Distribution of Exonic Keratin Variants.

<p>The table displays the number of patients with and without liver cirrhosis for the listed keratin variants.</p>*<p>The highlighted variant is considered to be a “polymorphism” rather than a “mutation” that is likely to have biologic significance.</p>†<p>Novel variants which were not previously described.</p>$<p>One female patient harbored 2 independent amino acid altering KRT8 variants (R341H+A319S). N.T.not tested.</p

Distribution of Non-Coding Keratin Variants.

<p>The table displays the number of patients with and without liver cirrhosis for the listed keratin variants.</p>*<p>IVS7+10delC variant completely associates with KRT8 R341H variant and is not included in the count of total intronic variants.</p>†<p>The highlighted variant was found only in one control subject (out of 234).</p>**<p><b><i>p</i></b><b> = 0.02</b>.</p

The K8 G62C variant does not influence the extent of hepatocellular iron accumulation.

<p>To test, whether hepatocellular iron accumulation is affected by the presence of keratin variants, transgenic mice overexpressing wild-type human keratin 8 (K8 WT) or K8 G62C variant were fed with 3% carbonyl iron-containing diet for one month (Iron) and compared to age-matched animals kept on standard diet (Control) (n = 4 mice/group). Iron-feeding did not lead to an increase in serum ALT (<b>A</b>), but caused a significant elevation in hepatic iron concentration (HIC) (<b>B</b>). There was no significant difference in ALT or HIC levels between K8 WT and G62C mice after iron feeding. (<b>C</b>) Perl's Prussian blue staining revealed a similar pattern of iron deposition in both mouse lines. Scale bar = 200 µm.</p

The Clinical Features of Hemochromatosis Patients Harboring Exonic and Intronic Keratin 8 Variants.

<p>Abbreviations: LBx:liver biopsy; SD: standard deviation; HIC: hepatic iron concentration; HII: hepatic iron index.</p><p>Note that intronic keratin variants were preferentially found in male subjects (<i>p = 0.09</i> for distribution among the sexes).</p

Patient Demographics and Biochemical Values.

<p>Abbreviations: LBx:liver biopsy; SD: standard deviation; HIC: hepatic iron concentration; HII:hepatic iron index.</p

The K8 G62C variant does not affect iron toxicity in ex vivo cultured hepatocytes.

<p>To study the hepatocellular iron toxicity, primary hepatocytes from transgenic animals overexpressing K8 WT or K8 G62C were cultured in normal media (Control) or media supplemented with 100 µM nitrilotriacetic acid (NTA) or 100 µM iron-NTA (FeNTA) for 24 hours. FeNTA, but not NTA treatment led to a significant increase in LDH (<b>A</b>) and ALT levels (<b>C</b>) in cell culture supernatants together with significant decrease in cell viability as assessed by MTT assay (<b>B</b>). The enzyme levels and cell viability did not differ significantly between K8 WT and K8 G62C hepatocytes.</p

CXCL1 serum levels in patients with alcoholic cirrhosis and healthy controls with distinct <i>CXCL1</i>

<div><p><b>rs4074 genotypes</b>. </p> <p>This figure shows CXCL1 serum concentrations in 66 healthy controls (grey columns) and in 66 patients with alcoholic cirrhosis (black columns) stratified with respect to rs4074 variants G/G, G/A and A/A. Results are shown as means ± standard errors. Groups were compared with the Mann-Whitney U test. </p></div

Upregulation of fibrosis markers in human HSC after stimulation with CXCL1.

<p>After incubation with and without 250pg/ml recombinant CXCL1 for 16 hours, human HSC were stained for α-SMA and Collagen type I and analysed by flow cytometry. This representative set of histograms shows that α-SMA and Collagen type I expression increases after CXCL1 stimulation (solid lines) as compared to the unstimlated controls (dotted line).</p

Frequency of the rs4074 A allele in the study groups.

<p>This figure illustrates that carriers of the <i>CXCL1 </i><i>rs4074</i> A allele were equally frequent in patients with alcohol related cirrhosis without and with HCC, but overrepresented in comparison to patients with alcohol abuse without liver damage and to healthy controls. Statistical significances refer to Fisher´s exact test.</p

Metabolic Signature of Electrosurgical Liver Dissection

<div><p>Background and Aims</p><p>High frequency electrosurgery has a key role in the broadening application of liver surgery. Its molecular signature, <i>i.e.</i> the metabolites evolving from electrocauterization which may inhibit hepatic wound healing, have not been systematically studied.</p><p>Methods</p><p>Human liver samples were thus obtained during surgery before and after electrosurgical dissection and subjected to a two-stage metabolomic screening experiment (discovery sample: N = 18, replication sample: N = 20) using gas chromatography/mass spectrometry.</p><p>Results</p><p>In a set of 208 chemically defined metabolites, electrosurgical dissection lead to a distinct metabolic signature resulting in a separation in the first two dimensions of a principal components analysis. Six metabolites including glycolic acid, azelaic acid, 2-n-pentylfuran, dihydroactinidiolide, 2-butenal and n-pentanal were consistently increased after electrosurgery meeting the discovery (p<2.0×10⁻⁴) and the replication thresholds (p<3.5×10⁻³). Azelaic acid, a lipid peroxidation product from the fragmentation of abundant sn-2 linoleoyl residues, was most abundant and increased 8.1-fold after electrosurgical liver dissection (p_replication = 1.6×10⁻⁴). The corresponding phospholipid hexadecyl azelaoyl glycerophosphocholine inhibited wound healing and tissue remodelling in scratch- and proliferation assays of hepatic stellate cells and cholangiocytes, and caused apoptosis dose-dependently <i>in vitro</i>, which may explain in part the tissue damage due to electrosurgery.</p><p>Conclusion</p><p>Hepatic electrosurgery generates a metabolic signature with characteristic lipid peroxidation products. Among these, azelaic acid shows a dose-dependent toxicity in liver cells and inhibits wound healing. These observations potentially pave the way for pharmacological intervention prior liver surgery to modify the metabolic response and prevent postoperative complications.</p></div