Hypoxia enhances H2O2-mediated upregulation of hepcidin: Evidence for NOX4-mediated iron regulation.

The exact regulation of the liver-secreted peptide hepcidin, the key regulator of systemic iron homeostasis, is still poorly understood. It is potently induced by iron, inflammation, cytokines or H2O2 but conflicting results have been reported on hypoxia. In our current study, we first show that pronounced (1%) and mild (5%) hypoxia strongly induces hepcidin in human Huh7 hepatoma and primary liver cells predominantly at the transcriptional level via STAT3 using two hypoxia systems (hypoxia chamber and enzymatic hypoxia by the GOX/CAT system). SiRNA silencing of JAK1, STAT3 and NOX4 diminished the hypoxia-mediated effect while a role of HIF1α could be clearly ruled out by the response to hypoxia-mimetics and competition experiments with a plasmid harboring the oxygen-dependent degradation domain of HIF1α. Specifically, hypoxia drastically enhances the H2O2-mediated induction of hepcidin strongly pointing towards an oxidase as powerful upstream control of hepcidin. We finally provide evidences for an efficient regulation of hepcidin expression by NADPH-dependent oxidase 4 (NOX4) in liver cells. In summary, our data demonstrate that hypoxia strongly potentiates the peroxide-mediated induction of hepcidin via STAT3 signaling pathway. Moreover, oxidases such as NOX4 or artificially overexpressed urate oxidase (UOX) can induce hepcidin. It remains to be studied whether the peroxide-STAT3-hepcidin axis simply acts to continuously compensate for oxygen fluctuations or is directly involved in iron sensing per se.

Carcinogenic Etheno DNA Adducts in Alcoholic Liver Disease: Correlation with Cytochrome P-4502E1 and Fibrosis.

BACKGROUND: One mechanism by which alcoholic liver disease (ALD) progresses is oxidative stress and the generation of reactive oxygen species, among others due to the induction of cytochrome P-4502E1 (CYP2E1). Experimental data underline the key role of CYP2E1 because ALD could be partially prevented in rats by the administration of the specific CYP2E1 inhibitor chlormethiazole. As CYP2E1 is linked to the formation of carcinogenic etheno DNA adducts in ALD patients, a causal role of alcohol-induced CYP2E1 in hepatocarcinogenesis is implicated. The purpose of this study was to investigate CYP2E1 induction in ALD, and its correlation with oxidative DNA lesions and with hepatic histology. METHODS: Hepatic biopsies from 97 patients diagnosed with ALD were histologically scored for steatosis, inflammation, and fibrosis. CYP2E1 and the exocyclic etheno DNA adduct 1,N6 -etheno-2'deoxyadenosine (εdA) were determined immunohistochemically. In addition, in 42 patients, 8-hydroxydeoxyguanosine (8-OHdG) was also evaluated using immunohistochemistry. RESULTS: A significant positive correlation was found between CYP2E1 and εdA (p < 0.0001) as well as between CYP2E1 and 8-OHdG (p = 0.039). Both CYP2E1 (p = 0.0094) and ɛdA (p < 0.0001) also correlated significantly with the stage of hepatic fibrosis. Furthermore, a significant correlation between the fibrosis stage and the grade of lobular inflammation (p < 0.0001) was observed. However, the amount of alcohol consumed did not correlate with any of the parameters determined. CONCLUSIONS: These data suggest an important role of CYP2E1 in the generation of εdA, in the fibrotic progression of ALD, and thus in alcohol-mediated hepatocarcinogenesis. CYP2E1 may be a target in the treatment of ALD and a potential prognostic marker for disease progression.

Detection of carcinogenic etheno-DNA adducts in children and adolescents with non-alcoholic steatohepatitis (NASH).

BACKGROUND: Carcinogenic exocyclic-DNA adducts like 1,N(6)-etheno-2'-deoxyadenosine (εdA) are formed through reactive intermediates of 4-hydroxynonenal (4-HNE) or other lipid peroxidation (LPO) products with the DNA bases A, C, methyl-C and G. High levels of hepatic etheno-DNA adducts have been detected in cancer prone liver diseases including alcoholic liver disease (ALD). In ALD εdA levels correlated significantly with cytochrome P-450 2E1 (CYP2E1) expression which is also induced in non-alcoholic steatohepatitis (NASH). We investigated the occurrence of εdA adducts in children with NASH as a DNA damage marker. METHODS: Liver biopsies from 21 children/adolescents with histologically proven NASH were analysed for hepatic fat content, inflammation, and fibrosis. εdA levels in DNA, CYP2E1-expression and protein bound 4-hydroxynonenal (HNE) were semi-quantitatively evaluated by immunohistochemistry. RESULTS: Among 21 NASH children, εdA levels in the liver were high in 3, moderate in 5, weak in 9 and not elevated in 4 patients. There was a positive correlation between CYP2E1 and protein-bound 4-HNE (r=0.60; P=0.008) and a trend for a positive relationship for CYP2E1 vs. staining intensity of εdA (r=0.45; P=0.06). Inflammatory activity and fibrosis correlated significantly (r=0.49, P=0.023). CONCLUSIONS: Our results demonstrate for the first time the presence of elevated carcinogenic etheno-DNA lesions (εdA) in the majority (17/21) of liver biopsies from young NASH patients. Our data suggest that LPO-derived etheno-adducts are implicated in NASH. Whether these adducts may serve as predictive risk markers in NASH children to develop hepatocellular cancer later in life remains to be investigated.

Holes in gastric mucosa in upper gastrointestinal endoscopy.

Gastritis cystica profunda (GCP) is a rare disease that shows multiple cystic gastric glands dispersed within the submucosa of the stomach. GCP occurs most commonly in patients who have undergone previous gastric surgery and presents as subepithelial tumor or a polypoid lesion. Here, we report the case of GCP in a 79-year-old patient who had undergone Billroth II gastric resection. During upper gastrointestinal endoscopy multiple lesions like tiny holes in the mucosa were observed. Endoscopic ultrasound showed cystic structures in the gastric submucosa. Biopsies finally proved the dispersed mucosal glands in the submucosa, which are pathognomonic for GCP. So far, in all published cases, GCP presented as polypoid lesions with no mucosal damage in upper gastrointestinal endoscopy. It is for the first time that GCP has been diagnosed with cystic lesions connected to the gastric lumen with a porus in each of the cysts.

Sex-specific differences in the development of acute alcohol-induced liver steatosis in mice.

AIMS: Results of several animal studies suggest that similar to humans, female rodents are more susceptible to chronic alcohol-induced liver disease (ALD). The aim of the present study was to determine whether female mice are more susceptible to acute alcohol-induced steatosis than male mice and to investigate possible mechanisms involved. METHODS: Male and female C57BL/6J mice received one single dose of ethanol (6 g/kg bodyweight) or isocaloric maltose-dextrin solution intragastrically. Plasma alcohol concentration, markers of hepatic steatosis, activation of the TLR-4 signaling cascade and triglyceride export as well as lipid peroxidation and of iron metabolism were measured 12 h after acute alcohol intake. RESULTS: In male and female ethanol-treated mice, plasma alcohol concentrations were still markedly increased 12 h after the alcohol challenge, which was associated with a significant accumulation of lipids in the liver and increase of transaminases in plasma; however, lipid accumulation was ∼3-fold higher in females in comparison with male animals. Expression of MyD88 was only found to be significantly induced in livers of female alcohol-exposed mice, whereas protein levels of ApoB were found to be significantly lower only in livers of female mice exposed to ethanol. Levels of 4-HNE protein adducts and ferritin were induced in livers of male and female ethanol-treated mice. CONCLUSION: Taken together, these data suggest that female mice are also more susceptible to acute alcohol-induced liver steatosis and that this involves an increased activation of TLR-4-dependent signaling pathways in the liver.

Exposing cells to H2O2: a quantitative comparison between continuous low-dose and one-time high-dose treatments.

Most studies investigating the influence of H2O2 on cells in culture apply nonphysiological concentrations over nonphysiological time periods (i.e., a one-time bolus that is metabolized in minutes). As an alternative, the glucose oxidase/catalase (GOX/CAT) system allows application of physiologically relevant H2O2 concentrations (300nM-10µM) over physiologically relevant time periods (up to 24h). Recent findings suggest that bolus and GOX/CAT treatments can lead to opposing cellular responses, thus warranting a quantitative comparison between the two approaches. First, we established a reaction-diffusion model that can predict the behavior of the GOX/CAT system with spatiotemporal resolution, thus aiding selection of optimal experimental conditions for its application. Measurements of H2O2 concentration in the cellular supernatant with the luminol/hypochlorite system were consistent with the predictions of the model. Second, we compared the impact of bolus and GOX/CAT treatments on cytosolic H2O2 levels over time. Intracellular H2O2 was monitored by the response of the thiol peroxidase Prx2 and the H2O2 sensor roGFP2-Orp1. We found that Prx2 rapidly and reversibly responds to submicromolar H2O2 levels and accurately reflects kinetic competition with cellular catalase. Our measurements reveal fundamental differences in the dynamic response of cellular H2O2 concentrations following either bolus or GOX/CAT treatments. Thus, different, or even opposing, biological outcomes from differing means of H2O2 delivery may be expected. Cellular responses induced by bolus treatment may not occur under GOX/CAT conditions, and vice versa.

Assessment of renal allograft fibrosis by transient elastography.

Transient elastography (TE, Fibroscan) has been established as a noninvasive assessment tool of liver fibrosis. We evaluated potentials and limitations of TE for identifying renal allograft fibrosis. The technical possibility of kidney examination by TE was assessed in two 10-week-old German landrace pigs and kidney stiffness (KS) was evaluated in 164 renal transplant patients. KS could be determined in all animals at the pole and pars media (29 ± 10 kPa vs. 31 ± 17 kPa). In human renal allografts KS was successfully performed in 94.5% of the test series with reliable results in 72% of the measurements. Mean KS at the pole or pars media were comparable (35.0 ± 19.9 kPa vs. 33.2 ± 18.6 kPa). Significantly higher KS was detected in renal allografts with histologically confirmed advanced fibrosis. Body-mass-index, skin-allograft distance, and peri or intrarenal fluid accumulation were important confounders of successful KS measurements (BMI: r = -0.31; P < 0.001; distance: r = -0.50; P < 0.001). Notably, KS did not correlate with renal function. TE represents a noninvasive approach in selected transplant recipients to identify allografts with severe fibrosis. The heterogeneous kidney morphology and several other confounding factors negatively affect measurability of KS by TE. Further technical modifications are required to improve applicability of TE for kidney assessment.

Association of liver stiffness with hepatic expression of pharmacokinetically important genes in alcoholic liver disease.

BACKGROUND: Enhanced drug elimination in alcoholics remains largely indefinable. In contrast, the reduced elimination of drugs in patients with advanced alcoholic liver disease (ALD) is normally owing to hepatic end-stage disease such as cirrhosis. We here study the mRNA expression of various hepatic drug metabolizing enzymes and transporters in association with liver stiffness (LS) being a novel noninvasive parameter for the assessment of cirrhosis to unravel the dynamic relationship between ALD and determinants of pharmacokinetics such as drug metabolizing enzymes and transporters. METHODS: We quantified mRNA expression levels of various cytochrome P-450 isoenzymes (CYPs) and drug transporters in 26 liver specimens of chronic alcoholics and 5 controls by quantitative polymerase chain reaction. In addition, liver histology, clinical data, and LS evaluated by transient elastography (Fibroscan) were obtained. RESULTS: Eighteen patients had a normal or moderate LS < 8 kPa (69.2%), while in the remaining 8 patients (30.7%) advanced F3 or F4 fibrosis could be established with an LS > 8 kPa. Overall, CYP3A4, CYP2E1, and solute carrier organic anion transporter 1B1 (SLCO1B1) were negatively correlated with increasing LS. CYPs and drug transporters tended to be up-regulated in alcoholics without advanced fibrosis (LS < 8.0 kPa) compared to healthy controls supporting data of boosted drug elimination in alcoholics without advanced ALD. However, in alcoholics with severely increased LS (>8 kPa), expression levels of CYP2E1, SLC22A2, and SLCO1B1 were significantly lower. CONCLUSIONS: In conclusion, CYPs and drug transporters seem to be induced in chronic alcoholics without irreversible liver damage but decline in case of manifest cirrhosis. Our study also suggests that noninvasive measurements of LS could be useful for pharmacokinetic predictions and individualized pharmacotherapy.

Sustained submicromolar H2O2 levels induce hepcidin via signal transducer and activator of transcription 3 (STAT3).

The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H(2)O(2) (0.3-6 µM), concentrations that are comparable with levels of H(2)O(2) released by inflammatory cells. In contrast, bolus treatment of H(2)O(2) has no effect at low concentrations and even suppresses hepcidin at concentrations of >50 µM. H(2)O(2) treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H(2)O(2)-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H(2)O(2) responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H(2)O(2) released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H(2)O(2). Thus, similar to cytokines, H(2)O(2) provides an important regulatory link between inflammation and iron metabolism.

Fulminant liver failure in Wilson's disease with histologic features of postinfantile giant cell hepatitis; cytomegalovirus as the trigger for both?

Giant cell hepatitis is a well-known histological feature of several neonatal and infantile liver diseases. In contrast, postinfantile giant cell hepatitis is rarely identified in adult liver biopsies. It has been associated with varying etiologies, mainly viral infections, drug toxicity, and autoimmunity. Here, we report an 18-year-old, previously healthy man with acute liver failure, who showed giant cell hepatitis in a liver biopsy. There was no evidence of viral hepatitis A-E, autoimmunity, and no drug history. Diagnostic work-up revealed Wilson's disease as the underlying disease. As syncytial giant cell formation is thought to be a uniform reaction pattern not related to any specific etiology, copper toxicity in Wilson's disease might cause giant cell formation. In contrast, our patient recalled a recent cytomegalovirus infection, which was confirmed serologically. Therefore, the giant cell formation might also be a fingerprint of an intercurrent cytomegalovirus infection as the common trigger for both giant cell hepatitis and decompensation of Wilson's disease.

Systemic mastocytosis: a rare case of increased liver stiffness.

Assessment of liver stiffness (LS) by transient elastography (Fibroscan) has significantly improved the noninvasive diagnosis of liver fibrosis. We here report on a 55-year-old patient with drastically increased LS due to previously unknown systemic mastocytosis. The patient initially presented with increased weight loss, nocturnal pruritus, increased transaminases, bilirubinemia, and thrombocytopenia. Abdominal ultrasound showed ascites, hepatomegaly, and splenomegaly. In addition, LS was 75 kPa (IQR 0 kPa) clearly exceeding the cut-off value for F4 cirrhosis of 12.5 kPa. However, histological analysis of the liver specimen indicated liver involvement by systemic mastocytosis and excluded liver cirrhosis. An additional CT scan detected disseminated bone lesions. After three months of treatment with Midostaurin, LS slightly decreased down to 31.9 kPa (IQR 8.3 kPa). This case illustrates that diffused sinusoidal neoplastic infiltrates are a pitfall in the non-invasive diagnosis of liver cirrhosis. In conclusion, refined clinical algorithms for increased LS should also include mastocytosis in addition to inflammation, congestion, and biliary obstruction.

Transient elastography with the XL probe rapidly identifies patients with nonhepatic ascites.

BACKGROUND: In contrast with other elastographic techniques, ascites is considered an exclusion criterion for assessment of fibrosis stage by transient elastography. However, a normal liver stiffness could rule out hepatic causes of ascites at an early stage. The aim of the present study was to determine whether liver stiffness can be generally determined by transient elastography through an ascites layer, to determine whether the ascites-mediated increase in intra-abdominal pressure affects liver stiffness, and to provide initial data from a pilot cohort of patients with various causes of ascites. METHODS AND RESULTS: Using the XL probe in an artificial ascites model, we demonstrated (copolymer phantoms surrounded by water) that a transient elastography-generated shear wave allows accurate determination of phantom stiffness up to a water lamella of 20 mm. We next showed in an animal ascites model that increased intra-abdominal pressure does not affect liver stiffness. Liver stiffness was then determined in 24 consecutive patients with ascites due to hepatic (n = 18) or nonhepatic (n = 6) causes. The cause of ascites was eventually clarified using routine clinical, imaging, laboratory, and other tools. Valid (75%) or acceptable (25%) liver stiffness data could be obtained in 23 patients (95.8%) with ascites up to an ascites lamella of 39 mm. The six patients (25%) with nonhepatic causes of ascites (eg, pancreatitis, peritoneal carcinomatosis) had a significantly lower liver stiffness (<8 kPa) as compared with the remaining patients with hepatic ascites (>30 kPa). Mean liver stiffness was 5.4 kPa ± 1.3 versus 66.2 ± 13.3 kPa. CONCLUSION: In conclusion, the presence of ascites and increased intra-abdominal pressure does not alter underlying liver stiffness as determined by transient elastography. We suggest that, using the XL probe, transient elastography can be used first-line to identify patients with nonhepatic ascites at an early stage.

Cannabinoid receptor type I modulates alcohol-induced liver fibrosis.

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H2O2, endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H2O2, as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 µmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

Investigation of tumor hypoxia using a two-enzyme system for in vitro generation of oxygen deficiency.

BACKGROUND: Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for in vitro studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for in vitro approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for in vitro investigation of oxygen deficiency. METHODS: Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. 14C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system. RESULTS: The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. 14C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation. CONCLUSION: The results of our study indicate that the enzymatic model can find application for in vitro investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.

Ethanol-mediated carcinogenesis in the human esophagus implicates CYP2E1 induction and the generation of carcinogenic DNA-lesions.

Chronic alcohol consumption is a major risk factor for esophageal cancer. Various mechanisms may mediate carcinogenesis including the genotoxic effect of acetaldehyde and oxidative stress. Ethanol exerts its carcinogenic effect in the liver among others via the induction of cytochrome P450 2E1 (CYP2E1) and the generation of carcinogenic etheno-DNA adducts. Here we investigated if such effects can also be observed in the human esophagus. We studied nontumorous esophageal biopsies of 37 patients with upper aerodigestive tract cancer and alcohol consumption of 102.3 ± 131.4 g/day (range: 15-600 g) as well as 16 controls without tumors (12 teetotalers and 4 subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno-DNA adducts and Ki67 as a marker for cell proliferation were determined immunohistologically. Chronic alcohol ingestion resulted in a significant induction of CYP2E1 (p = 0.015) which correlated with the amount of alcohol consumed (r = 0.6, p < 0.001). Furthermore, a significant correlation between CYP2E1 and the generation of the carcinogenic exocyclic etheno-DNA adducts 1,N(6)-ethenodeoxyadenosine (r = 0.93, p < 0.001) and 3,N(4)-ethenodeoxycytidine (r = 0.92, p < 0.001) was observed. Etheno-DNA adducts also correlated significantly with cell proliferation (p < 0.01), which was especially enhanced in patients who both drank and smoked (p < 0.001). Nonsmokers and nondrinkers had the lowest rate of cell proliferation, CYP2E1 expression and DNA lesions. Our data demonstrate for the first time an induction of CYP2E1 in the esophageal mucosa by ethanol in a dose dependent manner in man and may explain, at least in part, the generation of carcinogenic DNA lesions in this target organ.

Hyperferritinaemia-cataract syndrome: worldwide mutations and phenotype of an increasingly diagnosed genetic disorder.

The hereditary hyperferritinaemia-cataract syndrome (HHCS) is characterised by an autosomal dominant cataract and high levels of serum ferritin without iron overload. The cataract develops due to L-ferritin deposits in the lens and its pulverulent aspect is pathognomonic. The syndrome is caused by mutations within the iron-responsive element of L-ferritin. These mutations prevent efficient binding of iron regulatory proteins 1 and 2 to the IRE in L-ferritin mRNA, resulting in an unleashed ferritin translation. This paper reviews all 31 mutations (27 single nucleotide transitions and four deletions) that have been described since 1995. Laboratory test showing hyperferritinaemia, normal serum iron and normal transferrin saturation are indicative for HHCS after exclusion of other causes of increased ferritin levels (inflammation, malignancy, alcoholic liver disease) and should prompt an ophthalmological consultation for diagnostic confirmation. Invasive diagnostics such as liver biopsy are not indicated. HHCS is an important differential diagnosis of hyperferritinaemia. Haematologists, gastroenterologists and ophthalmologists should be aware of this syndrome to spare patients from further invasive diagnosis (liver biopsy), and also from a false diagnosis of hereditary haemochromatosis followed by venesections. Patients diagnosed with HHCS should be counselled regarding the relative harmlessness of this genetic disease, with early cataract surgery as the only clinical consequence.

Increased liver stiffness in alcoholic liver disease: differentiating fibrosis from steatohepatitis.

AIM: To test if inflammation also interferes with liver stiffness (LS) assessment in alcoholic liver disease (ALD) and to provide a clinical algorithm for reliable fibrosis assessment in ALD by FibroScan (FS). METHODS: We first performed sequential LS analysis before and after normalization of serum transaminases in a learning cohort of 50 patients with ALD admitted for alcohol detoxification. LS decreased in almost all patients within a mean observation interval of 5.3 d. Six patients (12%) would have been misdiagnosed with F3 and F4 fibrosis but LS decreased below critical cut-off values of 8 and 12.5 kPa after normalization of transaminases. RESULTS: Of the serum transaminases, the decrease in LS correlated best with the decrease in glutamic oxaloacetic transaminase (GOT). No significant changes in LS were observed below GOT levels of 100 U/L. After establishing the association between LS and GOT levels, we applied the rule of GOT < 100 U/L for reliable LS assessment in a second validation cohort of 101 patients with histologically confirmed ALD. By excluding those patients with GOT > 100 U/L at the time of LS assessment from this cohort, the area under the receiver operating characteristic (AUROC) for cirrhosis detection by FS improved from 0.921 to 0.945 while specificity increased from 80% to 90% at a sensitivity of 96%. A similar AUROC could be obtained for lower F3 fibrosis stage if LS measurements were restricted to patients with GOT < 50 U/L. Histological grading of inflammation did not further improve the diagnostic accuracy of LS. CONCLUSION: Coexisting steatohepatitis markedly increases LS in patients with ALD independent of fibrosis stage. Postponing cirrhosis assessment by FS during alcohol withdrawal until GOT decreases to < 100 U/mL significantly improves the diagnostic accuracy.

Non-invasive diagnosis of liver diseases by breath analysis using an optimized ion-molecule reaction-mass spectrometry approach: a pilot study.

Breath composition is altered in liver diseases. We tested if ion-molecule-reaction mass spectrometry (IMR-MS) combined with a new statistical modality improves the diagnostic accuracy of breath analysis in liver diseases. We analysed 114 molecules in the breath of 126 individuals (healthy controls, and patients with non-alcoholic and alcoholic fatty liver disease and liver cirrhosis) by IMR-MS. Characteristic exhalation patterns were identified for each group. Combining two to seven molecules in the new stacked feature ranking model reached a diagnostic accuracy (area under the curve) for individual liver diseases between 0.88 and 0.97. IMR-MS followed by sophisticated statistical analysis is a promising tool for liver diagnostics by breath analysis.

Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.

Liver stiffness is directly influenced by central venous pressure.

BACKGROUND & AIMS: Liver stiffness (LS) as measured by transient elastography [Fibroscan] offers a novel non-invasive approach to assess liver cirrhosis. Since Fibroscan seems to be unreliable in patients with congestive heart failure, it remains to be determined whether hemodynamic changes affect LS irrespective of fibrosis. METHODS & RESULTS: Using landrace pigs, we studied the direct relationship between the central venous pressure and LS measured by Fibroscan. Clamping of the inferior caval vein increased LS from 3.1 to 27.8kPa while reopening reversed LS within 5min to almost normal values of 5.1kPa. We then studied LS as a function of venous pressure in the isolated pig liver by clamping the upper and lower caval, portal vein and hepatic artery. The stepwise increase of intravenous pressure to 36cm of water column (3.5kPa) linearly and reversibly increased LS to the upper detection limit of 75kPa. We finally measured LS in 10 patients with decompensated congestive heart failure before and after recompensation. Initial LS was elevated in all patients, in 8 of them to a degree that suggested liver cirrhosis (median 40.7kPa). Upon recompensation with a median weight loss of 3.0kg, LS decreased in all 10 patients down to a median LS of 17.8kPa. Inflammation could not account for increased LS since initial liver enzyme counts were only slightly elevated and did not change significantly. CONCLUSION: LS is a direct function of central venous pressure which should be considered when assessing the degree of fibrosis.