bluuebunny/crossref_metadata_2025 huggingface dataset:
Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma
Authors: Roncalli Massimo , Bianchi Paolo , Bruni Barbara , Laghi Luigi , Destro Annarita , Di Gioia Sonia , Gennari Leandro , Tommasini Maurizio , Malesci Alberto , Coggi Guido
Published: June 2003

One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14ARF
, p15INK4B
, p16INK4A
, p18INK4C
, and RB1 genes in a series of HCCs and associated cirrhosis with the goal of ascertaining their pattern of inactivation by gene methylation. Thirty-three HCCs, adjacent nonneoplastic cirrhotic tissues, and 6 HCC cell lines were studied. Cirrhoses (25 of 33, 76%), HCCs (31 of 33, 94%), and 3 of 6 (50%) cell lines showed 1 or more methylated genes. Cirrhoses (17 of 33, 51%) had more frequently than HCCs (11 of 33, 33%, P = .01) only 1 methylated gene. With the exception of p18INK4C
the genes under study showed promoter methylation with frequency ranging from 82% (p16INK4A
in HCC) to 33% and 39% (p15INK4B
and p16INK4A
in cirrhoses). In cases with only 1 methylated gene, p15INK4B
in cirrhosis (8 of 17, 47%) and p16INK4A
in HCC (10 of 11, 91%) were the more frequently altered. An optimal correlation was found between p15 and p16 gene methylation and complete protein loss in HCC detected by immunocytochemistry, whereas a partial loss of the same proteins was a feature of methylated cirrhoses. Inactivation by DNA methylation of several genes of the RB1 pathway is common to cirrhosis and HCC. An early pattern of methylatory events (1 methylated gene) is a feature of cirrhosis rather than HCC, whereas an advanced one (≥3 methylated genes) is characteristic of malignancy. Early methylation changes seem to involve p15INK4B
and p16INK4A
in cirrhosis and p16INK4A
in HCC. In conclusion, a stepwise progression of methylating events is a feature of the sequence cirrhosis-HCC and contributes to the process of hepatic carcinogenesis with potential clinical implications.
Incidence of drug-induced hepatic injuries: A French population-based study
Authors: Sgro Catherine , Clinard François , Ouazir Kader , Chanay Henry , Allard Christian , Guilleminet Christian , Lenoir Claude , Lemoine Alain , Hillon Patrick
Published: June 2003

The incidence of hepatic adverse drug reactions (ADRs) remains unknown in the general population. The goal of this population-based study was to assess the incidence and seriousness of hepatic ADRs. All new cases of symptomatic drug-induced hepatic injuries were collected by 139 trained physicians (general practitioners [GPs] and specialists) between November 1997 and November 2000 in an area containing 81,301 inhabitants who could not go elsewhere for medical care. Over 3 years, 34 cases of hepatic ADRs were collected, 82% of them in outpatients. Global crude annual incidence rate was 13.9 ± 2.4 per 100,000 inhabitants; corresponding standardized annual global rate was 8.1 ± 1.5. There was no difference between urban and rural areas. Standardized incidence female/male ratio was 0.86 (0.26-2.90) until 49 years of age and 2.62 (1.00-6.92) after this age. Diagnosis was carried out by GPs in half of the cases. The outcome was recovery for 32 patients and death for 2. The main drugs implicated were anti-infectious, psychotropic, hypolipidemic agents, and nonsteroidal anti-inflammatory drugs (NSAIDs). Our results suggest that the number of hepatic ADRs in the French population would be 16 times greater than the number noted by spontaneous reporting to French regulatory authorities. In conclusion, the incidence and seriousness of drug-induced hepatitis are largely underestimated in the general population. These results may be useful for further evaluation of drug-induced hepatotoxicity.
Ursodeoxycholic acid (UDCA) prevents DCA effects on male mouse liver via up-regulation of CXP and preservation of BSEP activities
Authors: Paolini Moreno , Pozzetti Laura , Montagnani Marco , Potenza Giuseppa , Sabatini Laura , Antelli Alessandra , Cantelli-Forti Giorgio , Roda Aldo
Published: June 2003

To investigate whether ursodeoxycholic acid (UDCA) can prevent metabolic impairment induced by deoxycholic acid (DCA), we evaluated the effects of these bile acids on murine CYP enzymes and the relationship with canalicular bile salt export pump (Bsep) expression. In Swiss Albino CD1 mice, UDCA and DCA were injected intraperitoneally either singly, concurrently, or sequentially (UDCA 1 hour before DCA) at equimolar 24.4 mg/kg body weight (BW) doses. CYP content, NADPH-CYP-c-reductase, and individual mixed function oxidases (MFO) were measured 24 hours later. Modulations were observed mainly in males: whereas DCA decreased MFO activities to various isoenzymes with respect to controls (up to 43%, CYP1A2-linked activity), UDCA boosted them (up to 6-fold, testosterone 16β-hydroxylase); concurrent administration of UDCA and DCA provided a preventive effect, enhancing MFO activity with respect to single administration of DCA by up to 4.4-fold in the CYP3A1/2 and CYP2B1/2 (6β-hydroxylase) and by 2.1-fold in the CYP2E1 (p-nitrophenol hydroxylase). In males (but not females), sequential administration (UDCA then DCA) produced a rather similar protective pattern, but the extent of recovery was generally smaller. Western immunoblotting results for the most affected isoenzymes (CYP3A1/2 and CYP2E1) and Bsep confirmed that UDCA can both prevent and reduce the CYP-dependent MFO inactivation and Bsep down-regulation caused by DCA. These findings may shed further light on the mechanisms responsible for UDCA’s protective role in the treatment of cholestatic liver disease.
p53 may positively regulate hepatocyte proliferation in rats
Authors: Inoue Yukiko , Tomiya Tomoaki , Yanase Mikio , Arai Masahiro , Ikeda Hitoshi , Tejima Kazuaki , Ogata Itsuro , Kimura Satoshi , Omata Masao , Fujiwara Kenji
Published: June 2003

p53, known as a tumor suppressor gene, is a transcription factor that regulates various cellular functions. Recently, several growth factor gene promoters, including that of transforming growth factor α (TGF-α), were shown to be direct targets of p53-mediated transcription. Hepatic p53 mRNA is up-regulated during liver regeneration in rats. The aim of this study is to examine the role of p53 in hepatocyte proliferation. p53 protein levels were examined in rat hepatocytes cultured in the medium containing hepatocyte growth factor (HGF). p53 levels began to increase after 6 hours of incubation, reached a maximum at 18 hours, and decreased thereafter. DNA synthesis increased at 12 hours and peaked at 30 hours. When hepatocytes were incubated with p53 antisense oligonucleotide in addition to HGF, increases of p53 and TGF-α levels were suppressed, and DNA synthesis was reduced. The increases of TGF-α levels and DNA synthesis were also suppressed by a chemical inhibitor of p53, pifithrin-α. In rats after two-thirds partial hepatectomy, hepatic p53 increased and reached maximal levels around 16 hours when hepatic HGF levels have been shown to reach a maximum followed by an increase in hepatic TGF-α levels or hepatocyte proliferation. In contrast, sham-operated rats showed minor elevations of hepatic p53 levels. In conclusion, p53 production is stimulated by HGF and may contribute to the proliferation of rat hepatocytes. Considering previous findings indicating the importance of endogenous TGF-α for the proliferation of hepatocytes stimulated by HGF, TGF-α might play a role in HGF-p53 mediated hepatocyte proliferation.
Modification of
Pseudomonas aeruginosa
Interactions with Corneal Epithelial Cells by Human Tear Fluid
Authors: Fleiszig Suzanne M. J. , Kwong Mary S. F. , Evans David J.
Published: June 2003

Both cytotoxic and invasive strains of
Pseudomonas aeruginosa
can damage corneal epithelial cells in vitro, but neither can infect healthy corneas in vivo. We tested the hypothesis that whole human tear fluid can protect corneal epithelia against
P. aeruginosa
virulence mechanisms. Cultured corneal epithelial cells were inoculated with 10
6
CFU of one of 10 strains of
P. aeruginosa
(five cytotoxic, five invasive)/ml with or without reflex tear fluid collected from the conjunctival sacs of human volunteers. Cytotoxicity was assessed by observation of trypan blue staining and measurement of lactate dehydrogenase release; invasion was quantified by using gentamicin survival assays. Tear fluid retarded growth of only 50% of the
P. aeruginosa
strains (three of five invasive strains, two of five cytotoxic strains) yet protected corneal cells against invasion by or cytotoxicity of 9 of 10 strains. The only strain resistant to the tear cytoprotective effects was susceptible to tear bacteriostatic activity. Dilution of tear fluid threefold significantly reduced cytoprotection, while bacteriostatic activity prevailed with dilutions beyond 100-fold. Sulfacetamide (1 mg/ml) with bacteriostatic activity matching that of tear fluid was less cytoprotective than tear fluid (80% protection with tear fluid, 48% with sulfacetamide). Video microscopy revealed bacterial chain formation in both tear fluid and sulfacetamide, but tear fluid also blocked bacterial swimming motility. After prolonged tear contact, bacteria regained normal growth rates, swimming motility, and cytotoxic activity, suggesting a breakdown of protective tear factors. Boiled tear fluid lost bacteriostatic activity and effects on bacterial motility but retained cytoprotective function. These results suggest that human tear fluid can protect corneal epithelial cells against
P. aeruginosa
virulence mechanisms in a manner not dependent upon bacteriostatic activity or effects on bacterial motility. Whether overlapping tear film components are involved in these defense functions is to be determined.
Development of DNA Vaccines against Hemolytic-Uremic Syndrome in a Murine Model
Authors: Capozzo Alejandra V. E. , Creydt Virginia Pistone , Dran Graciela , Fernández Gabriela , Gómez Sonia , Bentancor Leticia V. , Rubel Carolina , Ibarra Cristina , Isturiz Martín , Palermo Marina S.
Published: June 2003

Shiga toxin type 2 (Stx2) produced byEscherichia coliO:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2ΔA). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.
Immune Responses Induced by theLeishmania(Leishmania)donovaniA2 Antigen, but Not by the LACK Antigen, Are Protective against ExperimentalLeishmania(Leishmania)amazonensisInfection
Authors: Coelho Eduardo Antonio Ferraz , Tavares Carlos Alberto Pereira , Carvalho Fernando Aécio Amorim , Chaves Karina Figueiredo , Teixeira Kadima Nayara , Rodrigues Rafaela Chitarra , Charest Hugues , Matlashewski Greg , Gazzinelli Ricardo Tostes , Fernandes Ana Paula
Published: June 2003

Leishmania amazonensisis one of the major etiologic agents of a broad spectrum of clinical forms of leishmaniasis and has a wide geographical distribution in the Americas, which overlaps with the areas of transmission of many otherLeishmaniaspecies. The LACK and A2 antigens are shared by variousLeishmaniaspecies. A2 was previously shown to induce a potent Th1 immune response and protection againstL. donovaniinfection in BALB/c mice. LACK is effective againstL. majorinfection, but no significant protection againstL. donovaniinfection was observed, in spite of the induction of a potent Th1 immune response. In an attempt to select candidate antigens for an American leishmaniasis vaccine, we investigated the protective effect of these recombinant antigens (rLACK and rA2) and recombinant interleukin-12 (rIL-12) againstL. amazonensisinfection in BALB/c mice. As expected, immunization with either rA2-rIL-12 or rLACK-rIL-12 induced a robust Th1 response prior to infection. However, only the BALB/c mice immunized with rA2-rIL-12 were protected against infection. Sustained gamma interferon (IFN-γ) production, high levels of anti-A2 antibodies, and low levels of parasite-specific antibodies were detected in these mice after infection. In contrast, mice immunized with rLACK-rIL-12 displayed decreased levels of IFN-γ and high levels of both anti-LACK and parasite-specific antibodies. Curiously, the association between rA2 and rLACK antigens in the same vaccine completely inhibited the rA2-specific IFN-γ and humoral responses and, consequently, the protective effect of the rA2 antigen againstL. amazonensisinfection. We concluded that A2, but not LACK, fits the requirements for a safe vaccine against American leishmaniasis.
Inflammatory Cytokine Response to
Vibrio vulnificus
Elicited by Peripheral Blood Mononuclear Cells from Chronic Alcohol Users Is Associated with Biomarkers of Cellular Oxidative Stress
Authors: Powell Jan L. , Strauss Kathy A. , Wiley Cynthia , Zhan Min , Morris J. Glenn
Published: June 2003

Vibrio vulnificus
is the leading cause of death in the United States associated with the consumption of raw seafood, particularly oysters. In epidemiological studies, primary septicemia and inflammation-mediated septic shock caused by
V
.
vulnificus
is strongly associated with liver disease, often in the context of chronic alcohol abuse. The present study was undertaken to determine whether clinical biomarkers of liver function or cellular oxidative stress are associated with peripheral blood mononuclear cell inflammatory cytokine responses to
V
.
vulnificus
. Levels of interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha elicited in response to
V
.
vulnificus
and measured in cell supernatants were not associated with the liver biomarkers aspartate aminotransferase (AST) or alanine aminotransferase (ALT) or the AST/ALT ratio. In contrast, reduced glutathione (GSH) levels were associated with the release of all four cytokines (IL-1β [
R
2
= 0.382;
P
= 0.006], IL-6 [
R
2
= 0.393;
P
= 0.005], IL-8 [
R
2
= 0.487;
P
= 0.001], and TNF-α [
R
2
= 0.292;
P
= 0.021]). Those individuals with below-normal GSH levels produced significantly less proinflammatory cytokines in response to
V
.
vulnificus
. We hypothesize that persons with markers for cellular oxidative stress have increased susceptibility to
V
.
vulnificus
septicemia.
Angiotensin II-induced inotropism requires an endocardial endothelium-nitric oxide mechanism in the in-vitro heart of Anguilla anguilla
Authors: Imbrogno Sandra , Cerra Maria Carmela , Tota Bruno
Published: June 2003

Using an isolated working heart preparation we show that angiotensin II(ANG II), at concentrations of 10-10–10-7 mol l-1, elicits negative chronotropism and inotropism in the freshwater eel Anguilla anguilla. The negative inotropism was insensitive to losartan and CGP42112 (AT1 and AT2 ANG II receptor antagonists, respectively), and was abrogated by the AT1receptor antagonist CV11974, the G protein blocker pertussis toxin (PTx) and the muscarinic antagonist atropine. In contrast, it was not affected by the adrenoceptor antagonists propanolol, sotalol and phentolamine. Using donors(l-arginine) and inhibitors[NG-monomethyl-L-arginine (l-NMMA), l-N5(1-iminoethyl)ornithine (L-NIO)] of nitric oxide synthase (NOS), and haemoglobin as NO scavenger, we demonstrate that NO signalling is involved in ANG II-mediated inotropism. Pretreatment with Triton X-100, a detergent that damages the endocardial endothelium (EE),or with 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase, or with the cGMP-activated protein kinase (PKG) inhibitor KT5328, abolished ANG II-mediated inotropism. Thus, ANG II-mediated inotropism occurs via an EE-NO-cGMP-PKG mechanism. ANG II did not affect the mechanical performance influenced by preload changes (i.e. the Frank–Starling response), which in the eel heart is modulated by NO. This EE-paracrine-mediated cardio-suppressive action of endoluminal ANG II suggests that the hormone plays an important intracardiac role in the fish heart.
Pilot study to assess the effects of early flea exposure on the development of flea hypersensitivity in cats
Authors: Kunkle G.A , McCall C.A , Stedman K.E , Pilny A , Nicklin C , Logas D.B
Published: June 2003

This pilot study was to determine if early oral flea exposure reduces the incidence of flea allergy dermatitis (FAD) in cats. Eighteen kittens, assigned to three groups, received no flea exposure, oral flea exposure or flea infestation for 12 weeks. Then all the kittens were exposed continually to fleas for 31 weeks. Sensitization was monitored using intradermal testing (IDT), in vitro measurement of anti-flea saliva immunoglobulin E (IgE) and development of FAD. There was no statistically significant difference between groups in IDT reactions, in vitro data or clinical scores. The development of FAD was not associated with the presence of anti-flea saliva IgE. However, the development of a delayed reaction to flea bite was associated with symptoms after flea exposure. Although not statistically significant, the FAD scores in the oral group were lower than in the controls. Further studies are required to determine the role of oral flea exposure in the development of FAD in cats.
Conservation of feline semen: Part II: Cold-induced damages on spermatozoal fertilizing ability
Authors: Luvoni G.C , Kalchschmidt E , Marinoni G
Published: June 2003

During the conservation of feline semen, the freeze–thaw procedure in particular is responsible for inducing severe spermatozoal damage, which diminishes fertilizing ability. Therefore, cold-induced damage represents a limiting factor for the conservation of semen, particularly semen from felids, which are often affected by teratospermia. In this article, feline sperm characteristics are reported, with special reference to motility and morphology, which are more likely to be affected by conservation protocols; and moreover, the causes of cold-induced damages are described.

Attention has been focused on methods to evaluate functional integrity of spermatozoa, and those applied to cat semen are reviewed. Among these, a rather recently developed technique involves fluorescent staining methods, and in particular chlortetracycline. The chlortetracycline assay applied to cryopreserved cat epididymal sperm shows that it is suitable to evaluate the functional status of cat sperm.
Serum leptin in NASH correlates with hepatic steatosis but not fibrosis: A manifestation of lipotoxicity?
Authors: Chitturi Shivakumar , Farrell Geoffrey , Frost Linda , Kriketos Adamandia , Lin Rita , Liddle Christopher , Samarasinghe Dev , George Jacob
Published: June 2003

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic steatosis, inflammation, and fibrosis. Leptin is an adipocyte-derived antiobesity hormone that in rodents prevents “lipotoxicity” by limiting triglyceride accumulation and also regulates matrix deposition (fibrosis) during wound healing. We therefore determined serum leptin levels in patients with NASH to determine whether relationships existed between leptin levels and severity of hepatic steatosis or fibrosis. We used a radioimmunoassay to determine serum [total] leptin concentrations in 27 men and 20 women with NASH and 47 controls matched for gender and body mass index (BMI; and partly for age). Serum leptin values were correlated with hepatic steatosis, fibrosis, and inflammation (each categorized semiquantitatively on liver histology), and with anthropometric indices, serum lipids, glucose, insulin, c-peptide, and alanine aminotransferase (ALT) levels. Compared with the controls, mean serum leptin levels were raised in both men and women with NASH (men 14 ± 11 ng/mL vs. 7.2 ± 4.1 ng/mL, P = .003; women 35 ± 16 ng/mL vs. 15 ± 8.2 ng/mL, P < .001). Leptin values correlated with serum c-peptide levels but not with BMI. In a multivariate analysis, serum leptin (P = .027), serum c-peptide (P = .001), and age (P = .027) were selected as independent predictors of the severity of hepatic steatosis. However, serum leptin was not an independent predictor of hepatic inflammation or fibrotic severity. In conclusion, hyperleptinemia occurs in NASH and is not explained simply by gender, obesity, or the presence of type 2 diabetes. Furthermore, leptin levels correlate directly with the severity of hepatic steatosis but not with inflammation or fibrosis. We propose that the relationship between leptin and steatosis reflects a pathogenic role of leptin in hepatic insulin resistance and/or a failure of the antisteatotic actions of leptin (“peripheral leptin resistance”).
Reduction in muscle fibre number during the adaptive radiation of notothenioid fishes: a phylogenetic perspective
Authors: Johnston Ian A. , Fernández Daniel A. , Calvo Jorge , Vieira Vera L. A. , North Anthony W. , Abercromby Marguerite , Garland Theodore
Published: June 2003

The fish fauna of the continental shelf of the Southern Ocean is dominated by a single sub-order of Perciformes, the Notothenioidei, which have unusually large diameter skeletal muscle fibres. We tested the hypothesis that in fast myotomal muscle a high maximum fibre diameter (FDmax) was related to a reduction in the number of muscle fibres present at the end of the recruitment phase of growth. We also hypothesized that the maximum fibre number (FNmax) would be negatively related to body size,and that both body size and size-corrected FNmax would show phylogenetic signal (tendency for related species to resemble each other). Finally, we estimated ancestral values for body size and FNmax. A molecular phylogeny was constructed using 12S mitochondrial rRNA sequences. A total of 16 species were studied from the Beagle Channel, Tierra del Fuego (5-11°C), Shag Rocks, South Georgia(0.5-4°C), and Adelaide Island, Antarctic Peninsula (-1.5 to 0.5°C). The absence of muscle fibres of less than 10 μm diameter was used as the criterion for the cessation of fibre recruitment. FDmaxincreased linearly with standard length (SL), reaching 500-650 μm in most species. Maximum body size was a highly significant predictor of species variation in FNmax, and both body size and size-corrected FNmax showed highly significant phylogenetic signal (P<0.001). Estimates of trait values at nodes of the maximum likelihood phylogenetic tree were consistent with a progressive reduction in fibre number during part of the notothenioid radiation, perhaps serving to reduce basal energy requirements to compensate for the additional energetic costs of antifreeze production. For example, FNmax in Chaenocephalus aceratus (12 700±300, mean ± s.e.m., N=18) was only 7.7% of the value found in Eleginops maclovinus (164 000±4100, N=17), which reaches a similar maximum length (85 cm). Postembryonic muscle fibre recruitment in teleost fish normally involves stratified followed by mosaic hyperplasia. No evidence for this final phase of growth was found in two of the most derived families(Channichthyidae and Harpagiferidae). The divergence of the notothenioids in Antarctica after the formation of the Antarctic Polar Front and more recent dispersal north would explain the high maximum diameter and low fibre number in the derived sub-Antarctic notothenioids. These characteristics of notothenioids may well restrict their upper thermal tolerance, particularly for Champsocephalus esox and similar Channichthyids that lack respiratory pigments.
Decrease of Cd56+T Cells and Natural Killer Cells in Cirrhotic Livers With Hepatitis C May Be Involved in Their Susceptibility to Hepatocellular Carcinoma
Authors: Kawarabayashi Nobuaki , Seki Shuhji , Hatsuse Kazuo , Ohkawa Takashi , Koike Yuji , Aihara Tsukasa , Habu Yoshiko , Nakagawa Ryusuke , Ami Katsunori , Hiraide Hoshio , Mochizuki Hidetaka
Published: June 2003

CD56+T cells and CD56+natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti–CD3 Ab, IL–2, or a combination of IL–2 and IL–12 and their IFN–γ production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (-) patients contained 20% CD56+T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN–γ production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH–7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (-) patients. CD56+T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH–7 cells. These results suggest that a decreased number of CD56+T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.
Evolution of Hepatitis B Virus Polymerase Gene Mutations in Hepatitis B E Antigen-Negative Patients Receiving Lamivudine Therapy
Authors: Lok Anna Suk-Fong , Hussain Munira , Cursano Carmela , Margotti Marzia , Gramenzi Annagiulia , Grazi Gian Luca , Jovine Elio , Benardi Mauro , Andreone Pietro
Published: June 2003

Lamivudine has been shown to be effective in patients with hepatitis B e antigen (HBeAg)–positive chronic hepatitis B, but its long–term efficacy and the rate of resistant mutations in patients with HBeAg–negative chronic hepatitis B is less clear. Twenty–nine patients with HBeAg–negative chronic hepatitis B, who have received lamivudine for at least 1 year were studied to determine the antiviral response, the rate and pattern of lamivudine–resistant mutations, and the effect of lamivudine–resistant mutations on HBeAg status. The mean duration of treatment was 21 ± 7 months. Before treatment, core promoter variant was detected in 16 (55%) patients and precore stop codon variant in 18 (62%) patients. Serum hepatitis B virus (HBV) DNA was detected by solution hybridization assay in 62%, 4%, and 24% and by polymerase chain reaction (PCR) assay in 100%, 31%, and 40% at months 0, 6, and 24, respectively. The cumulative rates of detection of lamivudine–resistant mutations after 1 and 2 years of treatment were 10% and 56%, respectively. In addition to the duration of treatment, core promoter mutation was associated with the selection of lamivudine–resistant mutants. Three patients with lamivudine–resistant mutations had reversion of the precore stop codon mutation; in 2 patients this was accompanied by the reappearance of HBeAg. We found that lamivudine–resistant mutants were detected at similar rates in patients with HBeAg–negative as in patients with HBeAg–positive chronic hepatitis B. Additional changes in other parts of the HBV genome may restore the replication fitness of lamivudine–resistant mutants.
Hyperhomocysteinemia in liver transplant recipients: Prevalence and multivariate analysis of predisposing factors
Authors: Herrero Ignacio J. , Quiroga Jorge , Sangro Bruno , Beloqui Oscar , Pardo Fernando , Cienfuegos Javier A. , Prieto Jesús
Published: June 2003

Liver transplant recipients have an increased risk for cardiovascular disease because of a high incidence of obesity, arterial hypertension, diabetes mellitus, and hyperlipidemia. Hyperhomocysteinemia has been found to be an important risk factor for cardiovascular disease in large studies. Fasting serum levels of homocysteine were measured in 105 liver transplant recipients, and hyperhomocysteinemia was defined as a fasting serum homocysteine level greater than 13 μmol/L. Patients with versus without hyperhomocysteinemia were compared. The possible association of hyperhomocysteinemia with age, sex, cause of liver disease, time elapsed since liver transplantation, immunosuppressive therapy, folic acid level, liver function test results, renal function, and other cardiovascular risk factors was investigated. Patients with serum homocysteine levels greater than 15 μmol/L were treated with folic acid, 10 mg/d, and serum homocysteine levels were measured again 1 to 3 months later in 10 patients. Hyperhomocysteinemia was detected in 28 patients (27%). In univariate analysis, it was associated with hepatitis C virus infection, treatment with mycophenolate mofetil, and greater serum levels of alkaline phosphatase, γ-glutamyl transpeptidase, urea, and creatinine. In multivariate analysis, only greater serum levels of creatinine (P = .006) were associated with hyperhomocysteinemia. Treatment with folic acid resulted in a decrease in fasting serum homocysteine levels in 9 of the 10 patients tested (P = .01). Hyperhomocystinemia, associated with renal dysfunction, is a frequent finding in liver transplant recipients. Treatment with folic acid may reduce fasting homocysteine levels.
Successful liver transplantation in a patient with budd-chiari syndrome caused by homozygous factor V leiden
Authors: Tan Henkie P. , Markowitz Jay S. , Maley Warren R. , Merritt William T. , Klein Andrew S.
Published: June 2003

Budd-Chiari syndrome (BCS) is a rare form of portal hypertension characterized by hepatic venous outflow obstruction. Although hematologic disorders are the most common cause of this syndrome, to date, 30% of the cases have been classified as idiopathic. Resistance to activated protein C caused by factor V Leiden is the most common cause of thrombophilia; its role in the pathogenesis of BCS is now becoming apparent. We report successful liver transplantation in a patient with BCS caused by homozygous factor V Leiden. The patient was administered standard heparin anticoagulation until activated protein C resistance was normalized by the liver allograft. Liver transplantation corrected the thrombophilic state. The patient has excellent graft function, is not on anticoagulation therapy, and has had no recurrent venous thrombosis at 5 months posttransplantation. Activated protein C resistance caused by the factor V Leiden mutation may be responsible for idiopathic cases of BCS. To avoid unnecessary long-term anticoagulation after liver transplantation, factor V Leiden should be considered as a pathogenic factor in BCS. In addition, because of the high prevalence of factor V Leiden in the world population, cadaveric organ donors with a history of venous thrombosis should be screened for activated protein C resistance lest thrombophilia be transmitted to the recipient.
Endoscopic management of biliary leaks after T-tube removal in liver transplant recipients: Nasobiliary drainage versus biliary stenting
Authors: Saab Sammy , Martin Paul , Soliman George Y. , Machicado Gustavo A. , Roth Bennett E. , Kunder Gregg , Han Steven-Huy B. , Farmer Douglas G. , Ghobrial Mark R. , Busuttil Ronald W. , Bedford Rudolph A.
Published: June 2003

This study presents the long-term sequelae of endoscopic retrograde cholangiopancreatography (ERCP)-managed biliary leakage in patients who underwent orthotopic liver transplantation (OLT) and compares the relative efficacy, safety, and charges of nasobiliary drainage (NBD) versus biliary stenting (BS). We identified all orthotopic liver transplant recipients from January 1, 1993, to December 31, 1997, who had undergone ERCP for biliary leakage. Clinical outcome and charges were calculated on an intention-to-treat basis according to initial endoscopic therapy. Of the 1,166 adult OLTs performed during the study period, 442 patients underwent elective T-tube removal. ERCP was attempted in 69 patients (16%) who developed biliary leakage after T-tube removal. Three patients (5%) in whom initial ERCP was unsuccessful underwent surgery. NBD and BS were used as primary therapy in 45 (68%) and 21 patients (32%), respectively. Three patients initially treated with NBD required reendoscopy or surgery compared with 6 patients initially treated with BS (P < .05). Although not statistically significant, there was a trend toward greater expense in the BS group compared with the NBD group. ERCP is a safe and effective method of managing biliary leakage after T-tube removal in orthotopic liver transplant recipients. However, our results suggest NBD is the preferred method because recurrent leaks were more common in patients treated initially with BS. With prompt use of ERCP, surgery is rarely needed for this complication of OLT.
Hepatic localization of endothelin-1 in patients with idiopathic portal hypertension and cirrhosis of the liver
Authors: Kamath Patrick S. , Carpenter Herschel A. , Lloyd Ricardo V. , McKusick Michael A. , Steers Jeffery L. , Nagorney David M. , Miller Virginia M.
Published: June 2003

Endothelin-1 (ET-1) may mediate increased resistance to hepatic sinusoidal blood flow. We evaluated the hepatic distribution of ET-1 in patients with idiopathic portal hypertension (IPH), in which liver architecture may be normal, and in patients with cirrhosis, in which distortion of hepatic sinusoidal architecture is prominent. Immunohistochemistry and in situ hybridization were used to localize ET-1 in hepatic tissue of patients with IPH and cirrhosis. ET-1 was measured in plasma from a peripheral vein, the hepatic vein, and the portal vein of patients with cirrhosis of the liver and controls. On immunohistochemistry and in situ hybridization, ET-1 was localized to periportal hepatocytes and sinusoidal cells in patients with IPH and cirrhosis. Minimal positive staining for ET-1 was observed in control livers. Plasma ET-1 levels were significantly greater in patients with cirrhosis than in controls. In patients with cirrhosis, ET-1 was greater in the hepatic vein compared with the portal vein. However, the level of plasma ET-1 in patients with cirrhosis did not correlate with either the presence of ascites or portacaval pressure gradient. We conclude that in IPH, ET-1 is localized to sites in which it can modulate intrahepatic resistance. In late stages of cirrhosis, ET-1 may not modulate resistance. We speculate that vascular resistance in late stages of cirrhosis probably results from distortion of hepatic architecture.
Does liver transplantation affect growth pattern in alagille syndrome?
Authors: Quiros-Tejeira Ruben E. , Ament Marvin E. , Heyman Melvin B. , Martin Martin G. , Rosenthal Philip , Gornbein Jeffrey A. , McDiarmid Sue V. , Vargas Jorge H.
Published: June 2003

Alagille syndrome (AGS) is frequently associated with growth failure, which has been attributed to concurrent congenital anomalies, cholestasis, and malabsorption and/or malnutrition. However, the underlying cause of the growth failure is not well understood. Our objective is to analyze the growth pattern in 26 patients with AGS and the possible effect that orthotopic liver transplantation (OLT) may have on this pattern. The standardized height, weight, and growth velocity of 26 pair-matched patients with AGS were compared. Thirteen patients underwent OLT. Repeated-measure ANOVA methods were used for the statistical analysis. The overall mean standardized height (z score) was -2.92 in the OLT group versus -1.88 in the non-OLT group (P = .03). The overall mean standardized weight was -1.21 in the non-OLT group and -1.67 in the OLT group (P = .23). In 15 patients, birth weight was 2.82 ± 0.4 kg, for a mean standardized weight of -0.95, and weight at diagnosis was 4.53 ± 2.12 kg, for a mean standardized weight of -1.56. Bone age was delayed in the 9 patients who underwent bone-age analysis. Growth hormone therapy administered to 2 patients did not improve growth. Patients with AGS had growth failure secondary to other factors in addition to liver disease. Growth failure beginning in the prenatal period supports a genetic basis for this feature. Growth improvement up to normal levels should not be expected as a benefit of OLT in these patients. Growth failure as a primary indication for OLT should be cautiously examined in patients with AGS.
Involvement of the fas system in hepatitis C virus recurrence after liver transplantation
Authors: Crespo Javier , Rivero Monteserrat , Mayorga Marta , Fabrega Emilio , Casafont Fernando , Gomez-Fleitas Manuel , Pons-Romero Fernando
Published: June 2003

To date, there have been no reports of the involvement of the Fas system in recurrent hepatitis C virus (HCV) infection after orthotopic liver transplantation (OLT). In 25 patients who underwent OLT for HCV-related liver cirrhosis, we evaluated the expression of the Fas antigen (FasAg) on hepatocytes, apoptic hepatocytes, and serum levels of soluble Fas (sFas). The level of HCV viremia and HCV genotype were determined by polymerase chain reaction. Serum sFas levels were determined by an enzyme immunoassay procedure. DNA fragmentation was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) technique on deparaffinized liver samples. FasAg expression was evaluated by an immunoperoxidase method. Sixteen patients had evidence of recurrent HCV disease. The number of hepatocytes expressing FasAg and the percentage of apoptotic hepatocytes was greater among patients who developed recurrent hepatitis than among those who did not (P < .01 and P < .0001, respectively). There was a correlation between hepatic expression of FasAg, intensity of lobular inflammation (P = .007), and TUNEL index (P < .001). The levels of sFas were greater among the patients with recurrent HCV hepatitis than those without recurrent hepatitis (P < .04). We conclude that (1) Fas expression is up-regulated in recurrent HCV after OLT and is related to the grading of liver disease; likewise, levels of sFas were greater in the patients with recurrent HCV hepatitis; and (2) the demonstration of hepatocytes with FasAg expression and the labeling of the nuclei by TUNEL assay suggest that hepatic apoptosis mediated by the Fas system may have a role in the pathogenesis of recurrent HCV hepatitis after OLT.
Retransplantation for hepatic allograft failure: Prognostic modeling and ethical considerations
Authors: Biggins Scott W. , Beldecos Athena , Rabkin John M. , Rosen Hugo R.
Published: June 2003

Retransplantation already accounts for 10% of all liver transplants performed, and this percentage is likely to increase as patients live long enough to develop graft failure from recurrent disease. Overall, retransplantation is associated with significantly diminished survival and increased costs. This review summarizes the current causes of graft failure after primary liver transplant, prognostic models that can identify the subset of patients for retransplantation with outcomes comparable to primary transplantation, and ethical considerations in this setting, i.e., outcomes-based versus urgency-based approaches.
The management of trust‐based working time in Germany
Authors: Singe Ingo , Croucher Richard
Published: June 2003

New developments in trust‐based working time systems (i.e. systems whereby managers formally devolve their responsibilities for monitoring working time) in Germany are examined. A picture of these systems is presented and the main debates reviewed. It is argued that the successful introduction of such systems is contingent on a number of inter‐related factors. These are: company size and management style, external and internal pressures and effective employee representation. It is concluded that such systems are most likely to be successful in larger organisations and that effective employee representation is a key requirement. Current circumstances are not necessarily conducive to the rapid spread of trust‐based working time systems.
Time and management: the need for hora management
Authors: Karsten Luchien , Leopold John
Published: June 2003

Working time patterns are moving away from the traditional pattern of regularity, standardisation and co‐ordination to a new triptych of individualism, heterogeneity and irregularity. Seeks to make sense of these changes through the concept of hora management as an approach to manage the interface between temporally asymmetric domains of organisational and domestic space mediated through the space of professional relations. Drawing on the societal approach, defines the three spaces – professional relations, organisational and domestic – and builds a model of their inter‐relationship that incorporates the supranational impact of the European Union. Organisational citizenship will require managers to pursue family‐friendly policies and recognise that time spent in one domain cannot be equated with time spent in another. Hora management offers a way of managing these tensions and contradictions.
Family‐friendly policies in The Netherlands
Authors: Remery Chantal , van Doorne‐Huiskes Anneke , Schippers Joop
Published: June 2003

The article reports on research among Dutch employers concerning the arrangements they provide for employees to help them with the reconciliation of work and family life. The research not only answers the question of to what extent different employers offer arrangements like childcare facilities, flexible working hours or leave schemes, but it also tries to explain employers’ policies. The explanatory analysis includes organisational characteristics and employers’ opinions with respect to costs and benefits of different arrangements. The empirical analysis is based on a survey among 871 organisations in the profit sector and the non‐profit sector. One major conclusion is that family‐friendly arrangements have become rather common among organisations; employers are aware of the fact that the reconciliation of work and care has become an issue for an increasing number of workers.
Functional Similarities between theicm/dotPathogenesis Systems ofCoxiella burnetiiandLegionella pneumophila
Authors: Zusman Tal , Yerushalmi Gal , Segal Gil
Published: June 2003

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular pathogen, whereasLegionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. During infection of humans both of these pathogens multiply in alveolar macrophages inside a closed phagosome.L. pneumophilaintracellular multiplication was shown to be dependent on theicm/dotsystem, which probably encodes a type IV-related translocation apparatus. Recently, genes homologous to all of theL. pneumophila icm/dotgenes (besidesicmR) were found inC. burnetii.To explore the similarities and differences between theicm/dotpathogenesis systems of these two pathogens, interspecies complementation analysis was performed. NineC. burnetii icmhomologous genes (icmT,icmS,icmQ,icmP,icmO,icmJ,icmB,icmW, andicmX) were cloned under regulation of the correspondingL. pneumophila icmgenes and examined for the ability to complementL. pneumophilamutants with mutations in these genes. TheC. burnetii icmSandicmWhomologous genes were found to complement the correspondingL. pneumophila icmmutants to wild-type levels of intracellular growth in both HL-60-derived human macrophages andAcanthamoeba castellanii. In addition, theC. burnetii icmThomologous gene was found to completely complement anL. pneumophilainsertion mutant for intracellular growth in HL-60-derived human macrophages, but it only partially complemented the same mutant for intracellular growth inA. castellanii. Moreover, as previously shown forL. pneumophila, the proteins encoded by theC. burnetii icmSandicmWhomologous genes were found to interact with one another, and interspecies protein interaction was observed as well. Our results strongly indicate that the Icm/Dot pathogenesis systems ofC. burnetiiandL. pneumophilahave common features.
Intestinal and Systemic Immune Responses to an Oral Cholera Toxoid B Subunit Whole-Cell Vaccine Administered during Zinc Supplementation
Authors: Karlsen Tom H. , Sommerfelt Halvor , Klomstad Skjalg , Andersen Per Kragh , Strand Tor A. , Ulvik Rune J. , Åhrén Christina , Grewal Harleen M. S.
Published: June 2003

Zinc plays a critical role in the normal functioning of the immune system. We investigated whether zinc sulfate administered orally to adult zinc-replete volunteers modulates systemic and intestinal immune responses to an oral killed cholera toxoid B subunit (CTB) whole-cell cholera vaccine. The 30 participants were immunized twice, with a 17-day interval. The vaccinees in the intervention group ingested 45 mg of elemental zinc thrice daily for 9 days starting 2 days before each vaccine dose. The median serum anti-CTB immunoglobulin A (IgA) and IgG responses from day 0 to day 30, i.e. after two vaccine doses, were 13-fold lower(Pvalue for identical distribution, <0.005) in the zinc-supplemented compared to the nonsupplemented vaccinees. The median serum vibriocidal responses from baseline to after one (day 0 to day 17) and two (day 0 to day 30) vaccine doses were at least sixfold(P= 0.033) and fourfold(P= 0.091) higher, while the median fecal anti-CTB IgA response after two doses was estimated to be fourfold higher(P= 0.084) in the zinc-supplemented vaccinees. These observations show that zinc reduces the antitoxin and may enhance the antibacterial responses in serum. Zinc may also improve the intestinal antitoxin immune response. Oral zinc administration has the potential to modify critical immune responses to antigens applied to mucosal surfaces.
Identification of CD8 T-Lymphocyte Epitopes in OmpB ofRickettsia conorii
Authors: Li Zhen , Díaz-Montero C. Marcela , Valbuena Gustavo , Yu Xue-Jie , Olano Juan P. , Feng Hui-Min , Walker David H.
Published: June 2003

The 1.2-kb DNA fragment of theRickettsia conoriiouter membrane protein B gene (OmpB451-846) was subcloned using site-specific PCR primers and expressed as six smaller fragments: OmpB458-652, OmpB595-744, OmpB595-654, OmpB645-692, OmpB689-744, and OmpB739-848. NCTC cells transfected with a mammalian expression vector expressing the fragments OmpB689-744and OmpB739-848stimulated immune anti-R.conoriiCD8 T lymphocytes, suggesting the presence of CD8 T-lymphocyte-stimulating epitopes on these fragments. In order to further characterize the CD8 T-lymphocyte-stimulatory elements, CD8 T-lymphocyte epitopes on OmpB689-744and OmpB739-848were mapped by overlapping synthetic peptides. The ability of these synthetic peptides to stimulate immune CD8 T lymphocytes was determined by gamma interferon (IFN-γ) production and cell proliferation after incubation with simian virus 40-transformed murine vascular endothelial cells in the presence of a 20 μM solution of each synthetic peptide. Five synthetic peptides, SKGVNVDTV (OmpB708-716), ANVGSFVFN (OmpB735-743), IVSGTVGGQ (OmpB749-757), ANSTLQIGG (OmpB789-797), and IVEFVNTGP (OmpB812-820), induced secretion of IFN-γ at significantly higher levels than the controls. Three of these five peptides, SKGVNVDTV (OmpB708-716), ANSTLQIGG (OmpB789-797), and IVEFVNTGP (OmpB812-820), also stimulated the proliferation of immune CD8 T lymphocytes. Significantly higher levels of specific cytotoxic T-lymphocyte killing were observed with the same three synthetic peptides, SKGVNVDTV (OmpB708-716), ANSTLQIGG (OmpB789-797), and IVEFVNTGP (OmpB812-820).