Paraoxonase activity in sera of patients with non-alcoholic fatty liver disease.

The results of our study showed significantly decreased levels of PON1 in patients with chronic liver diseases (controls 185 ± 14 U/l, NAFLD 160 ± 15 U/l, chronic hepatitis 99 ± 18 U/l, cirrhosis 52 ± 11 U/l). There were significant correlations of PON activities with standard liver function tests (Tab. 1, Ref. 5).

Loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation.

AIM: To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1. METHODS: We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver. Immunohistochemistry and Western blotting were performed to detect Dicer1 expression. We performed hematoxylin and eosin staining, Periodic acid-Schiff staining, Oil Red O staining, and Masson's trichrome staining to detect histological changes in Dicer1-deficient livers. Ki67 immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and Western blotting were used to determine hepatocyte proliferation and apoptosis. Serum biochemistry, cytokine assays, and flow cytometric analysis were performed to quantity liver necrosis and inflammation. Fibrogenic markers were determined by Western blotting and qPCR. CK19, CD133, and OV6 immunofluorescence were used to observe liver progenitor cells. Immunofluorescence and qPCR were performed to reveal embryonic gene expression. We also performed histological staining and Western blotting to analyze hepatocellular carcinoma (HCC) development. RESULTS: Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruption in the liver. Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis. In contrast to previous reports, the mutant liver exhibited chronic inflammation and progressive fibrosis, and could not be repopulated by Dicer1-positive cells. In addition, extensive activation of hepatic progenitor cells was observed. Primary HCC was observed as early as 4 mo after birth. CONCLUSION: Hepatic loss of Dicer1 results in complex chronic pathological processes, including hepatocyte death, inflammatory infiltration, chronic fibrosis, compensatory proliferation, progenitor activation, and spontaneous hepatocarcinogenesis.

Phosphate-dependent glutaminase response to liver injury and hyperbaric oxygenation.

Activity of phosphate-dependent glutaminase was determined in hepatocytes of white female rats, both in healthy animals and in rats with chronic CCl4-hepatitis on day 3 after liver resection and hyperbaric oxygenation. In healthy animals, activity of phosphate-dependent glutaminase was not altered after hepatic resection, but it was elevated in animals with chronic CCl4-hepatitis. Hyperbaric oxygenation inhibited activity of hepatocytic phosphate-dependent glutaminase in non-operated healthy rats but stimulated it after hepatic resection. In animals with chronic CCl4-hepatitis; hyperbaric oxygenation restricted the stimulating effect of hepatic resection on phosphate-dependent glutaminase activity.

Hepatic necro-inflammation and elevated liver enzymes: evaluation with MRI perfusion imaging with gadoxetic acid in chronic hepatitis patients.

AIM: To evaluate liver necro-inflammation and function by using gadoxetic acid-enhanced dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with histological analysis as the reference standard. MATERIALS AND METHODS: Seventy-nine subjects (21 healthy subjects; 58 chronic hepatitis patients) who received gadoxetic acid-enhanced DCE-MRI were divided into three subgroups: no (A0, n = 31), mild (A1, n = 27), and moderate-severe (A2-A3, n = 21) activities. Two DCE-MRI models were measured: (1) a dual-input single-compartment model to obtain absolute arterial, portal venous, and total blood flow, arterial fraction (ART), distribution volume, and mean transit time; (2) a curve analysis method to obtain peak, slope, and AUC (area under curve). The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels also obtained. Statistical testing included Kruskal-Wallis tests for continuous data, Pearson's correlation tests, and multiple linear regression analyses. RESULTS: Hepatic necro-inflammatory activity grades were significantly correlated with fibrotic stages, serum ALT level, ART and AUC. ART was helpful to predict the mild activity (≤ A1 versus >A1; Az = 0.728), whereas AUC could differentiate no activity from any activity (A0 versus >A0; Az = 0.703). Peak, slope and AUC were all associated with AST and ALT (p < 0.05). CONCLUSION: Gadoxetic acid-enhanced DCE-MRI parameters may be used to evaluate the severity of hepatic necro-inflammation and function.

Pediatric celiac disease, cryptogenic hypertransaminasemia, and autoimmune hepatitis.

OBJECTIVE: The association between celiac disease (CD) and liver disease in pediatrics is widely recognized, but its prevalence is unknown. This study aims to conduct a systematic review and meta-analysis to evaluate the prevalence of CD in children with cryptogenic persistent hypertransaminasemia (HTS) or autoimmune hepatitis (AIH), and vice versa. METHODS: We searched MEDLINE/PubMed, the Cochrane Library, Web of Science, and MD Consult from 1977 to May 2012 for studies reporting either CD and HTS or AIH. Pooled prevalences with 95% confidence intervals (CI) and relative risk (RR) were calculated. RESULTS: Nine studies (2046 patients) were identified. Pooled prevalences of CD in children with mild, nonspecific cryptogenic persistent HTS and vice versa were 12.0% (95% CI 4.17-29.96) and 36.0% (95% CI 32.15-40.11), respectively. A gluten-free diet normalized transaminase levels in 77% to 100% of patients with CD within 4 to 8 months. Pooled prevalences of CD in children with AIH and vice versa were 6.3% (95% CI 3.87-11.73) and 1.4% (95% CI 0.84-2.15), respectively. The RR of HTS in children with CD versus the general population, and of CD in children with HTS was 6.55 (95% CI 5.65-7.60) and 11.59 (95% CI 3.80-35.33), respectively. The corresponding RR of AIH in children with CD was 188.54 (95% CI 92.23-385.43). The RR of CD in children with AIH was 6.63 (95% CI 3.86-11.40). CONCLUSIONS: CD is associated with elevated transaminase levels in about one-third of newly diagnosed children. Cryptogenic persistent HTS may signal gluten-dependent nonspecific mild hepatitis (12.0% of cases) or more rarely (6.3%) severe CD-related autoimmune hepatopathy. RRs confirm these trends in the considered associations.

The role of cyclins and cyclin dependent kinases in development and progression of hepatitis C virus-genotype 4-associated hepatitis and hepatocellular carcinoma.

UNLABELLED: Altered cell cycle regulatory genes expression contributes to HCV-associated liver disease. We sought to assess the role of cyclins and cyclin dependent kinases (CDKs) in HCV-associated CH and HCC. Aberrant expression of cyclins A, E, D1, CDK2 and CDK4 was assessed by immunohistochemistry and differential PCR in HCV-associated CH and HCC with pericarcinomatous foci (PCF). S phase fraction (SPF) was determined by flow cytometry. Results were correlated with overall survival (OS) in HCC patients. In HCC, cyclins A, E, D1, CDK2 and CDK4 protein overexpression was detected in 52.8%, 52.8%, 69%, 47% and 58% compared to 36.1%, 33%, 56%, 27.8%, 55.6% for CH and 36.1%, 27%, 30.6%, 27%, 50% for PCF. Gene amplification was detected in 38.9%, 33% 66%, 33%, 44% of HCC compared to 27.8%, 25%, 44%, 27.8%, 36% in CH and 25%, 22.2%, 38.9%, 27%, 33% in PCF. A significant difference was reported between HCC, CH, NHT regarding cyclins A, E, D1, CDK2 (p=0.007, p=0.002, p=0.047, p=0.002) protein expression (ADD) and cyclin D1 amplification (p=0.009). Cyclins A, E, CDK2 expression was associated with fibrosis in CH (p=0.004, p=0.02, p=0.012). Reduced OS was (ADD) associated with cyclin D1 and cyclin A, grade, stage and metastasis (p=0.001, p=0.02, p=0.018, p=0.01, p=0.001). CONCLUSIONS: Increased cyclins A, E, D1, CDK2 and CDK4 expression is important for HCV-associated CH and HCC. Cyclin D1 and cyclin A are prognostic biomarkers associated with reduced OS in HCC. Cyclin D1 aberration could identify high risk groups of CH patients prone to develop HCC.

Serum paraoxonase-3 concentration is associated with the severity of hepatic impairment in patients with chronic liver disease.

OBJECTIVES: Research on paraoxonase-3 (PON3) has been hampered by the lack of methods for measurement. This is a pilot study aimed at exploring whether chronic liver impairment is associated with changes in serum PON3 concentrations, and to know whether this measurement may provide useful information to investigate this derangement. DESIGN AND METHODS: We studied 110 patients with chronic liver disease (21 minimal changes, 79 chronic hepatitis, 10 cirrhosis) and 356 healthy volunteers. Serum PON3 concentration was determined by ELISA using polyclonal antibodies generated against a synthetic peptide with a sequence specific to PON3. RESULTS: Serum PON3 concentrations were increased in patients with chronic hepatitis or cirrhosis and showed significant direct correlations with the degree of periportal abnormalities including fibrosis, and with serum FAS (a marker of antiapoptosis) concentrations. CONCLUSION: These results suggest that PON3 may play a hepatoprotective role against histological alterations and hepatic cell apoptosis leading to liver disease.

Increased expression of cyclooxygenase-2 is associated with the progression to cirrhosis.

BACKGROUND/AIMS: To investigate the degree of cyclooxygenase-2 (COX-2) protein expression in chronic hepatitis and cirrhosis. METHODS: COX-2 protein expression was evaluated in 43 cases of chronic hepatitis and 24 cases of cirrhosis using immunohistochemical techniques. The COX-2 immunohistochemical staining score was assessed using the scoring systems of Pazirandeh et al and Qiu et al. and each scoring system was based on a sum of the parameters of staining intensity and distribution. RESULTS: The mean COX-2 expression scores in chronic hepatitis and cirrhosis were 2.5 ± 1.3 vs. 3.3 ± 1.1 (p = 0.008), and 3.2 ± 2.0 vs. 4.5 ± 1.7 (p = 0.006), respectively, based on the Pazirandeh et al. and Qiu et al. scoring systems. The percentage samples of high COX-2 expression score (4 to 5) in chronic hepatitis and cirrhosis were 16.3% vs. 45.8% (p = 0.022), and 23.3% vs. 50% (p = 0.021), respectively, based on the two scoring systems. The mean COX-2 expression scores based on the severity of hepatic fibrosis scored using Ishak's modified staging system (fibrosis score 0 to 3 vs. 4 to 6) were 2.4 ± 1.3 vs. 3.2 ± 1.1 (p = 0.009), and 3.1 ± 2.0 vs. 4.3 ± 1.8 (p = 0.009), respectively, based on the two scoring systems. CONCLUSIONS: COX-2 expression was significantly higher in liver cirrhosis group than in chronic hepatitis. COX-2 expression scores according to Ishak's staging was significantly higher in the advanced fibrosis group. COX-2 may play a role in the progression of hepatic fibrosis.

Clinical significance and expression of cyclin kinase subunits 1 and 2 in hepatocellular carcinoma.

BACKGROUND: The mammalian cyclin kinase subunit (Cks) family has two members, Cks1 and Cks2, which were identified based on the protein sequence homology to yeast Cks. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and a poor prognosis in various malignancies, including gastric, breast and prostate carcinomas. Yet, whether Cks1 and Cks2 are overexpressed in hepatocellular carcinoma (HCC) remains uncharacterized. AIMS: To investigate whether overexpression of the Cks family is clinically relevant to HCC, and whether expression patterns of Cks1 and Cks2 in HCC have diagnostic and prognostic value. METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blot analyses were used to detect the expression of Cks1 and Cks2 at the mRNA and protein levels respectively. The associations between Cks1 and Cks2 expressions and clinical features, as well as the association between Cks1 or Cks2 and p27(kip1) expressions in HCC, were analysed. RESULTS: Expressions of Cks1 and Cks2 at both mRNA and protein levels were significantly higher in HCC than those in the adjacent noncancerous tissues (including chronic hepatitis and cirrhosis) and normal liver tissues. Overexpressions of Cks1 and Cks2 in HCC were closely associated with poor differentiation features. The expressions of both Cks1 and Cks2 were negatively associated with p27(kip1) at the protein level. CONCLUSIONS: Overexpression of Cks1 and Cks2 is associated with the aggressive tumour behaviours of HCC, and thus has diagnostic and prognostic value. Further efforts are needed to develop novel biomarkers for HCC based on CKs1 and Cks2 expressions.

Peptidyl arginine deiminase: A novel immunohistochemical marker for liver fibrosis in patients with chronic hepatitis.

Peptidylarginine deiminase (PAD) is an enzyme known to be involved in the pathogenesis of rheumatoid arthritis (RA). Since many of the molecular events present in the joints in RA also take place in the injured liver, we postulated in this study that PAD may be involved in liver fibrosis. The objectives of this study therefore were to find out if PAD could be demonstrated immunohistochemically in liver biopsies of patients with chronic hepatitis and if it is associated with METAVIR activity and fibrosis scores. Liver biopsies were obtained from 100 patients with chronic liver diseases between September 2006 and 2007. The biopsies were scored by two histopathologists according to the METAVIR activity and fibrosis scores after histological preparation. Immunohistochemistry for PAD was performed on the biopsies using a monoclonal antibody against PAD. PAD could not be demonstrated in normal liver biopsies but was found in the hepatocytes of patients with chronic hepatitis. PAD labeling could distinguish patients with no fibrosis from either F1 or F2 or F3 or F4 fibrosis. Similarly, PAD labeling could separate patients with no inflammatory activity from those with mild or moderate or severe activity. We concluded that PAD could be demonstrated immunohistochemically in liver biopsies of patients with chronic hepatitis and that its immunodetection was significantly associated with Metavir activity and fibrosis scores.

c-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) plays a pivotal role in the development of the metabolic syndrome including nonalcoholic fatty liver disease. However, the mechanism underlying the contribution of JNK to the progression from simple steatosis to steatohepatitis and liver fibrosis is unresolved. METHODS: Hepatic steatosis, inflammation, and fibrosis were examined in wild-type, jnk1(-/-), or jnk2(-/-) mice fed a choline-deficient L-amino acid-defined (CDAA) diet for 20 weeks. The functional contribution of JNK isoforms in Kupffer cells was assessed in vitro and in vivo using chimeric mice in which the hematopoietic compartment including Kupffer cells was replaced by wild-type, jnk1(-/-), or jnk2(-/-) cells. RESULTS: CDAA diet induced significantly less hepatic inflammation and less liver fibrosis despite a similar level of hepatic steatosis in jnk1(-/-) mice as compared with wild-type or jnk2(-/-) mice. CDAA diet-induced hepatic inflammation was chronic and mediated by Kupffer cells. Pharmacologic inhibition of JNK or gene deletion of jnk1 but not jnk2 repressed the expression of inflammatory and fibrogenic mediators in primary Kupffer cells. In vivo, CDAA diet induced less hepatic inflammation and liver fibrosis despite an equivalent level of hepatic steatosis in chimeric mice with jnk1(-/-) hematopoietic cells as compared with chimeric mice with wild-type or jnk2(-/-) hematopoietic cells. CONCLUSIONS: jnk1(-/-) mice are resistant to diet-induced steatohepatitis and liver fibrosis. JNK1 in hematopoietic cells, especially in Kupffer cells, contributes to the development of liver fibrosis by inducing chronic inflammation.

Retrospective evaluation of serum markers APRI and AST/ALT for assessing liver fibrosis and cirrhosis in chronic hepatitis B and C patients with hepatocellular carcinoma.

OBJECTIVE: Aspartate aminotransferase-to-platelet ratio index (APRI), aspartate aminotransferase-to-alanine aminotransferase (AST/ALT) ratio, platelet count, AST, albumin, bilirubin and alkaline phosphatase were retrospectively evaluated for the prediction of advanced liver fibrosis and cirrhosis in patients with resectable hepatocellular carcinoma in this study. PATIENTS: In total, the 97 selected patients consisted of 9 (9.3%) patients with non-B, non-C chronic hepatitis, 48 (49.5%) patients with chronic hepatitis B (CHB) and 40 (41.2%) patients with chronic hepatitis C (CHC). RESULTS: The APRI, but not AST/ALT or other serum markers, showed a significant correlation with advanced liver fibrosis and cirrhosis (p<0.05). The area under receiver operating characteristic curves (AUROC) for predicting advanced fibrosis was 0.69 in CHB patients and 0.87 in CHC patients, whereas AUROC for predicting cirrhosis was 0.75 in CHB patients and 0.84 in CHC patients. In addition, the sensitivity and specificity of APRI were greater than 80% for predicting advanced fibrosis and cirrhosis in the CHC patients. CONCLUSION: APRI is a simple and non-invasive biochemical marker of liver fibrosis and cirrhosis, particularly in CHC patients. APRI potentially could be used to decrease the number of liver biopsies.

[Effect of hemoperfusion treated plasma from patients with chronic severe hepatitis on the activity and expression of CYP4503A in C3A cells].

AIM: To investigate the changes of CYP4503A, a key enzyme of diazepam metabolism, and explore the effect of hemoperfusion treated plasma from patients with chronic severe hepatitis on the activity and expression of CYP4503A in C3A cells. METHODS: Plasma was prepared and C3A cells were cultured. There were four groups in the experiment: normal fetal bovine plasma (NFBP) group, normal human plasma (NHP) group, hemoperfusion plasma (HPP) group, chronic severe hepatitis plasma (CSHP) group. The activity of erythromycin N-demethylase (ERD), namely the activity of CYP4503A, was measured by spectrophotometer and the expression of CYP4503A4 was detected by Western blot. RESULTS: The activity of ERD in CSHP group and HPP group was lower than that in NFBP group (P<0.05) and NHP group (P<0.05) but the activity of ERD in HPP group was higher than that in CSHP group (P<0.05). The expression of CYP4503A4 increased in NFBP group (P<0.05) and NHP group (P<0.05) but the expression of CYP4503A4 in CSHP group decreased compared with HPP group(P<0.05). CONCLUSION: The activity of ERD and the expression of CYP4503A decreased in CSHP group while the activity of ERD and the expression of CYP4503A in HPP group increased, which may cause the changes of the diazepam metabolic rates of C3A cells. This finding will provide some references for future researches on cell construction.

Correlative analysis of histological profile of the adjoining liver parenchyma with liver enzyme levels in hepatocellular carcinoma and their comparison with chronic liver disease in autopsy cases.

UNLABELLED: The exact mechanism and aetiological factor for hepatocarcinogenesis is not yet well defined. Besides genomic integration of hepatitis B viral particles, persistent chronic inflammation is postulated to be important initiating factor in viral related hepatocellular carcinoma (HCC). The objectives of the present study were--to correlate histological profiles of chronic liver disease in the adjoining non-tumor liver tissue in HCC with liver enzymes, to compare with those of non-carcinomatous chronic liver disease cases using the liver tissue and data collected at autopsy, and to correlate with hepatitis B and C positive status. Post mortem liver and data available at autopsy were used for the study. Changes of chronic liver disease was graded and staged according to Peter Scheur's (1991). In HCC, the non-malignant liver tissue was used for the study. Hepatitis B surface and core antibodies were demonstration by immunohistochemistry. HCV was documented by RT-PCR using the tissue extract of paraffin embedded liver tissue. HCC group had higher inflammatory grading and transaminases levels than non-HCC group. HBcAg alone and dual HBcAg and HCV positive cases were more in HCC group. Incidence of biliary epithelial cell injury was higher in HCV positive subgroup. CONCLUSION: higher incidence of inflammatory grading and enzyme level in alone HBcAg and dual HBcAg and HCV positivity in HCC group would suggest significant role of ongoing persistent chronic inflammation and actively replicating HBV and HCV infections in carcinogenesis.

Polymorphisms of glutathione S-transferase M1, T1, and P1 in patients with HBV-related liver cirrhosis, chronic hepatitis, and normal carriers.

OBJECTIVES: Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis and cirrhosis. The role of host genetic factors in chronic HBV infection is not fully understood. We studied the influence of glutathione S-transferase (GST) M1, T1, and P1 polymorphisms in patients with different stages of HBV infections. METHODS: The sample population included 41 HBV normal carriers, 37 patients with chronic hepatitis, and 38 patients with cirrhosis (infected with HBV) compared to a control group (n = 59). PCR-based procedures were performed in the studied populations to confirm the genotypes of GSTT1, M1, and P1. Odds ratio analysis tests were used for statistical evaluation. RESULTS: We found that the frequency of GSTP1-Val (105)/Val (105) genotype was significantly higher in patients with liver cirrhosis (27%) than HBV normal carriers (2.4%; OR 14.8, 95% CI 1.8-122.5) and the frequency GSTP1-Val (105)/Ile (105) genotype was significantly higher in patients with liver cirrhosis (59.5%) than HBV normal carriers (19.5%; OR 6.1, 95% CI 2.1-16.7). The genotype GSTP1-Val (105)/Val (105) was more frequent in patients with chronic hepatitis (19.4%) than HBV normal carriers (2.4%; OR 9.65, 95% CI 1.1-82.8). Patients with cirrhosis also had a higher frequency of the GSTM1 null genotype (71.1%) than HBV normal carriers (27.5%; OR 6.5, 95% CI 2.4-17.4) and the GSTM1 null genotype was more frequent in patients with chronic hepatitis (64.9%) than HBV normal carriers (27.5%OR 4.9, 95% CI 1.8-12.8). The frequency of GSTT1 genotype was similar in all groups. CONCLUSION: These results suggest that in HBV infection, inheritance of the null GSTM1 and GSTP1-Val (105) polymorphisms involves a host genetic factor that is relevant to disease progression.

Liver enzyme values in injection drug users with chronic hepatitis C.

BACKGROUND: Liver enzymes fluctuate in chronic hepatitis C virus infection. However, the range that can be attributed to the course of hepatitis C virus (versus an intercurrent cause of hepatitis) is unknown. AIMS: To characterise the range of liver enzyme values as a function of the upper limit of normal (ULN) of the assay among persons chronically infected with hepatitis C virus. PATIENTS: One thousand and fifty-nine hepatitis C virus chronically infected individuals with > or =5 semi-annual evaluations. METHODS: Alanine aminotransferase and aspartate aminotransferase levels were prospectively obtained. Potential causes of elevations were examined using serologic testing. RESULTS: Among 1059 individuals, 11,463 enzyme measurements were obtained over 6.5 years, of which 63.5% were <1.25x ULN, 26.5% were 1.25-2.5x ULN, 8.3% were 2.5-5x ULN, and 1.6% were 5-10x ULN; only 0.2% were >10x ULN. Elevations >10x ULN were transient, the alanine aminotransferase/aspartate aminotransferase ratio tended to be different at the time of the elevation compared to before and after and 24% were associated with acute viral hepatitis. On the other hand, subjects with elevations 5-10x ULN tended to have elevated levels throughout follow-up and only 8% were associated with acute viral hepatitis. CONCLUSIONS: Liver enzymes fluctuate up to 5x ULN in most hepatitis C virus-infected persons; clinicians should seek alternate explanations for those with higher alanine aminotransferase or aspartate aminotransferase levels, especially among hepatitis C virus-infected persons with greater than 10-fold elevations.