bluuebunny/crossref_metadata_2025 huggingface dataset:
The inhibitory effect of interleukin 1β on rat hepatocyte DNA synthesis is mediated by nitric oxide
Authors: Wang Ziqiu , Wang Meifang , Carr Brian I.
Published: July 2004

Interleukin 1β (IL-1β) and nitric oxide (NO) have potent growth-regulatory effects on different cell types. We found that epidermal growth factor-induced DNA synthesis in primary cultures of adult rat hepatocytes was inhibited by NO when it was provided by addition to the cultures of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, as well as by addition of IL-1β in a dose-dependent manner. IL-1β also induced NO production and inducible NO synthase (iNOS) gene expression. The inhibition of DNA synthesis by IL-1β was completely abrogated when NO production was inhibited by N-monomethyl-l -arginine (NMA), a competitive inhibitor of iNOS. IL-1β-receptor antagonist (IL-1ra), which interferes with the interaction of IL-1β with target cells, also abolished the inhibitory effects of IL-1β on hepatocyte DNA synthesis as well as IL-1β-induced iNOS gene expression. We also found that hepatocyte DNA synthesis inhibition by IL-1β was completely antagonized by providing deoxynucleosides to bypass the block in ribonucleotide reductase, a rate-limiting step in DNA synthesis, thus implicating this enzyme in the mechanism of growth inhibition by IL-1β. These experiments extended prior observations on the growth-inhibitory actions of IL-1β on hepatocyte DNA synthesis, involving the IL-1β receptor, NO production, and ribonucleotide reductase.
Tolerance and latent cellular rejection in long-term liver transplant recipients
Authors: Wong Terence , Nouri-Aria Kayhan T. , Devlin John , Portmann Bernard , Williams Roger
Published: July 2004

Tolerance develops in a proportion of long-term liver transplant recipients but currently cannot be identified before an attempt at withdrawal from immunosuppression therapy. In the present study, we have examined the immunophenotypic characteristics of the cellular infiltrate in portal tracts and lobules as observed in liver biopsy specimens in relation to the outcome of subsequent withdrawal from immunosuppression therapy. Cryostat biopsy specimens from 27 long-term recipients before drug withdrawal, and from 10 patients with recent transplants who were having acute rejection, were analyzed. Immunohistochemical staining was performed for CD3+ (pan T cell), CD8+ (cytotoxic), CD4+ (helper), CD45RO+ (memory), CD45RA+ (naive), CD56+ (natural killer), CD68+ (macrophage), and CD8+ perforin+ cells. Fewer CD8+ and CD3+ cells were present in the lobular areas of biopsy specimens from patients who were successfully withdrawn from immunosuppression therapy (n = 6) compared with biopsy specimens from patients with nontolerant grafts (n = 9; 15 vs. 23 cells/high-power field [hpf] [P < .01] and 16 vs. 26 cells/hpf [P < .03], respectively) or biopsy specimens obtained during acute rejection (15 vs. 31 cells/hpf [P < .01] and 16 vs. 32 cells/hpf [P < .01]). Cell frequencies in the biopsy specimens of nontolerant long-term patients were similar to those found with acute rejection. Immunophenotyping the lobular inflammation within long-term liver allografts assists in identifying those patients in whom drug withdrawal is likely to be unsuccessful and in whom it is postulated a form of inactive, latent cellular rejection exists.
Infiltrating CD45RC− T cells are associated with immunologic unresponsiveness induced by donor class I major histocompatibility complex antigens in rats
Authors: Yamaguchi Yasuo , Miyanari Nobutomo , Ichiguchi Osamu , Akizuki Eiji , Matsumura Fujio , Matsuda Teishi , Okabe Kazutoshi , Liang Jian , Ohshiro Hajime , Mori Katsutaka , Ogawa Michio
Published: July 2004

It has previously been shown that a single intravenous injection of freshly heparinized donor-specific blood transfusion (DST) before transplantation significantly prolongs the survival of fully allogeneic ACI (RT1a)-to-LEW(RT11) rat hepatic allografts. Additionally, we have shown that pretreatment of LEW rats with PVG.r1 blood, which shares only the RT1.A major histocompatibility complex (MHC) region with ACI, significantly prolongs the survival of ACI hepatic allografts. In this study, we report the cellular identity of hepatic allograft leukocyte infiltrates following transplantation. Fluorescence-activated cell sorting (FACS) analysis revealed that CD4+ T cells infiltrating liver allografts could be divided into two subsets, CD45RC−CD4+ and CD45RC+CD4+ T cells, and that the ratio of CD45RC−CD4+/CD45RC+ CD4+ T cells was significantly higher in hepatic allografts of recipients pretreated with DST or PVG.r1 blood as compared to untreated allografts. Further, CD8+ T cells that accumulated in the liver grafts could be similarly divided into two subsets, and the ratio of CD45RC−CD8+/CD45RC+ CD8+ T cells was also significantly higher in hepatic allografts of recipients pretreated with DST or PVG.r1 blood. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that CD45RC−CD4+ T cells harvested from hepatic allografts pretreated with PVG.r1 blood expressed interleukin-4 (IL-4) and interleukin-10 (IL-10), but not interleukin-2 (IL-2) or interferon-γ (IFN-γ). In contrast, CD45RC−CD8+ T cells from hepatic allografts pretreated with PVG.r1 blood expressed IL-4, IL-10, and IFN-λ, but not IL-2. These results indicate that the CD45RC leukocyte common antigen could be used to differentiate CD4+ and CD8+ T cells following pretreatment with DST or PVG.r1 blood. Persistent infiltration of CD45RC−CD4+ and CD45RC−CD8+ T cells, capable of secreting Th2-type cytokines may prevent allograft rejection by causing immunologic unresponsiveness.
Effect of tumor necrosis factor α and intercellular adhesion molecule-1 expression on immunogenicity of murine liver cells in mice
Authors: Bumgardner Ginny L. , Li Jiashun , APTE SACH , HEININGER MARIE , FRANKEL WENDY L.
Published: July 2004

Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been implicated in the pathogenesis of various inflammatory liver disease states, including viral and autoimmune hepatitis as well as liver allograft rejection. Tumor necrosis factor α (TNF-α) is an inflammatory cytokine known to up-regulate adhesion molecules as well as major histocompatibility complex (MHC) class I expression, and has been demonstrated to be important in the rejection of vascularized organ allografts. The current studies address the effect of TNF-α and the role of ICAM-1 expression on liver cell immunogenicity in vitro in mixed lymphocyte hepatocyte culture (MLHC), in vitro in mixed lymphocyte liver nonparenchymal cell culture (MLNPC), in vivo in hepatocyte sponge matrix allografts (HC-SMA), and in vivo in liver nonparenchymal cell sponge matrix allografts (NPC-SMA). Purified allogeneic hepatocytes (HC) and liver nonparenchymal cells (NPC) under naive, unstimulated conditions demonstrated different profiles of MHC antigen and adhesion molecule expression, but both liver cell populations stimulated the proliferation and development of allospecific cytotoxic effectors in vitro and in vivo. Despite significant up-regulation of MHC class I and ICAM-1 on both HC and liver NPCs by in vivo treatment with TNF-α, the immunogenicity of TNF-α-stimulated liver cells was not appreciably different from naive, unstimulated liver cells. In contrast, ICAM-1-negative HC and NPCs were significantly less immunogenic both in terms of lymphocyte proliferative responses and the generation of allospecific cytolytic effectors. These results suggest that constitutive expression of ICAM-1 enhances the immunogenicity of “donor” liver cells but is not absolutely required to elicit immune responses to allogeneic liver cells. Further studies to determine the role of adhesion molecule expression on trafficking of host immune cells to the liver and the role of adhesion molecule expression by host cells are required to clarify their role in immune responses to liver cells.
Circulating cellular fibronectin may be a natural ligand for the hepatic asialoglycoprotein receptor: Possible pathway for fibronectin deposition and turnover in the rat liver
Authors: Rotundo Robert F. , Rebres Robert A. , Mckeown-Longo Paula J. , Blumenstock Frank A. , Saba Thomas M.
Published: July 2004

It has been postulated that the in vivo removal of many plasma glycoproteins after desialylation is mediated by their interaction with a specific endocytic receptor on hepatocytes called the asialoglycoprotein receptor (ASGP-R), which is known to have a high affinity for specific carbohydrate residues, such as galactose. However, this mechanism has never been proven in vivo, nor has a naturally occurring ligand for the ASGP-R been identified. We investigated the influence of the terminal galactose residues on plasma fibronectin (pFn) on its liver deposition and turnover in adult rats, using neuraminidase to remove sialic acid residues to expose galactose residues. We also tested the hypothesis that the normal presence of a large amount of terminal galactose residues in cellular Fn (cFn) may allow cFn to serve as a natural ligand readily able to interact with the ASGP-R. In contrast to the slow clearance of normal pFn from the blood, cFn and desialylated pFn (aFn) displayed a rapid plasma clearance (P < .001) with greater than 50% of both the 125I-cFn or 125I-aFn depositing in the liver within 15 minutes. The enhanced plasma removal and liver deposition of both 125I-cFn and 125I-aFn was competitively inhibited (P < .01) by prior intravenous infusion of excess asialofetuin, which can selectively bind to the ASGP-R. The enzymatic addition of terminal sialic acid residues onto cFn to “mask” or “cap” the normally exposed galactose residues delayed the rapid plasma removal of cFn. Accelerated degradation of 125I-aFn and 125I-cFn as compared with 125I-pFn was demonstrated in vitro by both primary cultures of normal rat hepatocytes or incubated (37°C) tissue slices of livers harvested from normal rats after in vivo preloading with tracer 125I-Fn forms. Thus, the ASGP-R appears to directly participate in the rapid in vivo removal of cFn from the blood, while native pFn may be removed by an alternative pathway unless it can become desialylated in vivo. These findings suggest that cFn may be a naturally occurring ligand that does not require desialylation before removal by the ASGP-R on hepatocytes.
Clinical characteristics of hereditary hemochromatosis patients who lack the C282Y mutation
Authors: Shaheen Nicholas J. , Bacon Bruce R. , Grimm Ian S.
Published: July 2004

Approximately 85% of patients with typical hereditary hemochromatosis (HH) are homozygous for the C282Y mutation (C282Y/C282Y) in the recently identified candidate gene for HH. However, some HH patients are instead homozygous for the wild-type allele (wt/wt) at this locus. These wt/wt patients may represent a phenotypically similar, but genotypically different, heritable trait, or may be unrecognized cases of secondary iron overload. The purpose of this study is to provide an in-depth analysis of the wt/wt HH patients identified in the original description of the HH gene, and to compare them with 62 patients from the same analysis who were homozygous for the C282Y mutation. Eighteen of the 21 wt/wt HH patients from the original study were assessed for 14 historical and laboratory variables, including previously unrecognized causes of secondary iron overload, the heritability of iron overload and liver disease, and other clinical characteristics. Ten of these 18 wt/wt HH patients (55.6%) were found to have previously unrecognized causes for secondary iron overload compared with 3 of 62 (4.8%) of the C282Y/C282Y patients (P < .001). The remaining 8 wt/wt patients had no recognizable etiology of secondary iron overload. None of the 18 wt/wt patients had a family history of iron overload or liver disease, compared with 58% of the C282Y/C282Y patients (P < .001). When compared with C282Y homozygotes, the 8 wt/wt patients without secondary iron overload had a higher presenting hepatic iron index (HII) (9.5 vs. 4.7; P = .01). We conclude that, in this series of patients, over half of the wt/wt HH patients possessed previously unrecognized causes of secondary iron overload, and therefore, may have been misdiagnoses. If these cases are excluded, the number of false-negative tests is decreased, and the sensitivity of the mutational analysis is increased. However, there is a subgroup of wt/wt patients who have typical hemochromatosis without an identifiable cause of secondary iron overload. These patients may have more severe iron loading than C282Y homozygotes.
Hepatocyte-specific expression of the human MDR3 P -glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (−/−) mice
Authors: Smith Alexander J. , de Vree Marleen J. , Ottenhoff Roelof , Elferink Ronald P. , Schinkel Alfred H. , Borst Piet
Published: July 2004

Mice homozygous for a disruption in the Mdr2 gene (Mdr2 (−/−) mice) lack the Mdr2 P-glycoprotein (P-gp) in the canalicular membrane of the hepatocyte and are unable to excrete phosphatidylcholine into the bile. These mice develop a nonsuppurative cholestatic liver disease, presumably caused by the high concentrations of free cytotoxic bile acids in bile. We generated transgenic mice that express the human homolog of Mdr2, MDR3, specifically in the liver by the use of an albumin promoter. In these mice the MDR3P-gp is exclusively located in the canalicular membrane of hepatocytes and phospholipid excretion into bile is restored. Mice that contain the same amount of MDR3 P-gp as that of Mdr2 P-gp in wild-type mice, also excrete the same amount of phospholipids. No histopathological abnormalities were observed in the livers of these mice. In mice that express MDR3 at a higher or lower level, the phospholipid excretion correlated with the amount of MDR3 P-gp. We conclude that the human MDR3P-gp is functionally homologous to the murine Mdr2 P-gp and that it can fully replace this P-gp in Mdr2 (−/−) mice, restoring the excretion of phospholipids into the bile. The phospholipid excretion is limited by the amount of MDR3 or Mdr2 P-gp. The excretion of cholesterol is not tightly coupled to the excretion of phospholipids in these mice, because a very low phospholipid excretion level is sufficient to give almost wild-type cholesterol excretion into the bile.
Regulation of cyclin G1 during murine hepatic regeneration following dipin-induced DNA damage
Authors: Jensen Michael Rugaard , Factor Valentina M. , Thorgeirsson Snorri S.
Published: July 2004

Cyclin G1 has been linked to both positive and negative growth regulation. The expression of cyclin G1 is induced by transforming growth factor β1and p53, as well as by multiple mitogenic stimuli in mammalian cells in culture. However, the physiological role of cyclin G1 remains unclear. To examine the cell-cycle regulation of cyclin G1 in vivo, two models of coordinated cell proliferation induced by partial hepatectomy (PH) in the presence or absence of DNA damage were used. To introduce DNA damage, mice were treated with the alkylating drug, 1,4-bis[N,N′-di(ethylene)-phosphamide]piperazine (Dipin) 2 hours before PH. Cell-cycle progression was monitored by 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA, the frequency of mitoses, the expression of cell-cycle control genes, and by flow cytometry. Dipin treatment resulted in cell-cycle arrest at the G2/M boundary without affecting G0/G1 and G1/S transitions. While the hepatocytes progressively entered G2 phase arrest, the cyclin G1 mRNA and protein levels increased more than five- and eightfold, respectively. Cyclin G1 had a nuclear localization in all interphase cells with clear absence from nucleoli. In contrast, during mitosis, cyclin G1 was undetectable by immunohistochemistry. Taken together, our data provide evidence for a putative role of cyclin G1 in G2/M checkpoint control.
Incidence and clinical consequences of surface and polymerase gene mutations in liver transplant recipients on hepatitis B immunoglobulin
Authors: Terrault Norah A. , Zhou Shuan , McCory Robert W. , Pruett Timothy L. , Lake John R. , Roberts John P. , Ascher Nancy L. , Wright Teresa L.
Published: July 2004

Mutations in the “a” determinant of the surface gene have been associated with failure of hepatitis B immunoglobulin (HBIg) prophylaxis. We compared sequences from the surface and polymerase regions of hepatitis B virus (HBV) from 4 patients who failed high-dose HBIg therapy with two control groups: HBIg-treated patients who remained hepatitis B surface antigen (HBsAg)-negative (n = 4) and HBV-infected transplant recipients who never received HBIg (n = 4). Mutations within the surface and overlapping polymerase region were more common in patients failing HBIg than controls (P = .03), and mutations in the region of the “a” determinant were present only in patients failing HBIg. To examine the relationship between HBIg failure and duration of therapy, five additional treatment failures from a second transplantation center were sequenced (total with HBIg failure = 9). Mutations in the “a” determinant developed in 1 of 3 patients receiving HBIg for less than 6 months compared with 5 of 6 patients failing HBIg after 6 months of therapy (P = .23). The most frequently identified amino acid substitution was glycine to arginine at position 145 (present in 4 of 6 patients who failed HBIg after at least 6 months of treatment). A unique mutation within the YMDD motif (methionine to leucine) was present in 1 patient who failed HBIg treatment and who received a short course of ganciclovir. We conclude that the emergence of mutations in the “a” determinant accounts for some, but not all, treatment failures in patients receiving HBIg prophylaxis. Mutations in other regions of the S gene were more common in patients failing HBIg than controls, suggesting that domains other than the “a” determinant may be important.
Detection of widespread hepatocyte infection in chronic hepatitis C
Authors: Agnello Vincent , Ábel György , Knight Glenn B. , Muchmore Elizabeth
Published: July 2004

The controversial question of the extent of hepatocyte infection in chronic hepatitis C was re-examined in both chimpanzees and humans using a newly modifiedin situhybridization (ISH) method for detecting hepatitis C virus (HCV) RNA. The specificity of the methodology for distinguishing positive- and negative-strand synthetic HCV RNA was at least six magnitudes greater than the reverse-transcription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was l4 ± 2 genomic equivalents (gE) of HCV positive strand per cell, which was three magnitudes less sensitive than RT-PCR quantitation of HCV. In contrast to previous studies in both humans and chimpanzees with chronic hepatitis C, a high percentage of hepatocytes positive for both positive- and negative-strand HCV RNA was found in most specimens studied. In humans, the extent of hepatocyte infection varied with histological activity index (HAI). In the two chimpanzees studied, the liver biopsies showed minimal histological disease activity, but high percentages of hepatocytes were HCV-positive by ISH that correlated with hepatocyte ultrastructural changes associated with HCV infection. Hepatocyte infection was confirmed by RNA extraction and RT-PCR techniques for detecting HCV RNA that minimize the false detection of negative strands. In both human and chimpanzee liver biopsies showing minimal HAI, the hepatocyte concentration of HCV was estimated to be very low. These findings suggested the hypothesis that persistent infection in the liver may be caused in part by low-level HCV replication. The theoretical and clinical implications of these findings are discussed.
Effect of the prokinetic agent, erythromycin, in the richardson ground squirrel model of cholesterol gallstone disease
Authors: Xu Qi-Wei , Scott Brent R. , Tan Daimen T. M. , Shaffer Eldon A.
Published: July 2004

Impaired gallbladder motility and delayed intestinal transit contribute to cholesterol gallstone formation by impeding the enterohepatic circulation of bile salts and causing gallbladder stasis. The therapeutic value of erythromycin, a prokinetic motilin analog, was evaluated in an animal model of gallstone formation. Eighty ground squirrels were fed either a trace- (control) or a high- (1%) cholesterol diet. Half of each diet group received either erythromycin stearate or placebo orally twice daily for 4 weeks. Biliary lipid secretion and bile salt pool size were determined via common duct cannulation. Gallbladder contractile response to cholecystokinin (CCK) was studied in vitro. Intestinal transit was evaluated in vivo by 51Cr marker. In the placebo-treated group, fed the high- versus the trace-cholesterol diet, bile salt secretion decreased (trace-cholesterol + placebo, 21.0 ± 1.8 nmol/min/g liver vs. high-cholesterol + placebo, 9.3 ± 1.4 nmol/min/g liver), cholesterol saturation index (CSI) doubled (trace-cholesterol + placebo, 0.61 ± 0.06 vs. high-cholesterol + placebo, 1.30 ± 0.04), nucleation time shortened (trace-cholesterol + placebo, >21 days vs. high-cholesterol + placebo, 6.4 ± 1.0 days), cholesterol crystals formed, gallbladder contractility diminished, and intestinal transit was delayed (each P < .05). Erythromycin treatment of animals on the high-cholesterol diet restored gallbladder contractility and intestinal transit to control levels, increased bile salt secretion, reduced the total bile salt pool, lowered the cholesterol saturation of bile, lengthened the nucleation time, and so reduced crystal formation (each P< .05). Erythromycin enhances gallbladder motility and hastens intestinal transit, promoting more rapid enterohepatic cycling of bile salts. This increases bile salt secretion, improves cholesterol solubility, and reduces crystal development.
Acute hepatic allograft rejection: Incidence, risk factors, and impact on outcome †
Authors: Wiesner Russell H. , Demetris Jake A. , Belle Steven H. , Seaberg Eric C. , Lake John R. , Zetterman Rowen K. , Everhart James , Detre Katherine M.
Published: July 2004

Hepatic allograft rejection remains an important problem following liver transplantation, and, indeed, complications related to the administration of immunosuppressive therapy remain a predominant cause of posttransplantation morbidity and mortality. The Liver Transplantation Database (LTD) was used to study a cohort of 762 consecutive adult liver transplantation recipients and determined the incidence, timing, and risk factors for acute rejection. We also evaluated the impact of histological severity of rejection on the need for additional immunosuppressive therapy and on patient and graft survival. Four hundred ninety (64%) of the 762 adult liver transplantation recipients developed at least one episode of rejection during a median follow-up period of 1,042 days (range, 336-1,896 days), most of which occurred during the first 6 weeks after transplantation. Multivariate analysis revealed that recipient age, serum creatinine, aspartate transaminase (AST) level, presence of edema, donor/recipient HLA-DR mismatch, cold ischemic time, and donor age were independently associated with the time to acute rejection. An interesting observation was that the histological severity of rejection was an important prognosticator: the use of antilymphocyte preparations was higher, and the time to death or retransplantation was shorter, for patients with severe rejection. Findings from this study will assist in decision-making for the use of immunosuppressive regimens and call into question whether complete elimination of all rejection or alloreactivity is a desirable goal in liver transplantation.
Human leukocyte antigen-C genes and susceptibility to primary sclerosing cholangitis
Authors: Moloney Manus M. , Thomson Linda J. , Strettell Michael J. , Williams Roger , Donaldson Peter T.
Published: July 2004

Genetic susceptibility to primary sclerosing cholangitis (PSC) is associated with the extended HLA A1-B8-DR3 haplotype and also with the DRB3*0101-DRB1*0301-DQA1*0103-DQB1*0603 haplotype. However, very few studies have considered the role of HLA C which lies between HLA A and B, is highly polymorphic, and encodes proteins which play an important role in immunoregulation and in disease susceptibility. Traditional assignment of HLA Cw antigens by serology is both inaccurate and unreliable, with a high error rate. The aim of this study was to characterize the distribution of HLA C alleles in a large group of patients with primary sclerosing cholangitis by using a recently developed polymerase chain reaction-based genotyping technique. Ninety-three white adult patients of northern European origin with well characterized PSC and 100 geographically and racially matched controls were studied. HLA C and HLA DRB1 alleles were assigned by polymerase chain reaction-based genotyping, HLA A and B antigens by standard microlymphocytotoxicity test and extended haplotypes were constructed according to known patterns of linkage disequilibrium. The Cw*07 gene was found in 67.7% of patients versus 54% of controls (P = .051, OR = 1.79). This increase was a result of inheritance of the Cw*0701 allele which was found in 51.6% of patients compared with 34% of controls (P = .013, OR = 2.07). There were no significant differences in the frequencies of any of the other Cw alleles including the Cw*07 group: Cw*0702, Cw*0703, and Cw*0704. HLA-encoded genetic susceptibility to PSC is associated with the HLA Cw*0701 allele, but the association is weak and may simply reflect linkage disequilibrium with the HLA B8-DR3 haplotype. These findings indicate that the telomeric limit of HLA-encoded susceptibility to primary sclerosing cholangitis lies close to the HLA C locus.
Neuropeptide Y stimulates bile secretion via Y1 receptor in the left dorsal vagal complex in rats
Authors: Yoneda Masashi , Nakamura Kimihide , Yokohama Shiro , Tamori Keisuke , Sato Yoichi , Aso Kazunobu , Aoshima Masaru , Kono Toru , Makino Isao
Published: July 2004

Neuropeptide Y (NPY) injected into the cerebrospinal fluid and the left dorsal vagal complex enhances bile acid-independent and bicarbonate-dependent bile secretion through vagal muscarinic pathways in animal models. NPY binds to and activates six different receptor subtypes, and NPY Y1 and Y2 receptors are distributed in the dorsal vagal complex. We sought to determine which NPY receptor subtypes are involved in central stimulation of bile secretion by examining the effect of microinjection of specific NPY receptor agonists into the dorsal vagal complex. The bile duct was cannulated in urethane-anesthetized and bile acid-compensated rats. After measuring basal secretion, NPY, peptide YY (PYY), [Leu31, Pro34]NPY, NPY(13-36), or NPY(3-36) was microinjected into the either right or left dorsal vagal complex and bile secretion was observed for 100 minutes. Hepatic branch vagotomy was performed 2 hours before the peptide injection. Microinjection of NPY and PYY (8 pmol) into the left dorsal vagal complex increased bile secretion. [Leu31, Pro34]NPY microinjected into the left dorsal vagal complex also dose-dependently (1-8 pmol) stimulated bile acid-independent and bicarbonate-dependent bile secretion. Microinjection of NPY(13-36) into the left dorsal vagal complex did not stimulate and NPY(3-36) dose-dependently inhibited bile secretion. Stimulation of bile secretion by [Leu31, Pro34]NPY was abolished by hepatic branch vagotomy. NPY acts in the left dorsal vagal complex to stimulate bile acid-independent and bicarbonate-dependent bile secretion via Y1 receptor subtype.
Effects of propranolol on the hepatic hemodynamic response to physical exercise in patients with cirrhosis
Authors: Bandi Juan-Carlos , García-Pagán Joan Carles , Escorsell Angels , François Erik , Moitinho Eduardo , Rodés Joan , Bosch Jaume
Published: July 2004

Physical exercise increases portal pressure (hepatic venous pressure gradient [HVPG]) in patients with cirrhosis. It is unknown if this deleterious effect is associated with changes in gastroesophageal collateral blood flow and if these can be prevented by propranolol administration. The aim of this study was to characterize the effects of propranolol on the splanchnic hemodynamic response to exercise in patients with cirrhosis. Twenty-three patients with cirrhosis and portal hypertension had hemodynamic measurements in baseline conditions, and during moderate cycling exercise (40 W) under double-blind propranolol or placebo administration. In patients receiving placebo, HVPG significantly increased during exercise (from 16.7 ± 0.9 to 19.0 ± 1.0 mm Hg; P < .01), hepatic blood flow (HBF) decreased (-18% ± 4%; P < .01), while azygos blood flow (AzBF) was unchanged (4% ± 12%; ns). In patients receiving propranolol, portal pressure did not increase during exercise, but decreased from 16.3 ± 1.0 to 12.9 ± 1.1 mm Hg (P < .01). The lack of increase in HVPG in response to exercise in patients receiving propranolol may be related to a more pronounced decrease in HBF, as compared with patients receiving placebo, and to a blunted increase in cardiac output (CO). Moderate physical exercise adversely influences the hepatic hemodynamics in patients with cirrhosis, causing a significant increase in portal pressure. This is effectively prevented by propranolol pretreatment.
N -acetylcysteine prevents development of the hyperdynamic circulation in the portal hypertensive rat
Authors: Fernando Bimbi , Marley Richard , Holt Steve , Anand Radhi , Harry David , Sanderson Peter , Smith Roy , Hamilton George , Moore Kevin
Published: July 2004

Partial portal vein ligation (PPVL) leads to the development of a hyperdynamic circulation. It is associated with elevated levels of tumor necrosis factor (TNF-α) and nitric oxide (NO) production, both of which can result in oxidant injury. In this study, we have investigated whether PPVL is associated with the development of oxidative stress, by measuring urinary F2-isoprostanes. In addition, we have examined whether N-acetylcysteine (NAC) can ameliorate oxidant injury and prevent the development of the hyperdynamic circulation. Urinary excretion of F2-isoprostanes increased sixfold following PPVL together with a significant increase in plasma nitrite and nitrate. Treatment with NAC inhibited the formation of F2-isoprostanes as well as the increase in plasma nitrite and nitrate. Hemodynamic studies in anesthetized rats showed that following PPVL, cardiac output and portal pressure increased, and systemic vascular resistance decreased, consistent with the development of a hyperdynamic circulation. These changes were prevented by chronic administration of NAC. We conclude that NAC prevents the development of the hyperdynamic circulation and that the formation of reactive oxygen species may be important in the pathogenesis of these hemodynamic changes.
Detection of cholangiocarcinoma in primary sclerosing cholangitis by positron emission tomography
Authors: Keiding Susanne , Hansen Søren B. , Rasmussen Henrik H , Gee Antony , Kruse Aksel , Roelsgaard Klaus , Tage-Jensen Ulrik , Dahlerup Jens Frederik
Published: July 2004

Primary sclerosing cholangitis (PSC) predisposes to cholangiocarcinoma (CC), which usually is widespread in the liver at the time of the diagnosis and which has a median survival of approximately 6 months. Positron emission tomography (PET) is a noninvasive scanning method that allows the assessment of metabolism in vivo by means of positron-emitting radiolabeled tracers. [18F]Fluoro-2-deoxy-d-glucose (FDG) is a glucose analogue that accumulates in various malignant tumors because of their high glucose metabolic rates. The purpose of the study was to develop a PET method to detect small CC tumors in patients with PSC. PET scanning of the liver was performed after intravenous injection of 200 MBq FDG in 9 patients with PSC, 6 patients with PSC + CC, and 5 controls. The scanning was performed at successive time intervals for a total of 90 minutes with simultaneous successive arterial blood sampling for radioactivity concentration determination. In each of the PSC + CC patients, 2 to 7 “hot spots” were seen, with volumes of 1.0 to 45 mL (median, 4.4 mL). There were no hot spots in the two other patient groups. The localization of hot spots was confirmed by single-blind evaluation. Data were analyzed by the Gjedde-Patlak plot, yielding values of the net metabolic clearance of FDG, K [mL min−1 100 mL−1 tissue]. In the CC hot spots, maximum K values were 1.59 to 4.17 (median, 2.34; n = 6); in the reference liver tissues of these patients, K values were 0.40 to 0.69 (median, 0.49); in PSC patients, they were 0.23 to 0.53 (median, 0.36); and in controls, they were 0.20 to 0.34 (median, 0.31). The difference between K in CC hot spots and the other groups was statistically significant (P< .001). We conclude that FDG-PET seems to be able to detect small CC tumors and may be useful in the therapeutic management of PSC.
Retinoic acid treatment induces apoptosis or expression of a more differentiated phenotype on different fractions of cultured fetal rat hepatocytes
Authors: Falasca Laura , Favale Anna , Gualandi Giampiero , Maietta Gennaro , Devirgiliis Laura Conti
Published: July 2004

The present study reports the effects of retinoic acid (RA) on cultured fetal rat hepatocytes. We show that RA treatment induces both differentiation and apoptosis. Hepatocytes cultured for 48 hours in the presence of 5 μmol/L RA form junctional complexes in the areas of contact between neighboring cells and develop bile canaliculi, typical features of mature and well-differentiated cells. At the same time, about 20% of cells are induced to die by apoptosis, and the percentage of apoptotic cells increases according to the concentration of RA used and the duration of treatment. The induction of apoptosis, studied at the morphological and biochemical levels, revealed that, in our system, the classical compaction of chromatin occurs only during the final stages of the process; instead of the common marker of apoptosis, i.e., the “DNA ladder” pattern of fragmentation, megabase-sized fragments were found. These observations provide further evidence of the existence of fundamental differences in the mechanisms of apoptosis among cell types. To investigate the molecular mechanism of the effects of RA, we evaluated the expression of two proteins, c-myc and p53, which are known to be involved in both cell differentiation and apoptosis. The data obtained show that the amount of p53 remained unchanged after RA treatment. On the contrary, a dose-dependent reduction in c-myc levels was found, suggesting that RA action may be mediated by modulation of this oncogene. Our findings regarding the apoptosis-inducing effect of RA, which was not found in adult hepatocytes, suggest a possible relationship between this phenomenon and the proliferative capacity and/or differentiation state of hepatocytes.
Osmotic regulation of the heat shock response in primary rat hepatocytes
Authors: Kurz Anna Kordelia , Schliess Freimut , Häussinger Dieter
Published: July 2004

The influence of cell hydration and taurine on the heat shock response was studied in primary rat hepatocytes. Heat-induced accumulation of inducible heat shock protein 70 (HSP70) mRNA and protein was increased under hypo-osmotic conditions. In contrast, hyper-osmotic exposure blocked the HSP70 response during an 8-hour recovery, and this was paralleled by a reduction of overall protein synthesis and an impairment of thermotolerance. Taurine counteracted the hyper-osmotic inhibition of heat-induced HSP70 expression, but increased overall protein synthesis only slightly. A rapid and transient activation of the stress-activated protein kinase, JNK-2, was triggered by hyper-osmolarity, whereas the JNK-2 response to hypo-osmolarity was delayed. JNK-2 activation in response to heat was suppressed by hypo-osmolarity, but was markedly increased under hyper-osmotic conditions. The latter effect was blocked by taurine. A pronounced induction of the mRNA for the MAP-kinase phosphatase, MKP-1, in response to heat was observed during hypo- and normo-osmolarity, but no MKP-1 induction was found under hyper-osmotic conditions, although hyper-osmolarity itself led to accumulation of small levels of MKP-1 mRNA. Also, the block of heat-induced MKP-1 mRNA expression by hyper-osmolarity was abolished in the presence of taurine. The data provide evidence for a role of cellular hydration and taurine in the protection of liver parenchymal cells against heat injury via regulation of HSP70 expression and the balance between JNK-2 and MKP-1 activity.
Altered regulation of Src tyrosine kinase by transforming growth factor β1 in a human hepatoma cell line
Authors: Fukuda Kazuto , Kawata Sumio , Tamura Shinji , Matsuda Yukihiko , Inui Yoshiaki , Igura Takumi , Inoue Satoshi , Kudara Takahiko , Matsuzawa Yuji
Published: July 2004

Transforming growth factor βs (TGF-βs) are the potent growth inhibitors for various cell types. Certain transformed cells, however, show poor response to TGF-β-induced growth inhibition, which contributes to their uncontrolled proliferation. Recently, we have reported that TGF-β1 induces degradation of activated Src tyrosine kinase in rat fibroblasts. To elucidate the alteration in TGF-β signaling pathway in tumor cells that cannot respond to the cytokine, we compared the effects of TGF-β1 on Src kinase in two human hepatoma cell lines, TGF-β1-insensitive Mahlavu cells and TGF-β1-sensitive HepG2 cells. TGF-β1 decreased Src kinase activity in HepG2 cells, but increased cellular Src levels and Src kinase activity in Mahlavu cells. Co-incubation of Mahlavu cells with TGF-β1 and 12-O-tetradecanoyl phorbol 13-acetate (TPA) decreased Src protein levels and Src kinase activity, inducing TGF-β1sensitivity. TGF-β1 induced tyrosine dephosphorylation of Ras guanosine triphosphatase-activating protein (Ras-GAP) and Ras inactivation in HepG2 cells, but induced Ras-GAP phosphorylation and Ras activation in Mahlavu cells. The Src kinase inhibitor abolished the increase of Src kinase activity in TGF-β1-treated Mahlavu cells, and induced TGF-β1 sensitivity. These findings suggest that regulation of Src kinase by TGF-β1is altered in Mahlavu cells. The altered regulation of Src may contribute to TGF-β1 insensitivity in this cell line, at least in part through activation of Ras.
Impact of alcohol on the histological and clinical progression of Hepatitis C infection
Authors: Wiley Thelma E. , McCarthy Mary , Breidi Lama , McCarthy Monica , Layden Thomas J.
Published: July 2004

In patients infected with the hepatitis C virus (HCV), 20% to 30% will progress to cirrhosis in over two to three decades. Viral and host factors that are important in the clinical and histologic progression of HCV infection are not entirely certain. It has been suggested that liver disease is worse in alcoholics infected with HCV. In the present retrospective study, we examined the effect of moderate alcohol intake on the histologic and clinical progression of HCV infection and assessed whether other variables such as gender, length of exposure, mode of exposure, HCV RNA levels, and ferritin levels also independently impacted disease progression. Liver biopsies were analyzed for the degree of fibrosis, presence of cirrhosis, and histologic activity by using the Histologic Activity Index of Knodell. Patients were divided into two groups based on whether their alcohol intake was significant or not significant. Significant alcohol intake was defined as >40 g alcohol/day in women and >60 g of alcohol/day in men for >5 years. Groups were further divided based on the decades of exposure to HCV. There was no difference in the age or length of exposure to HCV in the alcohol and the alcohol-free group. HCV RNA serum levels, ferritin levels, and viral genotypes were similar in both groups. There was a two- to threefold greater risk of liver cirrhosis and decompensated liver disease in the alcohol group. Also, the rate to which subjects developed cirrhosis was faster in the alcohol group with 58% being cirrhotic by the second decade as opposed to 10% being cirrhotic in the nonalcohol group by the second decade. The histologic and clinical acceleration of liver disease was independent of the mode of exposure or sex. In summary, alcohol intake is an independent risk factor in the clinical and histologic progression of HCV infection.
The efficacy of prophylactic interferon alfa-2b in preventing recurrent hepatitis C after liver transplantation
Authors: Sheiner Patricia A. , Boros Peter , Klion Franklin M. , Thung Swan N. , Schluger Leona Kim , Lau Johnson Y. , Mor Eytan , Bodian Carol , Guy Stephen R. , Schwartz Myron E. , Emre Sukru , Bodenheer Henry C. , Miller Charles M.
Published: July 2004

Clinical recurrence of hepatitis C after liver transplantation can lead to cirrhosis, liver failure, and death. In patients undergoing liver transplantation for hepatitis C, we assessed the efficacy of interferon alfa-2b (IFN) in preventing recurrent hepatitis. We randomized 86 patients to either an IFN group (3 MU three times a week starting within 2 weeks after transplantation and continued for 1 year) or a control (no IFN) group. Recurrence, the primary end point, was diagnosed on biopsy performed at 1 year or for abnormal biochemistries. HCV RNA levels were measured by branched-chain DNA (bcDNA) assay and arbitrarily defined as low, moderate, or high (<10 × 105, 10-100 × 105, or >100 × 105Eq/mL, respectively). Data on 30 IFN patients and 41 no-IFN patients who survived ≥3 months were reviewed. Mean follow-up was 669 ± 228 days for IFN patients and 594 ± 254 days for no-IFN patients. IFN patients were less likely to develop recurrent hepatitis (8 IFN vs. 22 no-IFN patients, P= .017, log rank analysis). IFN and 1-month HCV RNA level were independent predictors of recurrence. IFN reduced the risk of recurrence by a factor of 0.4 (P= .04, Cox proportional hazards model); HCV RNA level >100 × 105 Eq/mL at 1 month after transplantation increased the risk by a factor of 3.1 (P = .01). Low, moderate, and high viral levels at 1 and 3 months were associated with significantly different rates of recurrence in IFN patients (P = .05 at 1 month and P = .003 at 3 months) but not in untreated patients (P = .28 at 1 month and P = .25 at 3 months). In patients with two or more rejections, the risk of recurrence was increased by a factor of 2.17 (P = .05). On 47 1-year biopsies (24 IFN; 23 no IFN), piecemeal necrosis was more common in untreated patients (P < .02). One- and 2-year patient survival, respectively, was 96% and 96% with IFN and 91.2% and 87.2% without (P = NS). Prophylactic IFN reduced the incidence of recurrent hepatitis after transplant. Although IFN was most effective in patients with low HCV RNA levels, we also noted an effect in patients with moderate levels. IFN did not prevent viremia, suggesting that it may work through alternative mechanisms.
Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton
Authors: Yee Hal F.
Published: July 2004

Hepatic stellate cell activation, thought to play a key role in fibrosis of the liver, is characterized by changes in cellular morphology. The intracellular signals regulating morphological alterations associated with stellate cell activation are uncertain. The ras-like guanosine triphosphate-binding protein, rho, has recently emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The aim of this study was to test the hypothesis that rho signaling pathways direct activation-associated morphological changes in stellate cells by regulating the actin cytoskeleton. The morphology and actin cytoskeleton of primary rat hepatic stellate cells were studied with phase contrast, differential interference contrast, and epifluorescence microscopy. Immunohistochemistry and immunoblot analysis were used to examine rho expression and activity, respectively. Quiescent and activated stellate cells were investigated in the absence and presence of C3 transferase, a bacterial toxin that specifically inhibits rho. Stellate cell activation was characterized by the development of prominent intracellular fibers, and the loss of dendrite-like processes and perinuclear retinoid droplets. Moreover, activation was accompanied by the formation of prominent actin stress fibers and focal adhesions. Both rho expression and activity were demonstrated in stellate cells. C3 transferase blocked and reversed, both activation-associated morphological alterations and activation-associated changes in the actin cytoskeleton, in quiescent and activated stellate cells, respectively. These results indicate that rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.
Cryptosporidium parvum is cytopathic for cultured human biliary epithelia via an apoptotic mechanism
Authors: Chen Xian-Ming , Levine Susan A. , Tietz Pamela , Krueger Eugene , McNiven Mark A. , Jefferson Douglas M. , Mahle Megan , LaRusso Nicholas F.
Published: July 2004

While the clinical features of sclerosing cholangitis secondary to opportunistic infections of the biliary tree in patients with acquired immunodeficiency syndrome (AIDS) are well known, the mechanisms by which microbial pathogens such as Cryptosporidium parvum associated with this syndrome actually cause disease are obscure. We established an in vitro model of biliary cryptosporidiosis employing a human biliary epithelial cell line. Using morphological and biochemical techniques, we examined the interaction of C. parvum with cultured human cholangiocytes. When the apical plasma membrane of polarized, confluent monolayers of human biliary epithelial cells was exposed to C. parvum oocysts that had been excysted in vitro, sporozoites attached to and invaded the cells in a time-, dose-, temperature-, and pH-dependent manner. The infectious process was both plasma membrane domain- and cell-specific, because no attachment or invasion occurred when the basolateral membrane of cholangiocytes was exposed to the parasite, or when a human hepatocyte cell line (HepG2) was used. Time-lapse video microscopy and scanning electron microscopy (SEM) showed that sporozoite attachment was rapid, involved extensive cholangiocyte membrane ruffling, and culminated in parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane similar to those found in naturally occurring infection in vivo. Transmission electron microscopy (TEM) showed that C. parvum organisms formed parasitophorus vacuoles and were able to undergo a complete reproductive cycle, forming both asexual and sexual reproductive stages. Unexpectedly, direct cytopathic effects were noted in infected monolayers, with widespread programmed cell death (i.e., apoptosis) of biliary epithelial cells as assessed both morphologically and biochemically beginning within hours after exposure to the organism. The novel finding of specific cytopathic invasion of biliary epithelia by C. parvum may be relevant to the pathogenesis and possible therapy of the secondary sclerosing cholangitis seen in AIDS patients with biliary cryptosporidiosis.