Concentration of Serum Matrix Metalloproteinase-3 in Patients With Primary Biliary Cholangitis.

Background and Aims: Metalloproteinases (MMPs) are involved in many distinct processes in the liver. Matrix metalloproteinase-3 (MMP-3) plays an important role in connective tissue remodeling, degradation of collagen (types II, III, IV, IX, and X), proteoglycans, fibronectin, laminin, and elastin. In addition, MMP-3 can also activate other MMPs such as MMP-1, MMP-7, and MMP-9. Primary biliary cholangitis (PBC) is a cholestatic, autoimmune liver disease, characterized by the progressive destruction of intrahepatic bile ducts, leading to cholestasis, fibrosis, cirrhosis, and liver failure. Fibrosis is the result of an imbalance between production and degradation of the extracellular matrix surrounding hepatocytes. Our aim in the present study was to determine whether the measurement of serum MMP-3 is clinically useful for assessing ongoing liver fibrosis in patients with PBC. Methods: The MMP-3 concentration was determined in 182 PBC patients and 80 non-PBC controls using a commercially available ELISA kit. Results: Higher concentrations of MMP-3 were found in 61% of PBC patients. PBC subjects had greater MMP-3 levels than controls: 68.9 ± 62.6 vs 21.3 ± 7.4 ng/mL, p < 0.001 for healthy subjects; 68.9 ± 62.6 vs 22.7 ± 7.6 ng/mL, p = 0.022 for autoimmune hepatitis controls; and 68.9 ± 62.6 vs 37.2 ± 17.4 ng/mL, p = 0.002 for primary sclerosing cholangitis controls. The serum MMP-3 concentration was significantly elevated in patients with higher bilirubin concentration (107.6 ± 85.8 vs 61.6 ± 46.1 ng/mL, p < 0.001) and was correlated with the level of antimitochondrial antibodies specific for PBC. The concentration of MMP-3 in sera of PBC patients was also found to correlate with the state of liver fibrosis (OR = 4.3; p < 0.01). Conclusions: Our study demonstrated significantly higher MMP-3 levels in PBC patients than in healthy and pathological controls. Increased MMP-3 concentrations were positively correlated with various clinical and immunological parameters, and advanced liver fibrosis. The level of MMP-3 was associated with hepatic dysfunction and could play a role in the pathophysiology of hepatic fibrosis in PBC.

Diagnostic and Clinical Value of Specific Autoantibodies against Kelch-like 12 Peptide and Nuclear Envelope Proteins in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by the presence of antimitochondrial and antinuclear antibodies in patients' serum. Here, we analyzed the reactivity of autoantibodies against a novel autoantigen, kelch-like 12 (KLHL12) protein, in a cohort of 138 PBC and 90 non-PBC patients. Additionally, we compared the reactivity of KLHL12 with antinuclear envelope antibodies: anti-gp210, anti-p62, and anti-LBR. Commercially available kits and an 'in-house' ELISA were used in the studies. Antinuclear envelope antibodies were detected in 65% of PBC patients and the presence of these antibodies was observed more frequently in patients diagnosed with later stages (III/IV) of PBC, according to Ludwig's classification (p < 0.05) and were found to correlate with a higher concentration of bilirubin. Overall, anti-KLHL12 antibodies were found more frequently in PBC patients than in non-PBC controls (p < 0.001). Anti-KLHL12 antibodies were detected in 36% of the tested PBC cohort, including PBC patients negative for antimitochondrial antibodies. Presence of anti-KLHL12 was also associated with a higher concentration of bilirubin and correlated with fibrosis (p < 0.05). Anti-KLHL12 antibodies were detected in 30% of PBC individuals positive for antinuclear envelope antibodies, while anti-KLHL12 and antinuclear envelope antibodies were found in 17% of all PBC cases. Concluding, our data confirm that antibodies against the KLHL12 protein are highly specific for PBC and when used in combination with other markers, may significantly increase the diagnosis of PBC.

Analysis of Autoantibodies against Promyelocytic Leukemia Nuclear Body Components and Biochemical Parameters in Sera of Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by immune-mediated destruction of intrahepatic bile ducts and the presence of specific antibodies. The aim of the study was to examine the diagnostic significance of antibodies against promyelocytic leukemia nuclear body (PML NB) components such as Sp100, Sp140, and PML in a cohort of PBC patients and compare the results with biochemical and histological parameters. Serum samples were collected from 93 PBC patients. Anti-Sp100 and anti-PML antibodies were assessed using commercially available kits, anti-Sp140 using developed "in-house" ELISA test. Anti-Sp140, anti-Sp100, and anti-PML antibodies were present in 25 (27%), 37 (40%), and 29 (31%) PBC patients, respectively. Anti-PML NB positive patients also showed increased concentration of bilirubin and alkaline phosphatase (p < 0.05). In the group with the presence of at least two types of these antibodies, more frequent deaths or transplantations were observed. A correlation between the presence of antibodies and histological grade (OR = 2.55 p = 0.039) was established. Patients with bilirubin > 1.1 mg/dL at the time of diagnosis had a significantly shorter time of survival than patients with bilirubin ≤ 1.1 mg/dL (HR 5.7; 95% C.I., 2.7, 12.3; p < 0.001). Our data confirm very high specificity of anti-PML NB antibodies, which can expand the laboratory diagnostic capabilities of PBC. We found an association between positive reactivity of autoantibodies directed against components of PML nuclear bodies and higher concentrations of bilirubin and alkaline phosphatase, but the main prognostic marker of survival remains serum bilirubin.

Common functional alterations identified in blood transcriptome of autoimmune cholestatic liver and inflammatory bowel diseases.

Primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are heterogeneous chronic autoimmune diseases that may share underlying pathogenic mechanisms. Herein, we compared simultaneously analyzed blood transcriptomes from patients with PBC, PSC, and IBD. Microarray-based measurements were conducted using RNA isolated from whole blood samples from 90, 45, 95 and 93 patients with PBC, PSC, CD, and UC, respectively, and 47 healthy controls. Expression levels of selected transcripts were analyzed by quantitative reverse-transcribed PCR using an independent cohort of 292, 71 and 727 patients with PBC, PSC, and IBD, respectively. Of 4026, 2650 and 4967 probe sets differentially expressed (adjusted p-value < 0.05) in samples from patients with PBC, PSC, and IBD, respectively, compared with healthy controls, 1946 were common to all three comparisons. Functional analyses indicated that most terms enriched for genes differentially expressed in PBC, PSC, and IBD patients compared with healthy controls were related to mitochondrial function, the vesicle endomembrane system, and GTPase-mediated processes. This study indicates that microarray-based profiling of blood gene expression supports research into the molecular mechanisms underlying disease, rather than being useful for selection of diagnostic biomarkers for use in clinical practice.

Detection of Autoantibodies Against Nucleoporin p62 in Sera of Patients With Primary Biliary Cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by specific autoantibodies. We evaluated the prevalence of autoantibodies against nucleoporin p62 (anti-p62) in PBC patients' sera to determine whether it can be a marker for PBC, in comparison with other immunological and biochemical parameters. We validated the performance of our in-house ELISA technique. METHODS: Serum samples were collected from 135 PBC patients. Thirty patients with primary sclerosing cholangitis (PSC) and 30 with autoimmune hepatitis (AIH) were included as pathological controls, and 40 healthy blood donors served as healthy controls. The presence of anti-p62 was determined by an in-house ELISA using a recombinant protein. We calculated the sensitivity, specificity, positive and negative predictive values (PPV and NPV), and positive and negative likelihood ratio (LR+ and LR-) of our in-house ELISA for diagnosing PBC based on anti-p62. Findings were correlated with biochemical data and survival. RESULTS: Anti-p62 was detected in 32 PBC patients (23.7%). Specificity and PPV of anti-p62 for PBC were 99% and 97%, respectively. The difference between proportions of anti-p62-positive patients and controls was 0.23 (95% confidence interval [CI]: 0.03-0.40; P<0.0001); LR+ and LR- were 23.7 and 0.77, respectively. The presence of anti-p62 was associated with higher levels of bilirubin and alkaline phosphatase (P<0.001). The odds ratio for survival was 2.44 (95% CI: 0.87-6.87; P=0.091). CONCLUSIONS: Anti-p62 may be regarded as a significant serological marker of PBC.

A heterozygous mutation in GOT1 is associated with familial macro-aspartate aminotransferase.

BACKGROUND & AIMS: Macro-aspartate aminotransferase (macro-AST) manifests as a persistent elevation of AST levels, because of association of the protein with immunoglobulins in the circulation. Macro-AST is a rare, benign condition without a previously confirmed genetic basis. METHODS: Whole exome sequencing (WES)-based screening was performed on 32 participants with suspected familial macro-AST, while validation of variants was performed on an extended cohort of 92 probands and 1,644 healthy controls using Taqman genotyping. RESULTS: A missense variant (p.Gln208Glu, rs374966349) in glutamate oxaloacetate transaminase 1 (GOT1) was found, as a putative causal variant predisposing to familial macro-AST. The GOT1 p.Gln208Glu mutation was detected in 50 (54.3%) of 92 probands from 20 of 29 (69%) families, while its prevalence in healthy controls was only 0.18%. In silico analysis demonstrated that the amino acid at this position is not conserved among different species and that, functionally, a negatively charged glutamate on the GOT1 surface could strongly anchor serum immunoglobulins. CONCLUSIONS: Our data highlight that testing for the p.Gln208Glu genetic variant may be useful in diagnosis of macro-AST. LAY SUMMARY: Higher than normal levels of aspartate aminotransferase (AST) in the bloodstream may be a sign of a health problem. Individuals with macro-AST have elevated blood AST levels, without ongoing disease and often undergo unnecessary medical tests before the diagnosis of macro-AST is established. We found a genetic variant in the GOT1 gene associated with macro-AST. Genetic testing for this variant may aid diagnosis of macro-AST.

Clinical Applicability of Whole-Exome Sequencing Exemplified by a Study in Young Adults with the Advanced Cryptogenic Cholestatic Liver Diseases.

BACKGROUND: The proper use of new medical tests in clinical practice requires the establishment of their value and range of diagnostic usefulness. While whole-exome sequencing (WES) has already entered the medical practice, recognizing its diagnostic usefulness in multifactorial diseases has not yet been achieved. AIMS: The objective of this study was to establish usability of WES in determining genetic background of chronic cholestatic liver disease (CLD) in young patients. METHODS: WES was performed on six young patients (between 17 and 22 years old) with advanced fibrosis or cirrhosis due to CLD and their immediate families. Sequencing was performed on an Ion Proton sequencer. RESULTS: On average, 19,673 variants were identified, of which from 7 to 14 variants of an individual were nonsynonymous, homozygous, recessively inherited, and considered in silico as pathogenic. Although monogenic cause of CLD has not been determined, several heterozygous rare variants and polymorphisms were uncovered in genes previously known to be associated with CLD, including ATP8B1, ABCB11, RXRA, and ABCC4, indicative of multifactorial genetic background. CONCLUSIONS: WES is a potentially useful diagnostic tool in determining genetic background of multifactorial diseases, but its main limitation results from the lack of opportunities for direct linkage between the uncovered genetic variants and molecular mechanisms of disease.

A novel approach to genome-wide association analysis identifies genetic associations with primary biliary cholangitis and primary sclerosing cholangitis in Polish patients.

BACKGROUND: Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are forms of hepatic autoimmunity, and risk for both diseases has a strong genetic component. This study aimed to define the genetic architecture of PBC and PSC within the Polish population. METHODS: Subjects were 443 women with PBC, 120 patients with PSC, and 934 healthy controls recruited from Gastroenterology Departments in various Polish hospitals. Allelotyping employed a pooled-DNA sample-based genome-wide association study (GWAS) approach, using Illumina Human Omni2.5-Exome BeadChips and the following novel selection criteria for risk loci: blocks of at least 10 single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium, where the distance between each adjacent SNP pair in the block was less than 30 kb, and each SNP was associated with disease at a significance level of P < 0.005. A selected index SNP from each block was validated using TaqMan SNP genotyping assays. RESULTS: Nineteen and twenty-one SNPs were verified as associated with PBC and PSC, respectively, by individual genotyping; 19 (10/9, PBC/PSC) SNPs reached a stringent (corrected) significance threshold and a further 21 (9/12, PBC/PSC) reached a nominal level of significance (P < 0.05 with odds ratio (OR) > 1.2 or < 0.83), providing suggestive evidence of association. The SNPs mapped to seven (1p31.3, 3q13, 6p21, 7q32.1, 11q23.3, 17q12, 19q13.33) and one (6p21) chromosome region previously associated with PBC and PSC, respectively. The SNP, rs35730843, mapping to the POLR2G gene promoter (P = 1.2 × 10-5, OR = 0.39) demonstrated the highest effect size, and was protective for PBC, whereas for PSC respective SNPs were: rs13191240 in the intron of ADGRB3 gene (P = 0.0095, OR = 0.2) and rs3822659 (P = 0.0051, OR = 0.236) along with rs9686714 (P = 0.00077, OR = 0.2), both located in the WWC1 gene. CONCLUSIONS: Our cost-effective GWAS approach followed by individual genotyping confirmed several previously identified associations and discovered new susceptibility loci associated with PBC and/or PSC in Polish patients. However, further functional studies are warranted to understand the roles of these newly identified variants in the development of the two disorders.

Primary biliary cirrhosis in the era of liver transplantation.

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by the presence of antimitochondrial antibodies (AMA) and progressive immune-mediated destruction of biliary ductules, which lead to cirrhosis. Theories of the PBC etiopathogenesis assume that the disease develops secondarily as an improper immunological reaction to undefined environmental and/or infectious factors in genetically predisposed individuals. Ursodeoxycholic acid (UDCA) is the only drug recommended to treat PBC; it delays the progression of liver disease, but remains only a symptomatic treatment. In the advanced stage of PBC, the treatment of choice is liver transplantation (LTx). Nowadays, PBC is the third indication for LTx, after viral-related and alcoholic liver cirrhosis. Unfortunately, PBC recurs in 21-37% of patients at 10 years after LTx, and in 43% at 15 years after LTx, with the median time to recurrence of 3-5.5 years. Diagnosis of recurrent PBC (rPBC) is based on the liver histopathology. Although various risk factors of rPBC have been investigated, the cause of the recurrence is not clear. There is no specific treatment of rPBC. Together with immunosuppression after LTx, UDCA remains the treatment of choice. New diagnostic technologies (e.g., genomics, proteomics, cell-based therapy, and clinical study of the rPBC patients) may be helpful in understanding the pathogenesis of PBC and the development of new treatment modalities.

Detection of anti-SP100 antibodies in primary biliary cirrhosis. Comparison of ELISA and immunofluorescence.

Primary biliary cirrhosis (PBC) is, which a chronic, autoimmune liver disease. Some patients have antinuclear antibodies anti-Sp100, which are considered to be disease-specific. We compared an enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) for detection of anti-Sp100. The sensitivity of anti-Sp100 determined by ELISA and IIF was 44% and 34%, respectively. Specificity was 99% for ELISA and 98% for IIF, respectively. The positive and negative predictive value (PPV, NPV) for anti-Sp100 determined by ELISA were 98%, 60% and 95%, 56% for IIF respectively. IIF required substantial experience in interpreting subjective patterns, whereas ELISA was more sensitive, cheaper, less time consuming, and produced clear-cut quantitative results.

Results of liver transplantation in the Department of General, Transplant and Liver Surgery at the Medical University of Warsaw in patients with chronic hepatitis B and C viruses infection.

INTRODUCTION: Cirrhosis related to hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is the most frequent indication for liver transplantation worldwide. Progress in prophylaxis of posttransplant HBV recurrence has led to major improvements in long-term outcomes of patients after liver transplantation. Conversely, impaired posttransplant survival of patients with HCV infection was reported in several studies, mainly due to recurrence of viral infection. The purpose of this study was to compare long-term results of liver transplantation between patients with HBV monoinfection, HCV monoinfection and HBV/HCV coinfection. MATERIAL AND METHODS: A total of 1090 liver transplantations were performed in the Department of General, Transplant and Liver Surgery in cooperation with the Department of Immunology, Internal Medicine, and Transplantology at the Transplantation Institute Medical University of Warsaw between December 1994 and May 2012. After exclusion of patients with cirrhosis of non-viral etiology, patients with malignant tumors, and patients with acute liver failure, the final study cohort comprised 209 patients with HBV (HBV+/HCV- subgroup; n = 56) or HCV (HBV-/HCV+ subgroup; n = 119) monoinfection or HBV/HCV coinfection (HBV+/HCV+; n = 34). These subgroups of patients were compared in terms of long-term results of transplantations, defined by 5-year patient and 5-year graft survival estimates. RESULTS: Overall and graft survival rates after 5-years for the whole study cohort were 74.5% and 72.6%, respectively. Five-year overall survival was 70.4% for patients within the HBV+/HCV- subgroup, 77.8% for patients within the HBV-/HCV+ subgroup, and 68.5% for patients within the HBV+/HCV+ subgroup. The corresponding rates of graft survival were 67.0%, 76.3%, and 68.5% for patients within the HBV+/HCV-, HBV-/ HCV+, and HBV+/HCV+ subgroups, respectively. Observed differences were non-significant, both in terms of overall (p = 0.472) and graft (p = 0.461) survival rates. CONCLUSIONS: Both overall and graft survival rates after liver transplantations performed in the Department of General, Transplant and Liver Surgery in cooperation with the Department of Immunology, Internal Medicine, and Transplantology at the Transplantation Institute Medical University of Warsaw in patients with HBV and HCV infection are comparable to those reported by other European and American centers. In contrast to other studies, obtained results do not confirm the negative impact of HCV infection on long-term outcomes of patients.

Laboratory-based scoring system for prediction of hepatic inflammatory activity in patients with autoimmune hepatitis.

BACKGROUND & AIMS: In autoimmune hepatitis (AIH), inflammation is closely related to fibrosis. Although transaminase levels are commonly used to assess hepatic inflammation, they may not relate directly to the histology. We developed a noninvasive diagnostic score as an alternative to liver biopsy to help optimize treatment for AIH and monitor disease progress. METHODS: Eighty-two participants with type 1 AIH who had undergone liver biopsy were included (44 in training and 38 in validation sets). Liver histology was assessed according to the histologic activity index (HAI; score 0-18) and Ishak's histologic fibrosis index (HFI; score 0-6). High inflammation was defined as HAI>4, and advanced fibrosis was defined as HFI>2. Routine laboratory test findings and stepwise linear regression were used to develop the best models predicting HAI and HFI. The best cut-off value to predict high inflammation and advanced fibrosis for these formulas was then calculated based on receiver-operating characteristic analysis. RESULTS: The cut-off value for a model predicting high inflammation was ≥3.57 (AUROC = 0.93; 95% CI: 0.86-1.00), with 100% sensitivity and 85% specificity. High inflammation was confirmed with an 81% positive predictive value and excluded with a 100% negative predictive value. In the validation set, the sensitivity, specificity, positive predictive value and negative predictive values were 100, 56, 88 and 100% respectively. The diagnostic yield of the fibrosis score was unsatisfactory. CONCLUSIONS: The noninvasive inflammatory score based on four routine laboratory parameters discriminated patients with and without significant hepatic inflammation and may facilitate follow-up of type 1 AIH patients.

Lack of evidence for association of primary sclerosing cholangitis and primary biliary cirrhosis with risk alleles for Crohn's disease in Polish patients.

BACKGROUND: Numerous papers have addressed the association of mutations and polymorphisms of susceptibility genes with autoimmune inflammatory disorders. We investigated whether polymorphisms that confer susceptibility to Crohn's disease could be classified also as predisposing factors for the development of primary sclerosing cholangitis and primary biliary cirrhosis in Polish patients. METHODS: The study included 60 patients with CD, 77 patients with PSC, of which 61 exhibited IBD (40 UC, 8 CD, and 13 indeterminate colitis), and 144 patients with PBC. All the patients were screened against Crohn's disease associating genetic polymorphisms. The polymorphisms were chosen according to previously confirmed evidence for association with Crohn's disease, including Pro268Ser, Arg702Trp, Gly908Arg and 1007fs in NOD2/CARD15, Leu503Phe/-207G>C in SLC22A4/OCTN1/SLC22A5/OCTN2, Arg30Gln in DLG5, Thr300Ala in ATG16L1, and Arg381Gln, His3Gln and exon-3'UTR in IL23R. Genotyping was carried out using TaqMan SNP genotyping assays. RESULTS: We confirmed a strong association between three NOD2/CARD15 gene variants (Pro268Ser, OR = 2.52, 95% CI = 1.34-4.75); (Arg702Trp, OR = 6.65, 95% CI = 1.99-22.17); (1007fs, OR = 9.59, 95% CI = 3.94-23.29), and a weak association between both the protective OCTN1/OCTN2 CC haplotype (OR = 0.28, 95% CI = 0.08-0.94), and a variant of ATG16L1 gene (Thr300Ala, OR = 0.468, 95% CI = 0.24-0.90) with Crohn's disease. In contrast, none of the polymorphisms exhibited association with susceptibility to primary sclerosing cholangitis and primary biliary cirrhosis, including a group of primary sclerosing cholangitis patients with concurrent IBD. CONCLUSION: Although the clinical data indicate non-random co-occurrence of inflammatory bowel disease and primary sclerosing cholangitis, consistently with the previously published studies, no genetic association was found between the genetic variants predisposing to Crohn's disease and hepatobiliary autoimmune disorders. However, since estimation of genetic variant disproportion is limited by sample size, these negative results may also indicate that eventually shared genetic predispositions are too little to be captured by small patient groups.

Measurement of gp210 autoantibodies in sera of patients with primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease with unknown etiology. Patients with PBC have antimitochondrial autoantibodies (AMA) and additionally 50% of them have antinuclear antibodies (ANA). A 15-amino acid fragment (DRKASPPSGLWSPAY) from the C-terminal part of the nuclear envelope glycoprotein gp210 has been proposed as one of the antigenic targets for ANA. The aim of this work was to develop an immunoenzymatic assay for determination of gp210 autoantibodies using for its binding a synthetic pentadecapeptide derived from the gp210 amino acid sequence and to determine level of these autoantibodies in sera of patients with PBC and other autoimmune liver diseases from Poland. Polystyrene microtitration plates coated with the synthetic peptide were consecutively incubated with diluted sera, anti-human immunoglobulin G (IgG) antibodies conjugated with horseradish peroxidase, and with tetramethylobenzidine. Optical density (OD) was read at 450 nm. The mean values of the intra- and interassay of variation coefficients of the test were 4.1 and 10.2%, respectively. Anti-gp210 was detected in 44% of PBC patients and in 6% of patients with PSC. The results were negative for healthy blood donors and other controls. The specificity of the test was 99%, so the anti-gp-210 autoantibodies estimated on DRKASPPSGLWSPAY can be a reliable marker of PBC.

Expression of genes encoding mitochondrial proteins can distinguish nonalcoholic steatosis from steatohepatitis.

In patients without substantial alcohol use, triglyceride accumulation in the liver can lead to nonalcoholic fatty liver disease (NAFLD) that may progress to nonalcoholic steatohepatitis (NASH). The differential diagnosis between NAFLD and NASH can be accomplished only by morphological examination. Although the relationship between mitochondrial dysfunction and the progression of liver pathologic changes has been described, the exact mechanisms initiating primary liver steatosis and its progression to NASH are unknown. We selected 16 genes encoding mitochondrial proteins which expression was compared by quantitative RT-PCR in liver tissue samples taken from patients with NAFLD and NASH. We found that 6 of the 16 examined genes were differentially expressed in NAFLD versus NASH patients. The expression of hepatic HK1, UCP2, ME2, and ME3 appeared to be higher in NASH than in NAFLD patients, whereas HMGCS2 and hnRNPK expression was lower in NASH patients. Although the severity of liver morphological injury in the spectrum of NAFLD-NASH may be defined at the molecular level, expression of these selected 6 genes cannot be used as a molecular marker aiding histological examination. Moreover, it is still unclear whether these differences in hepatic gene expression profiles truly reflect the progression of morphological abnormalities or rather indicate various metabolic and hormonal states in patients with different degrees of fatty liver disease.

The RANKL/OPG system and bone mineral density in patients with chronic liver disease.

BACKGROUND/AIMS: Osteopenia and osteoporosis are common complications of chronic liver disease (CLD). Receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) regulate osteoclastogenesis and bone remodelling, and are involved in several inflammatory diseases. This study investigated the activation state of the RANKL/OPG system and its association with bone loss in CLD. METHODS: Serum levels of OPG and sRANKL were determined in 193 patients with CLD and 56 age- and gender-matched healthy controls. Cellular sources of OPG and RANKL were determined immunohistochemically. Dual-energy x-ray absorptiometry was performed to determine bone mineral density (BMD) of lumbar spine and femoral neck. RESULTS: sRANKL serum levels were significantly elevated in non-cirrhotic, but not cirrhotic patients compared to healthy controls. OPG serum levels were elevated 1.6-fold in non-cirrhotic and 2.8-fold in cirrhotic CLD patients. RANKL+ cells were mainly confined to portal fields, while OPG was broadly expressed. In the cirrhotic subgroup (87 patients) we observed a significantly higher OPG/sRANKL ratio in patients with osteopenia or osteoporosis of the lumbar spine and femoral neck region (T-score < -1) compared to those with normal BMD (T-score > or = -1). CONCLUSIONS: CLD is associated with alterations in RANKL/OPG serum levels, which could modulate bone loss in CLD.

Serum leptin, soluble leptin receptor, free leptin index and bone mineral density in patients with primary biliary cirrhosis.

BACKGROUND/AIM: The pathophysiology of osteoporosis in chronic liver diseases is unknown. Recent data suggest that serum leptin is associated with bone mineral density (BMD). In animal studies leptin was found to be a potent inhibitor of bone formation. We investigated the relationship between serum leptin levels, soluble leptin receptor (sOB-R), free leptin index (FLI) and BMD in patients with primary biliary cirrhosis (PBC). PATIENTS AND METHODS: Ninety-four female patients with PBC were included in this study; 122 healthy women served as controls. Serum leptin levels were measured by radioimmunoassay, sOB-R by enzyme-linked immunosorbent assay. BMD was measured by dual energy X-ray absorptiometry in the lumbar spine and femoral neck. RESULTS: Serum leptin was significantly lower in patients with PBC compared with healthy controls. No difference was found between the body mass index (BMI) of patients and controls. There was a strong positive correlation between leptin and BMI. In PBC no association was found between leptin, sOB-R and liver function tests, histological stages or the presence of osteoporosis. Osteoporosis was present in 38 patients. A positive correlation was found between serum leptin and femoral neck z-score even after adjustment for BMI, whereas serum sOB-R correlated inversely with the serum leptin level. There was no difference in FLI between the subgroups of PBC patients according to the stages of the disease. CONCLUSIONS: We found a lower serum leptin level and a higher sOB-R in patients with PBC, which could not be explained by the difference in BMI. As leptin was associated with BMD, it may be hypothesized that leptin is involved in the complex regulation of bone metabolism in PBC.

Insulin-like growth factor I gene microsatellite repeat, collagen type Ialpha1 gene Sp1 polymorphism, and bone disease in primary biliary cirrhosis.

BACKGROUND: Genetic factors have been implicated in the pathogenesis of osteoporosis, a common disorder in primary biliary cirrhosis. Insulin-like growth factor I (IGF-I) gene microsatellite repeat polymorphism was found to be associated with osteoporosis in some studies, and collagen-Ialpha1 (COLIA1) Sp1 s allele was associated with lower bone mineral density in primary biliary cirrhosis. IGF-I treatment restored osteopenia and reduced fibrogenesis in experimental cirrhosis. We investigated IGF-I and COLIA1 gene polymorphisms and bone mineral density in Hungarian primary biliary cirrhosis patients. PATIENTS AND METHODS: Seventy female patients with primary biliary cirrhosis were enrolled (mean age 57.6 years, range 37-76 years; all anti-mitochondrial antibody M2-positive; stage II-IV). One hundred and thirty-nine age-matched female subjects served as controls (mean age 55.9 years, range 43-72 years). COLIA1 and IGF-I polymorphisms were determined by polymerase chain reaction. Bone mineral density was measured by dual-energy X-ray absorptiometry in the lumbar spine and femoral neck. RESULTS: The IGF-I was not different between primary biliary cirrhosis patients and controls. The genotype frequency of COLIA1 polymorphism was also not different between primary biliary cirrhosis patients and controls. However, the s allele was significantly less frequent in patients with primary biliary cirrhosis. Osteoporosis was detected in 22 patients. The IGF-I 192/192 genotype was associated with higher femoral-neck z-scores compared with other genotypes. CONCLUSION: In contrast to previous studies, the s allele was less frequent in patients with primary biliary cirrhosis, and its presence was not associated with bone mineral density. Since IGF-I polymorphism was associated with bone mineral density, it may be hypothesised that not COLIA1 but IGF-I together with other genetic and environmental factors may be involved in the complex regulation of bone mineral density in primary biliary cirrhosis.

Association of coeliac disease with primary biliary cirrhosis in Poland.

OBJECTIVES: In western and northern but not southern Europe, the prevalence of coeliac disease among patients with primary biliary cirrhosis (PBC) is higher than in the general population. We analysed the prevalence of coeliac disease among patients with PBC in Poland, a central European country. METHODS: In 115 patients with PBC, immunoglobulin A (IgA) antibodies against guinea-pig tissue transglutaminase (tTGA), monkey endomysium (EMA) and gliadin (AGA) were determined. In patients positive for tTGA and/or EMA, the DNA typing of HLA-DQB gene and small-bowel biopsy were performed. RESULTS: IgA EMA was found in one patient with PBC (0.9%, 95% CI 0-2.5%). tTGA was detected in seven patients (6%, 95% CI 1.8-10.3%). Small-bowel biopsy showed flat mucosa in one subject, who was EMA positive/tTGA negative/AGA negative, and normal histology in four tTGA-positive/EMA-negative patients. In the latter four patients, the positive tTGA result was caused by IgA reactivity to proteins other than transglutaminase. Prevalence of coeliac disease in our 115 patients with PBC was 0.9% (95% CI 0-1.9%). In one patient with silent coeliac disease presenting with the HLA-DQ2 allele, introduction of a gluten-free diet was followed by improvement in liver function tests, improvement in histology of the distal duodenum, and disappearance of EMA. CONCLUSIONS: Our results from Poland do not confirm a high prevalence of coeliac disease in PBC patients. Guinea-pig liver transglutaminase immunolinked assay cannot be used as a screening test for coeliac disease in PBC patients. A gluten-free diet may be helpful in restoration of liver function in patients with such an association.