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Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
The macrophage-inducible C-type lectin (mincle) is part of the innate immune system and acts as a pattern recognition receptor for pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs). Ligand binding induces mincle activation which consequently interacts with the signaling adapter Fc receptor, SYK, and NF-kappa-B. There is also evidence that mincle expressed on macrophages promotes intestinal barrier integrity. However, little is known about the role of mincle in hepatic fibrosis, especially in more advanced disease stages. Mincle expression was measured in human liver samples from cirrhotic patients and donors collected at liver transplantation and in patients undergoing bariatric surgery. Human results were confirmed in rodent models of cirrhosis and acute-on-chronic liver failure (ACLF). In these models, the role of mincle was investigated in liver samples as well as in peripheral blood monocytes (PBMC), tissues from the kidney, spleen, small intestine, and heart. Additionally, mincle activation was stimulated in experimental non-alcoholic steatohepatitis (NASH) by treatment with mincle agonist trehalose-6,6-dibehenate (TDB). In human NASH, mincle is upregulated with increased collagen production. In ApoE deficient mice fed high-fat western diet (NASH model), mincle activation significantly increases hepatic collagen production. In human cirrhosis, mincle expression is also significantly upregulated. Furthermore, mincle expression is associated with the stage of chronic liver disease. This could be confirmed in rat models of cirrhosis and ACLF. ACLF was induced by LPS injection in cirrhotic rats. While mincle expression and downstream signaling via FC receptor gamma, SYK, and NF-kappa-B are upregulated in the liver, they are downregulated in PBMCs of these rats. Although mincle expressed on macrophages might be beneficial for intestinal barrier integrity, it seems to contribute to inflammation and fibrosis once the intestinal barrier becomes leaky in advanced stages of chronic liver disease.
Background and Aims: In patients with Rat sarcoma proto-oncogene (RAS) wild-type metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor (EGFR) antibodies have been established in first- and further therapy lines. Due to limited treatment options upon disease progression, anti-EGFR re-exposure is increasingly employed in real-world oncology. The aim of this study was to assess clinical implementation and utility of anti-EGFR retreatment strategies in real-world mCRC patients. Methods: In this monocentric retrospective study, we included 524 patients with CRC and identified patients who received an anti-EGFR-based treatment as well as anti-EGFR rechallenge (progression on first-line anti-EGFR therapy) or reintroduction (discontinuation due to intolerance/toxicity/other). Results: In total, 143 patients received an anti-EGFR-based first- or second-line treatment, showing a similar overall survival (OS) compared to the non-anti-EGFR treatment group (38.3 vs. 39.6 months, p = 0.88). Thirty-three patients met the inclusion criteria for anti-EGFR re-exposure and were either assigned to rechallenge (n = 21) or reintroduction (n = 12) subgroups. The median FU after re-exposure was 45.8 months. Cetuximab and Panitumumab were used in 21 and 12 patients, respectively, and the main chemotherapy at re-exposure was FOLFIRI in 39.4%. Anti-EGFR re-exposure was associated with a distinct trend towards a better outcome (median OS 56.0 vs. 35.4 months, p = 0.06). In a subgroup comparison, reintroduction was associated with a higher OS and PFS in trend compared to the rechallenge (mOS 66 vs. 52.4, n.s., mPFS 7.33 vs. 3.68 months, n.s.). Conclusions: This retrospective study provides real-world evidence underscoring that anti-EGFR re-exposure strategies might benefit patients independently of the reason for prior discontinuation.
Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.
Background and Aims: The Roadmap concept is a therapeutic framework in chronic hepatitis B for the intensification of nucleoside analogue monotherapy based on early virologic response. The efficacy and safety of this approach applied to telbivudine treatment has not been investigated.
Methods: A multinational, phase IV, single-arm open-label study (ClinicalTrials.gov ID NCT00651209) was undertaken in HBeAg-positive, nucleoside-naive adult patients with chronic hepatitis B. Patients received telbivudine (600 mg once-daily) for 24 weeks, after which those with undetectable serum HBV DNA (<300 copies/mL) continued to receive telbivudine alone while those with detectable DNA received telbivudine plus tenofovir (300 mg once-daily). Outcomes were assessed at Week 52.
Results: 105 patients commenced telbivudine monotherapy, of whom 100 were included in the efficacy analysis. Fifty-five (55%) had undetectable HBV DNA at Week 24 and continued telbivudine monotherapy; 45 (45%) received tenofovir intensification. At Week 52, the overall proportion of undetectable HBV DNA was 93% (93/100) by last-observation-carried-forward analysis (100% monotherapy group, 84% intensification group) and no virologic breakthroughs had occurred. ALT normalization occurred in 77% (87% monotherapy, 64% intensification), HBeAg clearance in 43% (65% monotherapy, 16% intensification), and HBeAg seroconversion in 39% (62% monotherapy, 11% intensification). Six patients had HBsAg clearance. Myalgia was more common in the monotherapy group (19% versus 7%). No decrease in the mean glomerular filtration rate occurred in either treatment group at Week 52.
Conclusions: Telbivudine therapy with tenofovir intensification at Week 24, where indicated by the Roadmap strategy, appears effective and well tolerated for the treatment of chronic hepatitis B.
Trial Registration: ClinicalTrials.gov NCT00651209
Introduction: MDRO-colonization has been shown to impair survival in patients with hematological malignancies and solid tumors as well as in patients with liver disease. Despite the increasing spread of multidrug-resistant organisms (MDRO), its impact on patients with hepatocellular carcinoma (HCC) has not been studied. We conducted this retrospective study to analyze the impact of MDRO-colonization on overall prognosis in HCC patients.
Materials and methods: All patients with confirmed HCC diagnosed between January 2008 and December 2017 at the University Hospital Frankfurt were included in this study. HCC patients with a positive MDRO screening before or within the first 90 days after diagnosis of HCC were defined as colonized HCC patients, HCC patients with a negative MDRO screening were defined as noncolonized HCC patients.
Results: 59 (6%) colonized and 895 (94%) noncolonized HCC patients were included. Enterobacterales with extended-spectrum β-lactamase-like phenotype with or without resistance to fluoroquinolones (ESBL/ ± FQ) were the most frequently found MDRO with 59%, followed by vancomycin-resistant Enterococcus faecium with 37%. Colonized HCC patients had more severe cirrhosis and more advanced HCC stage compared to noncolonized HCC patients. Colonized HCC patients showed an impaired survival with a median OS of 189 days (6.3 months) compared to a median OS of 1001 days (33.4 months) in noncolonized HCC patients. MDRO-colonization was identified as an independent risk factor associated with survival in multivariate analysis.
Conclusion: MDRO-colonization is an independent risk factor for survival in patients with HCC highlighting the importance of regular MDRO screening, isolation measures as well as interdisciplinary antibiotic steward-ship programs to guide responsible use of antibiotic agents.
Background The inhibitor telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A has been tested in a recent phase 1b clinical trial in patients infected with HCV genotype 1. This trial revealed residue mutations that confer varying degrees of drug resistance. In particular, two protease positions with the mutations V36A/G/L/M and T54A/S were associated with low to medium levels of drug resistance during viral breakthrough, together with only an intermediate reduction of viral replication fitness. These mutations are located in the protein interior and far away from the ligand binding pocket. Results Based on the available experimental structures of NS3-4A, we analyze the binding mode of different ligands. We also investigate the binding mode of VX-950 by protein-ligand docking. A network of non-covalent interactions between amino acids of the protease structure and the interacting ligands is analyzed to discover possible mechanisms of drug resistance. We describe the potential impact of V36 and T54 mutants on the side chain and backbone conformations and on the non-covalent residue interactions. We propose possible explanations for their effects on the antiviral efficacy of drugs and viral fitness. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chains support our interpretations. Experimental data using an HCV V36G replicon assay corroborate our findings. Conclusion T54 mutants are expected to interfere with the catalytic triad and with the ligand binding site of the protease. Thus, the T54 mutants are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Mutations at V36 and/or T54 result in impaired interaction of the protease residues with the VX-950 cyclopropyl group, which explains the development of viral breakthrough variants.
Background and Aims: Chronic infection with the hepatitis B virus (HBV) is a major health issue worldwide. Recently, single nucleotide polymorphisms (SNPs) within the human leukocyte antigen (HLA)-DP locus were identified to be associated with HBV infection in Asian populations. Most significant associations were observed for the A alleles of HLA-DPA1 rs3077 and HLA-DPB1 rs9277535, which conferred a decreased risk for HBV infection. We assessed the implications of these variants for HBV infection in Caucasians.
Methods: Two HLA-DP gene variants (rs3077 and rs9277535) were analyzed for associations with persistent HBV infection and with different clinical outcomes, i.e., inactive HBsAg carrier status versus progressive chronic HBV (CHB) infection in Caucasian patients (n = 201) and HBsAg negative controls (n = 235).
Results: The HLA-DPA1 rs3077 C allele was significantly associated with HBV infection (odds ratio, OR = 5.1, 95% confidence interval, CI: 1.9–13.7; p = 0.00093). However, no significant association was seen for rs3077 with progressive CHB infection versus inactive HBsAg carrier status (OR = 2.7, 95% CI: 0.6–11.1; p = 0.31). In contrast, HLA-DPB1 rs9277535 was not associated with HBV infection in Caucasians (OR = 0.8, 95% CI: 0.4–1.9; p = 1).
Conclusions: A highly significant association of HLA-DPA1 rs3077 with HBV infection was observed in Caucasians. However, as a differentiation between different clinical courses of HBV infection was not possible, knowledge of the HLA-DPA1 genotype cannot be translated into personalized anti-HBV therapy approaches.
Background: Autotaxin (ATX) and its product lysophosphatidic acid (LPA) are considered to be involved in the development of liver fibrosis and elevated levels of serum ATX have been found in patients with hepatitis C virus associated liver fibrosis. However, the clinical role of systemic ATX in the stages of liver cirrhosis was unknown. Here we investigated the relation of ATX serum levels and severity of cirrhosis as well as prognosis of cirrhotic patients.
Methods: Patients with liver cirrhosis were prospectively enrolled and followed until death, liver transplantation or last contact. Blood samples drawn at the day of inclusion in the study were assessed for ATX content by an enzyme-linked immunosorbent assay. ATX levels were correlated with the stage as well as complications of cirrhosis. The prognostic value of ATX was investigated by uni- and multivariate Cox regression analyses. LPA concentration was determined by liquid chromatography-tandem mass spectrometry.
Results: 270 patients were enrolled. Subjects with liver cirrhosis showed elevated serum levels of ATX as compared to healthy subjects (0.814±0.42 mg/l vs. 0.258±0.40 mg/l, P<0.001). Serum ATX levels correlated with the Child-Pugh stage and the MELD (model of end stage liver disease) score and LPA levels (r = 0.493, P = 0.027). Patients with hepatic encephalopathy (P = 0.006), esophageal varices (P = 0.002) and portal hypertensive gastropathy (P = 0.008) had higher ATX levels than patients without these complications. Low ATX levels were a parameter independently associated with longer overall survival (hazard ratio 0.575, 95% confidence interval 0.365–0.905, P = 0.017).
Conclusion: Serum ATX is an indicator for the severity of liver disease and the prognosis of cirrhotic patients.