Volume 39, Issue 4 (April 2004)
Liver Failure & Liver Disease
Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction (p 909-914)
Leon A. Adams, Ariel Feldstein, Keith D. Lindor, Paul Angulo
Patients with hypopituitarism develop a phenotype similar to metabolic syndrome with central obesity and diabetes. Similarly, patients with hypothalamic damage may develop central obesity, insulin resistance, and hyperphagia. We sought to examine the clinical associations between hypopituitarism, hypothalamic dysfunction, and nonalcoholic fatty liver disease (NAFLD). A case series of patients seen at our institution with diagnoses of hypopituitarism, hypothalamic obesity, or craniopharyngioma and NAFLD was undertaken. Clinical, laboratory, and liver biopsy features were reviewed. Twenty-one patients were identified. NAFLD was diagnosed 6.4 ± 7.5 years (median 3 years) after the diagnosis of hypothalamic/pituitary dysfunction. Mean gain in body mass index (BMI) between diagnoses of hypothalamic/pituitary disease and NAFLD was 11.3 ± 8.9 kg/m 2at an average yearly rate of 2.2 ± 2.2 kg/m 2. The majority of patients developed elevated glucose levels and dyslipidemia by time of diagnosis of NAFLD. Of the 10 patients biopsied, six were cirrhotic, two had nonalcoholic steatohepatitis (NASH) with fibrosis, and two had simple steatosis. Long-term follow-up of 66 ± 33 months (range 12-120) was available for 18 patients. Two required liver transplantation. Six patients died, two from liver related causes. In conclusion, patients with hypothalamic and/or pituitary disease are at risk of excessive weight gain, impaired glucose tolerance, and dyslipidemia with subsequent development of NAFLD. This group has a high prevalence of cirrhosis placing them at risk for liver-related death. The novel evidence that hypothalamic/pituitary dysfunction may be accompanied by progressive NAFLD has important implications for the work-up and management of patients with hypothalamic/pituitary disease. (HEPATOLOGY 2004;39:909-914.)
A randomized controlled trial of colchicine plus ursodiol versus methotrexate plus ursodiol in primary biliary cirrhosis: Ten-year results (p 915-923)
Marshall M. Kaplan, Steven Cheng, Lori Lyn Price, Peter A. L. Bonis
Primary biliary cirrhosis frequently progresses despite treatment with ursodeoxycholic acid (UDCA), the only approved therapy. Previous studies suggested that colchicine and methotrexate may improve biochemical tests of liver function, symptoms, and liver histology. The aim of the present study was to determine if the addition of colchicine or methotrexate to UDCA would improve survival free of liver transplantation. Eighty-five patients with histologically confirmed primary biliary cirrhosis whose serum alkaline phosphatase levels were at least twice the normal level and who were not yet candidates for liver transplantation were randomly assigned to receive colchicine or methotrexate in a double-blind study. UDCA was administered to all patients after 2 years. The primary end point was survival free of liver transplantation. Patients were followed up for a total of up to 10 years or until treatment failure. Data were analyzed on an intention-to-treat basis. Transplant-free survival was similar in both groups: 0.57 for colchicine plus UDCA and 0.44 for methotrexate plus UDCA, results that are similar to those predicted by the Mayo prognostic model. Significant improvement in liver biochemical tests and liver histology was observed in a subset of patients in both treatment groups who remained in the study for all 10 years. In conclusion, neither colchcine plus UDCA nor methotrexate plus UDCA improved survival beyond that predicted by the Mayo prognostic model. However, clinical, histologic, and biochemical improvement observed among those who remained in the study for 10 years suggests a possible benefit of these drugs in a subset of patients. (HEPATOLOGY 2004;39:915-923.)
Abnormal hepatocystin caused by truncating PRKCSH mutations leads to autosomal dominant polycystic liver disease (p 924-931)
Joost P. H. Drenth, Esa Tahvanainen, Rene H. M. te Morsche, Pia Tahvanainen, Helena Kääriäinen, Krister Höckerstedt, Jiddeke M. van de Kamp, Martijn H. Breuning, Jan B. M. J. Jansen
This study aims to compare the therapeutic effectiveness of continuous catheter drainage versus intermittent needle aspiration in the percutaneous treatment of pyogenic liver abscesses. Over a 5-year period, 64 consecutive patients with pyogenic liver abscess were treated with intravenous antibiotics (ampicillin, cefuroxime, and metronidazole) and randomized into two percutaneous treatment groups: continuous catheter drainage (with an 8F multi-sidehole pigtail catheter); and intermittent needle aspiration (18G disposable trocar needle). There was no statistically significant difference between the two groups regarding patient demographics, underlying coexisting disease, abscess size, abscess number, number of loculation of abscess, the presenting clinical symptoms such as fever, abdominal pain, and pretreatment liver function test. Although not statistically significant, the duration of intravenous antibiotics treatment before percutaneous treatment was longer with the catheter group, and the change of antibiotics after the sensitivity test was more frequent with the needle group. The needle group was associated with a higher treatment success rate, a shorter duration of hospital stay, and a lower mortality rate, although this did not reach statistical significance. In conclusion, this study suggests that intermittent needle aspiration is probably as effective as continuous catheter drainage for the treatment of pyogenic liver abscess, although further proof with a large-scale study is necessary. Due to the additional advantages of procedure simplicity, patient comfort, and reduced price, needle aspiration deserves to be considered as a first-line drainage approach. (HEPATOLOGY 2004;39:932-938.)
Treatment of pyogenic liver abscess: Prospective randomized comparison of catheter drainage and needle aspiration (p 932-938)
Simon C.H. Yu, Simon S.M. Ho, Wan Y. Lau, Deacons T.K. Yeung, Edmund H.Y. Yuen, Paul S.F. Lee, Constantine Metreweli
This study aims to compare the therapeutic effectiveness of continuous catheter drainage versus intermittent needle aspiration in the percutaneous treatment of pyogenic liver abscesses. Over a 5-year period, 64 consecutive patients with pyogenic liver abscess were treated with intravenous antibiotics (ampicillin, cefuroxime, and metronidazole) and randomized into two percutaneous treatment groups: continuous catheter drainage (with an 8F multi-sidehole pigtail catheter); and intermittent needle aspiration (18G disposable trocar needle). There was no statistically significant difference between the two groups regarding patient demographics, underlying coexisting disease, abscess size, abscess number, number of loculation of abscess, the presenting clinical symptoms such as fever, abdominal pain, and pretreatment liver function test. Although not statistically significant, the duration of intravenous antibiotics treatment before percutaneous treatment was longer with the catheter group, and the change of antibiotics after the sensitivity test was more frequent with the needle group. The needle group was associated with a higher treatment success rate, a shorter duration of hospital stay, and a lower mortality rate, although this did not reach statistical significance. In conclusion, this study suggests that intermittent needle aspiration is probably as effective as continuous catheter drainage for the treatment of pyogenic liver abscess, although further proof with a large-scale study is necessary. Due to the additional advantages of procedure simplicity, patient comfort, and reduced price, needle aspiration deserves to be considered as a first-line drainage approach. (HEPATOLOGY 2004;39:932-938.)
Prognostic value of altered oral glutamine challenge in patients with minimal hepatic encephalopathy (p 939-943)
Manuel Romero-Gómez, Lourdes Grande, Inés Camacho
Oral glutamine challenge (OGC) has been found to be safe, and an altered response predicts elevated risk of overt hepatic encephalopathy (HE) in patients with minimal hepatic encephalopathy (MHE). We assessed the survival prognosis of patients with cirrhosis, but without current overt HE, who have an altered OGC and MHE. MHE was inferred using 3 neuropsychological tests. Venous ammonia concentrations were measured pre- and post-60 minutes of a 10 g oral glutamine load. The median follow-up was 25.2 months, by which time 22 patients had had bouts of overt HE and 18 had died from liver-related causes. The results in 126 patients with cirrhosis, indicated 25 with MHE and abnormal OGC response. Survival among patients who developed overt HE was 59% at 1 year and 38% at 3 years. In patients without HE, survival was 96% and 86% at 1 and 3 years, respectively (log-rank 50.9, P< .0001). The presence of MHE was not related to survival (log-rank 2.21, P= .23). Patients with MHE and abnormal OGC test had elevated mortality risk (log-rank 13.1, P= .0003). Multivariate analyses indicated Child-Pugh score (hazard ratio [HR] 1.46; 95% CI, 1.46-2.08), and MHE plus altered OGC response (HR 5.5; 95% CI, 1.81-16.6) were predictors of mortality, whether from liver-related or non-liver-related causes. In conclusion, a pathological OGC response in patients with MHE appears to be associated with lower survival rate and may prove useful in the selection of candidates for liver transplantation. (HEPATOLOGY 2004;39:939-943.)
Identification of discriminators of hepatoma by gene expression profiling using a minimal dataset approach (p 944-953)
Soek Ying Neo, Chon Kar Leow, Vinsensius B. Vega, Philip M. Long, Amirul F.M. Islam, Paul B.S. Lai, Edison T. Liu, Ee Chee Ren
The severity of hepatocellular carcinoma (HCC) and the lack of good diagnostic markers and treatment strategies have rendered the disease a major challenge. Previous microarray analyses of HCC were restricted to the selected tissue sample sets without validation on an independent series of tissue samples. We describe an approach to the identification of a composite discriminator cassette by intersecting different microarray datasets. We studied the global transcriptional profiles of matched HCC tumor and nontumor liver samples from 37 patients using cDNA (cDNA) microarrays. Application of nonparametric Wilcoxon statistical analyses ( P< 1 ? 10 -6 ) and the criteria of 1.5-fold differential gene expression change resulted in the identification of 218 genes, including BMI-1, ERBB3, and those involved in the ubiquitin-proteasome pathway. Elevated ERBB2 and epidermal growth factor receptor (EGFR) expression levels were detected in ERBB3-expressing tumors, suggesting the presence of ERBB3 cognate partners. Comparison of our dataset with an earlier study of approximately 150 tissue sets identified multiple overlapping discriminator markers, suggesting good concordance of data despite differences in patient populations and technology platforms. These overlapping discriminator markers could distinguish HCC tumor from nontumor liver samples with reasonable precision and the features were unlikely to appear by chance, as measured by Monte Carlo simulations. More significantly, validation of the discriminator cassettes on an independent set of 58 liver biopsy specimens yielded greater than 93% prediction accuracy. In conclusion, these data indicate the robustness of expression profiling in marker discovery using limited patient tissue specimens as well as identify novel genes that are highly likely to be excellent markers for HCC diagnosis and treatment.
Coordinate expression of regulatory genes differentiates embryonic and perinatal forms of biliary atresia (p 954-962)
Dong-Yi Zhang, Gregg Sabla, Pranavkumar Shivakumar, Greg Tiao, Ronald J. Sokol, Cara Mack, Benjamin L. Shneider, Bruce Aronow, Jorge A. Bezerra
The molecular basis for the embryonic and perinatal clinical forms of biliary atresia is largely undefined. In this study, we aimed to: 1) determine if the clinical forms can be differentiated at the transcriptional level, and 2) search for molecular mechanisms underlying phenotypic differences. To this end, we generated biotinylated cRNA probes from livers of age-matched infants with the embryonic (n = 5) and perinatal (n = 6) forms of biliary atresia at the time of diagnosis and hybridized them against the Affymetrix human HG-U133 A and B microarrays containing 44,760 gene products. Data filtering and two-way cluster analysis of the gene expression platform identified 230 genes with an expression profile that is highly distinctive of the clinical phenotypes. Functionally, the profile did not reveal a higher-order function for a specific cell type; instead, it uncovered a coordinated expression of regulatory genes. These regulatory genes were predominantly represented in the embryonic form (45% of genes), with a unique pattern of expression of genes involved in chromatin integrity/function ( Smarca-1, Rybp , and Hdac3 ) and the uniform overexpression of five imprinted genes ( Igf2, Peg3, Peg10, Meg3 , and IPW ), implying a failure to downregulate embryonic gene programs. In conclusion, embryonic and perinatal forms of biliary atresia are distinguished by gene expression profiling. The coordinate expression of regulators of chromatin structure/function and of imprinted genes provides evidence for a transcriptional basis for the pathogenesis of the embryonic form of biliary atresia. Further studies exploring these biological processes are required to determine the significance of these findings. Supplementary material for this article can be found at http://genet.cchmc.org . (HEPATOLOGY 2004;39:954-962.)
Immunohistochemical analysis of Mallory bodies in Wilsonian and non-Wilsonian hepatic copper toxicosis (p 963-969)
Thomas Müller, Cord Langner, Andrea Fuchsbichler, Peter Heinz-Erian, Helmut Ellemunter, Barbara Schlenck, Ashish R. Bavdekar, Avinash M. Pradhan, Anand Pandit, Josef Müller-Höcker, Michael Melter, Kunihiko Kobayashi, Hironori Nagasaka, Hideaki Kikuta, Wilfried Müller, M. Stuart Tanner, Irmin Sternlieb, Kurt Zatloukal, Helmut Denk
Patients with Wilson's disease (WD), Indian childhood cirrhosis (ICC), and idiopathic copper toxicosis (ICT) develop severe liver disease morphologically characterized by ballooning of hepatocytes, inflammation, cytoskeletal alterations, and Mallory body (MB) formation, finally leading to mostly micronodular cirrhosis. The pathogenesis of MBs in copper toxicosis is still unresolved. Immunohistochemical analysis of MBs in different types of copper intoxication revealed that keratin, p62, and ubiquitin are integral components. Thus MBs associated with copper intoxication resemble those present in alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH). p62 is a multifunctional immediate early gene product that, on the one hand, is involved in stress-induced cell signaling (particularly that of oxidative stress) by acting as an adapter protein linking receptor-interacting protein (RIP) with the atypical protein kinase C. On the other hand, p62 binds with high affinity to polyubiquitin and ubiquitinated proteins. In conclusion, p62 accumulation in WD, ICC, and ICT and deposition in MBs indicates a central role of protein misfolding induced by oxidative stress in copper-induced liver toxicity. By sequestering potentially harmful misfolded ubiquitinated proteins as inert cytoplasmic inclusion bodies ( e.g. , as MBs), p62 may be a major player in an important cellular rescue mechanism in oxidative hepatocyte injury. (HEPATOLOGY 2004;39;963-969.)
Variation of hepatic glucuronidation: Novel functional polymorphisms of the UDP-glucuronosyltransferase UGT1A4 (p 970-977)
Ursula Ehmer, Arndt Vogel, Jan Karl Schütte, Britta Krone, Michael P. Manns, Christian P. Strassburg
UDP-glucuronosyltransferases are a family of drug metabolizing enzymes contributing to hepatic drug metabolism and protection against environmental toxins. The aim of this study was to identify polymorphisms at the human UGT1A gene locus and to characterize their function and potential association with hepatocellular carcinoma (HCC). Genomic DNA from the blood of 363 subjects (128 patients with HCC, 235 blood donors) was analyzed for polymorphisms of the UGT1A3, UGT1A4, UGT1A8, UGT1A9, UGT1A10 genes using polymerase chain reaction, sequencing analysis. Recombinant variant UGT protein was analyzed by activity assays. In the UGT1A8 gene an A173G variant and a conserved G to A exchange at position 765 were detected in 25% and 15%. UGT1A9 exhibited two variants C3Y and M33T in 1% and 3%. UGT1A10 exhibited conserved nucleotide exchanges (128 G A and 696 C T) in 2% and 13%. In the UGT1A3 gene a W11R, a V47A variant, and a conserved G to A exchange at position 81 with an incidence of 65%, 58%, and 65%, respectively, were identified. UGT1A4 exhibited a P24T and an L48V variant in 8% and 9%. UGT1A SNPs were not associated with HCC. UGT1A4 P24T and L48V exhibited reduced glucuronidation activities: -naphthylamine 30% and 50%, and dihydrotestosterone 50% and 0%, respectively. In conclusion, the high prevalence of SNPs throughout the human UGT1A gene locus illustrates a genetic basis of interindividual variations of hepatic metabolism. Two polymorphisms of the hepatic UGT1A4 protein show a differential metabolic activity toward mutagenic amines and endogenous steroids, altering hepatic metabolism and detoxification. (HEPATOLOGY 2004;39:970-977.)
Viral Hepatitis
HLA and cytokine gene polymorphisms are independently associated with responses to hepatitis B vaccination (p 978-988)
Chengbin Wang, Jianming Tang, Wei Song, Elena Lobashevsky, Craig M. Wilson, Richard A. Kaslow
Variable immune responses to hepatitis B virus (HBV) infection and recombinant HBV vaccines have been associated with polymorphisms in several genes within the human leukocyte antigen (HLA) complex. Analyses of polymerase chain reaction (PCR)-based genotyping data from 164 North American adolescents vaccinated with recombinant HBV products confirmed that HLA-DRB1*07 (relative odds [RO] = 5.18, P< .0001) and human immunodeficiency virus type 1 (HIV-1) infection (RO = 3.91, P< .001) were both associated with nonresponse to full-dose vaccination. Further associations were observed with single nucleotide polymorphisms (SNPs) at the IL2 and IL4 loci along with insertion/deletion variants at the IL12B locus ( P= .003-.01). Host genetic associations were independent of one another as well as other HLA (A,B,C, and DQB1 ) and cytokine gene ( IL4R, IL6, IL10 , and TNF ) variants. Statistical adjustments for nongenetic factors (gender, ethnicity, age, HIV-1 infection, and vaccination protocols) did not substantially alter the strengths of the genetic relationships. The overall distribution pattern of genetic variations was similar between the analyzed vaccinees and additional adolescents ( n= 292) from the same cohort. In conclusion, DRB1*07 (or a closely linked allele) and immunoregulatory cytokine gene polymorphisms correlate with variable immune response to recombinant HBV vaccines. (HEPATOLOGY 2004;39:978-988.)
Treatment of chronic hepatitis C in HIV/HCV-coinfection with interferon -2b+ full-course vs. 16-week delayed ribavirin (p 989-998)
Norbert Bräu, Maribel Rodriguez-Torres, Dale Prokupek, Maurizio Bonacini, Carol A. Giffen, Jeffery J. Smith, Kevin R. Frost, Jay R. Kostman
Human immunodeficiency virus (HIV)-infected patients increasingly experience the consequences of chronic hepatitis C virus (HCV) coinfection. This trial randomized 107 patients coinfected with HIV and HCV to receive 48 weeks of interferon alfa-2b (IFN) 3 million units three times weekly plus either a full course of ribavirin (RBV) at 800 mg/day (group A; n = 53) or 16 weeks of placebo, followed by RBV (group B; n = 54). The primary endpoint of sustained viral response (SVR) rate (undetectable HCV RNA at posttreatment week 24) was not different between groups A (11.3%) and B (5.6%; P= .32). Within group A, the SVR rate was lower in genotype 1 (2.5%) than in genotypes 2 through 4 (41.7%; P= .002). Fifty-five patients discontinued therapy prematurely, mostly because of adverse events or patient decisions. At treatment week 12, the percentage of CD4+ cells rose in group A (+4.1%; P< .001), but not in group B (-0.3%). A significant proportion (22%) of patients who were HIV viremic at baseline had undetectable HIV RNA at week 12. By week 16, the hemoglobin level decreased more in group A (-2,52 g/dL) than in group B (-1.02 g/dL; P< .001). In group A, the hemoglobin decline was steeper in patients receiving zidovudine (azidothymidine [AZT], -3.64 g/dL vs. no AZT, -2.08 g/dL), and patients receiving zidovudine had more anemia-related RBV dose reductions (AZT, 60% vs. no AZT, 16%). In conclusion, HCV therapy with IFN plus RBV is relatively safe in patients coinfected with HIV and HCV, but frequent treatment discontinuations and anemia-related RBV dose reductions contribute to a poor SVR rate. Control of HIV infection improves rather than worsens during therapy. (HEPATOLOGY 2004;39:989-998.)
Hepatitis C virus-infected patients report communication problems with physicians (p 999-1007)
Susan Zickmund, Stephen L. Hillis, Mitchell J. Barnett, Laura Ippolito, Douglas R. LaBrecque
We examined the prevalence and nature of perceived problems in the interaction between physicians and patients diagnosed with hepatitis C virus (HCV) infection. This cross-sectional study included 322 outpatients diagnosed with chronic HCV infection and treated at a tertiary referral hospital's hepatology clinic. Patients were asked to provide demographic information and to complete a semistructured interview, the Sickness Impact Profile (SIP) and Hospital Anxiety Depression (HAD) scale. A team of two blinded coders analyzed the interviews. A total of 131 (41%) study patients reported communication difficulties with physicians involved in their care. The main difficulties were the poor communication skills of physicians (91 [28%]), physician incompetence regarding the diagnosis and treatment of HCV infection (74 [23%]), feelings of being misdiagnosed, misled, or abandoned (51 [16%]), or being stigmatized by their physician (29 [9%]). Patients were twice as likely to report difficulties with subspecialists as compared with generalists. Nonresponse with antiviral therapy correlated with perceived physician conflict even after adjusting for treatment in relation to the time of interview, whereas previous or ongoing substance abuse and mode of acquisition did not. In a multivariate model, patients' psychosocial problems were the best predictors of communication difficulties. In conclusion, a substantial number of patients with HCV infection report difficulties when interacting with physicians, which may be due to coexisting emotional or social problems. However, perceived stigmatization by physicians and a sense of abandonment reflect the need for further educational efforts. These should target both specialists and primary care providers to inform them about the psychosocial challenges facing these patients. (HEPATOLOGY 2004;39:999-1007.)
Integration of woodchuck hepatitis and N-myc rearrangement determine size and histologic grade of hepatic tumors (p 1008-1016)
James R. Jacob, Agnes Sterczer, Ilia A. Toshkov, Amy E. Yeager, Brent E. Korba, Paul J. Cote, Marie-Annick Buendia, John L. Gerin, Bud C. Tennant
ntegrations of woodchuck hepatitis virus (WHV) DNA and rearrangements of the N-myc 2 gene have been detected frequently in hepatocellular carcinoma (HCC) of Eastern woodchucks ( Marmota monax ) chronically infected with WHV. Fifty-five hepatocellular neoplasms and matched nontumor hepatic tissue specimens obtained postmortem from 13 chronic WHV carriers were analyzed and the frequency of WHV DNA integrations and of N-myc rearrangements compared in tumors of different size and histologic grade. Four small tumor nodules were classified histologically as adenomas and integrated sequences of WHV DNA were detected in two of the four tumor nodules. In one of the two nodules, there was evidence of N-myc rearrangement. Fifty-one neoplasms were classified as HCC. Seven were grade 1 HCCs. WHV DNA integrations were demonstrated in 43% but none had N-myc rearrangements. Twenty grade 2 HCCs had WHV DNA integrations in 80% and in 38% N-myc rearrangements were present. Twenty-four grade 3 HCCs had integrations of WHV DNA in 79% and N-myc rearrangements in 74%. In two other grade 3 HCCs, rearrangements of N-myc were detected in the absence of WHV DNA integrations. The 12 largest tumors in the series all were grade 2 or 3 HCCs, and in 83%, both WHV DNA integrations and N-myc rearrangements were demonstrated. In conclusion, molecular changes observed in this study suggest a progression of genetic alterations providing either a significant proliferative stimulation and/or a growth advantage in hepatocarcinogenesis of woodchucks with chronic WHV infection. (HEPATOLOGY 2004;39:1008-1016.)
Kupffer cells required for high affinity peptide-induced deletion, not retention, of activated CD8+ T cells by mouse liver (p 1017-1027)
Yuhshi Kuniyasu, Suhail Mohammed Marfani, Irteza Bin Inayat, Shehzad Zafar Sheikh, Wajahat Zafar Mehal
The immune response to foreign antigens in the liver is often suboptimal and this is clinically relevant in chronic persistence of hepatotropic viruses. In chronic infection with the hepatitis C virus, activated CD8+ T cells specific for viral epitopes are present in the peripheral blood and the liver, yet viral clearance is unusual. To define the fate of activated CD8+ entering the liver, we developed a mouse model of portal vein injection of activated CD8+ T cells in vivo . Activated CD8+ T cells are retained very efficiently by the liver and undergo an approximately 8-fold expansion in the first 48 hours. This expansion is followed by apoptosis and a decline in numbers of the retained cells over the next 4 days. The presence of high affinity (HA) antigen does not affect the initial retention by the liver but greatly limits the expansion in the first 48 hours by increasing apoptosis of the retained cells. In the absence of Kupffer cells, the initial retention and expansion are unchanged, but HA antigen does not limit the expansion of the liver CD8+ T cell pool. In conclusion, these data identify a previously unknown phase of CD8+ T cell expansion after entering the liver, demonstrate that HA antigen limits the hepatic CD8+ T cell pool by inducing apoptosis, and that this effect requires Kupffer cells. Interfering with antigen presentation by Kupffer cells may be a strategy to limit HA antigen-induced deletion of activated CD8+ T cells entering the liver. (HEPATOLOGY 2004;39;1017-1027.)
Liver Biology & Pathology
Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation (p 1028-1037)
Zichen Zhang, Guan-Hua Lai, Alphonse E. Sirica
Recently, we demonstrated that the cyclooxygenase-2 (COX-2) inhibitor celecoxib acts to significantly suppress the growth of rat C611B cholangiocarcinoma (ChC) cells in vitro . To establish a molecular mechanism for this growth suppression, we investigated the effects of celecoxib on apoptotic signaling pathways in cultured rat C611B ChC cells. Celecoxib and another COX-2 inhibitor, rofecoxib, at 5 M were almost equally effective in inhibiting prostaglandin E 2(PGE 2) production by these cells, but at this low concentration, neither inhibitor suppressed growth or induced apoptosis. Celecoxib at 50 M induced prominent apoptosis in these cells, whereas rofecoxib at 50 M was without effect in either suppressing growth or inducing apoptosis. Celecoxib (50 M) did not alter Bcl-2, Bcl-x L, or COX-2 protein levels, nor did it inhibit p42/44 mitogen-activated protein kinase (MAPK) phosphorylation; however, it significantly suppressed serine/threonine kinase Akt/PKB (Akt) phosphorylation and kinase activity in cultured C611B cells. This effect, in turn, directly correlated with Bax translocation to mitochondria, cytochrome crelease into cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Addition of 25 M PGE 2to C611B cell cultures blocked the apoptotic actions of celecoxib. Rofecoxib (50 M) was without effect in suppressing Akt phosphorylation and caspase-3 activation. In vivo , celecoxib partially suppressed tumorigenic growth of C611B ChC cells. In conclusion, our results indicate that celecoxib preferentially acts in vitro to induce apoptosis in ChC cells through a mechanism involving Akt inactivation, Bax translocation, and cytochrome crelease. Our in vivo results further suggest celecoxib might have potential therapeutic or chemopreventive value against ChC. (HEPATOLOGY 2004;39:1028-1037.)
Progression of HCC in mice is associated with a downregulation in the expression of hepatocyte nuclear factors (p 1038-1047)
Natalia L. Lazarevich, Oksana A. Cheremnova, Ekaterina V. Varga, Dmitry A. Ovchinnikov, Elena I. Kudrjavtseva, Olga V. Morozova, Daria I. Fleishman, Natalia V. Engelhardt, Stephen A. Duncan
Hepatocyte nuclear factors (HNF) play a critical role in development of the liver. Their roles during liver tumorigenesis and progression of hepatocellular carcinomas (HCC) are, however, poorly understood. To address the role of HNFs in tumor progression, we generated a new experimental model in which a highly differentiated slow-growing transplantable mouse HCC (sgHCC) rapidly gives rise in vivo to a highly invasive fast-growing dedifferentiated variant (fgHCC). This in vivo model has allowed us to investigate the fundamental mechanisms underlying HCC progression. A complete loss of cell polarity, a decrease in cell-cell and cell-extracellular matrix (ECM) adhesion, elevation of telomerase activity, and extinction of liver-specific gene expression accompanies tumor progression. Moreover, cells isolated from fgHCCs acquired the ability to proliferate rapidly in culture. These alterations were coupled with a reduced expression of several liver transcription factors including HNF4, a factor essential for hepatocyte differentiation. Forced re-expression of HNF4 1 in cultured fgHCC cells reversed the progressive phenotype and induced fgHCC cells to re-establish an epithelium and reform cell-ECM contacts. Moreover, fgHCC cells that expressed HNF4 1 also re-established expression of the profile of liver transcription factors and hepatic genes that are associated with a differentiated hepatocyte phenotype. Importantly, re-expression of HNF4 1 in fgHCC reduced the proliferation rate in vitro and diminished tumor formation in congenic recipient mice. In conclusion, loss of HNF4 expression is an important determinant of HCC progression. Forced expression of this factor can promote reversion of tumors toward a less invasive highly differentiated slow-growing phenotype. (HEPATOLOGY 2004;39;1038-1047.)
Analyses of hepatocellular proliferation in a mouse model of -1-antitrypsin deficiency (p 1048-1055)
David A. Rudnick, Yunjun Liao, Jae-Koo An, Louis J. Muglia, David H. Perlmutter, Jeffrey H. Teckman
Published Online: 25 Mar 2004
-1-Antitrypsin ( 1-AT) deficiency is the most common cause of metabolic pediatric liver disease. Hepatocellular injury is caused by toxicity of the mutant -1-antitrypsin Z ( 1-ATZ) molecule retained within hepatocytes. In these studies, we used the PiZ transgenic mouse model of 1-AT deficiency to examine hepatocellular proliferation in response to chronic liver injury resulting from this metabolic disease. The results showed increased hepatocellular proliferation and caspase 9 activation in male PiZ mice compared with female PiZ and wild-type mice. Hepatic 1-AT mRNA and protein expression also were increased in male PiZ mice, suggesting that greater hepatocellular proliferation and caspase activation in males results from increased hepatotoxicity associated with greater intracellular 1-ATZ accumulation. Testosterone treatment of female PiZ mice increased 1-ATZ expression and hepatocellular proliferation to a level comparable with that in males. In PiZ mice, hepatocytes devoid of intracellular 1-AT globules had a proliferative advantage compared with globule-containing hepatocytes. However, this advantage is relative because both globule-containing and globule-devoid hepatocytes exhibited comparable proliferation after partial hepatectomy. In conclusion, these data indicate that intracellular retention of mutant 1-ATZ is associated with a regenerative stimulus leading to increased hepatocellular proliferation, that gender-specific signals influence the degree of 1-AT expression and associated hepatic injury, and that hepatocytes devoid of 1-ATZ have a proliferative advantage over cells that accumulate the mutant protein. This selective proliferation suggests that hepatocellular transplantation may be applicable for treatment of this and other slowly progressive metabolic liver diseases (HEPATOLOGY 2004;39:1048-1055.)
Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration (p 1056-1065)
Christoph Köhler, Aaron W. Bell, William C. Bowen, Satdarshan P. Monga, Wolfgang Fleig, George K. Michalopoulos
The Notch/Jagged signaling pathway is important for cellular differentiation and proliferation. Its dysfunction is associated with human pathologies in several tissues including liver. Point mutations in Jagged-1 gene are the cause for Alagille syndrome, associated with paucity of intrahepatic bile ducts. To determine the putative role of the trans-membrane receptor Notch and its ligand Jagged-1 in liver regeneration, we investigated the expression of Notch and Jagged-1 in rat liver following 2/3 partial hepatectomy. Immunohistochemical staining of normal rat liver showed that Notch was expressed in hepatocytes, bile duct cells and endothelial cells, whereas Jagged-1 was expressed in bile duct cells and hepatocytes. Both Notch-1 and Jagged-1 proteins were upregulated in hepatocytes after partial hepatectomy up to day 4. After partial hepatectomy, nuclear translocation of the intracellular cytoplasmic domain of Notch (NICD) increased and peaked within 15 minutes, indicating the activation of Notch. Expression of the Notch-dependent target gene (HES-1) expression increased within 30-60 minutes. Addition of recombinant Jagged-1 protein to primary cultures of hepatocytes stimulated hepatocyte DNA synthesis. Furthermore, injection of silencing RNA for Notch and Jagged-1 to livers 2 days before partial hepatectomy significantly suppressed proliferation of hepatocytes at days 2 to 4 of the regenerative response. In conclusion, Notch/Jagged signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting cell growth and differentiation.
A murine model of type 2 autoimmune hepatitis: Xenoimmunization with human antigens (p 1066-1074)
Pascal Lapierre, Idriss Djilali-Saiah, Susana Vitozzi, Fernando Alvarez
Autoimmune hepatitis (AIH) is characterized by an immune-mediated injury of the hepatic parenchyma of unknown pathogenesis. Type 2 AIH is identified by the presence of anti-liver-kidney microsomes type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) autoantibodies. The current study shows that a murine model of AIH can be generated by DNA immunization against type 2 AIH self-antigens (P450 2D6 and formiminotransferase-cyclodeaminase). A pCMV plasmid containing the N-terminal region of mouse CTLA-4 and the antigenic region of human CYP2D6 (672-1,377 bp) and human formiminotransferase cyclodeaminase (FTCD; 1,232-1,668 bp) was used for DNA immunization of C57BL/6 female mice. Immunized mice showed elevated levels of alanine aminotransferase (ALT), with peaks at 4 and 7 months postinjection. Periportal, portal, and intralobular liver inflammatory infiltrates were observed at histology. Mainly CD4+ lymphocytes, but also CD8+ and B lymphocytes, were found in the liver. Cytotoxic-specific T cells were found in both the liver and spleen of these animals. Mice developed anti-LKM1 and anti-LC1 antibodies of immunoglobulin G2 (IgG2) subclass, against specific mouse autoantigens. The ALT levels correlated with both the presence of anti-LKM1/anti-LC1 antibodies and the presence of liver necroinflammation. In conclusion, in mice, DNA immunization against human autoantigens breaks tolerance and induces an autoimmune liver disease. Molecular mimicry between foreign and self-antigens explains the liver injury. This model of AIH resembles human type 2 AIH and will be helpful for the study of its pathogenesis. (HEPATOLOGY 2004;39:1066-1074.)
Increased vascular heme oxygenase-1 expression contributes to arterial vasodilation in experimental cirrhosis in rats (p 1075-1087)
Yung-Chang Chen, Pere Ginès, Jianhui Yang, Sandra N. Summer, Sandor Falk, Nash S. Russell, Robert W. Schrier
Vascular heme oxygenase (HO) regulates vascular tone in normal conditions and in some pathologic circumstances ( e.g ., sepsis). However, its possible role in the pathogenesis of arterial vasodilation in cirrhosis is unknown. To address this question, the expression and activity of HO in arterial vessels was studied in rats at 1, 2, and 4 weeks after bile duct ligation (BDL) or sham operation. A progressively increased expression of HO-1 was found in aorta and mesenteric arteries of BDL rats in a close chronologic relationship with the progression from acute cholestatic liver injury (1 week) to the fully developed cirrhosis with intense systemic arterial vasodilation (4 weeks). No changes were found in the expression of the constitutive isoform HO-2. HO-1 was mainly located in vascular smooth muscle cells of the arterial wall. Aortic HO activity increased in parallel with the expression of HO-1 (up to 600% in rats with cirrhosis compared with sham rats) and correlated with hemodynamic parameters. Increased expression of HO-1 and HO activity were also found in other organs, such as liver and spleen, though to a lesser extent compared with vascular tissue. The acute administration of an inhibitor of HO to cirrhotic rats, at a dose that normalized aortic HO activity, was associated with significantly greater effects on arterial pressure, total peripheral vascular resistance, and cardiac index, compared with effects in sham rats. In conclusion, these findings are consistent with a role for HO in the pathogenesis of arterial vasodilation in cirrhosis. (HEPATOLOGY 2004;39:1075-1087.)
5-methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes (p 1088-1098)
Henar Hevia, Marta Varela-Rey, Fernando J. Corrales, Carmen Berasain, María L. Martínez-Chantar, M. Ujue Latasa, Shelly C. Lu, José M. Mato, Elena R. García-Trevijano, Matías A. Avila
Although Kupffer cells (KCs) may play a crucial role in post-cold ischemic hepatocellular injury, their role in nonnecrotic graft dysfunction remains unknown. This study examined reveal the role of KC in post-cold ischemic liver grafts. Rat livers treated with or without liposome-encapsulated dichloromethylene diphosphonate, a KC-depleting reagent, were stored in University of Wisconsin (UW) solution at 4°C for 8 to 24 hours and reperfused while monitoring biliary output and constituents. The ability of hepatocytes to excrete bile was assessed through laser-confocal microfluorography in situ . Cold ischemia-reperfused grafts decreased their bile output significantly at 8 hours without any notable cell injury. This event coincided with impaired excretion of glutathione and bilirubin-IX (BR-IX ), suggesting delayed transport of these organic anions. We examined whether intracellular relocalization of multidrug resistance protein-2 (Mrp2) occurred. Kinetic analyses for biliary excretion of carboxyfluorescein, a fluoroprobe excreted through this transporter, revealed significant delay of dye excretion from hepatocytes into bile canaliculi. The KC-depleting treatment significantly attenuated this decline in biliary anion transport mediated through Mrp2 in the 8-hour cold ischemic grafts via redistribution of Mrp2 from the cytoplasm to the canalicular membrane. Furthermore, thromboxane A 2(TXA 2) synthase in KC appeared involved as blocking this enzyme improved 5-carboxyfluorescein excretion while cytoplasmic internalization of Mrp2 disappeared in the KC-depleting grafts. In conclusion, KC activation is an important determinant of nonnecrotic hepatocellular dysfunction, jeopardizing homeostasis of the detoxification capacity and organic anion metabolism of the post-cold ischemic grafts. (HEPATOLOGY 2004;39;1099-1109.)
Kupffer cells alter organic anion transport through multidrug resistance protein 2 in the post-cold ischemic rat liver (p 1099-1109)
Atsushi Kudo, Satoshi Kashiwagi, Mayumi Kajimura, Yasunori Yoshimura, Koji Uchida, Shigeki Arii, Makoto Suematsu
Although Kupffer cells (KCs) may play a crucial role in post-cold ischemic hepatocellular injury, their role in nonnecrotic graft dysfunction remains unknown. This study examined reveal the role of KC in post-cold ischemic liver grafts. Rat livers treated with or without liposome-encapsulated dichloromethylene diphosphonate, a KC-depleting reagent, were stored in University of Wisconsin (UW) solution at 4°C for 8 to 24 hours and reperfused while monitoring biliary output and constituents. The ability of hepatocytes to excrete bile was assessed through laser-confocal microfluorography in situ . Cold ischemia-reperfused grafts decreased their bile output significantly at 8 hours without any notable cell injury. This event coincided with impaired excretion of glutathione and bilirubin-IX (BR-IX ), suggesting delayed transport of these organic anions. We examined whether intracellular relocalization of multidrug resistance protein-2 (Mrp2) occurred. Kinetic analyses for biliary excretion of carboxyfluorescein, a fluoroprobe excreted through this transporter, revealed significant delay of dye excretion from hepatocytes into bile canaliculi. The KC-depleting treatment significantly attenuated this decline in biliary anion transport mediated through Mrp2 in the 8-hour cold ischemic grafts via redistribution of Mrp2 from the cytoplasm to the canalicular membrane. Furthermore, thromboxane A 2(TXA 2) synthase in KC appeared involved as blocking this enzyme improved 5-carboxyfluorescein excretion while cytoplasmic internalization of Mrp2 disappeared in the KC-depleting grafts. In conclusion, KC activation is an important determinant of nonnecrotic hepatocellular dysfunction, jeopardizing homeostasis of the detoxification capacity and organic anion metabolism of the post-cold ischemic grafts. (HEPATOLOGY 2004;39;1099-1109.)
Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver (p 1110-1119)
Pierre-Michel Huet, Marcia R. Nagaoka, Geneviève Desbiens, Esther Tarrab, Antoine Brault, Marie-Pierre Bralet, Marc Bilodeau
Cold ischemia-warm reperfusion (CI-WR) injury of the liver is characterized by marked alterations of sinusoidal endothelial cells (SECs), whereas hepatocytes appear to be relatively unscathed. However, the time course and mechanism of cell death remain controversial: early versus late phenomenon, necrosis versus apoptosis? We describe the occurrence and nature of cell death after different periods of CI with University of Wisconsin (UW) solution and after different periods of WR in the isolated perfused rat liver model. After 24- and 42-hour CI (viable and nonviable livers, respectively), similar patterns of liver cell death were seen: SEC necrosis appeared early after WR (10 minutes) and remained stable for up to 120 minutes. After 30 minutes of WR, apoptosis increased progressively with WR length. Based on morphological criteria, apoptotic cells were mainly hepatocytes within liver plates or extruded in the sinusoidal lumen. In addition, only after 42-hour CI were large clusters of necrotic hepatocytes found in areas of congested sinusoids. In these same livers, the hepatic microcirculation, evaluated by means of the multiple-indicator dilution technique, revealed extracellular matrix disappearance with no-flow areas. In conclusion, different time courses and mechanisms of cell death occur in rat livers after CI-WR, with early SEC necrosis followed by delayed hepatocyte apoptosis. These processes do not appear to be of major importance in the mechanism of graft failure because they are similar under both nonlethal and lethal conditions; this is not the case for the loss of the extracellular matrix found only under lethal conditions and associated with hepatocyte necrosis. (HEPATOLOGY 2004;39:1110-1119.)
Severe hyperthyroidism induces mitochondria-mediated apoptosis in rat liver (p 1120-1130)
Geeta Upadhyay, Rajesh Singh, Ashok Kumar, Sanjeev Kumar, Amit Kapoor, Madan M. Godbole
Thyrotoxicosis may be associated with a variety of abnormalities of liver function. The pathogenesis of hepatic dysfunction in thyrotoxicosis is unknown, but has been attributed to mitochondrial dysfunction. We studied the effect of altered thyroid function on the apoptotic index in rat liver. Extensive DNA fragmentation and significantly increased caspase-3 activity ( P< .001) and caspase-9 activation ( P< .005) were observed in hyperthyroid rat liver; cell death by apoptosis was confirmed. In hyperthyroid rat liver, 60% of mitochondria exhibited disruption of their outer membranes and a decrease in the number of cristae. These findings, along with significant translocation of cytochrome cand second mitochondria-derived activator of caspases to cytosol ( P< .005), suggest activation of a mitochondrial-mediated pathway. However, no change in the expression levels of Bcl-2, Bax, and Bcl-x Lwere found in hyperthyroidism. For in vitro experiments, rat liver mitochondria were isolated and purified in sucrose density gradients and were treated with triiodothyronine (T3; 2-8 M). T3 treatment resulted in an abrupt increase in mitochondrial permeability transition. Using a cell-free apoptosis system, the apoptogenic nature of proteins released from mitochondria was confirmed by observing changes in nuclear morphologic features and DNA fragmentation. Proteins released by 6 M T3 contained significantly increased amounts of cytochrome c(P< .01) and induced apoptotic changes in 67% of nuclei. In conclusion, using in vivo and in vitro approaches, we provide evidence that excess T3 causes liver dysfunction by inducing apoptosis, as a result of activation of a mitochondria-dependent pathway. Thus, the results of this study provide an explanation for liver dysfunction associated with hyperthyroidism. (HEPATOLOGY 2004;39:1120-1130.)
Liver Biology & Pathobiology
Electric foot shock stress-induced exacerbation of -galactosylceramide-triggered apoptosis in mouse liver (p 1131-1140)
Yoichi Chida, Nobuyuki Sudo, Junko Sonoda, Hiroshi Sogawa, Chiharu Kubo
Recently, liver natural killer T (NKT) cells, which are specifically stimulated by -galactosylceramide ( -GalCer), were found to play a critical role in intrahepatic immunity to several infections and certain hepatic disorders. However, the role of psychophysical stress on NKT cell-dependent liver injury induced by -GalCer still remains to be elucidated. In this study, we employed inescapable electric foot shock as the mode of psychophysical stress and evaluated its effect on -GalCer-induced hepatitis. Pre-exposure of 12 hours of foot shock stress before -GalCer administration significantly enhanced -GalCer-triggered increase in serum alanine aminotransferase levels, followed by increases in both liver caspase-3 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive hepatocytes, thus indicating that the liver NKT cell-dependent apoptotic response was exacerbated by stress. Foot shock stress also significantly increased both the number of liver NKT cells and Fas expression levels on hepatocytes. Pretreatment with RU-486, a glucocorticoid (GC) receptor antagonist, completely reversed such stress-induced enhancement of the -GalCer-triggered serum alanine aminotransferase and hepatocyte Fas antigen responses. In contrast, such a reversal effect was not found in the mice pretreated with naloxone, a -opioid receptor antagonist, which thus suggests that an elevation of endogenous GCs, but not -endorphin, as responsible for such stress-induced aggravation in mouse hepatitis models. In conclusion, foot shock stress-induced elevation of endogenous GCs exacerbates -GalCer-initiated hepatic apoptosis through the expansion of liver NKT cells and the up-regulation of hepatocyte Fas antigen. (HEPATOLOGY 2004;39:1131-1140.)
Save Article Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for Avril 2004 • Volume 126 • Number 4
Rapid Communication
A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis
Stuart J. Forbes, Francesco P. Russo, Virginia Rey, Patrizia Burra, Massimo Rugge, Nicholas A. Wright, Malcolm R. Alison
Background & aims: Myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. We have analyzed the origin of myofibroblasts within fibrotic liver in 2 scenarios: (1) 7 male patients (hepatitis B; hepatitis B and D; Wilson’s disease; hepatitis B, D, and C; and 3 with hepatitis C) who received liver transplants from female donors and subsequently developed liver fibrosis and (2) a female patient who received a bone marrow transplant from a male donor and subsequently developed hepatitis Cinduced cirrhosis. Methods: Through the use of in situ hybridization for the Y chromosome, we have tracked male cells of extrahepatic origin. The phenotype of these male cells was examined by immunohistochemistry using a panel of antibodies against smooth muscle actin ( SMA), vimentin, fibulin-2, and leukocyte common antigen (CD45). Confocal microscopy was performed to confirm the location of the Y chromosome probe within the myofibroblast nuclei. Results: Significant numbers of Y chromosomepositive cells in fibrotic areas were found to be positive for -SMA, vimentin, and fibulin-2 and negative for CD45, thus having a myofibroblast phenotype. In the liver transplant cases, 6.8%22.2% of -SMApositive myofibroblasts contained the Y chromosome. In the female recipient of a bone marrow transplant from a male donor, 12.4% of the myofibroblasts were Y chromosome positive, indicating a bone marrow origin. Conclusions: There is a significant contribution to liver cirrhosis in humans from extrahepatically derived myofibroblasts in liver disease of diverse etiology.
Clinical-alimentary Tract
Loss of imprinting of insulin growth factor II gene: A potential heritable biomarker for colon neoplasia predisposition
Marcia Cruz-Correa, Hengmi Cui, Francis M. Giardiello, Neil R. Powe, Linda Hylind, Angela Robinson, David F. Hutcheon, David R. Kafonek, Sheri Brandenburg, Yiqian Wu, Xiaobing He, Andrew P. Feinberg
Background & aims: Loss of genomic imprinting (LOI) of insulin-like growth factor II gene ( IGF2 ) involves abnormal activation of the normally silent maternally inherited allele. LOI of IGF2 has been associated with personal and family history of colorectal neoplasia (CRN), supporting a role for LOI in colorectal carcinogenesis. Whether LOI of IGF2 is associated with known environmental risk factors for CRN is unknown. Methods: We performed quantitative hot-stop PCR for imprinting analysis of IGF2 on normal peripheral blood lymphocytes (PBL) of individuals. Environmental exposures including tobacco, alcohol, NSAIDs, and nutrient consumption (calcium, folate, selenium, fiber, and fat) were correlated with LOI expression in PBL. Odds ratios (OR) and 95% CI were calculated. Results: The prevalence of LOI of IGF2 was examined in 172 individuals. Persons with CRN (adenomas/cancer) had 5.1-fold (95% CI: 1.9213.6) increased risk of having LOI of IGF2 in PBL compared with those without CRN. In contrast, tobacco smoking (OR = 0.96, 95% CI: 0.362.55), alcohol consumption (OR = 1.22, 95% CI: 0.453.3), and NSAIDs use (OR = 1.21, 95% CI: 0.383.94) were not significantly associated with LOI of IGF2 . Nutrient ingestion including calcium ( P= 0.61), folate ( P= 0.23), selenium ( P= 0.19), fiber ( P= 0.63), and fat ( P= 0.14) was not statistically correlated with LOI of IGF2 .Conclusions: Abnormal imprinting of IGF2 gene was strongly associated with CRN but not with any of the environmental exposures examined. LOI of IGF2 does not appear to be an environmentally acquired phenomenon but rather a hereditary risk factor for CRN.
G-protein 3 subunit 825 CC genotype is associated with unexplained (functional) dyspepsia
Gerald Holtmann, Winfried Siffert, Sebastian Haag, Norbert Mueller, Mathias Langkafel, Wolfgang Senf, Rainer Zotz, Nicholas J. Talley
Background & Aims: In patients with functional dyspepsia, altered -adrenoreceptor function and depression are prevalent, features that are linked to a G-protein 3(GNB3) subunit gene polymorphism (C825T). We aimed to assess the association of specific G-protein 3 subunit genotypes with functional dyspepsia. Methods: In study A, abdominal symptoms were assessed in 67 patients with unexplained, upper abdominal symptoms and 259 consecutive blood donors with and without abdominal symptoms. In study B, a further 56 patients with functional dyspepsia and 112 age- and sex-matched healthy controls from a blood donor population study were evaluated. Genomic DNA was isolated from buccal swabs and genotyping of the C825T polymorphisms was performed by polymerase chain reaction and restriction analysis. Results: In the blood donors with no abdominal symptoms in study A (controls, n = 161), genotype distribution was 17 TT, 77 TC, and 67 CC. In blood donors and patients with unexplained abdominal symptoms, genotype distribution was 22 TT, 54 TC, and 89 CC ( P= 0.007 vs. controls). In study B, the genotype distribution in functional dyspepsia patients was 4 TT, 18 CT, and 34 CC compared with 4 TT, 62 CT, and 46 CC in the controls ( P< 0.02). Combining studies A and B, the odds ratio (OR) adjusted for age and sex for upper abdominal symptoms associated with the CC genotype was 2.2 (95% confidence interval [CI]: 1.43.3), compared with subjects with TC and TT genotype carrying an allele. Conclusions: Homozygous GNB3 825C carrier status is associated with unexplained predominantly upper abdominal symptoms.
Helicobacter pylori eradication releases prolonged increased acid secretion following omeprazole treatment
Derek Gillen, Angela A. Wirz, Kenneth E.L. McColl
Background & Aims : Rebound increased acid secretion has been observed at 2 weeks after discontinuing omeprazole treatment in Helicobacter pylori -negative, but not H. pylori -positive, subjects. It is unknown whether this is a prolonged phenomenon or whether a similar phenomenon appears later in H. pylori positives or is released by eradication therapy. The aims of this study were to answer these 3 questions. Methods : Twelve H. pylori -negative and 20 H. pylori -positive subjects were studied. Each had a basal, submaximal, and maximal pentagastrin-stimulated acid secretion study before, during, and at 7, 14, 28, 42, and 56 days after a 56-day course of omeprazole 40 mg/day. Ten of the H. pylori -positive subjects had their infection eradicated during the last week of treatment. Results : In the H. pylori -negative subjects, there was rebound secretion of submaximal ( P< 0.003) and maximal ( P< 0.003) acid output, which persisted until at least 56 days after discontinuing omeprazole. The H. pylori -uneradicated subjects had no rebound increased secretion other than in maximal acid output at 28 ( P< 0.01) and at 42 days after treatment ( P< 0.02). In those eradicated of H. pylori close to the end of omeprazole, there was rebound increased secretion of submaximal acid output ( P< 0.04) lasting until 56 days and of maximal acid output ( P< 0.01) lasting until 28 days after treatment. Conclusions : Rebound increased acid secretion following omeprazole is a prolonged phenomenon in H. pylori -negative subjects. There is little evidence of it in H. pylori -infected subjects, but eradicating the infection releases the phenomenon. The accentuated H. pylori -related oxyntic gastritis induced by omeprazole is likely to protect against the rebound phenomenon.
A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active crohn’s disease
Hiroaki Ito, Masakazu Takazoe, Yoshihiro Fukuda, Toshifumi Hibi, Kazuo Kusugami, Akira Andoh, Takayuki Matsumoto, Takehira Yamamura, Junichi Azuma, Norihiro Nishimoto, Kazuyuki Yoshizaki, Takashi Shimoyama, Tadamitsu Kishimoto
Background & Aims: Interleukin-6 (IL-6) regulates immune response and inflammation. We carried out a pilot placebo-controlled study to investigate the efficacy, pharmacokinetics, and safety of MRA, a humanized monoclonal antibody to IL-6 receptor, in patients with active Crohn’s disease. Methods: Thirty-six patients with active Crohn’s disease (Crohn’s Disease Activity Index [CDAI] 150) were randomly assigned to receive biweekly intravenous infusion of either placebo, MRA, or MRA/placebo alternately for 12 weeks at a dose of 8 mg/kg. The study’s primary end point was a clinical response rate that was defined as a reduction of CDAI 70. Results: At the final evaluation, 80% of the patients (8 of 10) given biweekly MRA had a clinical response as compared with 31% of the placebo-treated patients (4 of 13; P= 0.019). Twenty percent of the patients (2 of 10) on this regimen went into remission (CDAI <150), as compared with 0% of the placebo-treated patients (0 of 13). The clinical response rate of the every-4-week regimen was 42% (5 of 12). The serum concentrations of MRA were detected at 2 weeks after every infusion, at which time acute phase responses were completely suppressed; however, they were not suppressed at 4 weeks. Endoscopic and histologic examination showed no difference between MRA and placebo groups. The incidence of adverse events was similar in all the groups. Conclusions: This is the first clinical trial of humanized anti-IL-6 receptor monoclonal antibody in Crohn’s disease. A biweekly 8 mg/kg infusion of MRA was well tolerated, normalized the acute-phase responses, and suggests a clinical effect in active Crohn’s disease.
Clinical-liver, Pancreas, and Biliary Tract
Prophylactic antibiotic treatment in patients with predicted severe acute pancreatitis: A placebo-controlled, double-blind trial
Rainer Isenmann, Michael Rünzi, Martina Kron, Stefan Kahl, Dietmar Kraus, Norbert Jung, Ludwig Maier, Peter Malfertheiner, Harald Goebell, Hans G. Beger
Background & Aims: Antibiotic prophylaxis in necrotizing pancreatitis remains controversial. Until now, there have been no double-blind studies dealing with this topic. Methods: A total sample size of 200 patients was calculated to demonstrate with a power of 90% that antibiotic prophylaxis reduces the proportion of patients with infected pancreatic necrosis from 40% placebo (PLA) to 20% ciprofloxacin/metronidazole (CIP/MET). One hundred fourteen patients with acute pancreatitis in combination with a serum C-reactive protein exceeding 150 mg/L and/or necrosis on contrast-enhanced CT scan were enrolled and received either intravenous CIP (2 ? 400 mg/day) + MET (2 ? 500 mg/day) or PLA. Study medication was discontinued and switched to open antibiotic treatment when infectious complications, multiple organ failure sepsis, or systemic inflammatory response syndrome (SIRS) occurred. After half of the planned sample size was recruited, an adaptive interim analysis was performed, and recruitment was stopped. Results: Fifty-eight patients received CIP/MET and 56 patients PLA. Twenty-eight percent in the CIP/MET group required open antibiotic treatment vs. 46% with PLA. Twelve percent of the CIP/MET group developed infected pancreatic necrosis compared with 9% of the PLA group ( P= 0.585). Mortality was 5% in the CIP/MET and 7% in the PLA group. In 76 patients with pancreatic necrosis on contrast-enhanced CT scan, no differences in the rate of infected pancreatic necrosis, systemic complications, or mortality were observed. Conclusions: This study detected no benefit of antibiotic prophylaxis with respect to the risk of developing infected pancreatic necrosis.
Increased survival of cirrhotic patients with a hepatocellular carcinoma detected during surveillance
Angelo Sangiovanni, Ersilio Del Ninno, Pierangelo Fasani, Cristina De Fazio, Guido Ronchi, Raffaella Romeo, Alberto Morabito, Roberto De Franchis, Massimo Colombo
Background & Aims: Significant improvements in management of hepatocellular carcinoma (HCC) have occurred in the last years, but their impact on surveillance outcome is unknown. To clarify this, we compared survival of HCC patients identified along 3 consecutive quinquennia of surveillance. Methods: A cohort of 417 HCC-free outpatients with compensated cirrhosis was prospectively followed for 148 months (range, 1213 months) with periodic ultrasound examinations. Results: HCC developed in 112 patients, at a 3.4% rate per year, and was the prime cause of death (n = 54). Forty-six (41%) patients had a single tumor, with a mean size of 3.7 cm, 3.0 cm, and 2.2 cm in the 3 quinquennia (first vs. second: ns; first vs. third: P= 0.017; second vs. third: P= 0.02), and 38 (44%) underwent radical therapy. Mortality rates in HCC patients fell from 45% in the first quinquennium to 37% in the second and 10% in the third (first vs. second: ns; first vs. third: P= 0.0009; second vs. third: P= 0.018) in parallel with a reduction in yearly mortality of treated patients (34%, 28%, and 5%, respectively; first vs. second: ns; second vs. third: P= 0.036; first vs. third: P= 0.0024). After stratification for quinquennium, tumor staging, according to Cancer of the Liver Italian Program (CLIP), was the only independent predictor of survival ( P= 0.015). Conclusions: Cirrhotic patients developing a HCC during the last 5 years of surveillance survived longer than previously, as a consequence of improved management of the tumor and complications of cirrhosis.
Peginterferon Alfa-2a and ribavirin in patients with chronic hepatitis C who have failed prior treatment
Mitchell L. Shiffman, Adrian M. Di Bisceglie, Karen L. Lindsay, Chihiro Morishima, Elizabeth C. Wright, Gregory T. Everson, Anna S. Lok, Timothy R. Morgan, Herbert L. Bonkovsky, William M. Lee, Jules L. Dienstag, Marc G. Ghany, Zachary D. Goodman, James E. Everhart
Background & Aims: The most effective therapy currently available for treatment of chronic hepatitis C virus (HCV) is the combination of peginterferon and ribavirin. This study evaluated the effectiveness of this treatment in patients who were nonresponders to previous interferon-based therapy. Methods: The first 604 patients enrolled in the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) Trial were evaluated. All were HCV RNA positive, previous nonresponders to interferon, with or without ribavirin, and had bridging fibrosis or cirrhosis on liver biopsy (Ishak fibrosis stage 36). Patients were retreated with peginterferon alfa-2a 180 µg/wk plus ribavirin 10001200 mg/day. Those with no detectable HCV RNA in serum at week 20 continued treatment for a total of 48 weeks and were then followed for an additional 24 weeks. Results: Thirty-five percent of patients had no detectable HCV RNA in serum at treatment week 20, and 18% achieved sustained virologic response (SVR). Factors associated with an SVR included previous treatment with interferon monotherapy, infection with genotypes 2 or 3, a lower AST:ALT ratio, and absence of cirrhosis. Reducing the dose of ribavirin from 80% to 60% of the starting dose during the first 20 weeks of treatment was associated with a decline in SVR from 21% to 11% ( P0.05). In contrast, reducing the dose of peginterferon or reducing ribavirin after week 20, when HCV RNA was already undetectable, did not significantly affect SVR. Conclusions: Selected nonresponders to previous interferon-based therapy can achieve SVR following retreatment with peginterferon alfa-2a and ribavirin.
Impact of acute hepatitis C virus superinfection in patients with chronic hepatitis B virus infection
Yun-fan Liaw, Yi-Cheng Chen, I-shyan Sheen, Rong-nan Chien, Chau-ting Yeh, Chia-ming Chu
Background & Aims: Superinfection in patients with chronic hepatitis B virus (HBV) infection is not uncommon. Acute hepatitis delta virus (HDV) superinfection is associated with severe and/or progressive liver disease. The natural course following acute hepatitis C virus (HCV) superinfection has not been well studied. The aim of this study was to investigate the impact of acute HCV superinfection. Methods: The clinical features during acute phase and long-term outcomes of acute HCV superinfection were studied and compared with a cohort of acute HDV superinfection and a matched control group of active chronic hepatitis B. Results: Acute HCV superinfection typically occurs as acute icteric hepatitis. The severity is similar to acute HDV superinfection in that hepatic decompensation developed in 34% of patients, hepatitis failure occurred in 11%, and 10% died. During a follow-up period of 121 years, patients with acute HCV superinfection had a significantly higher cumulated incidence of cirrhosis (48% at 10 years) and hepatocellular carcinoma (14% at 10 years, 21% at 15 years, and 32% at 20 years) than acute HDV superinfection or active chronic hepatitis B. Hepatitis B surface antigen (HBsAg) seroclearance occurred earlier in HCV superinfected patients. Continuing hepatitis after HBsAg seroclearance was observed only in HCV superinfected patients. Conclusions: Acute HCV superinfection in patients with chronic HBV infection is clinically severe during its acute phase. The long-term prognosis following acute HCV superinfection is much worse than that following HDV superinfection or active hepatitis B in terms of continuing hepatitis activity after HBsAg loss and the development of cirrhosis or hepatocellular carcinoma.
Basic-alimentary Tract
Role of interferon-stimulated responsive element-like element in interleukin-8 promoter in Helicobacter pylori infection
Yoshio Yamaoka, Takahiko Kudo, Hong Lu, Antonella Casola, Allan R. Brasier, David Y. Graham
Background & Aims: Gastric mucosal interleukin (IL)-8 levels are related to the presence of both the cag pathogenicity island (PAI) and OipA. We investigated the regions of the IL-8 promoter and the upstream signaling involved in IL-8 gene transcription. Methods: We cocultured parental Helicobacter pylori and isogenic oipA, hopZ, or cagE gene knockout mutants with gastric cancer cells. The regulatory sites in the IL-8 promoter were examined by luciferase reporter gene assay, electrophoretic mobility shift assays, and immunoblot analyses. Phosphorylated signal transducers and activators of transcription 1 (STAT1) levels in the antral gastric mucosa were measured by enzyme-linked immunosorbent assay. Results : Maximal H. pylori -induced IL-8 gene transcription required the presence of the interferon-stimulated responsive element (ISRE)-like element, nuclear factor (NF)- B and activator protein (AP)-1 binding sites. In vitro studies showed that OipA and the cag PAI were involved in inducing interferon regulatory factor (IRF)-1 to bind and activate the ISRE-like element and that the cag PAI, but not OipA, was involved in activating AP-1 and NF- B. Both in vitro and in vivo studies showed that OipA, but not the cag PAI, was involved in STAT1 phosphorylation, as upstream signaling of IRF-1. Conclusions : OipA and the cag PAI are both necessary for full activation of the IL-8 promoter but act via different pathways that diverge upstream of IRF-1 where only OipA is involved in the STAT1-IRF1-ISRE pathway. The mucosal inflammatory response to H. pylori infection is complex and involves different pathways converging on the IL-8 promoter.
Consequences of bile duct obstruction on intestinal expression and function of multidrug resistance-associated protein 2
Christoph G. Dietrich, Andreas Geier, Nina Salein, Frank Lammert, Elke Roeb, Ronald P.J. Oude Elferink, Siegfried Matern, Carsten Gartung
Background & aims: Multidrug resistance-associated protein 2 (MRP2), a transporter of organic anions in hepatocytes, renal epithelial cells, and enterocytes, is differentially regulated in liver and kidney during cholestasis, but little is known about its regulation in the intestine. Methods: We investigated duodenal protein expression of MRP2 in male Sprague-Dawley rats with bile duct ligation (BDL) or biliary diversion as well as in 20 cholestatic patients with biliary obstruction. Results: In biliary obstruction, but not biliary depletion, intestinal Mrp2 protein mass was reduced to 9.3% ± 5.5% of controls and mRNA to 40.5% ± 20.8% of controls after 7 days. Binding of RXR :RAR heterodimers to the Mrp2 promoter element was significantly reduced in BDL rats. Cytokine blockade identified IL-1 as the responsible inducer of Mrp2 down-regulation. In humans with obstructive cholestasis, intestinal MRP2 protein expression was reduced to 27.3% ± 20.3% of control patients; this reduction correlated with the duration of cholestasis and was reversible after reconstitution of bile flow by stenting of the common bile duct. However, no significant differences in MRP2 mRNA levels were detected by RT-PCR in humans. Intestinal protein expression of P-glycoprotein, breast cancer resistance protein (BCRP), and MRP3 was unchanged. In BDL rats, oral bioavailability of the Mrp2 substrate and food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was elevated 2.5 times compared with sham-operated rats. Conclusions: Cholestasis promotes down-regulation of MRP2 expression in the duodenum of rats and humans. Selective down-regulation during cholestasis might be the consequence of species-specific transcriptional and posttranscriptional mechanisms and contributes to higher bioavailability of a food-derived carcinogen.
Mechanisms of cross hyporesponsiveness to toll-like receptor bacterial ligands in intestinal epithelial cells
Jan-Michel Otte, Elke Cario, Daniel K. Podolsky
Background & Aims: Despite the ability to participate in immune responses and the continuous presence of bacteria and bacterial products, functional responses of intestinal epithelial cells (IEC) seem to be muted. Previously, tolerance to Toll-like receptors (TLRs) ligands has been described in monocytic cells. However, mechanisms in the intestine are unknown. Methods: The effect of purified lipopolysaccharide (LPS) and lipoteichoicacid (LTA) on expression and function of TLRs in intestinal epithelial cells (Colo205, SW480, T84) was assessed by Northern and Western blot and FACS analysis, kinase activity assays, immunohistochemistry, and ELISA. Results: Expression of TLRs except 10 was detected in primary IEC and TLR1-10 in the cultured cells. Short-term stimulation with LPS or LTA activated proinflammatory signaling cascades in IEC, including phosphorylation of IRAK and MAP kinases and increased IL-8 secretion, whereas prolonged incubation resulted in a state of hyporesponsiveness with no reactivation of the cells by a second challenge with either substance detected. The cells remained responsive to tumor necrosis factor (TNF). Hyporesponsive cells showed no alteration in expression of TLR or signaling molecules but revealed a decrease in TLR surface expression and IRAK activity. Toll-interacting protein (Tollip) mRNA and protein expression were increased in hyporesponsive cells, and overexpression of Tollip in IEC resulted in a significantly decreased proinflammatory response. Conclusions: Continuous presence of specific bacterial components results in a status of hyporesponsiveness in otherwise reactive IEC. Down-regulation of TLR surface expression and up-regulation of inhibitory Tollip with decreased phosphorylation of IRAK might all contribute to this hyporesponsiveness.
Four-dimensional elastic light-scattering fingerprints as preneoplastic markers in the rat model of colon carcinogenesis
Hemant K. Roy, Yang Liu, Ramesh K. Wali, Young L. Kim, Alexei K. Kromine, Michael J. Goldberg, Vadim Backman
Background & Aims: Identification of preneoplastic changes in histologically normal epithelium (the “field effect”) could provide a powerful screening tool for colorectal cancer. However, to date, reliable detection has not been possible. We have recently developed a new generation of optical technology, 4-dimensional elastic light-scattering fingerprinting (4D-ELF), which enables us to probe the nanoscale/microscale architecture of living cells. We therefore investigated whether 4D-ELF would be able to identify preneoplastic changes in the colonocytes of the azoxymethane (AOM)-treated rat model of colon carcinogenesis. Methods: Forty-eight Fisher 344 rats were randomized to either 2 weekly injections of AOM or saline. Animals were killed 220 weeks after the second injection of AOM. Colons were removed and subjected to 4D-ELF analysis, with a subset undergoing assessment of aberrant crypt foci (ACF). All AOM-treated animals were compared with age-matched saline-treated controls. Results: AOM-induced ACF became apparent at approximately 46 weeks and continued to increase over time. ACF were predominantly located in the distal colon. At 2 weeks (before development of ACF), there were marked changes in a number of 4D-ELF signatures. The relevance to carcinogenesis of these 4D-ELF-detected microarchitectural abnormalities is supported by their spatial and temporal correlation with subsequent development of ACF. All changes reported were highly statistically significant. Conclusions: We show that probing the nanoscale cellular architecture with 4D-ELF provided an unprecedented tool for detecting the earliest stages of colon carcinogenesis. Future studies are necessary to explore the clinical applicability of this technology and elucidate the biological determinants of these microarchitectural changes.
Altered visceral sensation in response to somatic pain in the rat
Adrian Miranda, Shachar Peles, Colin Rudolph, Reza Shaker, Jyoti N. Sengupta
Background & Aims: Patients with fibromyalgia commonly have symptoms of abdominal pain, suggesting that altered somatic afferent activity may influence visceral sensations. It is hypothesized that a noxious somatic stimulus increases input to the projection neurons in the dorsal horn, resulting in visceral hyperalgesia. Methods: Two injections (100 µL, pH 4.0) were given unilaterally in the gastrocnemius muscle 2 days apart in male Sprague-Dawley rats. Paw withdrawal reflex (PWR) was measured to assess somatic pain. The control group received pH 7.2 saline injections. Similar injections (pH 4.0) were given in the front leg in a different group. Electromyography (EMG) from the external oblique muscle was recorded to graded colorectal distention at different time intervals. NMDA receptor antagonist (CGS-19755, 20 nmol) or AMPA/kainate receptor antagonist (NBQX, 20 nmol) was injected intrathecally before low-pH injections. Results: A bilateral decrease in PWR threshold occurred 72 hours after the second low-pH injection. There was no decrease in the threshold in rats injected with pH 7.2 saline. A significant increase in EMG to colorectal distention ( 30 mm Hg) occurred at 72 hours and 2 weeks in the pH 4.0 group. No change in EMG was observed following 2 unilateral low-pH injections in the front leg. Both the visceral hyperalgesia and the decrease in somatic pain thresholds were prevented by prior intrathecal CGS-19755 or NBQX injections. Conclusions: Noxious somatic afferent input from the hind limb facilitates visceral hyperalgesia, which is due to viscerosomatic convergence in the lower spinal cord. This can be blocked by ionotropic glutamate receptor antagonists.
Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer
Helen L. Reeves, Goutham Narla, Olagunju Ogunbiyi, Asif I. Haq, Amanda Katz, Sharon Benzeno, Eldad Hod, Noam Harpaz, Shlomit Goldberg, Sigal Tal-kremer, Francis J. Eng, Michael J.P. Arthur, John A. Martignetti, Scott L. Friedman
Background & Aims: Kruppel-like factor 6 (KLF6) is a ubiquitous zinc finger tumor suppressor that is often mutated in prostate cancer. Our aims were to establish the frequency of KLF6 inactivation in sporadic and inflammatory bowel disease (IBD)-associated colorectal cancers (CRC); to correlate these abnormalities with mutation and/or loss of TP53 ,APC , and K-RAS ; and to characterize the behavior of mutant KLF6 in colon-derived cell lines. Methods: We analyzed DNA isolated from 50 microdissected CRC cases, including 35 sporadic and 15 IBD-associated tumors. Microsatellite analysis and direct sequencing were used to establish the incidence of microsatellite instability, KLF6 and TP53 allelic imbalance, and KLF6 ,K-RAS ,TP53 , and APC mutation. Loss of growth suppressive function of the CRC-derived KLF6 mutants was characterized by in vitro thymidine incorporation assays and Western blotting. Results: KLF6 was inactivated by loss and/or mutation in most sporadic and IBD-related CRCs. The KLF6 locus was deleted in at least 55% of tumors, and mutations were identified in 44%. Rates of KLF6 loss and mutation were similar to those of TP53 and K-RAS in the same samples. KLF6 mutations were present in tumors with either microsatellite or chromosomal instability and were more common, particularly in the IBD-related cancers, in the presence of wild-type APC . Unlike wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21 following transfection into cultured cells. Conclusions: Deregulation of KLF6 by a combination of allelic imbalance and mutation may play a role in the development of CRC.
Basolateral ClC-2 chloride channels in surface colon epithelium: Regulation by a direct effect of intracellular chloride
Marcelo Catalán, Mara Isabel Niemeyer, L. Pablo Cid, Francisco V. Sepúlveda
Background & Aims: The principal function of the colon in fluid homeostasis is the absorption of NaCl and water. Apical membrane Na +channels, Na +/H +, and Cl /HCO 3exchangers have been postulated to mediate NaCl entry into colonocytes. The basolateral exit pathway for Cl has recently been proposed to be via ClC-2 channels present in that membrane domain in surface epithelium. The aim of this report is to obtain functional data for a basolateral localization of ClC-2 and explore a possible direct regulation by intracellular Cl .Methods: Guinea pig colon epithelium with the apical membrane perforated with nystatin in Ussing chambers is used to show a basolateral Cl conductance. Gramicidin D perforated-patch configuration of the patch-clamp technique is used on isolated surface colonocytes. Heterologous expression of the recombinant channel and the whole-cell configuration are used to investigate a direct regulation by intracellular Cl .Results: A basolateral membrane conductance with the characteristics of ClC-2 channels, including Cd 2+ sensitivity, selectivity, and inhibition by extracellular alkalinization, is present in distal colon epithelium. The effect of intracellular Cl on this conductance suggests activation by the permeant anion. Using the recombinant ClC-2 channel, a strong dependence of its activity on intracellular Cl is shown, with a shift of activation to more positive voltages as [Cl ]iis increased. Conclusions: It is suggested that ClC-2 serves as an exit pathway for Cl in the basolateral membranes of the distal colon and that its dependence on [Cl ]imight provide a cross-talk mechanism to match fluxes at the apical and basolateral domains of these epithelial cells.
An innate cell-mediated, murine ulcerative colitis-like syndrome in the absence of nuclear factor of activated T cells
Andrea J. Gerth, Ling Lin, Markus F. Neurath, Laurie H. Glimcher, Stanford L. Peng
Background & Aims: Nuclear factor of activated T cells transcription factors plays a central role in immunity by regulating the expression of multiple cytokines and other regulatory molecules, many of which have been heavily implicated in the pathogenesis of inflammatory bowel disease. However, few studies have directly investigated the nuclear factor of activated T cells proteins in inflammatory bowel disease. We describe here a specific role for nuclear factor of activated T cells c2 in the pathogenesis of murine inflammatory bowel disease. Methods: Mice deficient for nuclear factor of activated T cells c2, recombinase activating gene-2, or both and transgenic or nontransgenic for an anti-ovalbumin T-cell receptor or an anti-hen egg lysozyme B-cell receptor were studied. Adoptive transfers were performed of T or B cells or both from nuclear factor of activated T cells c2-deficient mice into nuclear factor of activated T cells c2-deficient recombinase activating gene-deficient animals, in the presence or absence of antibodies that neutralize interleukin-10 activity. Results: Nuclear factor of activated T cells c2-deficient, recombinase activating gene-deficient animals spontaneously developed a severe inflammatory bowel syndrome that resembled ulcerative colitis but was composed entirely of nonlymphocytes. The disease was suppressed by the adoptive transfer of polyclonal B-cell populations, even on neutralization of interleukin-10, but not by the presence of monoclonal T or B cells. Conclusions: Nuclear factor of activated T cells plays a critical role in the regulation of bowel inflammation by nonlymphoid immune cells, and B cells suppress bowel inflammation by innate immune cells. Such findings indicate a novel, interleukin-10-independent role for nuclear factor of activated T cells in the regulation of innate immunity and in intestinal immune tolerance.
Basic-liver, Pancreas, and Biliary Tract
Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis
Fernando Siller-López, Ana Sandoval, Silvia Salgado, Adriana Salazar, Miriam Bueno, Jesus Garcia, Jose Vera, Javier Gálvez, Iván Hernández, Martha Ramos, Estuardo Aguilar-Cordova, Juan Armendariz-Borunda
Background & Aims : An extrahepatic human neutrophil collagenase complementary DNA (matrix metalloprotease-8) cloned in an adenovirus vector was used as a therapeutic agent in cirrhosis. Methods : A high titer of clinical-grade AdMMP8 was obtained. Results : HeLa cells transduced with AdMMP8 expressed recombinant matrix metalloprotease-8 messenger RNA and matrix metalloprotease-8 protein. Matrix metalloprotease-8 in culture sups showed enzymatic activity against native collagen type I, which was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline, and tissue inhibitor of metalloprotease-1. In vivo transduction showed matrix metalloprotease-8 activity, and studies to establish the efficacy of this characterized vector were performed in CCl 4and bile duct-ligated cirrhotic rats. Transduction with 3 ? 10 11 viral particles per kilogram resulted in hepatic detection of both messenger RNA and protein matrix metalloprotease-8. A consistent response in fibrosis reversal was observed in CCl 4rats. Liver fibrosis in bile duct-ligated cirrhotic animals was decreased in 45%, along with diminished hydroxyproline content, after AdMMP8 treatment. The expression of matrix metalloprotease-2 and matrix metalloprotease-3 was up-regulated in AdMMP8 rats. Free tissue inhibitor of metalloprotease-1, as an indirect measurement of active uncomplexed matrix metalloproteases, was also increased in the AdMMP8 groups. Transforming growth factor- messenger RNA was diminished, and matrix metalloprotease-9 and hepatocyte growth factor increased. Treatment in both models correlated with improvements in ascites, functional hepatic tests, and gastric varices, indicating diminished intrahepatic blood pressure in animals injected with AdMMP8. Conclusions : Therefore, therapy with the matrix metalloprotease-8 gene is promising for use in a clinical setting.
Beta-Catenin is temporally regulated during normal liver development
Amanda Micsenyi, Xinping Tan, Tamara Sneddon, Jian-hua Luo, George K. Michalopoulos, Satdarshan P.S. Monga
Background & Aims :-Catenin, a key component of the Wnt pathway, plays an important role in unregulated liver growth in liver tumors, in regulated growth during liver regeneration, and in ex vivo embryonic liver cultures. Methods : We used developing livers from several stages of gestational development to examine -catenin expression, protein-protein interactions, localization, and regulation in prenatal and postnatal livers. Results : Microarray, Northern, and protein analyses showed peak expression of -catenin during early liver development at Embryonic day 10 (E10)-E12, followed by a decrease and a complete loss of normal -catenin (97-kilodalton species) after E16 through the remaining prenatal period. At the early stages, -catenin localized to the cytoplasm and nuclei of resident cells in addition to its normal membranous localization, which was seen at all later stages and in adult liver. Decreases in -catenin levels at E14 onward coincided with its decreased gene expression and increased degradation, as seen by an increase in serine 45/threonine 41-phosphorylated -catenin and its other negative regulators, such as axin, adenomatous polyposis coli gene product (APC), and glycogen synthase kinase-3 . Finally, we showed an intact association of E-cadherin and -catenin despite the loss of -catenin at E16-E18, owing to the presence of membrane-associated smaller-molecular-weight -catenin species. Conclusions : We also identified a stage-specific expression and regulation of -catenin during liver development that might be crucial for physiological liver development. Nuclear and cytoplasmic -catenin corresponded to cell proliferation in liver development. Finally, a smaller-molecular-weight species of -catenin might be maintaining normal interactions at the membrane.
Origin and characterization of a human bipotent liver progenitor cell line
Romain Parent, Marie-Jeanne Marion, Laetitia Furio, Christian Trépo, Marie-Anne Petit
Background & Aims: Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells. Methods: Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages. Results: HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules. Conclusions: HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.
Involvement of chloride channels in hepatic copper metabolism: ClC-4 promotes copper incorporation into ceruloplasmin
Ting Wang, Steven A. Weinman
Background & Aims: Copper transport in hepatocytes is regulated by the interaction of multiple pumps, chaperones, and accessory proteins. Intracellular chloride channels are essential for copper metabolism in yeast but their role in Cu transport in hepatocytes is unknown. The aim of this study was to determine whether chloride channels are modulators of copper incorporation into ceruloplasmin (CP). Methods: The effects of chloride concentration and chloride channel expression on secretion of holoCp and apoCp was measured by gel electrophoresis and immunoblotting. ClC family chloride channel expression in hepatocytes was determined by Western blotting. The association of ClC-4 and the Wilson’s disease protein (ATP7B) was determined by co-immunoprecipitation. Results: Chloride substitution reduced total Cp secretion and the ratio of secreted holoCp to apoCp ( P= 0.038). The role of specific chloride channels was examined by cotransfection of ceruloplasmin and the chloride channel. Overexpression of ClC-4 doubled copper incorporation into ceruloplasmin ( P= 0.011), whereas identical overexpression of ClC-3 had no effect. The effect of ClC-4 was most pronounced under copper-limiting conditions in which it increased copper incorporation more than 4-fold ( P= 0.037). ClC-4 protein was abundant in hepatocyte membranes and was localized in intracellular vesicles containing ATP7B. Conclusions: ClC-4 is an intracellular chloride channel that stimulates copper incorporation into ceruloplasmin, probably by improving the efficiency of the ATP7B copper pump. It is thus an important component of the regulation of hepatic copper transport and may modulate Cu transport rates during copper deficiency, Wilson’s disease, and other copper toxicosis syndromes.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Table of Contents for Volume 40, Issue 4, April 2004
Biliary Tract and Cholestasis
Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat
Gerald U. Denk et al.
Background/Aims
Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis.
Methods
Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry.
Results
Western blot analysis revealed a progressive, more than seven-fold increase (P <0.05) of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26±4% of controls (P <0.005). Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings.
Conclusions
Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.
Keywords: ATP-binding cassette transporter ;Bile acids ;Bile duct ligation ;Cyclic adenosine monophosphate ;Cyclic nucleotides ;Multidrug resistance-associated protein 4
Isolated idiopathic bile ductular hyperplasia in patients with persistently abnormal liver function tests
Aurelio Sonzogni et al.
Background/Aims
In routine examination of liver biopsies isolated ductular hyperplasia (IDH) may be the only histopathological change. Here we describe the clinical and immunophenotypic features of a number of cases retrospectively identified reviewing consecutive liver biopsies from five Italian centers over 4 years.
Methods
We reviewed 1235 cases biopsied for chronic liver disease (1078 for viral hepatitis). Records of cases fulfilling the inclusion criteria for IDH were reviewed to identify possible aetiologies. Biopsies showing IDH and control biopsies were studied by immunohistochemistry for cytokeratin-7, epithelial-membrane-antigen (EMA), neural-cell-adhesion-molecule (NCAM), Ki-67.
Results
Out of 70 biopsies fulfilling IDH criteria, 16 (22.8%) were of unknown aetiology. Patients with idiopathic IDH (age 38.2±11 years) were asymptomatic with mild, long-lasting ALT and/or GT increases. A significant increase of well-differentiated (EMA-positive; NCAM-negative) bile ductules localized at the portal interface and inside the lobule was found in idiopathic IDH.
Conclusions
Idiopathic IDH was present in 10% of adults biopsied for persistent mild liver function test abnormalities unrelated to viral hepatitis. In contrast with the ductular reaction seen in many forms of liver disease, it is characterized by well-differentiated hyperplastic ductules in absence of significant inflammation, and may represent a non-specific pattern of reaction to mild liver damages.
Keywords: Isolated ductular hyperplasia ;Epithelial-membrane-antigen ;Biopsy
Cell Biology, Metabolism and Transport
The transport of triglycerides through the secretory pathway of hepatocytes is impaired in apolipoprotein E deficient mice
Arjen R. Mensenkamp et al.
Background/Aims
Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and secrete reduced levels of VLDL-TG.
Methods and results
We examined the effects of apoE-deficiency on intracellular lipid homeostasis and secretion of triglycerides (TG). We show that intracellular TG turnover and activities of diacylglycerol acyltransferase (DGAT) and microsomal triglyceride transfer protein (MTP) are similar in Apoe /and wild type mice. In addition, apoB synthesis was not decreased in Apoe /cells. Thus, the accumulation of lipid in these cells is not attributable to perturbed TG turnover, apoB synthesis, and the activities of DGAT and MTP. Inhibition of MTP had a more profound impact on the secretion of VLDL-TG from wild type hepatocytes than Apoe /hepatocytes, indicating that MTP was more limiting for the production of VLDL-TG from wild type cells. In marked contrast to the MTP-deficient model of fatty liver, electron microscopy of lipid-stained liver sections of Apoe /mice revealed an accumulation of lipid in numerous small, putative ER-derived vesicles and in the cytosol. No abnormalities were observed in the Golgi of Apoe /mice.
Conclusions
These results suggest that the removal of lipids from the early or intermediary compartments of the secretory pathway of hepatocytes is impaired in Apoe /mice.
Keywords: Apolipoprotein E ;Fatty liver ;VLDL secretion ;MTP ;Hyperlipidemia ;Triglycerides
Iron-induced mitochondrial permeability transition in cultured hepatocytes
Ursula Rauen, Frank Petrat, Reiner Sustmann and Herbert de Groot
Background/Aims
We previously described that the cold-induced apoptosis of cultured hepatocytes and liver endothelial cells is mediated by an increase in the cellular chelatable iron pool in the absence of any increase in O 2/H 2O2formation. As this is an unusual mechanism, we here set out to assess whether an increase in cellular chelatable iron per se is sufficient to trigger cell injury/apoptosis.
Methods
Cultured rat hepatocytes were acutely loaded with iron using the membrane-permeable complex Fe(III)/8-hydroxyquinoline and incubated under otherwise `physiological' conditions.
Results
Incubation with Fe(III)/8-hydroxyquinoline (15 µM/30 µM) increased the cellular chelatable iron and induced strong hepatocellular injury with morphological features of apoptosis, but also of necrosis. The iron-induced cell injury was oxygen-dependent, and although it was not inhibitable by extracellular catalase, it was strongly inhibited by the novel membrane-permeable catalase mimic TAA-1/Fe. The experimentally induced increase in cellular chelatable iron triggered a mitochondrial permeability transition (MPT) as assessed using double-staining with calcein and tetramethylrhodamine methyl ester. The MPT inhibitor cyclosporine A partially and the well-known inhibitor combination trifluoperazine+fructose completely inhibited the iron-induced cell injury/apoptosis.
Conclusions
These results show that iron per se can induce cell injury/apoptosis and that this injury is mediated via an MPT.
Keywords: Chelatable iron ;Mitochondrial permeability transition ;Fenton reaction ;Iron-oxygen species ;Ferryl species ;Mitochondria ;Catalase mimic
Up-regulation of IL-1 receptor through PI3K/Akt is essential for the induction of iNOS gene expression in hepatocytes
Shigeru Teshima et al.
Background/Aims
Nuclear translocation and DNA binding of NF- B is essential, as interleukin-1 (IL-1 ) stimulates the induction of inducible nitric oxide synthase (iNOS) gene expression in hepatocytes. However, recent evidence indicates that the activation of NF- B is not sufficient to induce the NF- B-dependent transcription, and the existence of a second signaling is postulated.
Methods
Primary cultured hepatocytes were treated with IL-1 , and the expression of iNOS and type 1 IL-1 receptor (IL-1R1) was analyzed in the presence of antisense of IL-1R1, phosphatidylinositol 3-kinase (PI3K) inhibitor, proteasome inhibitor and hypoxia. Moreover, the activities of Akt and NF- B were recorded and the cotransfection was carried out.
Results
Antisense experiment revealed that IL-1R1 was required for iNOS transcription. IL-1 markedly stimulated the induction of IL-1R1, which preceded the induction of iNOS. The IL-1R1 induction was found to be PI3K/Akt-dependent but NF- B-independent. The up-regulation of IL-1R1 was associated with the second activation of Akt, which accelerated the phosphorylation of NF- B p65 subunit. Cotransfection experiments revealed that Akt increased the transcriptional activity of iNOS gene promoter.
Conclusions
These results indicate that the up-regulation of IL-1R1 in concert with the activation of NF- B is required for the transcriptional activation of iNOS gene.
Cirrhosis and its Complications
Tumour necrosis factor-alpha expression by activated monocytes and altered T-cell homeostasis in ascitic alcoholic cirrhosis: amelioration with norfloxacin
Agustín Albillos et al.
Background/Aims
To investigate the distribution and activation state of circulating monocytes and T-cell subsets, their contribution to tumour necrosis factor-alpha (TNF ) production, and their potential relationship with bacterial products of enteric origin in alcoholic cirrhosis.
Methods
Peripheral blood monocytes and T-lymphocytes from 60 cirrhotic patients and 24 controls were characterized by four-color flow-cytometry after labelling of differentiation antigens and cytokines, before and after a 4-week course of norfloxacin or placebo.
Results
Monocytes from ascitic patients showed increased number, enhanced CD80 and HLA-DR surface levels, and spontaneous intracytoplasmic TNF expression, when compared to non-ascitic patients and controls. Blood TNF levels directly correlated with the amount of TNF expressed by monocytes. In ascitic patients, there was a collapse of virgin CD4 +and CD8 +T-cell subsets; and, an expansion of activated CD4 +T-cells. The above abnormalities were mainly restricted to ascitic patients with high serum levels of lypolysaccharide-binding-protein. Norfloxacin normalized the number of monocytes, reduced their activated phenotype and ability to produce TNF and improved the abnormal T-cell homeostasis.
Conclusions
In ascitic cirrhosis with high lipolysaccharide-binding-protein, monocytes are spontaneously activated to produce TNF and are major contributors to the elevated serum TNF . The T-cell compartment is profoundly depleted. Enteric bacterial products play a relevant role in these immune cellular abnormalities.
Keywords: Alcoholic cirrhosis ;Cytotoxic T cells ;Helper T cells ;Lypolysaccharide ;Lypolysaccharide binding protein ;Mononuclear cells
Inflammation and Fibrosis
N-Acetyl-cysteine modulates inducible nitric oxide synthase gene expression in human hepatocytes
Pedro Lorenzo Majano et al.
Background/Aims
A major role has been described for inducible nitric oxide (NO) synthase in several chronic inflammatory liver diseases. N-Acetyl-cysteine (NAC) is a sulfhydryl donor molecule with antioxidant and antiinflammatory effects. It attenuates NO generation following lipopolysaccharide injection in rats. Our goal was to study the effect of NAC on NO synthase induction in hepatocytes in response to proinflammatory cytokines.
Methods
The effect of NAC on NO synthase induction was studied in the human hepatocyte cell lines HepG2 and 2.2.15 treated with a mixture of proinflammatory cytokines. Interactions between NAC and cytokines on nuclear factor- B (NF- B) activation and NO synthase promoter transactivation were investigated.
Results
NAC dose-dependently modulated the induction of NO synthase mRNA expression, the release of nitrites and the formation of NF- B binding complexes in cytokine-treated hepatocytes. NAC also reduced the transactivation of the NO synthase promoter.
Conclusions
Our data show that exposure of hepatocytes to NAC modulated NO synthase expression and NF- B activity, the key responses of the hepatocyte to inflammatory mediators. These data constitute preliminary evidence that NAC might have hepatoprotective actions of potential relevance in chronic inflammatory liver diseases, mediated partially through the modulation of NO production.
Keywords: N-acetyl-cysteine ;Inducible nitric oxide synthase ;Nitric oxide ;Cytokines ;Nuclear factor B;Hepatocytes
Modulation of fibronectin gene expression in inflammatory mononuclear phagocytes of rat liver after acute liver injury
Thomas Armbrust, Michael Kreißig, Kiril Tron and Giuliano Ramadori
Background/Aims : In acute liver injury, fibronectin (FN) is deposited at the site of hepatocellular necrosis. We have previously shown that liver inflammatory mononuclear phagocytes (MNP), in contrast to quiescent hepatic macrophages, synthesize abundant amounts of FN. We now analyzed effects of agents known to influence macrophage functions to better understand liver damage and repair.
Methods : Acute rat liver injury was induced by CCl 4. Liver cellular FN ( cFN ) expression was analyzed by in situ-hybridization. Liver MNP were isolated and characterized immunocytochemically. Protein synthesis was studied by biosynthetic labeling, immunoprecipitation, and SDS-PAGE. RNA was analyzed by Northern Blotting.
Results : cFN gene expression was localized by in situ-hybridization and immunohistochemistry within the pericentral inflammatory infiltrate. Treatment of inflammatory MNP with dexamethasone, or interferon-gamma, or lipopolysaccharide induced a dose-dependent decrease in cFN gene expression, whereas transforming growth factor- increased cFN gene expression.
Conclusions : 1. Inflammatory MNP express cFN . 2. Downregulation of cFN expression by dexamethasone in inflammatory MNP may explain delayed wound healing after corticosteroid therapy. Interferon- and lipopolysaccharide could also delay the repair process in the liver. Transforming growth factor- may promote liver wound healing after acute liver injury by increasing local cFN synthesis in inflammatory mononuclear phagocytes of the inflammatory infiltrate.
Keywords: Acute liver injury ;Mononuclear phagocytes ;Fibronectin ;Interferon ;Dexamethasone ;Lipopolysaccharide ;Transforming growth factor-
Decreased fibrosis during corticosteroid therapy of autoimmune hepatitis
Albert J. Czaja and Herschel A. Carpenter
Background/Aims
Reduction in hepatic fibrosis and reversibility of cirrhosis has been described in chronic liver disease. Our goal was to determine changes in fibrosis and the frequency of histological cirrhosis in corticosteroid-treated autoimmune hepatitis (AIH).
Methods
Three hundred twenty-five liver specimens from 87 treated patients were reviewed in batch under code by one pathologist and graded by the Ishak method.
Results
Fibrosis scores improved (3.4±0.2 versus 2.6±0.2, P=0.0002) during 63±6 months, and histological activity indices decreased concurrently (6.8±0.5 versus 2.1±0.2, P<0.0001). Fibrosis scores improved in 46 patients (53%) during 57±7 months and did not progress in 23 patients during 62±12 months. The fibrosis score improved more commonly in patients who had improvement in the histological activity indices than in others (61 versus 32%, P=0.02), and the frequency of histological cirrhosis decreased from 16% (14 patients) to 11% (10 patients).
Conclusions
Fibrosis commonly improves or does not progress during corticosteroid therapy of AIH, and histological cirrhosis may disappear. Improvements in fibrosis are associated with suppression of inflammatory activity. Improvement or prevention of fibrosis may be a common but unheralded advantage of corticosteroid therapy.
Keywords: Fibrosis ;Cirrhosis ;Biopsy changes ;Corticosteroids ;Autoimmune hepatitis
Liver Growth and Cancer
Hepatitis B virus transmission and hepatocarcinogenesis: a 9 year retrospective cohort of 13676 relatives with hepatocellular carcinoma
Chien-Hung Chen et al.
Background/Aims
Familial clustering of hepatitis B virus (HBV) infection is related to perinatal transmission, and is the main cause of familial-type hepatocellular carcinoma (HCC). The route of HBV transmission differs between the children and siblings of patients with HCC. This study examined the differences in HBV carrier rates and HCC-related mortality between two generations in HCC families.
Methods
From 1992 to 1997, relatives of individuals with HCC were screened prospectively with ultrasonography, alpha-fetoprotein, liver biochemistry tests and viral markers. Total HCC-related deaths during a 9-year period were compared between the generations of index patients and their children.
Results
The study included a total of 13676 relatives in two generations. More HCC-related deaths occurred in the index patient generation than in the child generation. Furthermore, children of female index patients had higher rates of liver cancer related mortality than children of male index patients. The same was true when the analysis was limited to male HBV carriers. The prevalence of HBsAg in the offspring of HBsAg positive mothers was 66% in the child generation and 72% in the index patient generation. These high prevalences indicated high maternal HBV replication status.
Conclusions
Perinatal transmission and maternal viral load are important risk factors in hepatocarcinogenesis.
Keywords: Maternal-fetal exchange ;Taiwan ;Survival analysis ;Hepatitis B surface antigen ;Family
Multiple chromosomal abnormalities in human liver (pre)neoplasia
Maria Raidl et al.
Background/Aims
In human hepatocarcinogenesis the tumor precursor lesions and the sequence of genetic aberrations are not known. We therefore compared genetic alterations of different types of benign liver lesions to those of hepatocellular carcinoma.
Methods
By comparative genomic hybridisation (CGH) 40 cases, including cirrhotic liver (CL), focal nodular hyperplasia (FNHs), hepatocellular adenoma (HCAs), dysplastic nodules (DNs), primary hepatocellular carcinoma (HCCs), and hepatocellular metastases to the lung were studied.
Results
FNHs and HCAs exhibited few chromosomal abnormalities. Frequency and pattern of genetic alterations in DNs highly resembled those in HCCs: gains of DNA clustered in chromosome arms 1p/q, 7q, 15q, 16p, 17q, and 20q and losses were often found at 3p, 4q, 9p, and 11q. Aberrations on 1p, 6q, 8p/q, and 13q occurred almost exclusively in HCCs; the gain at 8q encompassed amplification of c-myc, as verified by fluorescence in situ hybridisation.
Conclusions
The pattern of genetic alterations in HCCs resembled more the alterations found in DNs than in FNHs and HCAs, suggesting that DNs may be the actual tumor precursors. Furthermore, alterations at 4q, 9p, 11q, 16p, and 17q appear as early genetic events being crucial for hepatocarcinogenesis.
Keywords: Human hepatocarcinogenesis ;Dysplastic nodule ;Hepatocellular carcinoma ;Comparative genomic hybridisation
Transplantation
Treatment of recurrent hepatitis C after liver transplantation: a pilot study of peginterferon alfa-2b and ribavirin combination
Jérôme Dumortier, Jean-Yves Scoazec, Philippe Chevallier and Olivier Boillot
Background/Aims
After liver transplantation (LT) for HCV-related cirrhosis, recurrence of HCV infection is universal and the risk of progression to cirrhosis is high. The modalities and efficacy of antiviral therapy in this indication are still controversial. We present here the results of a pilot study of a 12-month combination therapy by pegylated alfa2b-interferon (PEG-IFN) and ribavirin in 20 patients.
Methods
Twenty patients entered the study (13 male and 7 female, median age 53.8 years). In 80% of patients, HCV infection was of genotype 1. The delay between LT and antiviral therapy was 28 months. The doses were progressively increased from 0.5 to 1 µg/kg/week for PEG-IFN and from 400 to 1000-1200 mg/d for ribavirin. Follow-up was based on biochemical (ALT), virological (HCV-RNA) and histological (liver biopsy) examinations.
Results
Four patients (20%) were withdrawn due to adverse effects. In 6 patients the dose of PEG-IFN had to be reduced to 0.5 µg/kg/week. A reduction in the dose of ribavirin in 13/16 cases was due to the onset of anemia. Histological evidence of mild acute rejection increased the immunosuppressive regimen in 5/20 patients. At the end of the treatment, 75% of the patients had a biochemical response and 55% a virological response. The mean METAVIR score, according to activity and fibrosis, was A1.8 F2.2 before treatment and A0.3 F1.6 at the end of treatment. In 9/20 patients, virological response persisted 6 months after the end of the treatment. Conclusions
Our results suggest that combination therapy by PEG-IFN and ribavirin may be well tolerated and beneficial during recurrent hepatitis C in liver transplant recipients.
Keywords: HCV ;Recurrence ;Liver transplantation ;Treatment ;Pegylated interferon ;Ribavirin
Viral Hepatitis
The impact of peginterferon alfa-2a plus ribavirin combination therapy on health-related quality of life in chronic hepatitis C
Tarek Hassanein et al.
Background/Aims
Peginterferon alfa-2a plus ribavirin improves sustained virological responses compared with interferon alfa-2b and ribavirin, or peginterferon alfa-2a alone in chronic hepatitis C. We examined the impact of these treatments on health related quality of life (HRQOL).
Methods
Patients ( n=1121) were randomized to peginterferon alfa-2a weekly plus ribavirin or placebo, or interferon alfa-2b thrice weekly plus ribavirin. HRQOL was assessed with the SF-36 Health Survey and Fatigue Severity Scale (FSS).
Results
Patients receiving peginterferon alfa-2a plus ribavirin reported better HRQOL than those receiving interferon alfa-2b plus ribavirin. These differences were statistically significant for three SF-36 domains and both FSS scores ( p<=0.05). Patients receiving peginterferon alfa-2a plus placebo had the least impairment; adding ribavirin significantly decreased five domains of the SF-36 and both FSS scores. Sustained virological response was associated with improvement at follow-up on all SF-36 and FSS scores.
Conclusions
The effects of combination therapy on HRQOL and fatigue are less with peginterferon alfa-2a plus ribavirin than interferon alfa-2b plus ribavirin. Each medication in combination therapy with interferon and ribavirin, affects patients' quality of life differently. Understanding the relationship of specific therapeutic options to HRQOL may help physicians minimize the impact of therapy on HRQOL.
Keywords: Hepatitis C ;Interferon alfa-2b ;Peginterferon alfa-2a ;Ribavirin ;Combination therapy ;Health related quality of life
Intrahepatic hepatitis C virus RNA quantification in microdissected hepatocytes
Giovanna Vona et al.
Background/Aims: Debate continues on whether serum and intrahepatic HCV viral loads are correlated and if HCV viral load correlates with the severity of liver disease. These difficulties may at least in part be linked to liver cell heterogeneity, when total liver extracts from HCV-infected individuals are tested for HCV RNA quantification. We have therefore investigated the feasibility of quantifying HCV replication using a laser-based microdissection technique.
Methods: We compared the results with those obtained for serum HCV RNA quantification and immunochemistry in the case of HCV antigen detection in the liver. Twenty-one HCV-positive patients with chronic active hepatitis ( n=10) or cirrhosis ( n=11) were analyzed.
Results: A positive correlation ( P=0.0019) was observed between HCV RNA quantifications in sera and microdissected cells. Immunohistochemistry demonstrated that HCV antigen hepatocytes were randomly distributed within liver lobules. Their percentage varied in different patients (0-40%), but did not correlate with the HCV viral load.
Conclusions: We have designed a sensitive methodology to evaluate the intrahepatic HCV viral load by combining a standardized RNA quantification method with microdissected hepatocytes from frozen liver needle biopsies. Our results directly demonstrate a positive correlation between serum and intrahepatic viral loads, which therefore provides a reliable reflection of intrahepatic HCV replication.
Keywords: Hepatocytes ;Intrahepatic HCV ;Hepatitis C virus ;Laser microdissection
Reduction of relapse rates by 18-month treatment in chronic hepatitis C. A Benelux randomized trial in 300 patients
Johannes T. Brouwer et al.
Background/Aims
Treatment of chronic hepatitis C with interferon can be ineffective due to relapse. We aimed to reduce the 40% relapse rate of 6 months interferon-ribavirin combination therapy by prolonging treatment to 18 months.
Methods
Three hundred patients with treatment-naive hepatitis C, were randomized to 18 months combination therapy with interferon (3MU tiw) and ribavirin (1000-1200 mg/day), 18 months interferon combined with placebo, or 6 months combination therapy with interferon and ribavirin, in a double blinded manner. All 295 patients who received at least one dose of treatment were included in the intention to treat analysis.
Results
At the end of treatment, HCV RNA was undetectable in 55 and 49% of those on 6 and 18 months combination therapy, respectively, versus 26% of those on monotherapy ( P<0.001). The relapse rate was 38% for 6 months combination therapy, 38% for 18 months monotherapy, and only 13% for 18 months combination treatment ( P=0.002). The sustained response rates were 34% for 6 months combination therapy, 16% for 18 months monotherapy and 43% for 18 months combination therapy ( P<0.05).
Conclusions
Reduction of relapse rates to 15% or less is feasible by prolongation of interferon-ribavirin treatment to 18 months.
Keywords: Hepatitis C treatment ;Randomized controlled trial ;Interferon ;Ribavirin ;Relapse rate ;Logistic regression
Case Report
Autosomal dominant iron overload due to a novel mutation of ferroportin1 associated with parenchymal iron loading and cirrhosis
Daniel F. Wallace, Roslyn M. Clark, Hugh A.J. Harley and V. Nathan Subramaniam
We report the identification of a novel mutation in ferroportin1 in an Australian family with autosomal dominant iron overload. The phenotype of iron overload in one member of this family is associated with high serum ferritin concentration and elevated transferrin saturation. The pattern of iron overload in the liver shows accumulation predominantly in parenchymal cells with some Kupffer cell iron loading. Although some cases of type 4 haemochromatosis have been associated with the development of liver fibrosis this is the first report of a patient with fully established cirrhosis at a relatively young age (32 years). The coexistence of sarcoidosis in this patient may contribute to the more severe phenotype. This report highlights the phenotypic variability that can occur in type 4 haemochromatosis. Some patients have predominant reticuloendothelial iron loading and normal transferrin saturation whereas others have predominant parenchymal iron loading and elevated transferrin saturation. The reasons for this variability remain to be determined. Interestingly this is the third mutation to affect asparagine 144, reinforcing the important role for this amino acid in the function of ferroportin1.
Keywords: Haemochromatosis ;Iron overload ;Ferroportin1 ;Autosomal dominant ;Cirrhosis
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