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Background and Aims: It remains unclear whether obesity increases the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C who achieved a sustained virological response (SVR) with antiviral therapy. Methods: In this multicenter cohort study, we enrolled patients with chronic hepatitis C who achieved SVR with interferon (IFN)-based therapy (IFN group) or direct-acting antiviral (DAA) therapy (DAA group) between January 1, 1990, and December 31, 2018. The patients underwent regular surveillance for HCC. Cumulative incidence of and the risk factors for HCC development after SVR were assessed using the Kaplan-Meier method and Cox proportional hazard regression analysis, respectively. Results: Among 2,055 patients (840 in the IFN group and 1,215 in the DAA group), 75 developed HCC (41 in the IFN group and 34 in the DAA group) during the mean observation period of 4.1 years. The incidence rates of HCC at 1, 2, and 3 years were 1.2, 1.9, and 3.0%, respectively. Multivariate analysis revealed that in addition to older age, lower albumin level, lower platelet count, higher alpha-fetoprotein level, and absence of dyslipidemia, obesity (body mass index ≥25 kg/m2) and heavy alcohol consumption (≥60 g/day) were independent risk factors for HCC development, with adjusted hazard ratio (HR) of 2.53 (95% confidence interval [CI]: 1.51–4.25) and 2.56 (95% CI: 1.14–5.75), respectively. The adjusted HR was not significant between the 2 groups (DAA vs. IFN; HR 1.19, 95% CI: 0.61–2.33). Conclusions: Obesity and heavy alcohol consumption increased the risk of HCC development after SVR.

AIM Opportunities to treat older patients with hepatitis C virus infection have increased. We investigated the efficacy and safety of glecaprevir/pibrentasvir in patients with HCV infection aged ≥75 years. METHODS We retrospectively evaluated 131 patients with hepatitis C virus infection treated with glecaprevir/pibrentasvir at nine institutions in Japan. The patients were divided into two groups according to their age: the elderly group (n = 43, aged ≥75 years) and younger group (n = 88, aged

Several major complications from endoscopic retrograde cholangiopancreatography (ERCP), including pancreatitis, cholangitis, and hemorrhage have been discussed in detail; however, a few uncommon but severe complications have been reported. We encountered an unusual case of post-ERCP intrahepatic subcapsular biloma. An 89-year-old woman with a 25-mm mass located at the hepatic hilum, suggestive of cholangiocarcinoma, underwent ERCP which demonstrated complete stricture of the common hepatic duct. Subsequently, two plastic stents were placed from the common bile duct to the right and left intrahepatic branches. On day 3, serum inflammatory markers were elevated and computed tomography revealed a large subcapsular fusiform fluid collection in the right liver, consistent with biloma. On day 6, the biloma ruptured and 500 ml of biliary ascites were removed. On day 8, endoscopic nasobiliary drainage via the right intrahepatic branch was performed because of recurrence of biliary ascites. After the procedure, 150 ml of bile was collected through the drain every day and no ascites recurred. We believe that minor injury to the right intrahepatic bile duct due to guidewire manipulation caused the biloma. Biloma may become apparent several days after ERCP, and endoscopic biliary drainage placement adjacent to the bile duct rupture site can stop bile leakage.

A 65-year-old male visited our hospital because of fever and difficulty in walking. He was suffering from left-sided hypochondrial pain for a month. Laboratory tests performed on admission revealed a white blood cell count of 1700/μl and C-reactive protein level of 9.51mg/dl, which were suggestive of severe inflammation. Contrast-enhanced computed tomography revealed a subphrenic abscess around the spleen, which we considered to be caused by gastric penetration into the gastrosplenic ligament. Upper esophagogastroduodenoscopy revealed a gastric ulcer together with a fistula that connected to the left subphrenic abscess. We thus performed endoscopic transgastric drainage through the fistula. Antibiotics and a proton pump inhibitor were administered, and drainage was continued. The patient's clinical and inflammatory symptoms subsequently improved. We thus consider that endoscopic transgastric drainage is an appropriate treatment option for subphrenic abscesses.

— Rats were pair-fed nutritionally adequate liquid diets, containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4–6 weeks. Ruffle formation of hepatic macrophages in the periportal area observed with a transmission electron microscope (which reflects their extent in activation) was more remarkable in ethanol-fed rats than in control rats. The ability of hepatic macrophages to produce superoxide anions assessed in situ by formazan deposition after liver perfusion with nitro-blue tetrazolium and phorbol myristate acetate was enhanced after such ethanol feeding. A similar result was seen 24 h after withdrawal of ethanol feeding. These findings suggest that long-term ethanol consumption may activate hepatic macrophages in secretory function.

Hepatocyte growth factor (HGF) has been shown to enhance albumin production as well as stimulate DNA synthesis in hepatocytes. The mode of action of HGF in exerting both effects is to be elucidated. We previously observed that hepatocyte proliferation occurred in normal rats given recombinant human HGF (rhHGF) intravenously. When rats received rhHGF similarly, serum albumin levels were significantly increased compared to controls. In primary culture of rat hepatocytes, albumin concentration in culture medium was significantly increased by rhHGF added at 24 h of culture compared to controls, increasing with time of culture. This effect of rhHGF was dose-related. When actinomycin D was added to the medium, the albumin concentration was reduced in a dose-related manner, but its enhancement by rhHGF was maintained. Albumin mRNA levels were not increased by rhHGF. When rhHGF was added similarly to the medium, immunocytochemically positive hepatocytes for 5-bromo-2'-deoxyuridine (BrdU) incorporation appeared 30 h later. Of these labeled hepatocytes, 12.5% were concomitantly stained for albumin. In contrast, albumin-positive hepatocytes were seen in 77% of BrdU-non-labeled hepatocytes (p

Background & Aims: Sphingosine 1-phosphate (S1P), a ligand for G protein-coupled endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5, Edg-6, and Edg-8, elicits a variety of responses by cells. Prominent among these is cell proliferation. S1P is abundantly stored in platelets and released upon their activation, suggesting that S1P plays a pathophysiologic role in vivo. Because the major part of injected S1P was distributed into the liver in mice, we wondered whether the liver would be one of its targets. The effects of S1P on hepatocytes, the major constituent cells in the liver, were examined. Methods & Results: Northern blot analysis revealed the expression of Edg-1 and Edg-5 messenger RNA (mRNA) in cultured rat hepatocytes, in which S1P decreased DNA synthesis induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting total protein synthesis. This inhibitory effect was attenuated by inactivation of small GTPase Rho with C3 exotoxin but not by inactivation of G i with pertussis toxin. Moreover, in the presence of JTE-013, a newly developed and specific binding antagonist for Edg-5, the inhibitory effect was also cancelled. Finally, the administration of S1P after 70% partial hepatectomy in rats reduced the peak of DNA synthesis in hepatocytes with increased Rho activity. Furthermore, Edg-5 but not Edg-1 mRNA expression was enhanced in hepatocytes 24–72 hours after partial hepatectomy, which coincides with decreasing hepatocyte proliferation. Conclusions: S1P has an antiproliferative property in rat hepatocytes by activating Rho via Edg-5. Our results raise the possibility that S1P is a negative regulator in liver regeneration.

Colonoscopy is routinely performed worldwide, but it is still a physically stressful screening method for colorectal cancer, particularly in elderly individuals. No previous study has evaluated physical stress from colonoscopy itself and polypectomy using blood-derived biomarkers. This study aimed to evaluate physical stress from colonoscopy and polypectomy in elderly individuals using white blood cell counts (WBCs) and C-reactive protein (CRP) levels, which are widely used blood-based biomarkers that respond quickly to tissue damage. In this single-center, retrospective observational study, 109 consecutive patients, aged ≥ 80 years, were enrolled. In all patients, WBCs and CRP levels were measured before and half-a-day after colonoscopy. The changes were subsequently compared. Patients were divided into two groups: those with polypectomy (P+) and those without polypectomy (P-). The changes in WBCs and CRP levels were determined and compared between the two groups. In the P + group, correlations between the number of resected polyps and changes in WBCs and CRP levels were also evaluated. Of the 109 patients, there were 48 men and 61 women with a mean age of 83.9 ± 3.1 years (range, 80-91 years); 58 patients received polypectomy. In all patients, the mean WBC after colonoscopy showed no significant change from that before colonoscopy (before: 5400/μL, after: 5200/μL, P = 0.092), and the mean CRP level showed a slight elevation after colonoscopy (before: 0.05 mg/dL, after: 0.08 mg/dL, P < 0.001). Compared to before colonoscopy, the mean WBC after colonoscopy decreased slightly in the P- group (before 5400/μL, after 4900/μL, P = 0.002), but showed no significant difference in the P+ group (5400/μL, 5400/μL, P = 0.48). Conversely, the mean CRP level increased very slightly in both the P- and P+ groups (from 0.06 mg/dL to 0.07 mg/dL, P = 0.003; from 0.04 mg/dL to 0.08 mg/dL, P < 0.001, respectively), but it was not significantly different between the groups. The elevation of both WBCs and CRP levels showed only minor correlations with the number of resected polyps. In conclusion, colonoscopy can be well tolerated even by elderly individuals because physical stress from colonoscopy, with respect to WBCs and CRP levels, was minimal even if polypectomy was performed.

p53, known as a tumor suppressor gene, is a transcription factor that regulates various cellular functions. Recently, several growth factor gene promoters, including that of transforming growth factor α (TGF-α), were shown to be direct targets of p53-mediated transcription. Hepatic p53 mRNA is up-regulated during liver regeneration in rats. The aim of this study is to examine the role of p53 in hepatocyte proliferation. p53 protein levels were examined in rat hepatocytes cultured in the medium containing hepatocyte growth factor (HGF). p53 levels began to increase after 6 hours of incubation, reached a maximum at 18 hours, and decreased thereafter. DNA synthesis increased at 12 hours and peaked at 30 hours. When hepatocytes were incubated with p53 antisense oligonucleotide in addition to HGF, increases of p53 and TGF-α levels were suppressed, and DNA synthesis was reduced. The increases of TGF-α levels and DNA synthesis were also suppressed by a chemical inhibitor of p53, pifithrin-α. In rats after two-thirds partial hepatectomy, hepatic p53 increased and reached maximal levels around 16 hours when hepatic HGF levels have been shown to reach a maximum followed by an increase in hepatic TGF-α levels or hepatocyte proliferation. In contrast, sham-operated rats showed minor elevations of hepatic p53 levels. In conclusion, p53 production is stimulated by HGF and may contribute to the proliferation of rat hepatocytes. Considering previous findings indicating the importance of endogenous TGF-α for the proliferation of hepatocytes stimulated by HGF, TGF-α might play a role in HGF-p53 mediated hepatocyte proliferation. (H EPATOLOGY 2002;36:336-344.)

Both transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) induce DNA synthesis in hepatocytes in vitro and in vivo. Hepatic and circulating levels of HGF have been reported to increase before an increase in TGF-alpha levels in several rat models of liver regeneration. In addition, serum TGF-alpha levels increase after an increase in serum HGF levels in patients with either partial hepatectomy or acute hepatitis. In this study, we investigate the significance of TGF-alpha in hepatocyte proliferation. TGF-alpha contents and DNA synthesis in cultured rat hepatocytes increased in response to HGF addition to the culture medium in a dose-related manner. These increases were suppressed by the addition of anti-sense TGF-alpha mRNA oligonucleotide. Furthermore, the addition of anti-TGF-alpha rabbit IgG suppressed the increase in DNA synthesis. When the anti-TGF-alpha antibody was administered to rats after partial hepatectomy, the number of mitotic hepatocytes was reduced in comparison to rats treated with normal rabbit IgG. These results were observed even though hepatic HGF levels were increased equally in rats given either anti-TGF-alpha antibody or normal rabbit IgG. Our results suggest that HGF stimulates TGF-alpha production in rat hepatocytes, and that the mitogenic activity of HGF depends on endogenous TGF-alpha activity.

We studied the effect of lysophosphatidic acid (LPA) on collagen gel contraction by cultured rat hepatic stellate cells (HSCs) in association with the function of Rho-kinase, one of the target molecules of small GTPase Rho. Binding studies showed a single class-binding site of LPA on HSCs. LPA enhanced the contraction of a collagen lattice seeded with HSCs. LPA increased the number of HSCs with polygonal morphology that contained actin stress fibers, and enhanced the phosphorylation of myosin light chain and the assembly of focal adhesion kinase and RhoA around fibronectin-coated beads seeded on HSCs. The electric cell-substrate impedance sensor system showed that LPA enhanced adhesion of HSC to extracellular substrate. All the effects of LPA were suppressed by Y-27632, Rho-kinase inhibitor. These data support the notion that LPA is involved in modulating HSC morphology, its attachment to surrounding extracellular matrix and its contraction by a mechanism involving Rho-kinase.

The efficacy of therapy with interferon (IFN)- β or - α for chronic hepatitis C is limited in long-term viral eradication and normalization of serum alanine transferase (ALT) levels. IFN- β, used intravenously, is shown to disappear from the circulation within 12 h after the injection, suggesting that the efficacy might be improved if IFN- β is injected at shorter intervals. Patients with chronic hepatitis C, diagnosed by liver histology and positive serum hepatitis C virus (HCV)-RNA, were randomly assigned to two groups and given IFN- β for 6 weeks. Patients received 6 MU every 24 h in group A and 3 MU every 12 h in group B. Fifty-four patients were enrolled in the trial, 28 in group A and 26 in group B. Demographic and clinical features of the patients before therapy did not differ between both groups. Proteinuria, thrombocytopenia and marked elevation of serum ALT levels developed more frequently in group B than in group A. Of seven patients in whom the therapy was ceased within 1 month in group B due to such adverse effects, three showed negative serum HCV-RNA during the follow-up period up to 12 months. Serum HCV-RNA disappeared in 95% of the patients in group B and 74% in group A at the end of the therapy. Multivariate logistic regression analysis revealed that the interval of IFN- β injections as well as serum HCV-RNA level were the important variables for determining the disappearance of serum HCV-RNA at the end of the therapy. IFN- β injected at 12-h intervals may be superior to the injections at 24-h intervals as a therapy with daily dose of 6 MU in the treatment of patients with chronic hepatitis C despite frequent adverse effects.

Transforming growth factor alpha (TGFalpha) and hepatocyte growth factor (HGF) are mitogens for hepatocytes in vitro and in vivo, produced by hepatocytes or nonparenchymal cells such as stellate cells in the liver. It is still uncertain whether TGFalpha and HGF are essential for liver regeneration. To assess the role of these growth factors in liver regeneration, their circulating and hepatic levels were studied in various rat models of liver regeneration. Hepatic and plasma HGF levels were increased with increased number of mitotic hepatocytes in rats after partial hepatectomy or carbon tetrachloride intoxication. However, hepatic HGF levels were decreased despite an increased number of mitotic hepatocytes and increased or unchanged plasma HGF levels in rats given phenobarbital and in rats after dimethylnitrosamine intoxication, which can induce hepatic necrosis after apoptosis of hepatic stellate cells. In contrast, hepatic and serum TGFalpha levels were increased in all of the models. In sham-operated rats with no increased number of mitotic hepatocytes, hepatic and circulating levels of HGF were increased, whereas those levels of TGFalpha were unchanged. The results indicate that TGFalpha levels in liver and blood more closely correlate with hepatocyte mitogenesis than HGF levels.

Activated Kupffer cells and hepatic macrophages can produce massive liver necrosis through microcirculatory disturbance due to sinusoidal fibrin deposition. This mechanism is involved in the development of liver injury after endotoxin administration in rats pretreated with heat-killed Propionibacterium acnes (P.acnes) or undergoing 70% liver resection. The significance of CD14, a receptor for lipopolysaccharide and its binding protein, was evaluated in both models in relation to the activation mechanisms of Kupffer cells and hepatic macrophages. Northern blot analysis revealed that CD14 mRNA expression was increased in the liver of rats following P.acnes administration. In these rats, hepatic macrophages immediately after isolation showed marked increased of CD14 mRNA expression compared to Kupffer cells from normal rats. In contrast, CD14 mRNA expression was minimal in partially resected liver. Interleukin (IL)-18 and IL-2 mRNA expression in the liver and interferon (IFN)-gamma mRNA expression in the spleen were significantly increased in P.acnes-treated rats compared to normal rats, while these increases were absent in partially hepatectomized rats. Thus, CD14 expressed on hepatic macrophages after activation through a cytokine network of IL-18, IFN-gamma, and IL-2 may contribute to endotoxin-induced liver injury in P.acnes-treated rats. In contrast, in partially hepatectomized rats, this network may not operate during Kupffer cell activation, and the liver injury might develop through endotoxin receptors other than CD14 on the cells.

The regulatory mechanisms of microcirculation might differ in the liver from other organs, because macrophages are resident in the hepatic sinusoids and sinusoidal endothelial cells are unique in shape and function. Thrombomodulin expression in endothelial cells and tissue factor activity in isolated macrophages were studied in the liver and lung of rats. In normal rats, the thrombomodulin expression was minimal in hepatic sinusoids, but prominent in pulmonary capillaries, while the tissue factor activity in the presence of endotoxin was higher in pulmonary macrophages than in Kupffer cells, although the levels in the absence of endotoxin were comparable in both cells. The tissue factor activity in hepatic macrophages was increased after priming of the cells with Corynebacterium parvum or after induction of liver necrosis or cirrhosis with carbon tetrachloride. In the necrotic or cirrhotic liver, increased thrombomodulin expression was seen along capillaries extending in necrotic areas and regenerating nodules, but this increase was minimal in the Corynebacterium parvum-treated rat liver. Blood coagulation equilibrium in microcirculation regulated by endothelial cells and macrophages may differ between the liver and lung. Such equilibrium in the liver may vary depending on pathological status.

Endothelial cell damage causes massive hepatic necrosis as a result of fibrin deposition in the hepatic sinusoids. When a stable analog of prostaglandin I2, beraprost sodium, was administered to rats given either dimethylnitrosamine, carbon tetrachloride, or endotoxin following Corynebacterium parvum administration, the hepatic necrosis produced in each was attenuated, but to a greater extent in the dimethylnitrosamine and endotoxin/Corynebacterium parvum models, where fibrin deposition in the hepatic sinusoids occurs, as compared to the carbon tetrachloride model, where such fibrin deposition does not occur. Beraprost sodium reduced the expected increase of portal venous pressure in the endotoxin/Corynebacterium parvum model without affecting plasma thrombin-antithrombin III complex levels. Beraprost sodium also significantly reduced cell killing of both isolated rat hepatocytes and hepatic sinusoidal endothelial cells exposed to tert-butyl hydroperoxide when compared to controls. Beraprost sodium could prove to be a therapeutic candidate for the treatment of hepatic necrosis, particularly in cases associated with fibrin deposition in the hepatic sinusoids because of its fibrin clot-clearing action.

A very elderly patient with primary non-Hodgkin lymphoma of the esophagus is reported. An 87-year-old woman presented with dysphagia. Endoscopy revealed an elevated lesion with ulceration in the middle and lower esophagus. Endoscopic biopsy demonstrated pathological diagnosis and immunochemistry typical of non-Hodgkin lymphoma (Ann Arbor stage IIIEA), diffuse large B-cell lymphoma on the WHO classification. Systemic chemotherapy with 6 courses of a 50% dose of R-CHOP followed by 8 courses of rituximab for maintenance, successfully resulted in complete remission. Complete remission has been maintained for 58 months.

Background: Massive hepatic necrosis caused by fibrin deposition in the hepatic sinusoids develops with hepatic macrophage activation in rats given endotoxin after administration of heat-killed Corynebacterium parvum . Targeted cells of such macrophages were investigated. In C. Parvum -treated rats, the pathological appe Methods: arance of liver cells was serially measured in serum following endotoxin administration and compared with the appearance in the perfusate during closed liver perfusion with endotoxin. Results: Serum activities of tumor necrosis factor, purine nucleoside phosphorylase present in both hepatocytes and sinusoidal endothelial cells, and levels of alanine aminotransferase were higher after 30 minutes, 1 hour, and 3 hours, respectively. Pretreatment of rats with gadolinium chloride, an inhibitor of macrophage function, reduced this liver injury. Although alanine aminotransferase activity remained almost unchanged in the liver perfusate, purine nucleoside phosphorylase activity increased. This increase was reduced when rats were pretreated with gadolinium chloride. There was sinusoidal endothelial cell damage around hepatic macrophages in the liver perfused with endotoxin. Conclusions: Activated hepatic macrophages may cause sinusoidal endothelial cell damage leading to hepatocyte necrosis in rats given C. parvum and endotoxin.

Fat-storing cells (FSC, lipocytes, or Ito cells) of liver store vitamin A and are the main producers of extracellular matrix in normal and cirrhotic liver. During liver injury, FSC undergo an activation process characterized by a decrease in vitamin A storage and an increase in cell proliferation and extracellular matrix deposition. This activation process also occurs upon culturing FSC from normal liver. In contrast to most cells of nonmuscle origin, activated FSC express two cytoskeletal proteins normally found in muscle, desmin, and smooth muscle α-actin. Based on their strategic perisinusoidal location, it has been hypothesized that FSC play a role in regulating blood flow. However, the nature of the contractile elements involved in this process remains to be determined. In this communication we demonstrate the presence of a sarcomeric myosin in proteins solubilized from liver biomatrix. In addition we demonstrate the expression of sarcomeric myosin heavy chain (MHC) mRNA and protein in two FSC clones derived from a CCl4-cirrhotic rat liver (CFSC). Through cloning the cDNA corresponding to the MHC gene expressed in these cells we demonstrate that it encodes fast IId skeletal MHC and thus represents a marker normally seen in adult muscle. The unexpected expression of an adult stage skeletal muscle molecular motor in FSC from cirrhotic liver is consistent with the proposed specialized contractile capacity of these cells. © 1993 Wiley-Liss, Inc.

When prostaglandin (PG) E1 was continuously administered to rats from 24 hours before giving a dose of carbon tetrachloride, deranged serum glutamic pyruvic transaminase levels and prothrombin time were significantly reduced 12 hours after intoxication compared with controls. A similar effect of PGE1 was seen at 24 hours in d-galactosamine-intoxicated rats. Liver histology showed a comparable attenuation of injury in these rats. These results were consistent with reported effects of PGE2, suggesting that both prostaglandins may share a common pathway in protection against liver injury. When PGE1 or 16,16′-dimethyl PGE2 was added to the medium of primary cultured rat hepatocytes, lipid peroxidation-dependent killing of the cells by tert-butyl hydroperoxide was significantly attenuated without affecting the extent of malondialdehyde accumulation compared with controls. Both prostaglandins significantly reduced the extent of increased plasma membrane microviscosity of these cells assessed by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene. PGE1 and PGE2 may possess cytoprotective effects on liver parenchymal cells through stabilization of membrane microviscosity, which may contribute to protection against liver injury.

When insulin and glucagon are administered to rats with severe liver injury, survival is enhanced with an attenuation of the liver injury compared to that of untreated controls. In rats with acute liver injury both hormones produce a rapid normalization of hepatic protein content following initiation of DNA synthesis. When rats receive both hormones after partial hepatectomy, the first burst of DNA synthesis reaches a maximum earlier than that seen in controls. Both hormones enhance the increment of hepatic putrescine essential for DNA synthesis through activation of ornithine decaroxylase and/or spermidine-N1-acetyltransferase. The enhancement of putrescine content by each hormone is additive. Putrescine supplementation promotes hepatic DNA synthesis after hepatectomy. Based on these data, we conclude that a combination of insulin, and glucagon is effective in the therapy of acute hepatic failure in rats. The restoration of liver function as well as the stimulation of liver cell proliferation via putrescine production may contribute to this effect.

Because hepatocellular carcinoma treated by transcatheter arterial embolization often regains its size, routine follow-up is necessary. The usefulness of pulsed Doppler ultrasound for detection of this type of recurrence was compared with ultrasonography and computed tomography in 21 such hepatocellular carcinomas. Of 15 hepatocellular carcinomas diagnosed by angiography as showing recurrence, four were detected with ultrasonography and five were detected with computed tomography. Doppler signals were obtained in the peripheral portions corresponding to tumor vessels or stains on angiograms in 14 of these 15 hepatocellular carcinomas, but they were undetectable in six hepatocellular carcinomas with no recurrence. All signals disappeared after transcatheter arterial embolization. One false-negative hepatocellular carcinoma with pulsed Doppler ultrasound showed faint tumor stains on angiograms; these were also negative on ultrasonography and computed tomography. Pulsed Doppler ultrasound may be superior to ultrasonography and computed tomography as a routine procedure to detect the recurrence of hepatocellular carcinoma treated by transcatheter arterial embolization. (Hepatology 1991;13:434–437.)

In order to investigate superoxide production by pulmonary macrophages in the rat, a route was created by ligating both the inferior and superior venae cavae and resecting the aorta after cannulation through the inferior vena cava into the right atrium of the heart. Lung perfusion was performed via this route with nitro blue tetrazolium. Although there was no formazan deposition throughout the lung, it became detectable in both alveolar and interstitial macrophages when phorbol myristate acetate was added to the perfusate. This deposition was markedly enhanced by previous injection of Corynebacterium parvum. The deposition disappeared after further addition of Cu(Lys)2, a scavenger of superoxide anions. This procedure may be useful for estimating in situ the ability of pulmonary macrophages to produce superoxide in the rat.

A 61-year-old male with hepatocellular carcinoma (HCC) complicating liver cirrhosis presented hypophosphatemia progressing with HCC expansion and serum alpha-fetoprotein elevation. These changes were associated with an increased fractional excretion of phosphate and decreased theoretical phosphate threshold. There was increased nephrogenous cyclic adenosine monophosphate despite normal serum parathyroid hormone. Serum 1,25-dihydroxyvitamin D levels were markedly reduced with normal 25-hydroxyvitamin D levels. There were no symptoms of osteomalacia, however, a slightly increased osteoid seam was elicited on autopsy. The hypophosphatemia could be explained by presumed secretion from HCC of humoral factors which have a phosphaturic effect and also inhibit 25-hydroxyvitamin D-1 alpha-hydroxylase in renal tubular cells.

When rats received endotoxin 48 hours after two-thirds liver resection, 50% of them died within 12 hours with massive hepatic necrosis at a dose that did not affect sham-operated rats. In the hepatic sinusoids, fibrin deposition and endothelial cell destruction occurred 5 hours after endotoxin administration. When antithrombin III concentrate was infused concomitantly with endotoxin administration, all rats survived 12 hours, and the extent of hepatic necrosis and the deranged serum glutamic pyruvic transaminase values were significantly attenuated at 5 hours compared with those in the control rats. Similar improvements in the incidence of mortality and liver injury were observed after treatment with gum arabic before hepatectomy. The stimulatory state of Kupffer cells based on the ability to produce Superoxide anions estimated by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was increased between 24 and 72 hours after operation. This increase disappeared after gum arabic treatment. It is concluded that massive hepatic necrosis can occur as a result of sinusoidal fibrin deposition provoked by endotoxin in partially hepatectomized rats. Activated Kupffer cells may contribute to this provocation.

Hantavirus is known to cause haemorrhagic fever with renal syndrome (HFRS). Although liver dysfunction has been reported in HFRS, hepatic manifestations of hantaviral infection have not been well described. We describe a case of autoimmune cholangitis in which an exacerbation of hepatitis was associated with hantaviral infection. Seroconversion of both IgG- and IgM-class antibodies to hantavirus was noted coincident with acute exacerbation of hepatitis, which was resolved promptly by treatment with corticosteroid. No extrahepatic manifestations were noted. This case suggests that hantavirus may trigger acute exacerbation of autoimmune liver disease without extrahepatic manifestations and that it may cause community-acquired hepatitis.

Using a rat myofibroblast-like hepatic stellate cell line, we studied the actomyosin-based cytoskeletal actions mediated by Rho-kinase and/or myosin light chain kinase (MLCK). Calmodulin/MLCK inhibitors W-7 and ML-7 attenuated cell migration dose-relatedly at concentrations from 10(-6) to 10(-4)M and collagen gel-contraction by the cells at 10(-4)M, respectively. Rho-kinase inhibitors Y-27632 and HA1077 attenuated the gel-contraction at concentrations from 10(-6) to 10(-4) M, respectively. These Rho-kinase inhibitors attenuated cell migration at 10(-7)M but enhanced the migration at 10(-4)M, respectively. They altered cell morphology showing prominent peripheral actin bundles and sparse central stress fibers, in comparison with the calmodulin/MLCK inhibitors. Both ML-7 and Y-27632 attenuated phosphorylation of myosin regulatory light chain and cell attachment to extracellular substrate. ML-7 attenuated the activation of GTP-binding protein Rac, while Y-27632 did not. These findings suggest that the actomyosin-based cytoskeletal actions can be functionally diverse depending on the Rho-kinase-mediated pathway and the MLCK-mediated pathway.

p53, known as a tumor suppressor gene, is a transcription factor that regulates various cellular functions. Recently, several growth factor gene promoters, including that of transforming growth factor alpha (TGF-alpha), were shown to be direct targets of p53-mediated transcription. Hepatic p53 mRNA is up-regulated during liver regeneration in rats. The aim of this study is to examine the role of p53 in hepatocyte proliferation. p53 protein levels were examined in rat hepatocytes cultured in the medium containing hepatocyte growth factor (HGF). p53 levels began to increase after 6 hours of incubation, reached a maximum at 18 hours, and decreased thereafter. DNA synthesis increased at 12 hours and peaked at 30 hours. When hepatocytes were incubated with p53 antisense oligonucleotide in addition to HGF, increases of p53 and TGF-alpha levels were suppressed, and DNA synthesis was reduced. The increases of TGF-alpha levels and DNA synthesis were also suppressed by a chemical inhibitor of p53, pifithrin-alpha. In rats after two-thirds partial hepatectomy, hepatic p53 increased and reached maximal levels around 16 hours when hepatic HGF levels have been shown to reach a maximum followed by an increase in hepatic TGF-alpha levels or hepatocyte proliferation. In contrast, sham-operated rats showed minor elevations of hepatic p53 levels. In conclusion, p53 production is stimulated by HGF and may contribute to the proliferation of rat hepatocytes. Considering previous findings indicating the importance of endogenous TGF-alpha for the proliferation of hepatocytes stimulated by HGF, TGF-alpha might play a role in HGF-p53 mediated hepatocyte proliferation.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.

Rat liver was kept at 4 degrees C or 37 degrees C in MEM, and reperfused through a closed circulation from the hepatic vein to the portal vein at 37 degrees C with the same solution. Although purine nucleoside phosphorylase and ALT activities were increased in the perfusate, depending on the duration of ischemia at both 4 degrees C and 37 degrees C, the ratio of the latter to the former was significantly higher after 37 degrees C-ischemia than after 4 degrees C-ischemia. The stimulation stage of Kupffer cells evaluated in situ by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was elevated after 4 degrees C-ischemia longer than 1 h, but not after 37 degrees C-ischemia. In contrast, the degree of oxidative stress in hepatocytes assessed by formazan deposition after liver perfusion with nitro blue tetrazolium alone was greater after 37 degrees C-ischemia than after 4 degrees C-ischemia. These results suggest that oxidative stress in hepatocytes and the stimulatory state of Kupffer cells after ischemia-reperfusion may differ between 4 degrees C-ischemia and 37 degrees C-ischemia, probably leading to different development of liver damage.

Contribution of collagen to cell proliferation was studied in cultured hepatocytes. When alpha,alpha'-dipyridyl, an iron chelator which blocks hydroxylation of prolyl and lysyl residues of procollagen and expression of procollagen mRNA, was added to the medium of rat hepatocytes in primary culture, DNA synthesis of the cells was reduced in a dose-related manner without changes in protein synthesis. The reduction of collagen synthesis was parallel with the changes of DNA synthesis. The addition of P-1894B or minoxidil, which inhibits specifically prolyl hydroxylase or lysyl hydroxylase, respectively, also produced similar results. However, the DNA synthesis was not affected by beta-aminopropionitrile fumarate, which inhibits cross linking in extracellular collagen maturation, nor by purified bacterial collagenase. Intracellular processing of collagen synthesis may be required for proliferation in cultured rat hepatocytes.

In rat hepatocytes in primary culture incubated with nitro blue tetrazolium, formazan content was increased by addition of t-butyl hydroperoxide, a potent oxidant, in a dose-related manner, but not by addition of valinomycin, which kills hepatocytes through mitochondrial damage. This increment after t-butyl hydroperoxide addition was not seen in hepatotyctes preincubated with deferoxamine mesylate, a ferric iron chelator which inhibits radical formation. Liver perfusion with nitro blue tetrazolium and t-butyl hydroperoxide in rats produced formazan deposition faintly on the surface of hepatocytes throughout the liver and prominently in the cytoplasm of some hepatocytes, which was attenuated when performed following deferoxamine mesylate perfusion. When liver perfusion with nitro blue tetrazolium was performed in carbon tetrachloride-intoxicated rats, formazan deposition appeared diffusely in hepatocytes in the centrilobular areas. Similar deposition was also observed on the surface and in the cytoplasm of hepatocytes in the periportal and mid-zonal areas in rats undergoing post-ischaemic reperfusion. Liver perfusion with nitro blue tetrazolium can detect in situ oxidative stress in hepatocytes and may be a useful tool for studying the role of lipid peroxidation in rat liver injury.

When epidermal growth factor was given to rats after partial hepatectomy, hepatic putrescine content was significantly increased at 4, 6 and 10 hr compared with control rats. Ornithine decarboxylase activity was also increased. Hepatic ornithine decarboxylase messenger RNA content was significantly greater than control levels at 2 hr after epidermal growth factor treatment, but not at 10 hr, when the amount of ornithine decarboxylase messenger RNA in control animals was four times that at 2 hr. When actinomycin D was administered 6 hr after partial hepatectomy, hepatic ornithine decarboxylase activity at 10 hr was reduced to half the control levels. This reduction was attenuated by epidermal growth factor treatment at 6 and 8 hr. Hepatic immunoreactive ornithine decarboxylase protein content showed a highly positive correlation with hepatic ornithine decarboxylase activity at 4, 6 and 10 hr, irrespective of epidermal growth factor treatment. Hepatic spermidine N1-acetyltransferase activity was significantly increased at 6 hr compared with control rats. These results suggest that, after partial hepatectomy in rats, exogenous epidermal growth factor may stimulate hepatic putrescine production by increasing ornithine decarboxylase messenger RNA content and altering posttranscriptional ornithine decarboxylase regulation, as well as enhancing spermidine N1-acetyltransferase activity. (HEPATOLOGY 1991;14:901–905).

Hepatocyte contribution to hepatic collagen production in vivo was estimated in rats, based on the fact that ornithine is used for protein synthesis in the liver as arginine after conversion by way of the urea cycle only by hepatocytes. From rats given a mixture of [14C] ornithine and [3H]arginine, hepatic collagen and serum albumin were obtained. The hepatocyte contribution was calculated from the 14C and 3H in arginine purified from collagen and albumin by high performance liquid chromatography. The contribution was less than 10% of total collagen production in normal and early fibrotic livers induced by a single dose of carbon tetrachloride or dimethylnitrosamine. We conclude that hepatocytes may play a minor role in collagen production in normal and early fibrotic rat livers. (HEPATOLOGY 1991;14:361–367.)

Hepatic ornithine decarboxylase (ODC) activity increases after partial hepatectomy and this activity is further stimulated by pharmacologic doses of glucagon and insulin. We now present data suggesting that glucagon and insulin stimulate ODC activity by distinct mechanisms. ODC activity increased progressively after partial hepatectomy and reached an initial peak at 4 h. Activity decreased to 50% of its peak value at 6 and 8 h and then rose progressively to a maximum at 12 h. Enzymatic activity was well correlated with the amount of hepatic immunoreactive ODC protein, thus suggesting that increased enzyme activity was due to increased amount of enzyme protein. Hepatic ODC mRNA increased gradually and continuously, reaching the maximal value by 12 h. In rats receiving glucagon after partial hepatectomy, ODC mRNA increased significantly by 2 h and enzyme immunoreactive protein and activity by 2 to 4 h as compared to controls. In contrast, insulin administration only induced a significant increase in enzyme immunoreactive protein and activity 10 to 12 h after partial hepatectomy. No significant changes in ODC mRNA level were observed. Our data suggest that the regulation mechanism of ODC induction following partial hepatectomy differs depending on the time after operation. Our data also suggest that while glucagon appears to regulate ODC activity by a transcriptional mechanism, insulin appears to operate at a post-transcriptional level.

Department of Internal Medicine, Kawakita General Hospital, Tokyo, JapanEmail: Mikio Yanase* - yanase-1IM@h.u-tokyo.ac.jp; Hitoshi Ikeda- ikeda-1IM@h.u-tokyo.ac.jp; Atsushi Matsui - atsushim@saitama-med.ac.jp; Eisei Noiri - noiri-1IM@h.u-tokyo.ac.jp; Tomoaki Tomiya -tomiya-1IM@h.u-tokyo.ac.jp; Masahiro Arai - arai-1IM@h.u-tokyo.ac.jp; Yukiko Inoue - yanase-1IM@h.u-tokyo.ac.jp; Kazuaki Tejima - yanase-1I M@h.u-tokyo.ac.jp; Kayo Nagashima- yanase-1IM@h.u-tokyo.ac.jp; Takako Nishikawa - yanase-1IM@h.u-tokyo.ac.jp; Satoshi Kimura - yanase-1IM@h.u-tokyo.ac.jp; Kenji Fujiwara - yanase-1IM@h.u-tokyo.ac.jp; Marcos Rojkind - marcos.rojkind@na.amedd.army.mil; Itsuro Ogata - itsuro@viola.ocn.ne.jp* Corresponding author

We describe a 59-year-old Japanese woman with a large mass of her liver encasing cystic components. Radiologic imaging showed the mass to be hypervascular, and surgical resection disclosed a white tumor. The solid portion was immunohistochemically characterized as a smooth muscle tumor. The cystic components were multilocular and lined with columnar epithelium, consistent with a hepatobiliary cystadenoma. The epithelium strongly stained for CA19-9. The subepithelial space was occupied by collagenous connective tissue interspersed with a small number of spindle-shaped cells. The cystic lesions lacked the mesenchymal stroma between the epithelium and connective tissue layer. There have been no previous reports of a hepatic smooth muscle tumor encasing a hepatobiliary cystadenoma. Because of the pathogenesis of the cystadenoma, it is possible to assume that the smooth muscle tumor also arose from the cells composing the biliary duct in association with the development of the cystadenoma.

Transcatheter arterial embolization (TAE) for unresectable hepatocellular carcinoma (HCC) was performed in cirrhotic patients with nonoccluded main portal trunk and total bilirubin levels less than 5 mg/dl. In 48 patients with tumor response to TAE, its benefit was evaluated by comparing their survival with that in 28 patients after conservative treatments, matched by clinical data. In TAE-treated patients, tumor findings on computerized tomography and angiogram were significantly different between those dying within 3 months and those surviving over 18 months. In cases of tumors occluding the first- or second-order portal branches or occupying more than 20% of the liver, or relatively hypovascular tumors with unclear boundaries, survival was not different between TAE- and non-TAE-treated patients, whereas it was significantly improved by TAE in cases without these findings. These results suggest that TAE is beneficial in cases of unresectable HCC complicating liver cirrhosis, but its benefit is limited under certain conditions.

The efficacy of exchange blood transfusion for acute hepatic failure was evaluated using rats. Rats receiving a dose of carbon tetrachloride died later than 24 h after dosing. When their blood was replaced with blood from normal rats at 24 h, survival time was prolonged, and hepatic protein synthesis was enhanced at 36 h with improved prothrombin time (PT). Those rats surviving 120 h showed better histological grade of recovery from injury after treatment. In contrast, dimethylnitrosamine (DMN)-mtoxicated rats showed no such improvement in survival time or PT. In rats given a nonfatal dose of carbon tetrachloride, serum glutamic-pyruvic transaminase values were five times higher than those in rats given DMN despite similar maximal PT. The benefit of exchange transfusion for acute hepatic failure may differ depending on the mechanism of development in rats. Enhanced hepatic protein synthesis may contribute to its effect.