from that in inhabitants HGV RNA negative, but HCV RNA positive. This indicates that a further study is needed about the relationship between liver damage and HGV infection.

We previously demonstrated that human TNF-alpha induces rapid apoptosis of human neutrophils. To understand better the in vivo significance of neutrophil apoptosis, we examined spontaneous, recombinant human and mouse TNF-alpha- or cycloheximide-induced apoptosis of normal peripheral blood neutrophils (PBN), PBN from rats injected i.p. with proteose peptone or a streptococcus preparation, OK-432 (inflammatory PBN), peritoneally exudated neutrophils (PEN) obtained after a proteose peptone injection, and normal bone marrow neutrophils. The following observations were made. 1) Normal PBN responded to TNF-alpha, but PEN, normal bone marrow neutrophils, and inflammatory PBN at 12 h after stimulation did not. 2) The sensitivity to TNF-alpha of the inflammatory PBN started to decrease at 3 h, was lowest at 12 h, and was almost restored at 52 h after stimulation. 3) Spontaneous apoptosis of normal and inflammatory PBN reached 25% at 12 h after in vitro incubation, but that of PEN and normal bone marrow neutrophils was very low over this period. 4) The sensitivity to cycloheximide (6 h incubation) was high for normal PBN and bone marrow neutrophils, but low for PEN and inflammatory PBN after 12 h. 5) 125I-rhTNF-alpha binding of bone marrow neutrophils was significantly lower than that of normal and inflammatory PBN and PEN. 6) TNF-alpha-induced apoptosis of normal or inflammatory PBN and bone marrow neutrophils was enhanced by treatment with low doses of cycloheximide that alone were barely able to induce neutrophil apoptosis; however, apoptosis of PEN was not. The mechanisms and in vivo significance of these phenomena are discussed.

Interferon (IFN) has been demonstrated to be effective against chronic hepatitis C. However, it has been believed that IFN treatment is generally ineffective in cases that have already progressed to cirrhosis. It is highly important to develop a treatment modality that will stop the progression of compensated to decompensated cirrhosis. We have carried out a randomized controlled study of human lymphoblastoid interferon in patients with compensated type C cirrhosis. There were no serious side effects, and treatment with 3 MU IFN daily for 2 wk and three times weekly for 24 wk thereafter is more effective than treatment with 1 MU, or no treatment. IFN administration can be effective and safe in patients with compensated type C cirrhosis, and it would be worthwhile to evaluate IFN therapy for such patients further.

To determine the efficacy of human lymphoblastoid interferon (L-IFN) in the treatment of compensated type C cirrhosis, 30 patients were assigned randomly to three groups, consisting of 10 patients each, who were treated as follows.The 1- and 3-megaunit (MU) groups received 1 or 3 MU of L-IFN, respectively, daily for 2 wk, and three times weekly for 24 wk thereafter. The control group received no treatment. All of the patients had positive C100-3 hepatitis C virus antibody (anti-HCV) titers.The serum alanine aminotransferase (ALT) levels decreased 26 wk after L-IFN treatment was began, in both treatment groups. In the 3-MU group, the ALT levels became normal [complete response (CR)] in 40%, improved to less than twice the upper limit of the normal value [partial response (PR)] in 10%, and remained unchanged [no change (NC)] in 50%. In the 1-MU group, a PR occurred in 30%. There was NC in 70%, and NC occurred in the control group. Twenty-four weeks after stopping L-IFN, the CR and NC rates in the 3-MU group were 10% and 90%, respectively, and NC was observed in all of the 1-MU and control patients. 2',5'-Oligoadenylate synthetase activities increased in both treatment groups (p0.05), but not in the control group. The anti-HCV titers decreased in the 3-MU group (p0.05), but not in the 1-MU and control groups. Higher doses of L-IFN were more effective. No serious side effects occurred.These findings suggest that IFN administration can be effective and safe in patients with compensated type C cirrhosis, and that it would be worthwhile to evaluate IFN therapy for cirrhotic patients further.

During health checks within the framework of the Geriatric Health Law, hepatic dysfunction was detected at a high incidence in inhabitants of K town. The factors affecting hepatic dysfunction in these cases were explored. The number of inhabitants who received health checks in 1989 in this town was 4,491 (78.5% of all inhabitants aged over 35 years). A diagnosis of hepatic dysfunction was made if GOT was equal to or higher than 41 IU/l or GPT was equal to or higher than 39 IU/l. On this basis, hepatic dysfunction was detected in 418 inhabitants (9.8%). The incidence of hepatic dysfunction was higher in males than in females. Inhabitants younger than 50 years had a higher incidence than those 50 years or older. The percentage of inhabitants with either a history of alcohol consumption or of receiving blood transfusion was significantly higher for inhabitants with hepatic dysfunction (29% and 10%, respectively) than for healthy inhabitants (10% and 3%, respectively). HBs antigen and anti HCV antibody positive rates for inhabitants with hepatic dysfunction were 3.3% and 36.1% respectively. These results suggest that the relatively high rate of hepatic dysfunction in this town is related to alcohol consumption, blood transfusion and hepatitis C virus (HCV) infection. Of these three factors, the influence of HCV infection seemed to be the most influential. The study additionally disclosed variations in the incidence of hepatic dysfunction among the different regions of the town probably related to regional differences in the anti-HCV antibody positive rate.

The prognosis of 135 cases with malignant peritoneal effusion from gastric cancer was analyzed clinically and cytologically. The histologic type of the primary gastric cancer or therapeutic differences had no significant influence on the prognosis. Previous gastrectomy or absence of distant metastases had no marked effect on survival. The formation of large clusters of cancer cells on smear preparations of peritoneal effusion was interpreted as a sign of good prognosis. The cancer cell number and the presence of lymphocytes on smear preparations bore no relationship to prognosis.

The ultrastructural localization of copper, zinc-superoxide dismutase (Cu, Zn-SOD) in the liver of patients with acute hepatitis, chronic hepatitis, liver cirrhosis and alcoholic fatty liver was studied by means of the indirect immunoperoxidase technique. In hepatocytes Cu, Zn-SOD was found to be localized in perinuclear cisternae, rough endoplasmic reticulum (rER), vesicles and Golgi apparatus. The Cu, Zn-SOD was also detected around the lipid droplets in hepatocytes as well as on the cytoplasmic membrane in cases of liver cirrhosis. These findings suggest that Cu, Zn-SOD is produced in the rER in hepatocytes and protects the cells from cellular injury caused by superoxide anion radical in various disorders of the liver.