The effect of serum from liver cancer patients on the growth and function of primary and immortalised hepatocytes.

A limiting factor in the efficacy of bioartificial liver (BAL) for the treatment of liver failure is the toxicity of the patients' serum to the hepatocytes in the device. This study investigates the interaction of liver cancer patient serum with primary and immortalised rat hepatocytes. Liver cancer serum increased the growth rate of immortalised hepatocytes, without affecting reduced glutathione levels. The activities of DT-diaphorase and pi glutathione-S-transferase (GST), enzymes associated with de-differentiation, were also increased. Exposure of primary hepatocytes to liver cancer serum resulted in a decrease in cytochrome P450 (CYP) content, and in P450 dependent metabolism of testosterone. Formation of 2-alpha- and 6-beta- hydroxy testosterone was decreased. These reactions are predominantly associated with CYP 2C11 and 3A1 respectively in normal rat liver. The activity of total GST was also decreased, although that of the pi isoenzyme of GST was not affected. Our results suggest that exposure of hepatocytes in a bioreactor to liver cancer patient serum will result in overgrowth of cells, if proliferating cells are being used, and in de-differentiation. The serum may have to be pretreated with adsorbants to remove toxins prior to BAL treatment.

Serum hyaluronan--a non-invasive test for diagnosing liver cirrhosis.

INTRODUCTION: Hyaluronan is a glucosaminoglycan synthesized by the mesenchymal cells and degraded by hepatic sinusoidal endothelial cells by a specific receptor-mediated process. Elevated levels are associated with the sinusoidal capillarization that is seen in cirrhosis. METHODOLOGY: Serum hyaluronan was measured, using a radiometric assay (Pharmacia, Sweden) in 221 patients with biopsy-proven chronic liver disease of a variety of aetiologies (alcohol n = 70, autoimmune chronic active hepatitis n = 23, primary biliary cirrhosis n = 17, hepatitis C n = 69, cryptogenic n = 15, various n = 27). All patients were fasted, and their liver function tests, full blood count, prothrombin time and Child-Pugh score were assessed at the time of the liver biopsy. RESULTS: Hyaluronan levels (microg/l) were significantly higher in patients with liver cirrhosis (cirrhosis n = 127, mean 440, 95% CI 367-515) (P < 0.0001) compared with hepatic fibrosis (n = 23, mean 144, 95% CI 69-190), chronic hepatitis (n = 60, mean 63, 95% CI 37-91) and fatty liver (n = 11, mean 107, 95% CI 37-177). Within the cirrhotic population, there was no significant difference in hyaluronan levels between different aetiologies, but hyaluronan level increased proportionally to the severity of cirrhosis. Overall, a hyaluronan level > 100 microg/l had a 78% specificity and 83% sensitivity for diagnosing cirrhosis, while the specificity was increased to 96% for all patients with hyaluronan levels > 300 microg/l. The highest specificity and sensitivity were seen at a cut-off value of 100 microg/l in patients with alcohol-associated liver disease (89%, 87%) and hepatitis C (93%, 72%) respectively. Within patient cohorts, there was a significant correlation (P < 0.01) between hyaluronan and albumin, platelet count and bilirubin, but not with alanine aminotransferase. CONCLUSION: Measurement of fasted serum hyaluronan reliably differentiated cirrhotic from non-cirrhotic liver disease and can be regarded as a useful test in the diagnosis of liver cirrhosis, particularly when a liver biopsy is contraindicated.

Liver transplantation of patients in end-stage cirrhosis.

The success of liver transplantation has resulted in its widespread use for end-stage liver disease; 1- and 5-year survival rates of 70-90% and 60-80% respectively have been reported. Indications for assessment for liver transplantation are now evidence-based and early referral is recommended, correlating with improved patient survival. The management of patients on the waiting list for liver transplantation is designed to prevent complications of liver disease and to avoid therapeutic misadventures. Following transplantation, rejection and infection dominate post-operative complications, and improvements in their prevention and treatment have also correlated with improved patient survival. The development and introduction into clinical practice of a variety of immunosuppressive agents has offered a bewildering array of therapeutic options but with a lack of evidence on which to select optimal immunosuppression. Similarly, difficulties remain in the treatment of some of the complications arising from liver transplantation such as recurrence of disease and complications of immunosuppression.

The effect of octreotide on gastroduodenal blood flow measured by laser Doppler flowmetry in rabbits and man.

OBJECTIVE: The somatostatin analogue, octreotide is valuable in the management of variceal bleeding, and it has been suggested that it may stop peptic ulcer hemorrhage by reducing gastroduodenal blood flow or increasing intragastric pH. The aim of this study was to determine the effect of intravenous octreotide infusion on gastroduodenal mucosal blood flow and gastric pH. METHODS: Seven New Zealand white rabbits and five healthy human volunteers were used. Mucosal blood flow was measured using a laser Doppler flowmeter (LDF). The Doppler probe was positioned in the upper gastrointestinal tract of the seven rabbits and five human volunteers. Blood flow was measured before and after octreotide infusion. RESULTS: In the animal experiments, mucosal blood flow was decreased in a dose dependent manner in the gastric body (209.1-56.3 U) (p < 0.008), antrum (143.3-33.3 U) (p < 0.02) and duodenum (254-67.6 U) (p < 0.016) by doses of octreotide ranging from 10-50 microg/kg of body weight. In the human studies, mucosal blood flow was decreased in the gastric body (p < 0.016) and antrum (p < 0.009) after octreotide infusion (dose 1-1.5 microg/kg). Intragastric pH was significantly increased (p < 0.05). The change was not associated with systemic hemodynamic changes. CONCLUSIONS: Gastroduodenal mucosal blood flow was reduced and intragastric pH increased by octreotide. This agent could be helpful in the management of gastroduodenal mucosal bleeding.

The impact of chronic hepatitis C virus infection on HIV disease and progression in intravenous drug users.

OBJECTIVE: It is unclear whether co-infection with hepatitis C virus (HCV) can influence HIV related morbidity or mortality, either by accelerating HIV-related disease progression, or by contributing to end stage liver disease. The aim of this study was to examine the effect of HCV infection on the severity and progression of HIV disease in a cohort of Edinburgh intravenous drug users (IDUs). METHODS: In 240 (47%) out of 508 patients in the Edinburgh IDU cohort both HIV seroconversion dates and anti-HCV serology were available. Demographic variables and HIV-related progression between anti-HCV positive and anti-HCV negative groups were compared. Parameters assessed included clinical endpoints (time of development of significant symptoms attributable to HIV (CDC stage IV), time of development of AIDS, and time of death) and immunological endpoints (time of CD4+ counts dropping below 200/mm3, 100/mm3 and 50/mm3). RESULTS: Two hundred and two out of 240 patients (84%) had positive anti-HCV serology. There was no significant difference in the frequency of clinical and immunological endpoints between the anti-HCV positive and negative groups. Progression analysis from HIV seroconversion to HIV related clinical endpoints indicated that anti-HCV serology was not a significant factor influencing the rate of HIV progression (relative risks (RR) for anti-HCV positive group: seroconversion to CDC IV, 1.01; seroconversion to AIDS, 1.05; seroconversion to death, 0.90). Likewise, HCV serostatus did not significantly affect progression to immunological endpoints (RR for anti-HCV positive group: seroconversion to CD4+ < 200/mm3, 1.04; seroconversion to CD4+ < 100/mm3, 1.13; seroconversion to CD4+ < 50/mm3, 0.97. Overall mortality from end stage liver failure was 4% in HCV-seropositive patients without AIDS. This suggests that HCV has had a clinically (though not statistically) significant impact on overall survival in this cohort. CONCLUSIONS: This study demonstrates that HCV co-infection does not influence the rate of progression to either clinical or immunological endpoints in our population of HIV-infected drug users. Further data are required to assess the effect of HIV on thge progression of HCV-related liver disease.

Prevalence, incidence, and clinical characteristics of hepatitis G virus/GB virus C infection in Scottish blood donors.

The prevalence, incidence, clinical features, and natural history of hepatitis G virus (HGV) or GB virus C (GBV-C) were investigated in a non-remunerated blood donor population to determine its clinical significance and its impact on blood safety. Of 1020 regular blood donors, 23 (2.25%) were positive for plasma HGV/GBV-C RNA. Alanine aminotransferase levels were lower than in uninfected donors (median, 20 IU/mL; 32 IU/mL in controls; P=.015). Clinical examination produced no other evidence for hepatitis or for shared nonhepatic diseases. Fifteen of 17 donors excreted HGV/GBV-C in saliva (mean level, 8x103 copies of RNA/mL). Testing of previous donations indicated an incidence of 170-200 new infections with HGV/GBV-C per 100,000 donor-years. The absence of further clinicopathologic data and the limitations of current polymerase chain reaction-based methods for screening suggests that it is neither necessary nor practical to commence screening.

Prediction of cirrhosis in patients with chronic hepatitis C infection by artificial neural network analysis of virus and clinical factors.

The diagnosis of cirrhosis in patients with hepatitis C virus (HCV) infection is currently made using a liver biopsy. In this study we have trained and validated artificial neural networks (ANN) with routine clinical host and viral parameters to predict the presence or absence of cirrhosis in patients with chronic HCV infection and assessed and interpreted the role of the different inputs on the ANN classification. Fifteen routine clinical and virological factors were collated from 112 patients who were HCV RNA positive by reverse transcriptase-polymerase chain reaction (RT-PCR). Standard and Ward-type feed-forward fully-connected ANN analyses were carried out both by training the networks with data from 82 patients and subsequently testing with data from 30 patients plus performing leave-one-out tests for the whole patient data set. The ANN results were also compared with those from multiple logistic regression. The performance of both ANN methods was superior compared with the logistic regression. The best performance was obtained with the Ward-type ANNs resulting in a sensitivity of 92% and a specificity of 98.9% together with a predictive value of a positive test of 95% and a predictive value of a negative test of 97% in the leave-one-out test. Hence, further validation of the ANN analysis is likely to provide a non-invasive test for diagnosing cirrhosis in HCV-infected patients.

The biology of interferon-alpha and the clinical significance of anti-interferon antibodies.

The therapeutic indications for Interferons (IFNs) have dramatically increased in number in recent years to include many different diseases of viral, malignant, angiogenic, allergic, inflammatory and fibrotic origin. In particular, the current pandemic of hepatitis C virus infection has further stimulated the requirement for a comprehensive understanding of both the mechanism of action of IFN and the reasons for therapeutic failure. The role of anti-IFN antibodies as a cause of treatment failure has been a particularly controversial area. In this review we will outline the biology and proposed mechanisms of action of IFN-alpha (IFN-alpha) and discuss the incidence, methods of detection and clinical significance of anti-IFN antibodies.

Detection of a novel DNA virus (TTV) in blood donors and blood products.

BACKGROUND: A newly discovered DNA virus, transfusion-transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). METHODS: We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. FINDINGS: TTV viraemia was detected in 19 (1.9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non-remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. INTERPRETATION: TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTV's aetiological role in hepatic and extra-hepatic disease, are urgently needed.

Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection.

BACKGROUND: The clinical significance of a single assessment of circulating hepatitis C virus (HCV) RNA and its relation to the level of intrahepatic HCV RNA remains unclear. AIMS: To investigate the relation between intrahepatic HCV levels and clinicopathological characteristics of chronic HCV infection. PATIENTS: Ninety eight consecutive patients with chronic HCV infection were studied; none had received alpha interferon therapy. Of these, 12 patients were repeatedly negative for HCV RNA in serum by reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: After diagnostic laparoscopy and liver biopsy, semiquantitative analysis of intrahepatic HCV RNA levels was carried out by limiting dilution of HCV cDNA. HCV genotypes were assessed in 96 patients by restriction fragment length polymorphism analysis of HCV cDNA. RESULTS: Ten out of 12 patients who were RT-PCR negative for HCV RNA in serum were RT-PCR positive in liver; however, this group had a significantly lower intrahepatic HCV level and serum aminotransferase level than the remaining 86 patients. Histological severity (cirrhosis: n = 10); histological activity index; HCV genotype (genotype 1: n = 41; genotype 2: n = 12; genotype 3: n = 36; genotype 4: n = 7); mode of infection (intravenous drug abuse: n = 58; post-transfusion: n = 10; haemophiliac: n = 4; sporadic: n = 26) and alcohol abuse did not affect the intrahepatic virus level. There was no correlation between patient age, duration of infection, and intrahepatic HCV level. CONCLUSIONS: Intrahepatic virus levels were not determined by host factors (age of patient, mode or duration of infection) or by virus factors (HCV genotype). Repeatedly negative RT-PCR for HCV RNA in serum does not indicate absence of HCV from the liver.

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.

Diagnostic laparoscopy by physicians: we should do it.

Only a few centres in the UK practise diagnostic laparoscopy and liver biopsy; in comparison, laparoscopy is widely practised by physicians in Europe, the Far East and in the US. We consider the role of diagnostic laparoscopy in the assessment of liver disease in the 1990s, and describe the technique of laparoscopy, including how it may be performed safely in the endoscopy suite, under local anesthesia with mild sedation and analgesia, enabling direct visualization of the liver. We examine potential complications and contraindications. Complication rate and mortality are similar to that for percutaneous liver biopsy. Finally, the invaluable role of laparoscopy in diagnosing and staging chronic hepatitis, cirrhosis, liver tumours, hepatic infiltration, infection and structural abnormalities is considered.

Association between chronic hepatitis C infection and hepatocellular carcinoma in a Scottish population.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The geographical prevalence varies considerably in different countries and Scotland is regarded as an area of low risk for the disease. AIMS: To assess the association between chronic hepatitis C infection (HCV) and HCC in a population of patients presenting to a single hospital. PATIENTS: One hundred and fourteen cases of histologically confirmed liver cancer presenting to the Royal Infirmary of Edinburgh between 1985 and 1994 were examined. METHODS: Of 114 cases of HCC, 80 samples of stored sera were available. Samples positive for HCV Ab were genotyped by restriction fragment length polymorphism analysis of HCV c-DNA. A population of 29 cirrhotic patients (diagnosed between 1985 and 1994) with chronic HCV infection was also genotyped. RESULTS: Chronic HCV infection was a major risk factor (30% of tested HCC patients) identified. HCV genotype 1b was predominant (16 of 20 patients). The time from HCV transmission to development of cancer ranged from 10 to 50 years (median 30). In the cirrhotic patient population, a broader distribution of genotypes was present (genotype 1a: 7; genotype 1b: 8; genotype 2b: 3; genotype 3a: 8 and genotype 4: 2). However, this population was significantly younger. (Mean (SD) 52 (14.5) years) (p = 0.0002) and demonstrated a significantly shorter duration of infection: range 10-40 years (median: 19). CONCLUSION: There is a strong association between chronic HCV infection, cirrhosis, and hepatocarcinogenesis in this Scottish population. The study was unable to distinguish whether the high prevalence of genotype 1b in the HCC population reflected increased oncogenicity in itself, or whether 1b was simply the most prevalent genotype in Scotland when these patients were infected.

The clinical significance of the detection of hepatitis GBV-C RNA in the serum of patients with fulminant, presumed viral, hepatitis.

In a significant number of cases of fulminant (presumed viral) hepatitis worldwide, no aetiological agent has been identified. Recently, it has been suggested that a newly described flavivirus, GBV-C, is responsible for some of these cases. This study aimed to assess the clinical significance of GBV-C RNA, demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR), in the serum of patients with fulminant non-A to E hepatitis. Twenty-three consecutive cases of non-A to E fulminant hepatitis were included in the study. GBV-C RNA was reverse transcribed and amplified using two RT-PCR based detection methods. Medical records were examined to assess clinical history, duration and mode of infection, transfusion history, liver histology and clinical outcome. Five (three female, two male; mean age 21.2 years) of 23 patients had GBV-C RNA detected in their serum by RT-PCR: all five patients were RT-PCR positive following amplification by primers specific for the 5' non-coding region (NCR), whilst four were positive by primers for the NS3 region. Prior to the onset of illness, two patients had risk factors for transmission of an infectious agent; however, all five patients had been transfused during their illness, prior to testing for GBV-C. Of these, two (of two in whom serum was available) were negative for GBV-C after the onset of fulminant hepatitis but before their first transfusion. This study does not support the hypothesis that the detection of hepatitis G virus (HGV)/GBV-C RNA in the serum of patients with fulminant hepatitis indicates a causal association. However, it does demonstrate that a careful transfusion history and screening of blood products is vital before the importance of GBV-C in the aetiology of fulminant hepatitis can be established.

Alteration in mRNA levels of Fas splice variants in hepatitis C-infected liver.

The Fas receptor (APO-1/CD95) is expressed on hepatocytes and is thought to be important in triggering apoptosis after ligation by the Fas ligand carried on cytotoxic T cells. Recent evidence has shown that several splice variants of Fas exist, the major one of which (FasTMDel) may produce a soluble protein which can modulate apoptosis by interacting with ligand. There are no data on the expression of splice variants of Fas in liver disease. RNA was extracted from needle biopsies from 13 patients with hepatitis C virus (HCV) infection and six normal liver samples. By reverse transcriptase polymerase chain reaction (RT-PCR) FasTMDel expression was demonstrated at the mRNA level, in both normal and HCV-infected liver. Quantitative PCR demonstrated an increase in Fas transcript relative to FasTMDel in HCV infection. This difference is due to an induction of Fas, with FasTMDel remaining at constant levels in the two groups. If translated into protein, liver cells may express more Fas and thus be susceptible to apoptosis inducible by ligand-bearing cytotoxic T cells. These findings suggest that mechanisms exist to regulate the differential splicing of Fas and FasTMDel dependent on the cell's environment. The degree of alteration in the levels of Fas relative to FasTMDel occurred independently of the ALT levels and histological grading of the HCV-infected cases. However, an association was noted between increasing Fas:FasTMDel ratio and log viral load in the liver, measured by competitive PCR.

Screening for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major worldwide health problem and the most common cause of cancer induced mortality. This review article examines the natural history of HCC and the known risk factors, as well as evaluating the current methods of detection of HCC and their impact on patient survival.

Assessment of liver histology in patients with hepatitis C and normal transaminase levels.

OBJECTIVE: To compare liver histology in patients with hepatitis C infection (HCV) between patients with normal and abnormal alanine aminotransferase (ALT) and examine its relationship with HCV genotype and route of infection. METHODS: One hundred consecutive patients with known HCV presenting for diagnostic laparoscopy and liver biopsy were studied. Route of infection was noted and ALT measured on the day of biopsy and HCV genotype determined. Hepatic pathology was analysed using the Edinburgh scoring system (ESS). RESULTS: Fifteen patients had normal ALT, of whom three (20%) had cirrhosis and 10 (66.7%) fibrosis. Of patients with raised ALT, cirrhosis and fibrosis were similarly found in 14 (16.5%) and 62 (72.9%) respectively. Severe, moderate and mild inflammation occurred in one (6.7%), four (26.7%) and 10 (66.6%) patients with normal ALT compared with 17 (20%), 50 (58.8%) and 18 (21.2%) of those with raised ALT (P < 0.01). There was no significant difference in age, route of infection or genotype between those with normal and raised ALT levels. CONCLUSION: Of patients with normal ALT at time of liver biopsy, a significant minority have cirrhosis or significant hepatic inflammation. Liver biopsy is essential in HCV infection as a guide to both therapy and prognosis.

Hypoxemia during diagnostic laparoscopy: a prospective study.

BACKGROUND: Laparoscopic liver biopsy can be safely performed using local anesthesia and intravenous sedation, but the frequency of hypoxemia is unknown. METHODS: We prospectively studied 68 patients undergoing diagnostic laparoscopy and liver biopsy managed by a standard protocol. RESULTS: The mean duration of laparoscopy was 27 +/- 5.53 minutes; the mean dose of diamorphine administered was 6.9 +/- 2.7 mg; diazepam, 7.05 +/- 3.52 mg. The baseline arterial oxygen saturation was 95.6% +/- 2.5% and trough was 85% +/- 5.1%. A fall of greater than 4% saturation from the baseline occurred in 64 out of 68 patients (94%). The mean decrease in saturation was 10.1% +/- 5.4%. An arterial oxygen saturation of less than 85% was seen in 32 patients (47%). There was no correlation between the fall in oxygen saturation and the dose of diamorphine or diazepam, the duration of procedure, body mass index, hemoglobin, or volume of pneumoperitoneum induced. One-way analysis of the variance failed to show a significant relationship between the degree of oxygen saturation and Child's class, etiology of liver disease, or smoking habit. CONCLUSIONS: In this study, we demonstrated that significant desaturation is common in diagnostic laparoscopy with liver biopsy and is likely due to a combination of different pharmacologic and physiologic effects. We recommend continuous monitoring of both arterial oxygen saturation and supplemental oxygen for all patients throughout laparoscopy.