Apoptotic cytokeratin 18 neoepitopes in serum of patients with chronic hepatitis C

In patients with chronic hepatitis C, alanine aminotransferase (ALT) levels do not accurately reflect the extent of liver inflammation. The discrepancy between ALT level and liver damage could be related to the mode of cell death. In the present study, we quantified serum levels of apoptotic cytokeratin 18 (CK-18) neoepitopes that are generated by activated caspases during apoptosis. Apoptotic CK-18 neoepitopes were quantified by enzyme linked immunosorbent assay in sera from patients with chronic hepatitis C and elevated ALT levels (n = 72), patients with chronic hepatitis C and persistently normal ALT levels (n = 27) and healthy controls (n = 19). Serum CK-18 neoepitope levels were strongly correlated with ALT (r = 0.659, P < 0.0001) and the histology activity index (r = 0.374, P < 0.001). Patients with chronic hepatitis C and persistently normal ALT levels had higher apoptotic CK-18 neoepitope levels than healthy controls (P = 0.03) but lower levels than patients with chronic hepatitis C and elevated ALT levels (P < 0.001). Highest serum CK-18 neoepitope levels were observed in patients with cirrhosis (P = 0.002). Hence apoptotic CK-18 neoepitopes in serum of patients with chronic hepatitis C are associated with ALT level and histological liver damage. Serum apoptotic CK-18 neoepitope levels are elevated both in patients with chronic hepatitis C and elevated ALT levels as well as in patients with normal ALT levels indicating that also patients with chronic hepatitis C and normal ALT have an increased hepatocyte loss by apoptosis.     

Apoptotic and anti-apoptotic seromarkers for assessment of disease severity of non-alcoholic steatohepatitis

Background and study aimsThis study aimed to find out non-invasive markers for the assessment of severity of non-alcoholic steatohepatitis (NASH) in an attempt to decrease the need for liver biopsy. It also aimed to evaluate the key role of apoptosis in the pathogenesis of the disease and the suggested role of anti-apoptotic factors in therapeutic modalities and disease prognosis.Patients and methodsThe serum levels of soluble Fas (s. Fas), s. Fas ligand, cytokeratin 18 (CK-18) fragment and Bcl-2 were measured in 80 patients and 15 non-hepatic subjects as control. The patients were divided based on histological examination of liver biopsy into three groups. Group I included 40 patients with NASH, group II had 40 patients with non-alcoholic fatty liver disease (NAFLD) non-NASH and group III had 15 non-hepatic subjects as control. Apoptosis of hepatocytes was assessed by morphological examination using a light microscope and expressed as number per square millimetre.ResultsThere was a significant increase in the serum levels of s. Fas, s. Fas ligand and CK-18 fragments in the NASH group. The anti-apoptotic protein Bcl-2 showed significantly low levels in NASH patients. Apoptosis of hepatocytes was significantly higher in the NASH group. The degree of apoptosis was inversely correlated with the level of Bcl-2. A significant correlation between both s. Fas and CK-18 fragment with liver histology with regard to lobular inflammation and ballooning was found.ConclusionsIncreased serum levels of s. Fas and CK-18 fragment in the NASH group and its correlation with the severity of disease suggested the key role of apoptosis in NASH pathogenesis which can be used for the assessment of the severity of NASH. A high level of anti-apoptotic Bcl-2 in NAFLD suggests its protective role in disease progress.     

The Clinical Significance of Serum Apoptotic Cytokeratin 18 Neoepitope M30 (CK-18 M30) and Matrix Metalloproteinase 2 (MMP-2) Levels in Chronic Hepatitis B Patients with Cirrhosis

Background

Serum apoptotic cytokeratine 18 neoepitope M30 (CK-18 M30) and matrix metalloproteinase 2 (MMP-2) have been popular markers for detecting liver fibrosis in recent years. CK-18 is a major intermediate filament protein in liver cells and one of the most prominent substrates of caspases during hepatocyte apoptosis. MMP-2 plays an important role in tissue remodeling and repairing processes during physiological and pathological states.

Objectives

The objective of this study was to investigate the significance of CK-18 M30 and MMP-2 levels for clinical use in patients with chronic hepatitis B (CHB), as well as their sensitivity in determining cirrhotic patients.

Patients and Methods

This study included 189 CHB patients and 51 healthy controls. A modified Knodell scoring system was used to determine the fibrosis level in chronic hepatitis B patients. CK-18 M30 levels were determined with an M30-Apoptosense ELISA assay. MMP-2 levels were determined with the ELISA assay.

Results

The study group consisted of 132 (69.8%) males and 57 (30.2%) females, and the control group consisted of 25 males (49.0%) and 26 females (51%). Patients’ CK-18 M30 levels were higher than values of the control group (308 [1–762] vs. 168 [67–287], P=0.001). Serum MMP-2 levels were found to be statistically higher in the patient group with respect to the controls (3.0 [1.1–6.8] vs. 2.0 [1.2–3.4], P=0.001). The highest serum CK-18 M30 and MMP-2 levels were measured in patients with cirrhosis. Serum apoptotic CK-18 M30 levels positively correlated with advanced age, fibrosis stage, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (P= 0.001, 0.033, 0.001, and 0.001, respectively). Serum MMP-2 levels positively correlated with fibrosis stage, serum ALT, and AST levels (P= 0.001, 0.001, and 0.001, respectively).

Conclusions

Our study indicated that CK-18 M30 and MMP-2 levels were higher in CHB patients compared to healthy controls and they were in association with significant hepatic fibrosis, especially cirrhosis.     

Oxidative stress and thyroid hormones in patients with liver diseases

BackgroundThe liver metabolizes the thyroid hormones and regulates their systemic endocrine effects so liver disease could affect thyroid hormone metabolism. Oxidative stress could play a role in the pathogenesis and progression of liver diseases. The objective of this study was to investigate serum levels of oxidative stress and antioxidant in liver diseases as prognostic markers and know the importance of these antioxidants level in relation to thyroid hormones.MethodsSerum nitric oxide (NO), malondialdehyde (MDA) and triiodothyronine (T₃), thyroxine (T₄), thyroid stimulating hormone (TSH), apolipoprotein-1 (APOA1) levels and erythrocyte reduced glutathione (GSH) level and glutathione peroxidase (GSHPx) and glutathione reductase (GR) activities were determined in 20 control subjects, 13 patients with non-alcoholic steatohepatitis (NASH), 18 patients with chronic HCV, 17 patients with compensated cirrhotic HCV and 42 patients with decompensated cirrhotic HCV.ResultsCirrhotic patients with HCV had higher NO and MDA levels while lower T₃ and erythrocyte GSH levels, and GSHPx activity than the chronic. Serum T₃ showed negative correlation with serum NO and MDA whereas positive correlation with APOA1, GSH, and GSHPx in cirrhotic patients with HCV.ConclusionThe measurement of the total T₃, NO, MDA, GSH reduced and GSHPx as biomarkers for liver diseases might be a beneficial tool, helping in monitoring the state of liver disease patients.     

Elevated Serum Levels of Cell Death Circulating Biomarkers,M30 and M65, In Patients with β-Thalassemia Major

Deposition of iron in visceral organs, mainly in the liver, causes tissue damage in β-thalassemia major (β-TM) patients. Keratin 18 (K18) represents one of the major caspase substrates during apoptosis of hepatocytes. To better characterize the hepatic apoptosis and/or necrosis in β-thal patients, the circulating levels of M65 (soluble intact K18) and M30 (the caspases-generated K18 fragment) were measured in 40 β-TM patients and compared with 40 healthy controls. The ratio of M30/M65 (caspase-cleaved to total K18) was also determined in thalassemic and normal subjects. Results of the ELISA assays revealed that the serum levels of hepatocyte death markers, M65 and M30, were significantly increased in β-thal patients compared to healthy controls (p <0.0001). M30 serum levels were also positively correlated with the serum levels of liver transaminases including aspartate aminotransferase (AST) (r = 0.337, p = 0.047) and alanine aminotransferase (ALT) (r =0.391, p = 0.02).     

Erectile dysfunction in patients with chronic hepatitis C virus infection

Background and study aimsHepatitis C virus (HCV) infection is a major public health problem worldwide and in Egypt. Several studies have suggested that chronic HCV infection may be associated with erectile dysfunction (ED) in men. The aim of our study was to detect the prevalence of ED among male patients with chronic HCV infection.Patients and methodsThe study included 150 male patients with chronic HCV infection (124 patients with chronic hepatitis and 26 patients with HCV-associated liver cirrhosis). The Child–Pugh score was used to assess the severity of cirrhosis. An Arabic validated version of the five-item International Index of Erectile Function (IIEF-5) was used to detect the presence and severity of ED.ResultsThe patients’ age ranged from 20 to 80 years with mean age ± standard deviation (SD; 50 ± 17.19) years. The prevalence of ED among patients with chronic HCV infection was found to be 29.3%. The prevalence was significantly higher in cirrhotic as compared to chronic hepatitis patients (p < 0.001) and the average ED score was significantly lower in patients with liver cirrhosis than in those with chronic hepatitis. There was a highly significant relation between the severity of ED and the severity of liver disease. There was a significant negative correlation between serum bilirubin and ED score and a significant positive correlation between serum albumin and ED score in patients with liver cirrhosis.ConclusionAbout 30% of patients with chronic HCV infection were found to have ED; so, given the high prevalence of HCV infection in Egypt, chronic HCV infection may be considered in the differential diagnosis of ED. There was a highly significant relation between the severity of ED and the severity of liver disease and the majority of patients with liver cirrhosis proved to be suffering from ED, which may be related to the associated hypoalbuminaemia.     

血清学诊断肝纤维化新指标--金属蛋白酶组织抑制因子检测方法学建立及应用评价

目的：自行建立一种改良固相致敏红细胞吸附技术（SPASE）用于快速检测肝硬化病人血清中金属蛋白酶组织抑制因子－1，2（TIMP－1和TIMP－2），同时研究TIMP－1和TIMP－2在肝化病人肝组织中的定位及表达状态，以及肝组织TIMPs与血清TIMPs的相关性。探讨血清中TIMP－1和TIMP－2可否作为肝纤维化的血清学诊断新指标。方法：应用自行建立的SPASE检测1082例肝病病人（其中肝硬化310例）血清标本中的TIMP－1和TIMP－2。用原位杂交及免疫组织化学技术分别检测40例肝硬化病人活检肝组织中TIMP－1和TIMP－2 mRNA及相关抗原的表达，并与病人自身血清检测结果对比。另检测20例正常肝组织作为对照。结果：SPASE法特异性中和抑制试验示中和抑制率均在70％以上，检测310例肝硬化病人血清中TIMP－1和TIMP－2的阳性率分别为79．68％和65．16％。40例肝硬化病人肝组织中TIMP－1和TIMP－2 mRNA及相关抗原表达阳性率为100％，TIMP－1表达强度高于TIMP－2（P＜0．01）；阳性信号主要位于肝细胞胞质内，未见细胞核表达。40例肝硬化肝活检病人血清检测结果，TIMP－1阳性率为100％，TIMP－1阳性率为77．5％（31／40）。正常肝组织无一例有TIMPs相关抗原表达。结论：TIMPs定位于肝硬化病人的肝细胞胞质内，在肝硬化组织内呈程度不等的阳性表达；血清中TIMPs与肝组织中TIMPs表达有明显相关性，因而血清中TIMP－1和TIMP－2可作为肝纤维化较为有用的血清学诊断新指标，尤其是TIMP－1的诊断意义更大。SPASE法检测血清中TIMPs具有较好的特异性。     

Diagnostic accuracy of hepatic vein arrival time performed with contrast-enhanced ultrasonography for HCV liver cirrhosis

Background and study aims: Contrast-enhanced ultrasonography (CEUS) has increased considerably the use of ultrasound for hemodynamical analyses and quantification. Bolus injection of microbubble agents is used to evaluate transit times. This study aimed to determine the diagnostic accuracy of arrival time (seconds) to the hepatic artery (HAAT), hepatic vein (HVAT), and portal vein (PVAT), based on CEUS used for the diagnosis of cirrhosis, and to correlate these arrival times with the liver stiffness and disease severity. Patients and methods: This study evaluated 29 HCV cirrhotic and 19 chronic hepatitis C patients. History, clinical examination, laboratory investigations, abdominal ultrasonography, point shear-wave elastography (pSWE), and CEUS were conducted. Results: The mean liver stiffness increased significantly in cirrhotic versus chronic HCV (22.7 versus 5.1; p-value < 0.001). The mean HAAT (p-value = 0.001), PVAT (p-value = 0.002), and HVAT values (p-value: 0.001) were significantly prolonged in cirrhotic compared with chronic HCV. The HVAT cut-off point of cirrhotic patients was 18 s with a sensitivity, specificity, and accuracy of 96.6%, 63.2%, and 83.3%, respectively (area under curve: 0.801). Significant positive correlation was found between liver stiffness (kPa) and HVAT (s) (r = 0.585; p-value = 0.005). No significant correlation was detected between HVAT (s) and the severity of liver disease, as assessed by the Child or MELD scores in cirrhotic patients. Conclusion: Measuring HVAT by CEUS yielded high-accuracy and correlation outcomes for cirrhosis detection. It could be a valuable noninvasive method for the diagnosis of cirrhosis.     

Quantitation of HCV RNA in liver of patients with chronic hepatitis C

BACKGROUND/AIMS: Liver HCV RNA has been quantitated in few studies and the feasibility and the role of this parameter in the evaluation of patients with chronic HCV hepatitis still warrant study. Our aim was to determine the concentrations of HCV RNA in the liver of chronic HCV patients and to correlate the results with serum viral load. We also studied the relation of levels of HCV RNA in the liver with serum aminotransferases levels and with the presence of cirrhosis. METHODS: Twenty patients (14 males, aged 28 to 61 years) were studied. Twelve were infected by HCV type 1, six by type 3 and one by type 5. Percutaneous liver biopsy samples were obtained from 14 patients, and the remainder from liver explant in patients undergoing OLT. Twelve had chronic hepatitis and eight cirrhosis. HCV RNA levels were determined by bDNA. RESULTS: HCV RNA levels below the detection limit were found in one liver and in five serum samples. HCV RNA (mean +/- SD) was 2.1 x 10(8) +/- 2.2 x 10(8) Eq/gm in the liver and 94 x 10(5) +/- 93 x 10(5) Eq/mL in serum, with a significant correlation between these values (r = 0.89; P < 0.0001). Serum HCV RNA levels were significantly lower (P = 0.001) in cirrhotic than in chronic hepatitis patients, while the groups did not differ in liver HCV RNA levels. No correlation was observed between liver or serum HCV RNA and serum ALT or AST. CONCLUSIONS: Quantitation of HCV RNA is possible even in small liver samples. Although average levels are more than one log higher than those observed in serum, hepatic concentrations correlate with those observed in serum. The application of this technology to monitoring antiviral therapy and understanding the pathogenesis of the disease remains to be determined.     

Quantitative liver-spleen scan using single photon emission computerized tomography (SPECT) for assessment of hepatic function in cirrhotic patients

BACKGROUND/AIMS: Accurate quantitative determination of liver function is critical in cirrhotic patients in order to predict outcome, particularly in patients who undergo hepatic resection or non-hepatic surgery. As colloid uptake by perfused Kupffer cells is proportional to perfused hepatocyte mass, quantitative liver spleen scan may be used as an index of perfused hepatocyte mass. Thus, this study was conducted to evaluate quantitative single photon emission computerized tomography (SPECT) of Tc-99mm-phytate colloid uptake by the liver as a test for hepatic function in cirrhotic patients. METHODS: Quantitative SPECT was used to measure liver volume, quantitative colloid uptake by the liver and percentage of injected dose/ml of liver tissue in cirrhotic patients (n=75), non-cirrhotic patients with chronic liver disease (n=52) and patients without liver disease (n=36). RESULTS: Although liver volume was similar among the three groups, the cirrhotic patients had significantly lower total quantitative uptake and quantitative uptake/ml compared to groups 2 and 3 (P<0.001). Quantitative liver uptake in the cirrhotic patients was highly correlated with Child-Pugh score (r=-0.64, P<0.0001) and with indocyanine green retention at 15 min (r=-0.84, P<0.0001). CONCLUSIONS: Quantitative SPECT of the liver may be an additional, useful, non-invasive quantitative test for assessment of hepatic function and severity of liver disease in cirrhotic patients.     

Quantitation of tissue polypeptide antigen (TPA) in hepatic and systemic circulation in patients with chronic liver diseases

Background and Aim: Abnormal serum tissue polypeptide antigen (TPA) values are commonly found in patients with chronic liver damage and liver cirrhosis even in the absence of malignancies. The aim of this study was to compare serum TPA levels in patients with cirrhosis, to examine correlations between TPA levels and the degree of portal hypertension, and to evaluate TPA concentrations in paired hepatic and peripheral blood samples. Methods: A total of 128 patients with chronic liver disease of various severity were studied prospectively. TPA concentrations in hepatic vein and peripheral blood were determined, and Hepatic Vein Pressure Gradient (HVPG) was measured. Results: TPA levels were significantly higher in patients with cirrhosis than in those with chronic hepatitis, and in systemic circulation than in hepatic vein blood. Peripheral but not hepatic TPA levels did correlate with the HVPG. Subjects with clinically significant portal hypertension (HVPG > 10 mmHg) showed significantly higher peripheral TPA levels than those with HVPG < 10 mmHg. Conclusions: Our data suggest that the increased TPA levels observed in cirrhotic patients and the high systemic‐to‐hepatic blood TPA gradient are probably due to the presence of portal‐systemic shunts rather than to hepatic necro‐inflammatory activity. In clinical practice, TPA determination could help us to identify and to follow up cirrhotic patients with more severe portal hypertension.     

Serum HCV-RNA levels in patients with chronic hepatitis C: Correlation with histological features

Background and study aimsLiver disease in chronic hepatitis C virus (HCV) infection ranges from minimal lesion to liver cirrhosis and sometimes eventually evolving hepatocellular carcinoma. Whether and how HCV determines the different clinical and histological manifestation of the disease is not fully understood. It has not been clearly elucidated whether the extent of liver injury induced by HCV is influenced mainly by direct cytopathic damage or by an immune-mediated response against HCV-infected hepatocytes. The aim of this study is to verify whether the amount of virus in individual patient’s serum could be related to the severity of liver injury.Patients and methodsThis study was carried out in the Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad. Serum levels of HCV-RNA were measured in 27 patients with chronic HCV using b-DNA assay. Core liver biopsies of the patients were evaluated according to Ishak histological activity index system.ResultsThe serum HCV RNA concentrations in the patients ranged from 3.2 × 10³ to 1.2 × 10⁷ copies/ml. In all patients no correlation was observed between the variable levels of viraemia and the age of the patients. Furthermore no correlations were observed between the serum HCV RNA concentrations and the biochemical liver function test levels: Total serum bilirubin, AST, ALT, and alkaline phosphatase. Histologically; patients were categorized into four subgroups: four patients (14.8%) had minimal activity, 17 patients (63%) had mild activity, and six patients (22.2%) had moderate activity. No significant correlation was found between viraemic levels and these histological findings or their individual components: Interface hepatitis, confluent necrosis, intralobular liver cell necrosis and portal inflammation. According to the stage of the fibrosis, the patients were categorized into seven subgroups: one patient (3.7%) with stage zero, seven patients with stage one (25.9%), four patients with stage two (14.9%), eight patients with stage three (29.6%), three patients with stage four (11.1%), two patients with stage five (7.4%), and two patients in cirrhotic stage six (7.4%). There was no correlation between the serum HCV RNA concentration and the stage of fibrosis. Hepatic steatosis was observed in 16/27 patients. It was mild in nine patients, moderate in five patients, and severe in two patients. Correlation has not been observed between the serum HCV RNA viraemic level and the severity of steatosis.ConclusionSerum HCV-RNA level does not determine the degree of hepatic injury precisely and liver biopsy is necessary to accurately evaluate the extent of liver damage.     

Metabolic bone disease of liver cirrhosis: Is it parallel to the clinical severity of cirrhosis?

Metabolic bone disease has long been recognized in chronic liver disease, especially cholestatic or alcoholic liver diseases. The aim of the present study was to investigate the prevalence and severity of osteodystrophy in cirrhotic men and the correlation of its incidence with the clinical severity of cirrhosis in an endemic area of post-necrotic hepatitis. We measured serum levels of osteocalcin, 25-hydroxyvitamin D, parathyroid hormone mid-molecule, calcium and testosterone in 74 cirrhotic men (Child-Pugh's classification grade A n= 30, B n= 21 and C n= 23) and 16 healthy controls. Standard X-rays and bone mineral densities of lumbar spine were performed in 30 patients with post-necrotic cirrhosis and 10 healthy controls. Serum levels of osteocalcin, parathyroid hormone and testosterone were significantly lower in patients with cirrhosis than in controls. Changes paralleling an increased severity of cirrhosis were found in the serum levels of 25–hydroxyvitamin D and testosterone, but not in the serum levels of osteocalcin and parathyroid hormone. The lumbar bone mineral density was significantly lower in patients with post-necrotic cirrhosis than in controls (0.97 ± 0.13 vs 1.07±0.12 g/cm², P<0.05) and was correlated with serum 25–hydroxyvitamin D levels (r = 0.467; P<0.005). There was no correlation between the bone mineral density and serum osteocalcin or the clinical severity of cirrhosis. The prevalence of spinal osteoporosis, as defined by a lumbar bone mineral density greater than two standard deviations below the mean value of the controls, was 20% in cirrhotic patients compared with 10% in controls. Two (6.7%) patients (both grade C) had spinal compression fractures compared with none in the control group. In conclusion, serum osteocalcin and lumbar bone mineral density were significantly lower in cirrhotic men than in controls. However, they were not correlated with each other or the clinical severity of cirrhosis.     

Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation

BACKGROUND: The alpha isotype of actin expressed by hepatic stellate cells reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis, and indirectly to human fibrosis in chronic liver disease. AIMS: To evaluate the changes in distribution and percentage of alpha-smooth muscle actin-positive hepatic stellate cells and the correlation with the degree of the fibrosis in cirrhotic livers, as well as in patients with recurrent HCV chronic hepatitis after liver transplantation. METHODS: Human liver biopsies were divided in four groups: (1) normal livers obtained from cadaveric liver donors (n=35), (2) cirrhosis post-HBV hepatitis (n=11), (3) cirrhosis post-HCV hepatitis (n=10), and (4) post-transplant recurrent HCV chronic hepatitis (n=13). Samples were stained with anti-alpha-smooth muscle actin antibody by immunoperoxidase method and semi-quantitatively evaluated. Liver fibrosis was assessed from specimens stained with Masson's trichrome and quantified by computer image analysis. RESULTS: The percentage of alpha-smooth muscle actin-positive hepatic stellate cells was significantly higher in the HBV cirrhosis, HCV cirrhosis and post-transplant HCV recurrent hepatitis groups (36.1+/-15.2, 23.8+/-19.7 and 27.8+/-16.4%, respectively) compared to the liver donor group (2.9+/-4.0%). The alpha-smooth muscle actin-positive hepatic stellate cells to fibrous tissue ratio were significantly higher in the post-transplant recurrent HCV hepatitis group (2.36+/-1.12) compared to both the donor livers and the HCV cirrhosis groups (0.74+/-1.09 and 1.03+/-0.91, respectively). The alpha-smooth muscle actin-positive hepatic stellate cell percentage and fibrosis correlated positively in the post-transplant recurrent HCV hepatitis group and negatively in the HCV cirrhosis group. No difference in the immunohistochemical and morphometrical variables was found between the HCV cirrhosis and HBV cirrhosis groups. CONCLUSIONS: These results indirectly confirm that, in vivo, alpha-smooth muscle actin expression is a reliable marker of hepatic stellate cells activation which precedes fibrous tissue deposition even in the setting of recurrent HCV chronic hepatitis after liver transplantation, and it could be useful to identify the earliest stages of hepatic fibrosis and monitoring the efficacy of the therapy. In the presence of advanced cirrhosis other factors, rather than alpha-smooth muscle actin-positive hepatic stellate cells, may sustain fibrosis deposition.     

Keratin 8 and 18 hyperphosphorylation is a marker of progression of human liver disease

Keratin 8 and 18 (K8/18) phosphorylation plays a significant and site-specific role in regulating keratin filament organization, association with binding proteins, and modulation of cell cycle progression. Keratin hyperphosphorylation correlates with exposure to a variety of stresses in cultured cells and in mouse models of liver, pancreatic, and gallbladder injury, and it is found in association with mouse and human Mallory bodies. We asked whether K8/18 phosphorylation correlates with human liver disease progression by analyzing liver explants and biopsies of patients with chronic noncirrhotic hepatitis C virus (HCV) or cirrhosis. We also examined the effect of HCV therapy with interleukin-10 on keratin phosphorylation. Using site-specific antiphosphokeratin antibodies we found keratin hyperphosphorylation on most K8/18 sites in all cirrhotic liver explants tested and in most liver biopsies from patients with chronic HCV infection. Immunofluorescence staining of precirrhotic HCV livers showed focal keratin hyperphosphorylation and limited reorganization of keratin filament networks. In cirrhotic livers, keratin hyperphosphorylation occurred preferentially in hepatic nodule cells adjacent to bridging fibrosis and associated with increased stress kinase activation and apoptosis. Histological and serological improvement after interleukin-10 therapy was accompanied by normalization of keratin hyperphosphorylation on some sites in 7 of 10 patients. In conclusion, site-specific keratin phosphorylation in liver disease is a progression marker when increased and a likely regression marker when decreased.     

Nitric Oxide and Chronic HCV and HIV Infections

High concentrations of nitric oxide (NO) are generated by the inducible form of the enzyme nitric oxide synthase (iNOS), which is expressed in activated macrophages and in hepatocytes. Increased expression of iNOS in hepatocytes or macrophages might be expected in chronic HCV liver disease and HIV infections. This might in turn be reflected in increased serum NO levels in these two conditions. In view of the discrepant findings in published reports, we measured serum NO levels in a large number of chronic HCV-infected patients and patients with chronic HIV infections with or without AIDS-related opportunistic infection. We also localized HCV and iNOS antigens by immunohistochemistry, in liver biopsy tissue from patients with chronic HCV-related hepatitis, HCV-related cirrhosis, and HCV-related hepatocellular carcinoma. A group of 121 subjects with serological evidence of HCV with or without HIV infection were studied. These were compared with 14 controls without HIV or HCV disease (group A). Among the subjects with HCV, 35 were negative for HIV (group B), 66 were HIV positive (group C), and 20 had AIDS-related opportunistic infection (group D). The serum NO concentration was determined by the Brucine method. A well-characterized commercially available antibody (HCV88) directed against a synthetic NS3 peptide fragment of HCV, which localizes to the hepatocyte nuclei, and an antibody to human macrophage iNOS, were both used to detect these proteins in liver biopsy tissue by immunohistochemistry. Mean serum NO values in HIV negative/HCV negative control patients (group A) (54.6 ± 12 M) were similar to those in HIV negative/HCV positive patients (group B) (55.0 ± 13 M) and HIV positive without AIDS-related disease/HCV positive patients (group C) (47.2 ± 25 M). By contrast, the mean serum NO (70.1 ± 24 M) was significantly increased in HCV-positive patients with AIDS-related infection (group D) compared to controls (P = 0.02). HCV NS3 and iNOS antibody staining hepatocytes were not detected in any of the control non-HCV-infected biopsy samples. In early chronic HCV hepatitis (fibrosis scores F0–F2), HCV NS3 antigen localized focally to only a small number of hepatocytes. In cirrhosis (fibrosis score F4) with or without hepatocellular carcinoma, the majority of hepatocyte nuclei stained positively with HCV NS3 antibody. The majority of hepatocytes in chronic HCV hepatitis expressed iNOS, irrespective of histological disease severity. The staining was present uniformly in the cytoplasm. In chronic HCV and HIV coinfection, the pattern and number of iNOS staining cells were similar to that in patients with chronic HCV infection alone. In conclusion, there is widespread expression of iNOS in hepatocytes in chronic HCV liver disease, irrespective of liver disease stage. However, elevated NO levels in serum were related only to active AIDS-related bacterial, protozoan, and fungal infections, rather than to chronic viral infection with HCV or HIV alone. NO may play a role in the local control of chronic viral infections at tissue level, but this is not reflected in serum levels.     

Fas-mediated hepatocyte apoptosis is increased by hepatitis C virus infection and alcohol consumption, and may be associated with hepatic fibrosis: mechanisms of liver cell injury in chronic hepatitis C virus infection

Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C virus (HCV) infection is associated with advanced liver disease, including cirrhosis. The aims of this study were to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in HCV-infected patients and to determine the role of Fas in HCV-mediated apoptosis. Liver tissue from 44 HCV-infected patients with variable alcohol consumption, and 10 normal control subjects who did not consume alcohol was examined for hepatocyte apoptosis, proliferation and Fas expression. Alcohol consumption was assessed using the 'Lifetime Drinking History' alcohol questionnaire. HCV RNA, alanine aminotransferase (ALT) and ferritin were also assessed in addition to demographic data. Hepatocyte apoptosis was significantly greater in HCV-infected patients compared to controls. Expression of Fas (CD95) was found in HCV patients but not in controls. The degree of Fas expression correlated with hepatocyte apoptosis as detected by terminal UTP nick end labelling (TUNEL). Active ethanol consumption led to a significant increase in hepatocyte apoptosis. Fas expression correlated with fibrosis in HCV-infected patients who were not actively drinking ethanol. In summary, HCV leads to increased apoptotic cell death in hepatocytes. Programmed cell death can be further up-regulated by active ethanol consumption. The correlation between Fas expression and TUNEL supports the hypothesis that the Fas–Fas ligand interaction is the major mechanism for HCV-induced hepatocyte apoptosis.