Martin Lenicek

The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance

Intestinal FGF19 has emerged as a novel endocrine regulator of hepatic bile salt and lipid metabolism. In patients with nonalcoholic fatty liver disease (NAFLD) hepatic lipid metabolism is deranged. A possible role of FGF19 in NAFLD has not been reported yet. In this study, we assessed intestinal FGF19 production and the hepatic response to FGF19 in NAFLD patients with and without insulin resistance [homeostasis model of assessment (HOMA) score > or =2.5 (n = 12) and HOMA score <2.5 (n = 8), respectively]. To this end, NAFLD patients received a standardized oral fat challenge. Postprandial excursions of triglycerides, bile salts, and FGF19 were monitored, and plasma levels of a marker for bile salt synthesis (7alpha-hydroxy-4-cholesten-3-one) were determined. Fasted FGF19 levels were comparable in a control group of healthy volunteers (n = 15) and in NAFLD patients (0.26 +/- 0.28 vs. 0.18 +/- 0.09 ng/ml, respectively, P = 0.94). Postprandial FGF19 levels in both controls and NAFLD patients peaked between 3-4 h and were three times higher than baseline levels. The areas under the postprandial FGF19 curve were similar in controls and in the HOMA score-based NAFLD subgroups. In NAFLD patients with HOMA score <2.5, the postprandial increase in plasma FGF19 was accompanied by a lowering of plasma levels of 7alpha-hydroxy-4-cholesten-3-one (-30%, P = 0.015). This anticipated decline was not observed in insulin-resistant NAFLD patients (+10%, P = 0.22). In conclusion, patients with NAFLD show an unimpaired intestinal FGF19 production. However, the hepatic response to FGF19 is impaired in NAFLD patients with insulin resistance (HOMA score > or =2.5). This impaired hepatic response to FGF19 may contribute to the dysregulation of lipid homeostasis in NAFLD.

Differences in antitumor effects of various statins on human pancreatic cancer

Statins are widely used for the treatment of hypercholesterolemia. However, their inhibitory action on HMG‐CoA reductase also results in the depletion of intermediate biosynthetic products, which importantly contribute to cell proliferation. The aim of the present study was to compare the effects of the individual commercially available statins on experimental pancreatic cancer. The in vitro effects of individual statins (pravastatin, atorvastatin, simvastatin, lovastatin, cerivastatin, rosuvastatin and fluvastatin) on the viability of human pancreatic cancer were evaluated in CAPAN‐2, BxPc‐3 and MiaPaCa‐2 cell lines. The in vivo experiments were performed on nude mice xenotransplanted with CAPAN‐2 cells. The mice received oral treatments either with a placebo, or with the statins mentioned earlier in a daily dose corresponding to a hypocholesterolemic dose in humans. The effect of these statins on the intracellular Ras protein, trafficking in MiaPaCa‐2 transfected cells, was also investigated. Substantial differences in the tumor‐suppressive effects of all statins were detected in both in vitro and in vivo experiments. While simvastatin exerted the highest tumor‐suppressive effects in vitro, rosuvastatin (p = 0.002), cerivastatin (p = 0.002) and fluvastatin (p = 0.009) were the most potent compounds in an animal model. All statins (except pravastatin) inhibited intracellular Ras protein translocation. In summary, substantial tumor‐suppressive effects of various statins on the progression of experimental pancreatic adenocarcinoma were demonstrated, with marked differences among individual statins. These results support greatly the potential of statins for the chemoadjuvant treatment of pancreatic cancer.

Bile acid malabsorption in inflammatory bowel disease: Assessment by serum markers

Background: Bile acid malabsorption (BAM) is a common feature of Crohns disease (CD). We aimed to determine whether BAM develops only in patients with a resected distal ileum or if it also occurs in patients who have not undergone surgery for CD. Methods: The study included 347 patients with CD or ulcerative colitis (UC) and 119 healthy subjects (controls). BAM was assessed by measurement of serum levels of 7&agr;‐hydroxycholest‐4‐en‐3‐one (C4) and fibroblast growth factor 19 (FGF19). We surveyed members of the European Crohns and Colitis Organization and International Organization for the Study of Inflammatory Bowel Disease to collect current information about BAM diagnosis. Results: The severity of BAM was associated with resection of the distal ileum. Compared with controls, patients who received moderate or extensive ileal resection had significantly increased levels of serum C4 (12 versus 62 versus 243 μg/L, respectively; P < 0.001). However, BAM was also present in a substantial number of the patients with CD who were not treated by surgery who had ileitis or colitis (14% and 11%, respectively). There was an indirect, proportional relationship between levels of C4 and FGF19 (P < 0.001). Conclusions: The most severe BAM occurs in CD patients after resection of the distal ileum, but BAM can occur in surgically untreated CD patients, regardless of disease localization. Laboratory tests for BAM should become a part of the algorithm for diagnosis of CD to identify patients who might respond to therapies such as bile acid sequestrants. FGF19 appears to be a reliable marker of BAM. (Inflamm Bowel Dis 2011;)

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues.

Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75+/-5%. The detection limit was 10pmol UCB/g wet tissue. Variance was +/-2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (</=40pmol UCB/g tissue) in non-jaundiced rats.

Determination of Beta-Defensin Genomic Copy Number in Different Populations: A Comparison of Three Methods

Background There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohns disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories. Findings In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations. Conclusions QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.

Effect of carvedilol on portal hypertension depends on the degree of endothelial activation and inflammatory changes.

Objective. Bleeding from esophageal varices is a major complication of liver cirrhosis. Non-selective beta-blockers exert an influence on the functional part of portal hypertension, thereby reducing the risk of bleeding. Direct measurement of this functional part is not possible; nevertheless, pro-inflammatory markers as well as parameters of endothelial dysfunction might serve as surrogate markers. The aim of study was to assess the correlation between the therapeutic efficacy of carvedilol and markers of endothelial dysfunction and systemic inflammation in patients with liver cirrhosis and portal hypertension. Material and methods. Thirty-six patients with cirrhosis and portal hypertension were given carvedilol, 25 mg q.i.d. for 30 days. Hepatic venous pressure gradient (HVPG) and biochemical determinations were performed prior to and after the treatment. Eight healthy individuals served as controls for comparison of biochemical markers. Results. In the whole group of cirrhotic patients, HVPG decreased from 17.7±3.8 to 14.9±4.8 mmHg (p<0.001). Complete response was seen in 15 patients (42%). Baseline serum levels of E-selectin were significantly higher in responders than in non-responders (119.8±70.6 versus 52.6±25.7 ng/ml; p=0.023) and in controls (28.8±22.2 ng/ml; p=0.004). Furthermore, baseline TNF-α levels were significantly higher in responders than in non-responders (22.8±15.7 versus 7±8.9; p=0.047) and in controls (5.5±5.9 pg/ml; p=0.005). Serum levels of ICAM-1 showed the same trend (4360±2870 versus 2861±1577 versus 651±196 ng/ml), although differences did not reach statistical significance. Conclusions. Markers of systemic inflammation and endothelial dysfunction seem to predict the hypotensive effect of carvedilol on portal hypertension in patients with liver cirrhosis and may be useful in the assessment of the efficacy of the therapy.

Infliximab dependency in children with Crohn's disease

Backgroundu2002 Recently, infliximab dependency has been described.

Variants of CARD15, TNFA and PTPN22 and susceptibility to Crohn’s disease in the Czech population: high frequency of the CARD15 1007fs

Crohns disease (CD) has been shown to be associated with the variants in the CARD15 gene as well as in other genes involved in the immune response. The frequencies of the variants profoundly differ among populations and so does the associated risk. We examined the associations of variants in the CARD15, TNFA and PTPN22 genes with pediatric-onset and adult-onset CD in the Czech population. Genotype, phenotype and allelic frequencies were compared between 345 patients with CD (136 pediatric-onset and 209 adult-onset patients) and 501 unrelated healthy controls. At least one minor allele of the CARD15 gene was carried by 46% patients and only 21% control subjects (OR = 3.2, 95% CI 2.4-4.4). In a multiple logistic regression model, the strongest association with CD was found for the 1007fs variant (OR = 4.6, 95% CI 3.0-7.0), followed by p.G908R (OR = 2.9, 95% CI 1.5-5.7) and p.R702W (OR = 1.7, 95% CI 1.0-2.9), while no independent association was found for the remaining variants in the CARD15 gene (p.268S, p.955I and p.289S), for the p.R620W variant in the PTPN22 gene or for the g.-308G>A variant in the TNFA gene. The age at CD onset was strongly modified by positivity for the 1007fs allele: it was present in 42% pediatric-onset and only 25% adult-onset patients. In conclusion, we report a high frequency of the minor allele of the CARD15 1007fs polymorphism in the Czech population and a strong effect of this allele on the age at disease onset.

The CTLA4 variants may interact with the IL23R- and NOD2-conferred risk in development of Crohn's disease

BackgroundThe CTLA4 (cytotoxic T-lymphocyte antigen 4) gene is associated with several immunopathologic diseases and because of its important immuno-regulatory role it may be considered also a plausible candidate for a genetic association with inflammatory bowel diseases. Previously published studies found no association of CTLA4 with Crohns disease itself, but some indicated an association with its subphenotypes. The aim of this study was to assess the association in the Czech population, using a set of markers shown to associate with other diseases.MethodsSix polymorphisms within the CTLA4 region were investigated in 333 patients with Crohns disease and 482 unrelated healthy controls, all Caucasians of Czech origin. The genotypes of the SNPs were determined using the TaqMan SNP genotyping assays. Haplotypes were reconstructed using an expectation-maximization algorithm, and their association with the condition was assessed using log-linear modeling. Then, potential interactions were tested between the CTLA4 variants and other genetic factors known to confer the disease susceptibility.ResultsNo crude associations with Crohns disease were found for the tested CTLA4 variants under the log-additive or dominant models. However, when stratified for the genetic risk conferred by the variants in the NOD2 (the p.Leu1007fsX1008, rs5743293) or the IL23R (p.R381Q, rs11209026), a significant negative association emerged for the minor alleles of CTLA4 CT60 (rs3087243), JO31 (rs11571302), JO27-1 (rs11571297) polymorphisms. This negative association with CTLA4 was apparent only in the strata defined by presence minor alleles at the NOD2 rs5743293 (here the CTLA4 CT60 A coffered an OR = 0.43, 95%CI 0.19 - 0.95 for the presence of CT60 A), or IL23R rs11209026 (here the OR for presence of CT60 A was 0.23, 95%CI 0.07 - 0.71). We observed this effect also for the haplotype consisting of minor alleles of the three tightly linked CTLA4 markers. Furthermore, this haplotype was associated with the younger age at diagnosis (OR 1.52, 95%CI 1.09 - 2.11, p = 0.014).ConclusionsA protective effect of a CTLA4 haplotype was unmasked after stratification for the risk variants in the NOD2 and IL23R genes, and may point towards the biological relevance of the molecule in the pathogenesis of the disease.

Cholestatic effect of epigallocatechin gallate in rats is mediated via decreased expression of Mrp2

Epigallocatechin gallate (EGCG) has been shown to be protective in various experimental models of liver injury, although opposite effects have also been reported. Since its effect on biliary physiology has not been thoroughly investigated, the present study evaluated effect of EGCG on bile flow and bile acid homeostasis in rats. Compared to controls, EGCG treatment decreased bile flow by 23%. Hepatic paracellular permeability and biliary bile acid excretion were not altered by EGCG administration, but biliary glutathione excretion was reduced by 70%. Accordingly, the main glutathione transporter on the hepatocyte canalicular membrane, multidrug resistance-associated protein 2 (Mrp2), was significantly decreased at the protein level. EGCG administration also doubled plasma bile acid levels compared to controls. While protein levels of the main hepatic bile acid transporters were unchanged, the rate-limiting enzyme in the bile acid synthesis, Cyp7a1, was significantly increased by EGCG. Enhanced bile acid synthesis in these animals was also confirmed by a 2-fold increase in plasma marker 7α-hydroxy-4-cholesten-3-one. In contrast, EGCG markedly downregulated major bile acid transporters (Asbt and Ostα) and regulatory molecules (Shp and Fgf15) in the ileum. When EGCG was coadministered with ethinylestradiol, a potent cholestatic agent, it did not show any additional effect on the induced cholestasis. This study shows ability of EGCG to raise plasma bile acid concentrations, mainly through Cyp7a1 upregulation, and to decrease bile production through reduction in Mrp2-mediated bile acid-independent bile flow. In conclusion, our data demonstrate that under certain conditions EGCG may induce cholestasis.