Implications of anaemia and response to anaemia treatment on outcomes in patients with cirrhosis.

Background & Aims: Anaemia is frequently observed in patients with cirrhosis and was identified as a predictor of adverse outcomes, such as increased mortality and occurrence of acute-on-chronic liver failure. To date, the possible effects of iron supplementation on these adverse outcomes are not well described. We therefore aimed to assess the role of iron supplementation in patients with cirrhosis and its capability to improve prognosis. Methods: Laboratory diagnostics were performed in consecutive outpatients with cirrhosis admitted between July 2018 and December 2019 to the University Hospital Essen. Associations with transplant-free survival were assessed in regression models. Results: A total of 317 outpatients with cirrhosis were included, of whom 61 received a liver transplant (n = 19) or died (n = 42). In multivariate Cox regression analysis, male sex (hazard ratio [HR] = 3.33, 95% CI [1.59, 6.99], p = 0.001), model for end-stage liver disease score (HR = 1.19, 95% CI [1.11, 1.27], p <0.001) and the increase of haemoglobin levels within 6 months (ΔHb6) (HR = 0.72, 95% CI [0.63, 0.83], p <0.001) were associated with transplant-free survival. Regarding the prediction of haemoglobin increase, intake of rifaximin (beta = 0.50, SD beta = 0.19, p = 0.007) and iron supplementation (beta = 0.79, SD beta = 0.26, p = 0.003) were significant predictors in multivariate analysis. Conclusions: An increase of haemoglobin levels is associated with improvement of transplant-free survival in patients with cirrhosis. Because the prediction of haemoglobin increase significantly depends on rifaximin and iron supplementation, application of these two medications can have an important impact on the outcome of these patients. Impact and implications: Anaemia is very common in patients with cirrhosis and is known to be a predictor of negative outcomes, but little is known about the effect of iron substitution in these individuals. In our cohort, increase of haemoglobin levels improved transplant-free survival of patients with cirrhosis. The increase of haemoglobin levels was mainly induced by iron supplementation and was even stronger in the case of concomitant use of iron and rifaximin. Clinical trial registration: UME-ID-10042.

Vitamin A Plasma Levels in COVID-19 Patients: A Prospective Multicenter Study and Hypothesis.

COVID-19 is a pandemic disease that causes severe pulmonary damage and hyperinflammation. Vitamin A is a crucial factor in the development of immune functions and is known to be reduced in cases of acute inflammation. This prospective, multicenter observational cross-sectional study analyzed vitamin A plasma levels in SARS-CoV-2 infected individuals, and 40 hospitalized patients were included. Of these, 22 developed critical disease (Acute Respiratory Distress Syndrome [ARDS]/Extracorporeal membrane oxygenation [ECMO]), 9 developed severe disease (oxygen supplementation), and 9 developed moderate disease (no oxygen supplementation). A total of 47 age-matched convalescent persons that had been earlier infected with SARS-CoV-2 were included as the control group. Vitamin A plasma levels were determined by high-performance liquid chromatography. Reduced vitamin A plasma levels correlated significantly with increased levels of inflammatory markers (CRP, ferritin) and with markers of acute SARS-CoV-2 infection (reduced lymphocyte count, LDH). Vitamin A levels were significantly lower in hospitalized patients than in convalescent persons (p < 0.01). Of the hospitalized patients, those who were critically ill showed significantly lower vitamin A levels than those who were moderately ill (p < 0.05). Vitamin A plasma levels below 0.2 mg/L were significantly associated with the development of ARDS (OR = 5.54 [1.01-30.26]; p = 0.048) and mortality (OR 5.21 [1.06-25.5], p = 0.042). Taken together, we conclude that vitamin A plasma levels in COVID-19 patients are reduced during acute inflammation and that severely reduced plasma levels of vitamin A are significantly associated with ARDS and mortality.

Silymarin as Supportive Treatment in Liver Diseases: A Narrative Review.

Silymarin, an extract from milk thistle seeds, has been used for centuries to treat hepatic conditions. Preclinical data indicate that silymarin can reduce oxidative stress and consequent cytotoxicity, thereby protecting intact liver cells or cells not yet irreversibly damaged. Eurosil 85® is a proprietary formulation developed to maximize the oral bioavailability of silymarin. Most of the clinical research on silymarin has used this formulation. Silymarin acts as a free radical scavenger and modulates enzymes associated with the development of cellular damage, fibrosis and cirrhosis. These hepatoprotective effects were observed in clinical studies in patients with alcoholic or non-alcoholic fatty liver disease, including patients with cirrhosis. In a pooled analysis of trials in patients with cirrhosis, silymarin treatment was associated with a significant reduction in liver-related deaths. Moreover, in patients with diabetes and alcoholic cirrhosis, silymarin was also able to improve glycemic parameters. Patients with drug-induced liver injuries were also successfully treated with silymarin. Silymarin is generally very well tolerated, with a low incidence of adverse events and no treatment-related serious adverse events or deaths reported in clinical trials. For maximum benefit, treatment with silymarin should be initiated as early as possible in patients with fatty liver disease and other distinct liver disease manifestations such as acute liver failure, when the regenerative potential of the liver is still high and when removal of oxidative stress, the cause of cytotoxicity, can achieve the best results.

Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper.

Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 µM CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a >20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O2vs. 21% O2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy.

BACKGROUND: Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD. METHODS: Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy. RESULTS: Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells. CONCLUSION: We conclude that repeated AGAL activity measurements in patients' white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

Spatial investigation of the elemental distribution in Wilson's disease liver after d-penicillamine treatment by LA-ICP-MS.

At present, the copper chelator d-penicillamine (DPA) is the first-line therapy of Wilson's disease (WD), which is characterized by an excessive copper overload. Lifelong DPA treatments aim to reduce the amount of detrimental excess copper retention in the liver and other organs. Although DPA shows beneficial effect in many patients, it may cause severe adverse effects. Despite several years of copper chelation therapy, discontinuation of DPA therapy can be linked to a rapidly progressing liver failure, indicating a high residual liver copper load. In order to investigate the spatial distribution of remaining copper and additional elements, such as zinc and iron, in rat and human liver samples after DPA treatment, a high resolution (spotsize of 10µm) laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method was applied. Untreated LPP-/- rats, an established animal model for WD, appeared with a high overall copper concentration and a copper distribution of hotspots distributed over the liver tissue. In contrast, a low (>2-fold decreased) overall copper concentration was detected in liver of DPA treated animals. Importantly, however, copper distribution was highly inhomogeneous with lowest concentrations in direct proximity to blood vessels, as observed using novel zonal analysis. A human liver needle biopsy of a DPA treated WD patient substantiated the finding of an inhomogeneous copper deposition upon chelation therapy. In contrast, comparatively homogenous distributions of zinc and iron were observed. Our study indicates that a high resolution LA-ICP-MS analysis of liver samples is excellently suited to follow efficacy of chelator therapy in WD patients.

Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells.

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds.

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis.

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO) and small interfering RNA (siRNA) designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

Concentrated green tea extract induces severe acute hepatitis in a 63-year-old woman--a case report with pharmaceutical analysis.

UNLABELLED: ETNOPHARMACOLOGICAL RELEVANCE: The popularity of concentrated green tea extracts as dietary supplements for a wide range of applications is increasing due to their health-promoting effects attributed to the high amounts of catechins they contain. The most important of the green tea catechins is (-)-epigallocatechin-3-O-gallate (EGCG). While their beneficiary effects have been studied extensively, a small number of adverse events have been reported in the medical literature. Here we present a typical reversible course of severe hepatitis after green tea consumption. MATERIALS AND METHODS: The case study describes in a 63-year old woman during treatment with green tea-capsules upon recommendation of a cancer support group. RESULTS: The histological finding was consistent with drug induced hepatitis, and other possible causes of hepatitis were excluded. According to the CIOMS/RUCAM score the causality was assessed as "probable". After discontinuation of medication, followed by extracorporal albumin dialysis, rapid and sustained recovery occurred. Pharmaceutically analysis (HPLC) of the green tea capsules did not give evidence for contaminants but revealed the two typical compounds of green tea, namely (-)-epigallocatechin-3-O-gallate (EGCG, 93.2%) and epicatechin (EC, 6.8%) at a very high dose level. CONCLUSION: The present case highlights the fact that such concentrated herbal extracts from green tea may not be free of adverse effects under certain circumstances. There is still a lack of a uniform European Union-wide surveillance system for adverse drug reactions of herbal products. Therefore this case underlines the importance of public awareness in the potential risks in use of herbal products.

Liver transplantation versus supraselective transarterial chemoembolization in palliative patients with hepatocellular carcinoma exceeding the Milan Criteria - is it time for a more individual approach?

BACKGROUND: This study evaluated the effect of liver transplantation (LTX) and STACE on overall survival in palliative patients with HCC exceeding Milan criteria. MATERIAL AND METHODS: At a single center 63 HCC patients exceeding Milan criteria were retrospectively analyzed. Forty patients underwent STACE as palliative therapy modality and 23 palliative patients were scheduled for LTX. The primary endpoint was overall patient survival. Statistical analysis included Kaplan-Meier method, log rank, chi squared tests and Cox regression model for the identification of prognostic factors. RESULTS: There was no significant difference when comparing the 2 groups (LTX vs. no LTX) in terms of Child classification, co-morbidities, underlying disease, and sex. Overall survival was significantly prolonged after LTX was performed (p=0.012). In the Cox regression model, LTX (p=0.021), LTX <3Mo (p=0.047), CHILD stage (p=0.007), AFP (p=0.020), and tumor size of largest HCC nodule <40 mm (p=0.028) were independent prognostic factors for survival. CONCLUSIONS: Palliative patients beyond Milan have a significant survival benefit after they received early liver transplantation in comparison with STACE. The current approach to waiting list candidacy based on Milan criteria should be modified with a more individualized approach that considers age, AFP level, and tumor size.

Efficacy and safety of oral chelators in treatment of patients with Wilson disease.

BACKGROUND & AIMS: Wilson disease is a genetic copper storage disorder that causes hepatic and neurologic symptoms. Chelating agents (D-penicillamine, trientine) are used as first-line therapies for symptomatic patients, but there are few data from large cohorts. We assessed the safety of D-penicillamine and trientine therapy and outcomes of patients with Wilson disease. METHODS: We performed a retrospective analysis of data on 380 patients with Wilson disease from tertiary care centers in Germany and Austria, and 25 additional patients from the EUROWILSON registry. Chelator-based treatment regimens were analyzed for their effect on neurologic and hepatic symptoms and for adverse events that led to discontinuation of therapy (Kaplan-Meier estimation; data were collected for a mean of 13.3 y after therapy began). RESULTS: Changes in medication were common, resulting in analysis of 471 chelator monotherapies (326 patients receiving D-penicillamine and 141 receiving trientine). Nine of 326 patients treated with D-penicillamine and 3 of 141 patients given trientine underwent liver transplantation. Adverse events leading to discontinuation of treatment were more frequent among those receiving D-penicillamine than trientine (P = .039). Forty-eight months after therapy, hepatic deterioration was reported in only 4 of 333 patients treated initially with a chelating agent. Hepatic improvements were observed in more than 90%, and neurologic improvements were observed in more than 55%, of therapy-naive patients, and values did not differ significantly between treatments. However, neurologic deterioration was observed less frequently in patients given D-penicillamine first (6 of 295) than those given trientine first (4 of 38; P = .018). CONCLUSIONS: Chelating agents are effective therapies for most patients with Wilson disease; D-penicillamine and trientine produce comparable outcomes, although D-penicillamine had a higher rate of adverse events. Few patients receiving chelation therapy had neurologic deterioration, which occurred more frequently in patients who received trientine.

Longitudinal analysis of serum miR-122 in a rat model of Wilson's disease.

PURPOSE: MicroRNA-122 (miR-122) has recently been shown to represent a novel biomarker of liver disease. However, the presence of serum miR-122 after liver injury was mostly studied at singular time points. The course of serum miR-122 was determined at consecutive time points during the onset of disease. METHODS: Fulminant hepatitis was induced by a high-copper diet in Long-Evans Cinnamon (LEC) rats that were used as models for Wilson's disease (WD). Levels of serum miR-122, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and liver histology were determined. RESULTS: Toxic copper given to isolated hepatocytes induced release of miR-122 into the tissue culture medium. Levels of serum miR-122 were highly elevated (21.9 ± 5) in LEC rats after high-copper diet in fulminant hepatitis, whereas healthy rats showed low (<0.6) baseline levels of miR-122. Levels of miR-122 in the serum of LEC rats after high-copper diet continuously increased for about 4 weeks prior to the onset of fulminant hepatitis. In most of the animals (77.8%), significantly increased levels of miR-122 were detected about 2 weeks (13.7 ± 2 days) earlier as compared to hepatitis-associated serum markers ALT, AST, and bilirubin. Analysis of miR-122 in survivors after cell-based therapy of WD demonstrated a rapid decrease of miR-122 levels following hepatocyte transplantation. miR-122 expression in the serum was normalized to baseline levels in most of the (4/5) survivors. CONCLUSION: Our results suggest that longitudinal analysis of miR-122 allows detection of severe liver disease at an early stage and might be excellently suited to monitor therapy, at least when severe liver disease can be restored as observed after cell-based therapy of WD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12072-012-9348-5) contains supplementary material, which is available to authorized users.

A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses.

Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM(20K)) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium. Through subsequent shift to newly developed active Schneider's medium (SM(active)), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity. We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM(active). This is confirmed by patch-clamp recordings, which in addition show that SM(active)-cultured neurons display different spiking patterns. Furthermore, we demonstrate that transmission can be evoked in SM(active) cultures, revealing the existence of synaptic plasticity. Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila.