bluuebunny/crossref_metadata_2025 huggingface dataset:
Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute self-limited hepatitis B
Authors: Penna A , Del Prete G , Cavalli A , Bertoletti A , D'Elios M M , Sorrentino R , D'Amato M , Boni C , Pilli M , Fiaccadori F , Ferrari C
Published: July 2004

The cytokine pattern secreted by T cells on viral antigen recognition is believed to exert a profound influence on both the type of disease caused by the infecting agent and the final outcome of the viral infection. To characterize the cytokine pattern associated with spontaneous resolution of acute hepatitis B, we analyzed interferon gamma (IFN-γ), interleukin (IL)-4, and IL-5 production by a wide series of hepatitis B virus (HBV) nucleocapsid-specific T-cell lines (34 lines) and T-cell clones (71 clones) derived from the peripheral blood of 13 patients during the acute or recovery phase of hepatitis B (2 and 7 of them were studied only in the recovery or the acute phase, respectively, and 4 during both). Most T-cell lines (67%) and clones (77%) isolated during the acute phase of infection expressed a T-helper (Th) 1 cytokine profile dominated by the production of IFN-γ. A larger proportion (74%) of T-cell lines produced several years after resolution of hepatitis was able to secrete not only IFN-γ, but also IL-4 and IL-5 (Th0-like cells). Results indicate that the antigen-specific fraction of peripheral blood T cells in acute self-limited hepatitis B selectively secrete Th1-type eytokines, suggesting that Th1-mediated effects may contribute not only to liver cell injury, but probably also to recovery from disease and successful control of infection.
Long-term evolution of fibrosis from chronic hepatitis to cirrhosis in patients with hepatitis C: Morphometric analysis of repeated biopsies
Authors: Kage M , Shimamatu K , Nakashima E , Kojiro M , Inoue O , Yano M
Published: July 2004

To clarify the characteristics of fibrosis developed in the process from chronic hepatitis C to cirrhosis, a morphometric analysis of liver biopsy samples was conducted on 25 chronic hepatitis C patients and 20 chronic hepatitis B patients (controls). Hepatitis C patients were followed up for 3 to 23 years. The mean number of liver biopsies performed on these patients was 3.8. Each biopsy was evaluated for the degree of fibrosis by using two methods: a semiquantitative method with a staging scoring system, and morphometry using a computed image analysis system. A significant correlation was observed between the Stage and the area of fibrosis (AF = the ratio of the area of fibrosis to that of the entire tissue specimen). The AF in cirrhosis was significantly higher in hepatitis C patients than in hepatitis B patients. The ratio of AF in the last biopsy sample to AF in the initial biopsy sample was significantly higher in hepatitis C patients than in hepatitis B patients. Evolution from chronic hepatitis C to cirrhosis occurred more frequently in patients aged ≥ 50 years, and this time period was 1.8 times shorter than that in patients aged < 50 years. AF in the initial biopsy related significantly to the period of evolution from chronic hepatitis C to cirrhosis. AF in the initial biopsy might be a predictive factor for prognosis.
APF/CBP, the small, amphipathic, anionic protein(s) in bile and gallstones, consists of lipid-binding and calcium-binding forms
Authors: Lafont H , Domingo N , Groen A , Kaler E W , Lee S P , Koehler R , Ostrow J D , Veis A
Published: July 2004

Two very similar small anionic, amphipathic proteins, a phospholipid-binding apoprotein (anionic polypeptide fraction [APF]) and a calcium-binding polypeptide (CBP), are found abundantly in bile and all types of gallstones. The often disparate properties among various preparations of APF/CBP could reflect different sources and separation procedures, leading to partly degraded and/or denatured protein and varied association of bile salts, lipids, bile pigments, and detergents. The present study presents new methods for isolation and purification of APF/CBP, and characterizes the preparations thus obtained. It was found that isolation by selective precipitation of proteins from fresh T-tube bile by added calcium chloride, followed by demineralization with ethylenediaminetetraacetic acid (EDTA), removal of salts, lipids, and some pigment by Sephadex LH-20, and serial ultrafiltration yields the purest preparations. Though free of lipids, bile salts, detergents, and most pigments, these new preparations all show the same 7-kd and 12-kd bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the same major peaks on hydrophobic high-performance liquid chromatography (HPLC), and retain the self-associative, lipid- and calcium-binding functions, typical of older preparations obtained by potentially denaturative procedures. The varied properties among APF/CBP preparations are thus apparently related mainly to their content of different proportions of two major components, lipid-binding APF and calcium-binding CBP. Immunologic cross-reactions indicate common epitopes, and amino acid analyses are also similar, suggesting that APF and CBP may have the same polypeptide backbone, but differ because of posttranslational modification(s). Sufficiently pure APF and CBP have now been obtained to permit possible structural identification by sequencing and molecular biological techniques, though such attempts have thus far been unsuccessful.
Histopathologic analysis of transjugular intrahepatic portosystemic shunts
Authors: Ducoin H , El-Khoury J , Rousseau H , Barange K , Peron J , Pierragi M , Rumeau J , Pascal J , Vinel J , Joffre F
Published: July 2004

This prospective study was undertaken in 17 patients treated with 22 transjugular intrahepatic portosystemic shunt (TIPS) procedures and aimed to evaluate the nature and causes of short- and long-term shunt abnormalities. Specimens were collected after autopsy in 6 patients and after liver transplantation in 11 patients; the time from shunting ranging from 4 to 385 days. During the first 2 weeks the shunt surface was covered by poorly organized fibrin platelet clots that were mixed with inflammatory and red blood cells. Thereafter, a pseudointima developed, initially made of loose granulation tissue that was formed by edema, myofibroblasts, neo-capillaries, collagen fibers, and inflammatory cells. This pseudo-intima extended with time as the myofibroblasts increased in number, as the collagen fibers thickened, and as the inflammatory cells grew more scarce. Its thickness ranged from 50 to 3,500 microm, the maximal values being observed in the middle of the shunt. The shunt was obstructed by a clot in 4 patients (18%). In 7 shunts (31.8%) the pseudo-intima was thicker than 1,000 microm and was referred to as hyperplastic pseudo-intima. It showed more dense inflammatory infiltrate and anarchic deposition of more scarce collagen fibers. Pseudo-intima hyperplasia was associated with previously diagnosed thrombosis, which had been treated by dilatation in 2 cases and by biliary deposits in 3 cases, while it remained unexplained in 2 cases. Accordingly, this study confirms the following: 1) that early TIPS obstruction results from thrombosis; 2) that 2 weeks after insertion, the stent is covered by a smooth pseudo-intima; 3) that thereafter, pseudo-intimal hyperplasia is the main cause of TIPS stenosis or occlusion; and 4) that biliary secretions and previous thrombosis could be important triggering mechanisms for this pseudo-intima overgrowth.
Nuclear DNA fragmentation and expression of Bcl-2 in primary biliary cirrhosis
Authors: Koga H , Sakisaka S , Ohishi M , Sata M , Tanikawa K
Published: July 2004

It is uncertain whether or not apoptosis is involved in the pathogenesis of primary biliary cirrhosis (PBC). The aims of this study were to assess the nuclear DNA fragmentation and expression of Bcl-2 in the biliary epithelial cells (BECs) and in the hepatocytes of PBC. Additionally, the effects of ursodeoxycholic acid (UDCA) on DNA fragmentation and Bcl-2 expression in PBC were evaluated. Liver tissue specimens from 35 PBC patients were examined by in situ nick-end labeling to detect any nuclear DNA fragments, and by immunohistochemistry for Bcl-2. Ten of these patients underwent a second liver biopsy after the treatment with UDCA. Sixteen histologically normal liver tissues and 17 chronic viral hepatitis C (CVHC) samples were chosen as controls. DNA fragmentation in BECs was more frequently found in PBC than in CVHC and more frequently than in the normal controls (both P < .05), and fragmentation in the hepatocytes of PBC more frequently than in normal controls (P < .01). Bcl-2 expression was more frequently found in both the BECs and hepatocytes of PBC than in the controls. UDCA significantly decreased the DNA fragmentation in BECs (P < .05) and the positivity for Bcl-2 in BECs (P < .01), although no significant decrease was found in hepatocytes. In conclusion, de novo Bcl-2 expression in hepatocytes of PBC was shown, and the increased nuclear DNA fragmentation and the Bcl-2 expression both in BECs and in hepatocytes may reflect apoptotic stress, although nuclear DNA fragmentation in BECs did not necessarily represent apoptosis. UDCA showed a potential effect of reducing nuclear DNA fragmentation in BECs.
Comparative studies of antimitochondrial autoantibodies in sera and bile in primary biliary cirrhosis
Authors: Nishio A , Van de Water J , Leung P S , Joplin R , Neuberger J M , Lake J , Bjorkland A , Totterman T H , Peters M , Worman H J , Ansari A A , Coppel R L , Gershwin M E
Published: July 2004

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of intrahepatic bile ducts. Although the pathogenesis of this disease is still unknown, high titers of antimitochondrial autoantibodies (AMA) have long been recognized in patient sera. However, little is known about the presence of AMA in bile. In this study, we investigated bile and sera from patients with PBC and healthy controls for the presence of AMA and mitochondrial autoantigens. AMA were detected in the bile of 17 of 19 patients (89.4%) with PBC; they were specifically directed against the pyruvate dehydrogenase complex (PDC-E2) in 15 of 19 patients (78.9%), to the branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) in 6 of 19 patients (31.6%), and to the 2-oxoglutarate dehydrogenase complex E2 (OGDC-E2) in 1 of 19 patients (5.3%). In a comparative study of sera from the same patients, anti-PDC-E2 antibodies were found in 19 of 19 patients (100%), anti-BCOADC in 9 of 19 patients (47.3%), and anti-OGDC-E2 in 4 of 19 patients (21.1%) patients. AMA in bile were always found together with antibodies of corresponding specificities in the serum from the same patient. Immunoglobulin (Ig)A AMA were found in the bile of 9 of 19 patients (47.7%) with PBC; they were specifically directed against PDC-E2 in 8 of 19 patients (42.1%) and to BCOADC in 2 of 19 patients (10.5%). Epitope mapping of IgA anti-PDC-E2 antibodies indicated that, like serum autoantibodies, the immunodominant epitope is directed against the inner lipoyl domain of PDC-E2. The prevalence and antigen reactivity of IgA AMA in sera correlated completely with IgA AMA in bile. Autoantibodies against nuclear envelope pore proteins (gp210) were found in 1 of 8 (12.5%) sera of patients with PBC, but not in bile. Furthermore, and of particular interest, we detected the autoantigens, PDC-E2, OGDC-E2, and BCOADC-E2, in the bile of 12 of 19 patients (63.2%), 9 of 19 patients (47.4%), and 9 of 19 patients (47.4%), respectively; PDC-E2 was found in only 1 of 17 (5.9%) disease controls. Although the presence of AMA in bile may merely reflect the presence of these antibodies in sera, the simultaneous detection of mitochondrial autoantigens in bile suggests an increase of mitochondrial autoantigens at inflammatory sites. Such autoantigens, coupled with AMA, may augment the local immune response and disease progression.
Comparison of the clinical features and clinical course of antimitochondrial antibody-positive and -negative primary biliary cirrhosis
Authors: Invernizzi P , Crosignani A , Battezzati P M , Covini G , De Valle G , Larghi A , Zuin M , Podda M
Published: July 2004

Reports from North America and Northern Europe have suggested that antimitochondrial antibody (AMA) negative primary biliary cirrhosis (PBC) is a distinct chronic cholestatic liver disease with high prevalence of serum non-organ-specific autoantibodies other than AMA. To evaluate if such a peculiar serum immunoreactivity is associated with clinically relevant characteristics, we reviewed our experience with 297 Italian patients who have had a clinical and histological diagnosis of PBC and were regularly followed-up at our Center from June 1974 to June 1994. AMA-negative and AMA-positive patients were compared in terms of biochemical and clinical features, and clinical outcome of the disease. At presentation, 30 of 297 patients (10%) tested negative for AMA by indirect immunofluorescence. Six of them tested positive for antimitochondrial M2 antibodies (AMA-M2) by immunoblotting analysis, therefore, diagnosis of AMA-negative PBC was made in 24 patients (8%). At the initial visit, AMA-negative and AMA-positive patients were similar in terms of biochemical and clinical features. Antinuclear and anti-smooth-muscle antibodies (ANA and ASMA) were more frequently positive in the AMA-negative patients (71% vs. 31%, and 37% vs. 9%; both P = .0002). Incidence of complications of cirrhosis and development of liver failure resulting in death or referral for liver transplantation did not differ significantly between the two populations. In conclusion, data from this historical cohort study suggest that the distinct serological features of AMA-negative PBC are not associated with substantial differences in the clinical spectrum or course of the disease.
Emergency banding ligation versus sclerotherapy for the control of active bleeding from esophageal varices
Authors: Lo G , Lai K , Cheng J , Lin C , Huang J , Hsu P , Chiang H
Published: July 2004

Active bleeding varices are a great challenge to endoscopists. In this study, we compared the short-term efficacy and safety of banding ligation with injection sclerotherapy in the arresting of active bleeding from esophageal varices. Seventy-one cirrhotic patients with active variceal bleeding were randomized to receive banding ligation (37 patients) or sclerotherapy (34 patients) immediately after endoscopic examinations. Primary success rate (bleeding stopped for 72 hours) was 97% in the ligation group and 76% in the sclerotherapy group (P = .009). The efficacy of ligation was similar to sclerotherapy in the control of oozing varices (100% vs. 89%, P = .23), whereas ligation was superior to sclerotherapy in the control of spurting varices (94% vs. 62%, P = .012). The requirement of vasoconstrictors after emergency endoscopic treatment was lower in the ligation group than in the sclerotherapy group (11% vs. 41%, P = .007). Treatment failure within 1 month was 8% in the ligation group vs. 30% in the sclerotherapy group (P = .02). Blood transfusion requirements were significantly lower in the ligation group than in the sclerotherapy group (3.2 ± 1.2 vs. 4.5 ± 1.8 units, P < .01). Rebleeding rate within 1 month was 17% in the ligation group and 33% in the sclerotherapy group (P = .19). Significant complications were encountered in 5% of the ligation group and 29% of the sclerotherapy group (P = .007). Mortality rates within 1 month were 19% in the ligation group and 35% in the sclerotherapy group (P = .19). Banding ligation and sclerotherapy were comparable in the arresting of oozing varices, whereas ligation was superior to sclerotherapy in the control of spurting varices. Patients treated with ligation required fewer vasoconstrictors and fewer transfusion units than patients treated with sclerotherapy. Furthermore, banding ligation was associated with a lower complication rate than sclerotherapy.
Anti-neutrophil cytoplasmic antibodies in type 1 and 2 autoimmune hepatitis
Authors: Zauli D , Ghetti S , Grassi A , Descovich C , Cassani F , Ballardini G , Muratori L , Bianchi F B
Published: July 2004

Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) have been recently defined as the most sensitive autoantibody of type 1 autoimmune hepatitis (AIH-1). Their prevalence in type 2 autoimmune hepatitis (AIH-2) has not yet been evaluated. The aim of the present study was to verify the association of pANCA with AIH-1 in an Italian series and to investigate the prevalence of the antibodies in AIH-2 and in proper control groups represented by cases of chronic hepatitis C (CH-C) with similar autoimmune features. pANCA were found in 30 of 46 (65%) AIH-1 and in 4 of 30 (13%) ANA/smooth muscle antibody (SMA) positive CH-C (P = .0000006). Nineteen AIH-2, 29 liver kidney microsomal antibody type 1/liver cytosol antibody type 1 (LKM1/LC1) positive CH-C cases and 50 healthy controls were all negative. In AIH- 1, pANCA were significantly (P = .009) more frequent in males (8 of 9, 89%) than in females (22 of 37, 59%). All pANCA positive sera showed SMA of the antiactin type. The present data confirm that pANCA, although less prevalent in our series than in other reports, do associate with AIH-1 also in the Mediterranean area and show that it can identify a small subgroup (13%) of ANA/SMA positive chronic hepatitis C, in which autoimmune reactions might play a more relevant role than viral infection. They also show the antibodies are absent in AIH-2. In conclusion, pANCA appear to be mutually exclusive of LKM1 positivity, either hepatitis C virus-related or not, thus representing a further valuable tool to differentiate the two types of autoimmune hepatitis.
Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma
Authors: Tatsumi T , Takehara T , Katayama K , Mochizuki K , Yamamoto M , Kanto T , Sasaki Y , Kasahara A , Hayashi N
Published: July 2004

Costimulation mediated by costimulatory molecules, such as B7-1 and B7-2, which are ligands for the CD28/cytolytic T lymphocyte associated antigen (CTLA)-4 counter-receptor, plays an important role in the induction of T cell-mediated antitumor immunity. We investigated the expressions of B7-1, B7-2, and human leukocyte antigen (HLA) class I in seven human hepatocellular carcinoma (HCC) cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. RT-PCR showed that all these human HCC cell lines were positive for B7-1 and B7-2 at the messenger RNA (mRNA) level. Flow cytometric analysis revealed that they all expressed B7-1, B7-2, and HLA class I on the cell surface. However, the expression levels of B7-1 and B7-2 were very low whereas those of HLA class I were high. B7-1 and B7-2 expression could be increased by treatment with interferon α and interferon γ in a dose-dependent manner, although the expression levels of B7-1 and B7-2 after interferon treatment remained low. By transfecting Hep3B cells with a plasmid containing human B7-1 complementary cDNA (cDNA), we were able to establish Hep3B cell lines strongly expressing B7-1. From mixed lymphocytes and tumor cultures analysis, the primary cytolytic activity against parental Hep3B cells could be induced effectively by B71-transfected Hep3B cells. These findings suggested that B7-1 gene transfer is the best way to induce strong expression of this molecule and this might be useful for immuno-gene therapy against human HCC.
Expression of α-fetoprotein and stem cell factor/c-kit system in bile duct ligated young rats
Authors: Omori M , Evarts R P , Omori N , Hu Z , Marsden E R , Thorgeirsson S S
Published: July 2004

The existence of a facultative hepatic stem cell compartment in bile ductules that participates in the renewal process of epithelial cell populations in the liver is well documented. The present study was undertaken to determine whether the immature bile epithelium responds differently to growth stimulus induced by bile stasis to that seen in the adult animal. In addition, the possible involvement of the growth factor/receptor systems associated with early activation of hepatic stem cells in bile duct proliferation was also examined. Bile duct ligation was used to induce the proliferation of bile epithelial cells. The expression of full-length alpha-fetoprotein (AFP) was used as an indicator for activation of the stem cell compartment. AFP was highly and selectively expressed in small bile ducts 7 days after bile duct ligation in immature rats up to 5 weeks of age. Although no significant increase in the expression of stem cell factor (SCF) c-kit, hepatocyte growth factor (HGF), and transforming growth factor-α (TGF-α) was observed 7 days after bile duct ligation in adult rats, the expression of all these growth factors was increased in bile duct ligated rats up to 5 weeks of age. These results suggest that the bile ductular epithelium in the young rats responds to bile stasis in a fashion that is phenotypically similar to that seen during early activation of hepatic stem cells in adult liver.
The effect of changes in hepatocyte membrane potential on immediate-early proto-oncogene expression following partial hepatectomy in rats
Authors: Minuk G Y , Kren B T , Xu R , Zhang X , Burczynski F , Mulrooney N P , Fan G , Gong Y , Steer C J
Published: July 2004

The stimulus responsible for inducing hepatocytes to enter the cell cycle following partial hepatectomy (PHx) remains to be identified. One suggested candidate is the change in hepatocyte membrane potential (PD) that occurs immediately following PHx. To test this possibility, we monitored changes in hepatocyte PD and immediate-early proto-oncogene expression in rats pretreated with saline or gamma aminobutyric acid (GABA), an amino-acid neurotransmitter that hyperpolarizes isolated hepatocytes. Intraperitoneal injections of saline or GABA (500 μg/g body weight) were administered to adult, male Sprague-Dawley rats 1 hour prior to 70% PHx. Rats were sacrificed and the livers excised at various times until 180 minutes post-PHx for messenger RNA (mRNA) and protein analyses. In additional groups of saline- and GABA-treated rats, PD changes were recorded continuously from −260 to 180 minutes post-PHx. Serum GABA concentrations were determined by ion-exchange chromatography with orthopthaldehyde fluorescence detection. Hepatocyte PD's were recorded in situ by intracellular microelectrodes with an Axoprobe-1A amplifier. Steady-state levels of c-fos, c-jun, jun-B, and c-myc transcripts and proteins were documented by Northern blots of poly(A)-enriched RNA derived from resected livers and Western blots of total nuclear protein, respectively. Serum GABA concentrations remained unchanged in saline-treated controls but increased 10- to 20-fold above baseline values in GABA-treated rats. In saline-treated controls, hepatocyte depolarization occurred immediately and was maintained throughout the 180 minutes post-PHx period (PD pre-PHx, −36.8 ± 5.1; 15 minutes post-PHx, −27.5 ± 5.7; and 180 minutes post-PHx, −28.3 ± 4.4 mV, mean ± SD); whereas in GABA-treated rats, hepatocyte PD remained unchanged (-37.0 ± 1.1; −36.4 ± 3.1 and −39.2 ± 2.7 mV, respectively). Despite abrogation of hepatocyte PD changes, proto-oncogene mRNA and protein levels in saline- and GABA-treated rats were either similar or, in the case of c-fos and c-jun, increased five- to sevenfold in GABA-treated rats. The results of this study indicate the following: 1) hepatocytes depolarize immediately post-PHx and remain depolarized throughout the priming phase of the cell cycle; 2) elevated serum GABA concentrations prevent PHx-induced hepatocyte depolarization; and 3) depolarization is not the stimulus responsible for priming hepatocytes into replicative competence.
Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases
Authors: Bradham C A , Stachlewitz R F , Gao W , Qian T , Jayadev S , Jenkins G , Hannun Y , Lemasters J J , Thurman R G , Brenner D A
Published: July 2004

The injury resulting from cold ischemia and warm reperfusion during liver transplantation is a major clinical problem that limits graft success. Kupffer cell activation plays a pivotal role in reperfusion injury, and Kupffer cell products, including free radicals and tumor necrosis factor α (TNF-α), are implicated as damaging agents. However, the second messengers and signaling pathways that are activated by the stress of hepatic ischemia/reperfusion remain unknown. The purpose of this study is to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the activating protein 1 (AP-1) transcription factor in response to ischemia and reperfusion in the transplanted rat liver. There was a potent, sustained induction of c-jun N-terminal kinase (JNK), but not of the related MAPKs extracellular signal-regulated kinases (ERK) or p38, upon reperfusion after transplantation. TNF-α messenger RNA (mRNA) levels and transcription factors AP-1 and nuclear factor-κB (NF-κB) were induced in the liver after 60 minutes of reperfusion. Finally, there was an elevation of ceramide, but not diacylglycerol or sphingosine, in the transplanted liver. Ceramide is a second messenger generated by TNF-α treatment and is an activator of JNK. Because JNK activation preceded the elevations in ceramide and TNF-α mRNA, these results suggest that increased hepatic TNF-α and ceramide may perpetuate JNK induction, but that they are not the initiating signals of JNK activation during reperfusion injury in the transplanted liver.
Urinary transforming growth factor β1 levels in hepatitis C virus-related chronic liver disease: Correlation between high levels and severity of disease
Authors: Tsai J , Jeng J , Chuang L , Chang W
Published: July 2004

To assess the clinical relevance of transforming growth factor β1 (TGF-β1) in chronic liver disease, urinary TGF-β1 and circulating aminoterminal propeptides of type III procollagen (PIIINP) levels were determined by radioimmunoassay in 100 cirrhotic patients, 44 patients with chronic hepatitis, and 50 healthy controls. TGF-β1 and PIIINP levels in cirrhotic patients were higher than those in patients with chronic hepatitis (each P < .0001) or healthy controls (each P < .0001), respectively. There was a correlation between TGF-β1 and PIIINP levels in patients (r = .858, P < .0001). The higher the urinary TGF-β1 level, the worse the severity of chronic liver disease (P < .001). TGF-β1 levels in cirrhotic patients with antibodies to hepatitis C virus (anti-HCV) were higher than in those without (P <.0001). Compared with cirrhotic patients with hepatitis B surface antigen (HBsAg) alone, those with HBsAg and anti-HCV had higher TGF- β1 levels (P < .001), a higher frequency of raised TGF-β1 (P < .005), and a higher frequency of patients with Child-Pugh C (P < .005). Multivariate analysis indicated that the TGF-β1 level was significantly correlated with the presence of cirrhosis. In conclusion, urinary TGF-β1 levels may be used as a marker for hepatic fibrogenesis. Higher urinary TGF-β1 levels correlate with more severe liver disease.
Heat shock protein induction in murine liver after acute treatment with cocaine
Authors: Salminen W F , Roberts S M , Fenna M , Voellmy R
Published: July 2004

The effect of cocaine on heat shock protein (hsp) induction in murine liver was examined using Western blotting and immunohistochemistry. A single dose of cocaine (50 mg/kg, intraperitoneal [i.p.]) was administered to naive, phenobarbital (PB)-induced or β-naphthoflavone (βNF)-induced mice, and the level of hsps in the liver analyzed 3, 6, and 24 hours after the cocaine dose. As measured by Western blotting, hsp70i levels were increased at all time points, and hsp25 levels at the 6- and 24-hour time points. Levels of hsp60, hsc70, and hsp90 remained unchanged. Pretreatment of mice with the cytochrome P-450 inhibitor SKF-525A eliminated both cocaine hepatotoxicity and the induced accumulation of hsp25 and hsp70i. Immunohistochemical localization of hsp25 and hsp70i in the liver showed that concentrations of both hsps were elevated only in cells with altered morphology. As has been observed previously, hepatic enzyme induction with PB or βNF shifted the location of the necrotic lesion within the lobule from zone 2, as observed in naive mice of this strain, toward zone 1 (PB) or zone 3 (βNF), respectively. Localization of induced accumulation of hsp25 and hsp70i was found to shift within the lobule in parallel with the necrotic lesion in these animals. Immunostaining of cocaine reactive metabolites bound to proteins was superimposable on the areas with hsp accumulation and cells with altered morphology. Our observations indicate a strong spatial correlation within the lobule between cocaine reactive metabolite formation, induced accumulation of hsp25 and hsp70i, and cytotoxicity (necrosis).
Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells
Authors: Castillo M B , Berchtold M W , Rulicke T , Schwaller B , Gotzos V , Pinzani M , Reichen J , Celio M R
Published: July 2004

To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased in normal mice but virtually not affected in the transgenic animals. This suggests that ectopically expressed parvalbumin is involved in the regulation of Ca2+ signals in the sinusoidal endothelial cells. This animal model could be of interest to those working on the physiology of liver circulation.
Pancreatic secretory trypsin inhibitor as a diagnostic marker for adult-onset type II citrullinemia
Authors: Kobayashi K , Horiuchi M , Saheki T
Published: July 2004

Adult-onset type II citrullinemia is characterized by which deficiency of argininosuccinate synthetase (ASS) protein is found specifically in the liver of patients. Our recent study using differential messenger RNA (mRNA) display showed that the expression of human pancreatic secretory trypsin inhibitor (hPSTI) mRNA increases significantly in the liver of all type II patients tested. In the present work, we found that the concentration of hPSTI protein was higher in the liver of type II patients than controls. Because it is well known that PSTI is a secretory protein and because serum PSTI has been proposed as a marker of pancreatic and nonpancreatic diseases, we measured the hPSTI level in the blood of type II patients and compared it with other serum markers such as elastase 1, trypsin, phospholipase A2, α-fetoprotein, CA19-9, and C-reactive protein (CRP). We found a significant increase in serum hPSTI level with no change in the other serum markers. These results suggest that serum hPSTI is useful as a diagnostic marker for adult-onset type II citrullinemia.
Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture
Authors: Greuet J , Pichard L , Ourlin J C , Bonfils C , Domergue J , Le Treut P , Maurel P
Published: July 2004

The influence of cell density and epidermal growth factor (EGF) on the expression and inducibility of cytochrome P450 (CYP) genes of the CYP3A and CYP1A families in adult human hepatocytes in primary culture has been evaluated. Only when cultured at subconfluence and in the presence of EGF did hepatocytes exhibit a proliferative response, assessed by measuring DNA synthesis and cyclin A accumulation. In the absence of EGF, the accumulation of CYP3A4 and CYP1A2 messenger RNAs (mRNAs) in response to their respective inducers (rifampicin and dioxin) was dramatically decreased in subconfluent culture with respect to confluent cultures. The presence of EGF only slightly decreased the accumulation of these mRNAs in both confluent and subconfluent cultures. The accumulation of CYP2D6 and CYP2E1 proteins, which are constitutively expressed in confluent cultures, and the production of fibrinogen and apolipoprotein (Apo) B100 exhibited similar behavior, while nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity was affected neither by cell density nor by EGF. In contrast, the accumulation of CYP1A1 mRNA in response to dioxin was similar in confluent and subconfluent cultures, irrespective of the presence of EGF. Interestingly, CYP3A7, a gene that is preferentially expressed in the fetal liver, was expressed constitutively neither in confluent nor in subconfluent cultures, irrespective of the presence of EGF. It is concluded that the loss of cell-cell contacts rather than the proliferative status of cells per se is responsible for the dramatic decrease in the expression of CYP genes, normally expressed in the adult human liver.
Hepatic basolateral sodium-dependent-bile acid transporter expression in two unusual cases of hypercholanemia and in extrahepatic biliary atresia
Authors: Shneider B L , Fox V L , Schwarz K B , Watson C L , Ananthanarayanan M , Thevananther S , Christie D M , Hardikar W , Setchell K D , Mieli-Vergani G , Suchy F J , Mowat A P
Published: July 2004

The recent cloning of a human sodium-dependent bile acid transporter (NTCP) permits analysis of its expression in human liver disease and investigation of potential primary defects in its expression. NTCP from normal human liver (NHL) was first characterized in detail. Northern blotting of RNA from NHL revealed a 1.8-kb NTCP transcript. Western blotting of crude NHL plasma membranes using a carboxyterminal antipeptide antibody showed that NTCP is a 39-kd polypeptide that is N- glycosylated to a final molecular weight of 56 kd. Indirect immunofluorescent analysis of NHL sections indicated that the NTCP protein is expressed on the basolateral surface of hepatocytes. We hypothesized that the clinical phenotype of a defect in NTCP might be hypercholanemia in the relative absence of liver disease. Accordingly, the coding region of the NTCP gene of two children with this phenotype was sequenced after reverse transcription/polymerase chain reaction (RT/PCR) amplification. No primary defects in the deduced NTCP amino acid sequence were found. Despite the extremely high serum bile salt levels (235 and 126 micromol/L) in these two patients, NTCP messenger RNA (mRNA) and protein expression were quantitatively normal, in contrast to the published observations in a rat model of cholestasis secondary to common bile duct ligation. Hepatic steady-state NTCP mRNA levels in a group of 23 pre- and postportoenterostomy biliary atresia patients were inversely related to total bilirubin, indicating that extrahepatic bile duct obstruction leads to down-regulation of NTCP mRNA levels, similar to that observed in rat common bile duct ligation. Therefore the lack of down-regulation in the two patients with hypercholanemia indicates that elevated serum bile salts are not sufficient to down-regulate NTCP expression, these two patients have abnormal responses to hypercholanemia, or these two patients have a defect in a gene other than NTCP that influences hepatic clearance of bile salts.
Endotoxin impairs biliary glutathione and HCO3 − excretion and blocks the choleretic effect of nitric oxide in rat liver
Authors: Trauner M , Nathanson M H , Rydberg S A , Koeppel T A , Gartung C , Sessa W C , Boyer J L
Published: July 2004

Cholestasis in patients with sepsis has been attributed to the effects of endotoxin (lipopolysaccharides, LPS) and LPS-induced cytokines, which are also potent stimulators of systemic and hepatic nitric oxide (NO) synthesis. NO donors stimulate bile acid-independent bile flow in normal rat liver, but the effects of LPS-induced NO on bile formation remain unclear. To address this question we examined the effects of NO and its mediator guanosine 3′,5′-cyclic monophosphate (cGMP) on bile flow and biliary HCO3
− and glutathione excretion in isolated perfused rat livers (IPRL) from LPS-treated rats. Portal and systemic NO2- + NO3- plasma levels were increased 47-fold in LPS-treated rats and were also elevated in perfusate (6-fold) and bile (9-fold) after isolating and perfusing livers from these animals. Bile flow, HCO3
−, and glutathione output were decreased by 33%, 25%, and 81% in these IPRL, respectively. Stimulation of NO synthesis with L-arginine or inhibition of inducible NO synthesis with aminoguanidine did not change bile flow, although pretreatment with aminoguanidine inhibited NO production by 85%. Moreover, the choleretic effects of infusions of the NO donors sodium nitroprusside (SNP) and S-nitroso-acetyl-penicillamine were markedly reduced in endotoxemic IPRL compared with normal controls, and SNP-induced HCO3
− and glutathione excretion were reduced by 61% and 86%, respectively. SNP-induced cyclic GMP production was 2.3-fold lower than in normals, but the choleretic effect of dibutyryl cGMP was only slightly reduced in endotoxemic livers. These findings indicate that LPS reduces bile acid-independent bile flow primarily by inhibiting biliary excretion of glutathione and to a lesser extent HCO3
−, whereas LPS-induced NO does not modulate bile formation in endotoxemia. Thus, impairment of the major determinants of bile acid-independent bile flow by LPS may contribute significantly to the pathogenesis of the cholestasis of sepsis.
Cell lines with heterogeneous phenotypes result from a single isolation of albumin-sv40 T-antigen transgenic rat hepatocytes
Authors: Bulera S J , Haas M J , Sattler C A , Li Y , Pitot H C
Published: July 2004

Several immortalized cell lines were established from the livers of two transgenic rats expressing the simian virus protein large T antigen under the control of the albumin promoter. Hepatocytes from transgenic rats were isolated by a two-step perfusion procedure from a normal- appearing liver and from a liver that contained a single neoplasm. Cells were also isolated from the dissociated liver neoplasm. After 5 weeks in culture, cell colonies were isolated and subcloned as individual cell lines. Electron microscopy revealed that all cell lines had a high nuclear-to-cytoplasmic ratio compared with normal hepatocytes. The cytoplasm contained numerous organelles, including smooth and rough endoplasmic reticulum, Golgi, mitochondria, peroxisomes, and annulate lamellae. However, the lines exhibited a variety of different cell morphologies. All cell lines, including those derived from neoplastic cells, exhibited a similar doubling time of 26 hours and the ability to grow in soft agar. Northern blot analysis revealed that the cell lines differentially expressed hepatocyte markers. Large T antigen was expressed in the cultured cell lines at much higher levels than was observed in transgenic hepatocytes in vivo. This suggests that the viral protein is required to maintain cell viability in culture. In addition, the tumor suppressor proteins, p53 and Rbp105, were detected in all cultured cell lines. In contrast, these same proteins were not detected by Western blot in transgenic hepatocytes in vivo. All cell lines expressed the oncogene c-myc, yet growth factor-dependent and independent growth were observed. The data presented show for the first time the establishment and characterization of a number of cell lines derived from hepatocytes isolated from an alb-SV40 large T-antigen transgenic rat. These cell lines exhibited varied morphological and biochemical hepatocellular characteristics in vitro, suggesting that the expression of the transgene in hepatocytes leads to considerable phenotypic diversity analogous to that seen in hepatocellular carcinomas induced by chemical carcinogenesis.
Hrp12, a novel heat-responsive, tissue-specific, phosphorylated protein isolated from mouse liver
Authors: Samuel S J , Tzung S P , Cohen S A
Published: July 2004

Previous studies from this laboratory identified a 28-kd nonreducible protein, liver-derived immunoinhibitory factor (LDIF) from the mouse liver. Isolation of this protein resulted in the co-purification of another unique protein called heat responsive protein 12 kd (Hrp12). In contrast to LDIF, Hrp12 was totally reducible to a protein of 12 kd suggesting a dimer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification, followed by sequencing of an in situ cyanogen bromide digest of membrane bound Hrp12, yielded an internal 20-amino acid polypeptide. Degenerate oligonucleotides made from this peptide were used to screen a murine liver complementary DNA (cDNA) library. A 1240-bp cDNA clone was obtained with an internal 521-bp open reading frame (ORF). Sequence analysis of the 173-amino acid ORF of mouse Hrp12 showed a high degree of homology with a 99 amino acid rat liver-kidney perchloric acid-soluble protein (LKPS) and a 136-amino acid perchloric acid soluble rat protein (PSP). Transcripts for Hrp12 were mainly restricted to the liver and kidney in mouse and man. The protein was estimated to be approximately 0.8% of the total liver-soluble cytosolic protein. A zoo-blot probed at moderate stringency with labeled cDNA revealed a strong conservation of the gene in all of the mammalian species tested. Analysis of the protein structure of Hrp12 revealed motifs predicted to be targets for protein kinase C (PKC). More importantly, purified mouse Hrp12 could be phosphorylated in vitro with PKC. The protein had significant similarity to DnaK heat shock protein (Hsp)70 and contained a 54-amino acid stretch with sequence similarity to Hsp90. This prompted us to investigate the heat shock response of Hrp12. Isolated hepatocytes and hepatoma cells were exposed to different heat shock temperatures (39.5 degrees C, 42.5 degrees C, and 44.5 degrees C); and then total RNA was extracted and Northern analysis carried out. The message for this novel protein responded atypically to heat shock. Although the steady-state level of the message increased after heat shock, a marked oscillatory pattern was superimposed on it. In contrast, the steady-state levels of Hsp90 and Hsp70 messenger RNA (mRNA) were found to respond to heat shock in the expected manner. Finally, the amount of Hrp12 protein was also found to increase after heat shock in a manner that was consistent with heat-responsive proteins.
Hepatitis C virus-related proteins in kidney tissue from hepatitis C virus-infected patients with cryoglobulinemic membranoproliferative glomerulonephritis
Authors: Sansonno D , Gesualdo L , Manno C , Schena F P , Dammacco F
Published: July 2004

Membranoproliferative glomerulonephritis (MPGN) may be a component of a generalized vasculitis as well as a component of the clinical expression of type-II mixed cryoglobulinemia (MC). Several studies have established a striking association between hepatitis C virus (HCV) infection and MC. The potential role of HCV in the pathogenesis of MPGN, which occurs in almost half of the cases of MC patients, has not been fully investigated, and the demonstration of HCV proteins as the antigenic constituent of the glomerular immune deposits has remained elusive. Kidney biopsy specimens were obtained from 12 HCV RNA, antibody to HCV (anti-HCV)-positive patients with MPGN and type-II MC, and from 8 controls (3 HCV RNA, anti-HCV-negative patients with MPGN and MC and 5 with noncryoglobulinemic “idiopathic” MPGN). Murine monoclonal antibodies developed against c22-3, E2/NS1, c33c, c100-3, and NS5 proteins were used to detect HCV-related antigens by indirect immunohistochemistry. Acid electroelution of tissue sections was performed to enhance the sensitivity of the immunohistochemical method. Specific HCV-related proteins were detected in glomerular and tubulo-interstitial vascular structures in 8 (66.7%) HCV-positive MC patients and in none of the HCV RNA, anti-HCV-negative controls. HCV immunoreactive deposits displayed the following two major patterns: 1) a linear, homogeneous deposition along glomerular capillary walls, including endothelial cells and sub-endothelial spaces; and 2) a granular bead-like appearance with distinct deposits in mesangial and paramesangial cells. Immunoglobulin G (IgG) and M (IgM) and C3 fraction deposition in adjacent kidney sections displayed features comparable with those found for HCV deposits. Patients with granular deposits showed more pronounced renal impairment and severe proteinuria. These findings indicate that in MC patients with HCV-associated MPGN, kidney deposits consist of HCV-containing immune complexes that are likely to play a direct pathogenetic role in the renal damage.
Detection of hepatitis GB virus type C RNA in serum and liver from children with chronic viral hepatitis B and C
Authors: Lopez-Alcorocho J M , Millan A , Garcia-Trevijano E R , Bartolome J , Ruiz-Moreno M , Otero M , Carreno V
Published: July 2004

The aim of this work was to study the presence of the hepatitis GB virus type C (HGBV-C) in liver and serum samples of children with chronic viral hepatitis, the time course of changes in viral RNA, and the possible acquisition routes of infection. Frozen serum and liver samples from 58 children with chronic hepatitis B (n = 33) or C (n = 25) were analyzed using polymerase chain reaction. Twenty-seven children had been included in different interferon trials. Two additional serum samples from the HGBV-C-positive children as well as serum samples from 29 of their relatives were also analyzed. HGBV-C RNA was detected in serum and liver samples from 9 of 58 (15%) of children as well as in serum samples from 3 of 29 of the relatives of the HGBV-C- infected children: the mother and the brother of one child (index case A) and the mother of another child (index case B). The homologies of the HGBV-C RNA sequences were 93% between index case A and his mother, 88% between index case A and his brother, and 94% between index case B and his mother. In the 3 children receiving alpha-interferon, HGBV-C RNA became undetectable during treatment although it reappeared in 2 of them after therapy. In conclusion, we found that 15% of children with chronic viral hepatitis were coinfected with HGBV-C. HGBV-C RNA was simultaneously present in serum and liver samples and tended to remain detectable even after alpha-interferon therapy. Our results suggest that vertical transmission of HGBV-C may occur.