Hepatoprotectant Ursodeoxycholyl Lysophosphatidylethanolamide Increasing Phosphatidylcholine Levels as a Potential Therapy of Acute Liver Injury

It has been long known that hepatic synthesis of phosphatidylcholine (PC) is depressed during acute such as carbon tetrachloride-induced liver injury. Anti-hepatotoxic properties of PC as liposomes have been recognized for treatment of acute liver damage. Ursodeoxycholate (UDCA) is a known hepatoprotectant in stabilizing cellular membrane. For therapeutic management of liver injury, we coupled UDCA with a phospholipid known as ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE). UDCA-LPE has been shown to first-in-class hepatoprotectant being superior to UDCA or PC. It inhibits mitochondrial damage and apoptosis, elicits survival signaling pathway, and promotes regeneration of hepatocytes. We herein report that a unique contribution of UDCA-LPE in increasing concentrations of PC in vitro and in vivo. UDCA-LPE-treated hepatocytes contained significantly increased PC levels. UDCA-LPE underwent the hydrolysis to LPE which was not the precursor of the increased PC. The levels of PC in the liver and blood were increased rapidly after intraperitoneally administration UDCA-LPE, and were found to be sustained even after 24 h. Among PC synthesis genes tested, UDCA-LPE treatment of mouse hepatocytes increased transcription of CDP-diacylglycerol synthase 1 which is an enzyme catalyzing phosphatidic acid to generate intermediates for PC synthesis. Thus, UDCA-LPE as a hepatoprotectant was able to induce synthesis of protective PC which would supplement for the loss of PC occurring during acute liver injury. This property has placed UDCA-LPE as a candidate agent for therapy of acute hepatotoxicity such as acetaminophen poisoning

HDAC inhibitor treatment of hepatoma cells induces both TRAIL-independent apoptosis and restoration of sensitivity to TRAIL

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Early virological response may predict treatment response in sofosbuvir-based combination therapy of chronic hepatitis c in a multi-center “real-life” cohort

Background: The combination of sofosbuvir (SOF), ribavirin (RBV) and peg-interferon-alfa-2a (peg-IFN-alfa-2a) as well as the combination of SOF and RBV for the treatment of patients infected with hepatitis c virus (HCV) has improved rates of sustained virological response (SVR) considerably in recent trials. However, there is only limited data concerning the efficacy and safety in a “real-life” cohort. Methods: We analyzed a cohort of 119 patients with chronic HCV infection treated at four investigational sites in Germany. All patients received either a combination treatment of SOF, RBV and peg-IFN-alfa-2a or SOF and RBV. Results: The rates of SVR at 12 weeks after end of treatment (SVR 12) were as follows: Among 76 patients with genotype 1 infection the SVR 12 rate was 74 % (n = 56), among 14 patients with genotype 2 infection the SVR 12 rate was 79 % (n = 11), among 24 patients with genotype 3 infection the SVR 12 rate was 92 % (n = 22) and among 5 patients with genotype 4 infection the SVR 12 rate was 80 % (n = 4). Of all 26 patients with a relapse in our cohort, 69 % (n = 18) of these patients presented with liver cirrhosis and 58 % (n = 15) were treatment experienced. Notably, the level of HCV-RNA after 4 weeks of treatment was a significant predictor of treatment response in genotype 1 patients. Patients with HCV-RNA levels ≥ 12 IU ml-1 after 4 weeks of treatment achieved SVR 12 only in 30 % (n = 17/56, p < 0.0001) of cases and treatment response was even lower with SVR 12 of 25 % (n = 5/20, p = 0.0016) in the subgroup of patients with cirrhosis. Conclusion: We observed a high rate of SVR 12 with SOF-based treatment regimes, however probably due to the high number of patients with liver cirrhosis and prior treatment experience, treatment response rates were lower than in previously published trials. In genotype 1 patients the analysis of early virological response may predict treatment response in SOF-based combination therapies

Programmed cell death ligand 1 (PD-L1, CD274) in cholangiocarcinoma – correlation with clinicopathological data and comparison of antibodies

Background: Cholangiocarcinoma (CCA) may arise in the intra- or extrahepatic biliary tract and is associated with a poor prognosis. Despite recent advances, to date there is still no established targeted therapeutic approach available. Non-surgical therapeutic agents are urgently needed, as most patients are non-eligible to surgical resection. Anti-PD-L1 therapy prevents cancer cells from evading the immune system and has emerged as a new treatment option in several cancer entities. Recently, PD-L1 expression has been analyzed in comparably small CCA patient cohorts. However, a systematic validation of different PD-L1 antibodies has not been performed in CCA so far. Methods: We stained a tissue microarray consisting of 170 patients, including 72 intrahepatic cholangiocarcinomas (iCCAs), 57 perihilar cholangiocarcinomas (pCCAs) and 41 distal cholangiocarcinomas (dCCAs) by immunohistochemistry and evaluated PD-L1 positivity in tumor and stromal cells. We analyzed three different PD-L1 antibodies (clones 28–8, SP142, and SP263) that are frequently used and recommended for predictive diagnostic testing in other cancer types. Results: For PD-L1 antibody clone SP263, 5% of iCCAs, 4% of pCCAs and 3% of dCCAs exhibited PD-L1 expression on tumor cells, thereby showing the highest frequencies of PD-L1 positivity. Accordingly, highest PD-L1 positivity rates of stromal cells with 31% in iCCA, 40% in pCCA and 61% in dCCA were detected for clone SP263. Agreement of PD-L1 positivity in tumor cells was moderate for clone 28–8 and SP263 (κ = 0.44) and poor between 28-8 and SP142 (κ = 0.13), as well as  SP142 and SP263 (κ = 0.11), respectively. Statistical analyses of PD-L1 expression (clone SP263) on tumor cells with clinicopathological data revealed a positive correlation with shortened overall survival in CCA patients. Conclusions: Selection of appropriate PD-L1 antibodies and careful evaluation of immunohistochemical staining patterns have a significant impact on PD-L1 testing in CCA. Clinical trials are necessary to investigate the putative beneficial effects of PD-L1 targeted immunotherapy in CCA patients

Low frequency of mismatch repair deficiency in gallbladder cancer

Background: DNA mismatch repair (MMR) deficiency is a major pathway of genomic instability in cancer. It leads to the accumulation of numerous mutations predominantly at microsatellite sequences, a phenotype known as microsatellite instability (MSI). MSI tumors have a distinct clinical behavior and commonly respond well to immune checkpoint blockade, irrespective of their origin. Data about the prevalence of MSI among gallbladder cancer (GBC) have been conflicting. We here analyzed a well-characterized cohort of 69 Western-world GBCs. Methods: We analyzed the mononucleotide MSI marker panel consisting of BAT25, BAT26, and CAT25 to determine the prevalence of MMR deficiency-induced MSI. Results: MSI was detected in 1/69 (1.4%) of analyzed GBCs. The detected MSI GBC had a classical histomorphology, i.e. of acinar/tubular/glandular pancreatobiliary phenotype, and showed nuclear expression of all four MMR proteins MLH1, MSH2, MSH6, and PMS2. The MSI GBC patient showed a prolonged overall survival, despite having a high tumor stage at diagnosis. The patient had no known background or family history indicative of Lynch syndrome. Conclusions: Even though the overall number of MSI tumors is low in GBC, the potentially therapeutic benefit of checkpoint blockade in the respective patients may justify MSI analysis of GBC

Successful direct acting antiviral (DAA) treatment of HCV/HIV-coinfected patients before and after liver transplantation

Objectives The aim of this multicenter retrospective study was to investigate safety and efficacy of direct acting antiviral (DAA) treatment in the rare subgroup of patients with HCV/HIV-coinfection and advanced liver cirrhosis on the liver transplant waiting list or after liver transplantation, respectively. Methods When contacting 54 German liver centers (including all 23 German liver transplant centers), 12 HCV/HIV-coinfected patients on antiretroviral combination therapy were reported having received additional DAA therapy while being on the waiting list for liver transplantation (patient characteristics: Child-Pugh A (n = 6), B (n = 5), C (n = 1);MELD range 7-21;HCC (n = 2);HCV genotype la (n = 8), 1 b (n = 2), 4 (n = 2)). Furthermore, 2 HCV/HIV-coinfected patients were denoted having received DAA therapy after liver transplantation (characteristics: HCV genotype 1a (n = 1), 4 (n = 1)). Results Applied DAA regimens were SOF/DAC (n = 7), SOF/LDV/RBV (n = 3), SOF/RBV (n = 3), PTV/r/OBV/DSV (n = 1), or PTV/r/OBV/DSV/RBV (n = 1), respectively. All patients achieved SVR 12, in the end. In one patient, HCV relapse occurred after 24 weeks of SOF/DAC therapy;subsequent treatment with 12 weeks PTV/r/OBV/DSV achieved SVR 12. One patient underwent liver transplantation while on DAA treatment. Analysis of liver function revealed either stable parameters or even significant improvement during DAA therapy and in followup. MELD scores were found to improve in 9/13 therapies in patients on the waiting list for liver transplantation;in only 2 patients a moderate increase of MELD scores persisted at the end of follow-up. Conclusion DAA treatment was safe and highly effective in this nation-wide cohort of patients with HCV/HIV-coinfection awaiting liver transplantation or being transplanted

Loss of aquaporin-4 expression and putative function in non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Aquaporins (AQPs) have been recognized to promote tumor progression, invasion, and metastasis and are therefore recognized as promising targets for novel anti-cancer therapies. Potentially relevant AQPs in distinct cancer entities can be determined by a comprehensive expression analysis of the 13 human AQPs.</p> <p>Methods</p> <p>We analyzed the presence of all AQP transcripts in 576 different normal lung and non-small cell lung cancer (NSCLC) samples using microarray data and validated our findings by qRT-PCR and immunohistochemistry.</p> <p>Results</p> <p>Variable expression of several AQPs (AQP1, -3, -4, and -5) was found in NSCLC and normal lung tissues. Furthermore, we identified remarkable differences between NSCLC subtypes in regard to AQP1, -3 and -4 expression. Higher transcript and protein levels of AQP4 in well-differentiated lung adenocarcinomas suggested an association with a more favourable prognosis. Beyond water transport, data mining of co-expressed genes indicated an involvement of AQP4 in cell-cell signalling, cellular movement and lipid metabolism, and underlined the association of AQP4 to important physiological functions in benign lung tissue.</p> <p>Conclusions</p> <p>Our findings accentuate the need to identify functional differences and redundancies of active AQPs in normal and tumor cells in order to assess their value as promising drug targets.</p

Treatment with tenofovir disoproxil fumarate or entecavir in chronic hepatitis B virus-infected patients with renal impairment: results from a 7-year, multicentre retrospective cohort study.

BackgroundLimited data exist regarding tenofovir disoproxil fumarate (TDF) safety and effectiveness in chronic hepatitis B virus-infected (CHB) patients with renal impairment (RI).AimsTo compare real-world data on renal safety and effectiveness of TDF vs entecavir (ETV) in CHB patients with moderate-to-severe RI.MethodsRetrospective, non-interventional, cohort study analysing medical records for TDF/ETV-treated CHB patients (54 European centres). Included patients experienced moderate-to-severe RI (creatinine clearance 20-60 mL/min [Cockcroft-Gault]) either before TDF/ETV initiation ('before' subgroup [baseline = treatment initiation]) or after TDF/ETV initiation ('after' subgroup [baseline = first RI occurrence]). The primary objective was TDF safety, particularly renal-related adverse events of special interest (AESI). TDF and ETV safety and effectiveness were compared and multivariate analyses were performed using inverse probability treatment weighting.Results'Before' subgroup included 107 TDF- and 91 ETV-treated patients; 'after' subgroup included 212 TDF- and 77 ETV-treated patients. Mean baseline creatinine clearance was higher for TDF- vs ETV-treated patients (both subgroups). Median follow-up was 3.1 years (both treatments). AESI were more frequent with TDF vs ETV ('before': 18.7% vs 8.8%; 'after': 9.9% vs 3.9%); however, differences were not significant by multivariate analysis. Only TDF-treated patients experienced renal tubular dysfunction (6.5% 'before'; 1.9% 'after') as well as renal adverse events leading to treatment discontinuation (8.4% 'before'; 7.1% 'after'). Effectiveness was similar between treatments.ConclusionsOverall safety was similar for TDF vs ETV (both subgroups). Given that renal tubular dysfunction occurred with TDF and not with ETV, renal safety concerns may be greater with TDF in CHB patients with RI

Epigenetic modulation by histone deacetylase inhibition as new therapeutic option against hepatocellular carcinoma

Acetylierung und Deacetylierung DNA-assoziierter Histonproteine durch das gegensätzlich arbeitende Enzympaar Histondeacetylase (HDAC) und Histonacetyltransferase (HAT) erlauben feinste Modulierungen der zellulären Genexpression auf epigenetischer Ebene. In malignen Zellen können HDAC-Inhibitoren (HDAC-I) eine Veränderung der Nukleosomenstruktur und damit eine Reaktivierung von Genen bewirken, welche zentrale Kontrollfunktionen bei Proliferation, Differenzierung und dem zellulären Selbstmordprogramm (Apoptose) ausüben. In der Konsequenz dieser epigenetisch vermittelten „Reprogrammierung“ können die entarteten Zellen im Wachstum gehemmt und in die Apoptose getrieben werden. In dieser Dissertation wurden zwei neuartige HDAC-I, das Antikonvulsivum Valproat (VPA) und der Wirkstoff ITF2357 unter Verwendung der humanen Hepatomzelllinien HepG2, PLC/PRF/5 und HuH7 und zusätzlich auch von primären humanen Hepatozyten (PHH) charakterisiert. Die HDAC-I Behandlung führte in den Hepatomzellen zu einer konzentrationsabhängigen Hemmung der Proliferation und Induktion von Apoptose. Im Gegensatz hierzu führte eine identische Behandlung bei PHH zu keinen meßbaren Zeichen einer Zytotoxizität. VPA und ITF2357 führten weiterhin zur einer erhöhten Expression des regulatorischen Zellzyklusproteins p21 und von pro-apoptotischen Mitgliedern der Bcl-2 Familie, wie Bid und Bax, jedoch auch zu einer Verminderung des anti-apoptotischen Bcl-XL Proteins. Eine artifizielle Überexpression von Bcl-XL durch Transfektion eines Bcl-XL kodierenden Plasmids konnte die durch HDAC-I hervorgerufene Zelltodrate deutlich reduzieren, so daß ein Übergewicht pro-apoptotischer Faktoren in den Hepatomzellen entscheidend zu der HDAC-I vermittelten Induktion von Apoptosephänomenen beizutragen scheint. Der zweite Teil der Dissertation befaßte sich mit einer Kombinationsbehandlung aus HDAC-I und dem Zytokin TNF-related apoptosis inducing ligand (TRAIL). Interessanterweise konnte gezeigt werden, daß eine durch HDAC-I hervorgerufene „Reprogrammierung“ der Tumorzellen in der Lage ist die TRAIL-Resistenz von Hepatomzellen zu überwinden und eine Sensibilisierung gegenüber TRAIL zu erreichen. Während TRAIL alleine keine relevante Apoptose auslösen konnte, führte eine Präinkubation mit einem HDAC-I zu einer deutlichen Zunahme der Zelltodrate nach TRAIL-Exposition in allen Hepatomzellen. Auf der Suche nach dem hierfür verantwortlichen Mechanismus zeigten sich zwar keine Veränderungen pro-apoptotischer TRAIL-Rezeptoren an der Zelloberfläche, jedoch konnte eine Herunterregulation des intrazellulären anti-apoptotischen Caspase-8 Inhibitors FLIP identifiziert werden. Die Kombination von HDAC-I und TRAIL zeigte in gesunden Leberzellen fünf verschiedener Spender ein ausgesprochen gutes Toleranzprofil ohne Zeichen der Toxizität, während gleichartig behandelte Hepatomzellen deutliche Zeichen eines massiven Zelluntergangs boten. Die Selektivität der HDAC-I gesteuerten TRAIL-Sensibilisierung korrelierte mit einer differentiellen Regulation von FLIP, welches in den PHH im Gegensatz zu den malignen Hepatomzellen hochreguliert war und somit die Apoptoseschwelle nicht maligner Zellen als Schutzfaktor zusätzlich erhöhte. Zusammenfassend eröffnet die untersuchte Kombinationsbehandlung von TRAIL mit einer HDAC-inhibitorisch wirkenden Substanz die Perspektive für eine innovative Therapiestrategie in der Behandlung des Hepatozellulären Karzinoms, die weiter klinisch evaluiert werden sollte.Due to a particular resistance against conventional chemotherapeutics, palliative treatment of hepatocellular carcinomas (HCC) is highly ineffective. Recent demonstration of both proliferation-inhibition and apoptosis of hepatoma cells by a histone deacetylase inhibitor (HDAC-I) treatment opens up a promising new approach. HDAC-I substances with favourable in vivo profiles, valproate (VPA) and ITF2357, were investigated on HCC cell lines and primary human hepatocytes (PHH). Histone acetylation and apoptosis-modulating proteins were investigated by western-blotting, proliferation by sulforhodamin B binding, toxicity by enzyme release, apoptosis by FACS analysis. VPA and ITF2357 inhibited proliferation in HCC cell lines. Both substances induced considerable cellular damage in HCC-derived cells, but PHH tolerated these substances well. A downregulation of anti- and upregulation of proapoptotic factors was found. Moreover, Bcl-X(L) transfection into HCC cells abrogated apoptosis induced by both substances, indicating that modulation of intracellular pro- and anti-apoptotic proteins is a key event in VPA or ITF2357 induced tumor-cell death.Preferential induction of cell death in HCC-derived cell lines, without toxicity in PHH, demonstrates the potential of VPA and ITF2357 to become promising new tools in the fight against HCC. Hepatocellular carcinoma displays further a striking resistance to innovative tumor cell apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In TRAIL-sensitive cancer cells, the mechanism of HDAC-I-induced cell death has been identified to be TRAIL-dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC-derived cells, a prototype of TRAIL-resistant tumor cells, we found a HDAC-I-mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC-I and TRAIL, increasing the fraction of apoptotic cells two- to threefold compared with HDAC-I treatment alone, whereas any premature HDAC-I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell-specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC-I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC-I. Importantly, combination HDAC-I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC-I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC

Circulating Phospholipid Patterns in NAFLD Patients Associated with a Combination of Metabolic Risk Factors

Background: Non-alcoholic fatty liver disease (NAFLD) is associated with inefficient macro- and micronutrient metabolism, and alteration of circulating phospholipid compositions defines the signature of NAFLD. This current study aimed to assess the pattern of serum phospholipids in the spectrum of NAFLD, and its related comorbidities and genetic modifications. Methods: 97 patients with diagnosed NAFLD were recruited at a single center during 2013&ndash;2016. Based on histological and transient elastography assessment, 69 patients were divided into non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) subgroups. 28 patients served as healthy controls. Serum phospholipids were determined by liquid-chromatography mass spectrometry (LC-MS/MS). Results: The total content of phosphatidylcholine (PC) and sphingomyelin in the serum was significantly increased in NAFL and NASH patients, compared to healthy controls. In addition, serum lysophospatidylethanolamine levels were significantly decreased in NAFL and NASH individuals. Circulating PC species, containing linoleic and &alpha;-linolenic acids, were markedly increased in NAFLD patients with hypertension, compared to NAFLD patients without hypertension. The pattern of phospholipids did not differ between NAFLD patients with diabetes and those without diabetes. However, NAFLD patients with hyperglycemia (blood glucose level (BGL) &gt;100 mg/dL) exhibited significantly a higher amount of monounsaturated phosphatidylethanolamine than those with low blood glucose levels. In addition, NAFLD patients with proven GG-genotype of PNPLA3, who were at higher risk for the development of progressive disease with fibrosis, showed lower levels of circulating plasmalogens, especially 16:0, compared to those with CC- and CG-allele. Conclusions: Our extended lipidomic study presents a unique metabolic profile of circulating phospholipids associated with the presence of metabolic risk factors or the genetic background of NAFLD patients