ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Impact of STAT6 Variants on the Response to Proton Pump Inhibitors and Comorbidities in Patients with Eosinophilic Esophagitis.

Proton pump inhibitors (PPIs) are the first-line drug for eosinophilic esophagitis (EoE), although it is estimated that there is a lack of histological remission in 50% of patients. This research aimed to identify pharmacogenetic biomarkers predictive of PPI effectiveness and to study their association with disease features. Peak eosinophil count (PEC) and the endoscopic reference score (EREFS) were determined before and after an eight-week PPI course in 28 EoE patients. The impact of the signal transducer and activator of transcription 6 (STAT6), CYP2C19, CYP3A4, CYP3A5, and ABCB1 genetic variations on baseline PEC and EREFS, their reduction and histological response, and on EoE symptoms and comorbidities was analyzed. PEC reduction was higher in omeprazole-treated patients (92.5%) compared to other PPIs (57.9%, p = 0.003). STAT6 rs12368672 (g.18453G>C) G/G genotype showed higher baseline PEC values compared to G/C and C/C genotypes (83.2 vs. 52.9, p = 0.027). EREFS reduction in STAT6 rs12368672 G/G and G/C genotypes was higher than in the C/C genotype (36.7% vs. -75.0% p = 0.011). However, significance was lost after Bonferroni correction. Heartburn incidence was higher in STAT6 rs167769 (g.27148G>A) G/G patients compared to G/A (54.55% vs. 11.77%, p = 0.030). STAT6 rs12368672G>C and rs167769G>A variants might have a relevant impact on EoE status and PPI response. Further research is warranted to clarify the clinical relevance of these variants.

Circulating immunome fingerprint in eosinophilic esophagitis is associated with clinical response to proton pump inhibitor treatment.

Objectives: The aim of the study was to characterize the circulating immunome of patients with EoE before and after proton pump inhibitor (PPI) treatment in order to identify potential non-invasive biomarkers of treatment response. Methods: PBMCs from 19 healthy controls and 24 EoE patients were studied using a 39-plex spectral cytometry panel. The plasmacytoid dendritic cell (pDC) population was differentially characterized by spectral cytometry analysis and immunofluorescence assays in esophageal biopsies from 7 healthy controls and 13 EoE patients. Results: Interestingly, EoE patients at baseline had lower levels of circulating pDC compared with controls. Before treatment, patients with EoE who responded to PPI therapy had higher levels of circulating pDC and classical monocytes, compared with non-responders. Moreover, following PPI therapy pDC levels were increased in all EoE patients, while normal levels were only restored in PPI-responding patients. Finally, circulating pDC levels inversely correlated with peak eosinophil count and pDC count in esophageal biopsies. The number of tissue pDCs significantly increased during active EoE, being even higher in non-responder patients when compared to responder patients pre-PPI. pDC levels decreased after PPI intake, being further restored almost to control levels in responder patients post-PPI. Conclusions: We hereby describe a unique immune fingerprint of EoE patients at diagnosis. Moreover, circulating pDC may be also used as a novel non-invasive biomarker to predict subsequent response to PPI treatment.

A New Method to Evaluate Lower Esophageal Distension Capacity in Eosinophilic Esophagitis by Using Functional Lumen Imaging Probe (EndoFLIP™).

Endoluminal functional lumen impedance planimetry (EndoFLIPTM) has become the gold standard to evaluate esophageal distensibility, although the study itself and its analysis present challenges. We propose here a new method to assess lower esophageal distension capacity that overcomes several limitations of prior approaches, including incomplete and corrupted EndoFLIPTM recordings. Esophageal distension capacity was evaluated with a 16-channel EndoFLIPTM in 10 controls and 14 patients with eosinophilic esophagitis (EoE). Controls were evaluated once. EoE patients were evaluated at baseline and after at least six weeks of treatment with orodispersible budesonide tablets, 1 mg bd. Balloon volumes were increased by 5 mL stepwise, either reaching a maximum volume of 60 mL or a maximum balloon pressure of 60 mmHg. Recordings were analyzed with a homemade R script. The mean esophageal diameter at 60 mL, D (60 mL), was calculated or extrapolated depending on whether the 60 mL volume was reached. By fitting a Michaelis-Menten curve across all measured diameters throughout all constant volume steps, the mean D (60 mL) was estimated. For control subjects, the mean ± SD value of D (60 mL) was 17.08 ± 1.69 mm, and for EoE patients at baseline, D (60 mL) was 14.51 ± 2.68 mm. After six weeks of treatment of EoE patients, D (60 mL) significantly increased to 16.22 ± 1.86 mm (paired Wilcoxon signed test: p = 0.0052), although the values for control subjects were not reached. The estimated mean esophageal diameter at 60 mL is a good proxy for esophageal distension capacity, which correlates with clinical outcomes in EoE. The method presented in this study overcomes difficulties encountered during the standard measurement protocol, allowing the analysis of recordings from incomplete and corrupted registries.

Biomecánica esofágica valorada mediante planimetría por impedancia (EndoFLIP™) en sujetos sanos y en pacientes con esofagitis eosinofílica. Valores de normalidad / Esophageal biomechanics assessed by impedance planimetry (EndoFLIPTM) in healthy subjects and in patients with eosinophilic esophagitis. Normality values

Antecedentes: la esofagitis eosinofílica (EEo) activa se asocia a alteraciones en el calibre, la distensibilidad y la motilidad esofágica que podrían revertir con el tratamiento. Objetivos: estudiar el diámetro, la distensibilidad y la contractilidad esofágica en sujetos sanos comparándolos con pacientes con EEo antes y después del tratamiento. Métodos: estudio cuasiexperimental. Mediante EndoFLIP™, se analizaron el cuerpo esofágico y la unión esofagogástrica (UEG) de los tres grupos, y se diseñó un programa para obtener los valores de diámetro, distensibilidad y contractilidad esofágica. Resultados: incluimos diez voluntarios sanos (24-61 años, seis hombres) y nueve pacientes con EEo (21-52 años, siete hombres). El índice de distensibilidad de la UEG fue de 5,07 mm2/Hg en controles, 2,40 mm2/Hg en EEo antes del tratamiento y 2,46 mm2/Hg después; la meseta de distensibilidad fue de 20,02 mm, 15,43 mm y 17,41 mm, respectivamente; y el diámetro, de 21,90 mm, 17,73 mm y 18,30 mm, con diferencias significativas (p < 0,05) excepto entre los diámetros de controles y pacientes tratados (p = 0,079). Las contracciones anterógradas repetitivas aparecieron en el 90 % de los controles, en el 66,7 % de EEo a antes del tratamiento y en el 88,9 % después (p > 0,05). Conclusiones: el índice de distensibilidad de la UEG, la meseta de distensibilidad y el diámetro en controles son mayores que en pacientes, aunque seis semanas de tratamiento parece poco tiempo para ver cambios significativos en la biomecánica esofágica. Las contracciones anterógradas repetitivas son el patrón predominante en sanos y en EEo. Aportamos valores de normalidad de la biomecánica esofágica medida mediante planimetría por impedancia en nuestro entorno.(AU)

Background: active eosinophilic esophagitis is associated with esophageal caliber, distensibility and motility changes that may be reversed with treatment. Objectives: to study esophageal diameter, distensibility and contractility in healthy subjects compared to patients with eosinophilic esophagitis, both before and after treatment. Methods: a quasi-experimental study, EndoFLIP™, was used to analyze the esophageal body and esophago-gastric junction (EGJ) in all three groups, and a program was designed to obtain esophageal diameter, distensibility and contractility values. Results: ten healthy volunteers (24-61 years, six men) and nine patients with eosinophilic esophagitis (21-52 years, seven men) were included. The esophagogastric junction distensibility index was 5.07 mm2/Hg in the control subjects, 2.40 mm2/Hg in the subjects with eosinophilic esophagitis before treatment and 2.46 mm2/Hg after treatment. The distensibility plateau was 20.02 mm, 15.43 mm and 17.41 mm, respectively, and the diameter was 21.90 mm, 17.73 mm and 18.30 mm, showing significant differences (p < 0.05), except between control subjects and patients after treatment (p = 0.079). Repetitive antegrade contractions developed in 90 % of control subjects, 66.7 % of eosinophilic esophagitis patients before treatment and 88.9 % of the latter after treatment (p > 0.05). Conclusions: esophago-gastric junction distensibility index, distensibility plateau and diameter values were higher in controls than in patients, although six weeks of treatment seems a short period to observe significant changes in esophageal biomechanics. Repetitive antegrade contractions are the predominant pattern in healthy subjects and eosinophilic esophagitis. We provide normality values for esophageal biomechanics, measured by impedance planimetry in our setting.(AU)

Esophageal biomechanics assessed by impedance planimetry (EndoFLIPTM) in healthy subjects and in patients with eosinophilic esophagitis. Normality values.

BACKGROUND: active eosinophilic esophagitis is associated with esophageal caliber, distensibility and motility changes that may be reversed with treatment. OBJECTIVES: to study esophageal diameter, distensibility and contractility in healthy subjects compared to patients with eosinophilic esophagitis, both before and after treatment. METHODS: a quasi-experimental study, EndoFLIP™, was used to analyze the esophageal body and esophago-gastric junction (EGJ) in all three groups, and a program was designed to obtain esophageal diameter, distensibility and contractility values. RESULTS: ten healthy volunteers (24-61 years, six men) and nine patients with eosinophilic esophagitis (21-52 years, seven men) were included. The esophagogastric junction distensibility index was 5.07 mm2/Hg in the control subjects, 2.40 mm2/Hg in the subjects with eosinophilic esophagitis before treatment and 2.46 mm2/Hg after treatment. The distensibility plateau was 20.02 mm, 15.43 mm and 17.41 mm, respectively, and the diameter was 21.90 mm, 17.73 mm and 18.30 mm, showing significant differences (p < 0.05), except between control subjects and patients after treatment (p = 0.079). Repetitive antegrade contractions developed in 90 % of control subjects, 66.7 % of eosinophilic esophagitis patients before treatment and 88.9 % of the latter after treatment (p > 0.05). CONCLUSIONS: esophago-gastric junction distensibility index, distensibility plateau and diameter values were higher in controls than in patients, although six weeks of treatment seems a short period to observe significant changes in esophageal biomechanics. Repetitive antegrade contractions are the predominant pattern in healthy subjects and eosinophilic esophagitis. We provide normality values for esophageal biomechanics, measured by impedance planimetry in our setting.

Proteomic analysis of the esophageal epithelium reveals key features of eosinophilic esophagitis pathophysiology.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic non-IgE-mediated allergic disease of the esophagus. An unbiased proteomics approach was performed to investigate pathophysiological changes in esophageal epithelium. Additionally, an RNAseq-based transcriptomic analysis in paired samples was also carried out. METHODS: Total proteins were purified from esophageal endoscopic biopsies in a cohort of adult EoE patients (n = 25) and healthy esophagus controls (n = 10). Differentially accumulated (DA) proteins in EoE patients compared to control tissues were characterized to identify altered biological processes and signaling pathways. Results were also compared with a quantitative proteome dataset of the human esophageal mucosa. Next, results were contrasted with those obtained after RNAseq analysis in paired samples. Finally, we matched up protein expression with two EoE-specific mRNA panels (EDP and Eso-EoE panel). RESULTS: A total of 1667 proteins were identified, of which 363 were DA in EoE. RNA sequencing in paired samples identified 1993 differentially expressed (DE) genes. Total RNA and protein levels positively correlated, especially in DE mRNA-proteins pairs. Pathway analysis of these proteins in EoE showed alterations in immune and inflammatory responses for the upregulated proteins, and in epithelial differentiation, cornification and keratinization in those downregulated. Interestingly, a set of DA proteins, including eosinophil-related and secreted proteins, were not detected at the mRNA level. Protein expression positively correlated with EDP and Eso-EoE, and corresponded with the most abundant proteins of the human esophageal proteome. CONCLUSIONS: We unraveled for the first time key proteomic features involved in EoE pathogenesis. An integrative analysis of transcriptomic and proteomic datasets provides a deeper insight than transcriptomic alone into understanding complex disease mechanisms.

Proton pump inhibitor therapy reverses endoscopic features of fibrosis in eosinophilic esophagitis.

BACKGROUND: Long-standing inflammation leads to esophageal remodeling with stricture formation in patients with eosinophilic esophagitis (EoE). The ability of proton pump inhibitors (PPI) to reverse endoscopic features of fibrosis is still unknown. OBJECTIVE: To investigate the effect of a short course of PPI treatment in reducing endoscopic findings indicative of esophageal fibrosis in EoE patients. METHODS: Cross-sectional analysis of the EoE CONNECT registry. Patients who received PPI to induce EoE remission were evaluated. Endoscopic features were graded using the EoE Endoscopic Reference Score (EREFS), with rings and strictures indicating fibrosis. Results were compared to those from patients treated with swallowed topic corticosteroids (STC). RESULTS: Clinico-histological remission was achieved in 83/166 adult patients treated with PPI (50%) and in 65/79 (82%) treated with STC; among responders, 60 (36%) and 57 (72%) patients respectively achieved deep histological remission (<5 eosinophils/hpf). At baseline, mean±SD EREFS was lower in patients treated with PPI compared to those who received STC (p < 0.001). Short term treatment significantly reduced EREFS scores in patients treated either with PPI or STC as well as rings and strictures. Among patients treated with PPI, deep histological remission (<5 eosinophils/hpf) provided further reduction in total EREFS score. CONCLUSION: Effective PPI therapy for EoE significantly reduced endoscopic esophageal fibrosis in the short term.

Naturally Occurring and Engineered Alphaviruses Sensitive to Double-Stranded-RNA-Activated Protein Kinase Show Restricted Translation in Mammalian Cells, Increased Sensitivity to Interferon, and Marked Oncotropism.

Alphaviruses are insect-borne viruses that alternate between replication in mosquitoes and vertebrate species. Adaptation of some alphaviruses to vertebrate hosts has involved the acquisition of an RNA structure (downstream loop [DLP]) in viral subgenomic mRNAs that confers translational resistance to protein kinase (PKR)-mediated eIF2α phosphorylation. Here, we found that, in addition to promoting eIF2-independent translation of viral subgenomic mRNAs, presence of the DLP structure also increased the resistance of alphavirus to type I interferon (IFN). Aura virus (AURAV), an ecologically isolated relative of Sindbis virus (SV) that is poorly adapted to replication in vertebrate cells, displayed a nonfunctional DLP structure and dramatic sensitivity to type I IFN. Our data suggest that an increased resistance to IFN emerged during translational adaptation of alphavirus mRNA to vertebrate hosts, reinforcing the role that double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays as both a constitutive and IFN-induced antiviral effector. Interestingly, a mutant SV lacking the DLP structure (SV-ΔDLP) and AURAV both showed a marked oncotropism for certain tumor cell lines that have defects in PKR expression and/or activation. AURAV selectively replicated in and killed some cell lines derived from human hepatocarcinoma (HCC) that lacked PKR response to infection or poly(I·C) transfection. The oncolytic activities of SV-ΔDLP and AURAV were also confirmed using tumor xenografts in mice, showing tumor regression activities comparable to wild-type SV. Our data show that translation of alphavirus subgenomic mRNAs plays a central role in IFN susceptibility and cell tropism, suggesting an unanticipated oncolytic potential that some naive arboviruses may have in virotherapy.IMPORTANCE Interferons (IFNs) induce the expression of a number of antiviral genes that protect the cells of vertebrates against viruses and other microbes. The susceptibility of cells to viruses greatly depends on the level and activity of these antiviral effectors but also on the ability of viruses to counteract this antiviral response. Here, we found that the level of one of the main IFN effectors in the cell, the dsRNA-activated protein kinase (PKR), greatly determines the permissiveness of cells to alphaviruses that lack mechanisms to counteract its activation. These naive viruses also showed a hypersensitivity to IFN, suggesting that acquisition of IFN resistance (even partial) has probably been involved in expanding the host range of alphaviruses in the past. Interestingly, some of these naive viruses showed a marked oncotropism for some tumor cell lines derived from human hepatocarcinoma (HCC), opening the possibility of their use in oncolytic therapy to treat human tumors.

Poor Sensitivity of Fecal Gluten Immunogenic Peptides and Serum Antibodies to Detect Duodenal Mucosal Damage in Celiac Disease Monitoring.

A lifelong gluten-free diet (GFD) is the only current treatment for celiac disease (CD), but strict compliance is complicated. Duodenal biopsies are the "gold standard" method for diagnosing CD, but they are not generally recommended for disease monitoring. We evaluated the sensitivity and specificity of fecal gluten immunogenic peptides (GIPs) to detect duodenal lesions in CD patients on a GFD and compared them with serum anti-tissue transglutaminase (tTG) IgA antibodies. A prospective study was conducted at two tertiary centers in Spain on a consecutive series of adolescents and adults with CD who maintained a long-lasting GFD. Adherence to a GFD and health-related quality of life were scored with validated questionnaires. Mucosal damage graded according to the Marsh-Oberhüber classification (Marsh 1/2/3) was used as the reference standard. Of the 97 patients included, 27 presented duodenal mucosal damage and 70 had normal biopsies (Marsh 0). The sensitivity (33%) and specificity (81%) of GIPs were similar to those provided by the two assays used to measure anti-tTG antibodies. Scores in questionnaires showed no association with GIP, but an association between GIPs and patients' self-reported gluten consumption was found (p = 0.003). GIP displayed low sensitivity but acceptable specificity for the detection of mucosal damage in CD.

Natural Plants Compounds as Modulators of Epithelial-to-Mesenchymal Transition.

Epithelial-to-mesenchymal transition (EMT) is a self-regulated physiological process required for tissue repair that, in non-controled conditions may lead to fibrosis, angiogenesis, loss of normal organ function or cancer. Although several molecular pathways involved in EMT regulation have been described, this process does not have any specific treatment. This article introduces a systematic review of effective natural plant compounds and their extract that modulates the pathological EMT or its deleterious effects, through acting on different cellular signal transduction pathways both in vivo and in vitro. Thereby, cryptotanshinone, resveratrol, oxymatrine, ligustrazine, osthole, codonolactone, betanin, tannic acid, gentiopicroside, curcumin, genistein, paeoniflorin, gambogic acid and Cinnamomum cassia extracts inhibit EMT acting on transforming growth factor-ß (TGF-ß)/Smads signaling pathways. Gedunin, carnosol, celastrol, black rice anthocyanins, Duchesnea indica, cordycepin and Celastrus orbiculatus extract downregulate vimectin, fibronectin and N-cadherin. Sulforaphane, luteolin, celastrol, curcumin, arctigenin inhibit ß-catenin signaling pathways. Salvianolic acid-A and plumbagin block oxidative stress, while honokiol, gallic acid, piperlongumine, brusatol and paeoniflorin inhibit EMT transcription factors such as SNAIL, TWIST and ZEB. Plectranthoic acid, resveratrol, genistein, baicalin, polyphyllin I, cairicoside E, luteolin, berberine, nimbolide, curcumin, withaferin-A, jatrophone, ginsenoside-Rb1, honokiol, parthenolide, phoyunnanin-E, epicatechin-3-gallate, gigantol, eupatolide, baicalin and baicalein and nitidine chloride inhibit EMT acting on other signaling pathways (SIRT1, p38 MAPK, NFAT1, SMAD, IL-6, STAT3, AQP5, notch 1, PI3K/Akt, Wnt/ß-catenin, NF-κB, FAK/AKT, Hh). Despite the huge amount of preclinical data regarding EMT modulation by the natural compounds of plant, clinical translation is poor. Additionally, this review highlights some relevant examples of clinical trials using natural plant compounds to modulate EMT and its deleterious effects. Overall, this opens up new therapeutic alternatives in cancer, inflammatory and fibrosing diseases through the control of EMT process.

Corrigendum to "T Helper 17/Regulatory T Cell Balance and Experimental Models of Peritoneal Dialysis-Induced Damage".

[This corrects the article DOI: 10.1155/2015/416480.].

Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture.

Hepatitis C virus (HCV) infection is a major biomedical problem worldwide. Although new direct antiviral agents (DAAs) have been developed for the treatment of chronic HCV infection, the potential emergence of resistant virus variants and the difficulties to implement their administration worldwide make the development of novel antiviral agents an urgent need. Moreover, no effective vaccine is available against HCV and transmission of the virus still occurs particularly when prophylactic measures are not taken. We used a cell-based system to screen a battery of polyanionic carbosilane dendrimers (PCDs) to identify compounds with antiviral activity against HCV and show that they inhibit effective virus adsorption of major HCV genotypes. Interestingly, one of the PCDs irreversibly destabilized infectious virions. This compound displays additive effect in combination with a clinically relevant DAA, sofosbuvir. Our results support further characterization of these molecules as nanotools for the control of hepatitis C virus spread.

Mesothelial-to-mesenchymal transition in the pathogenesis of post-surgical peritoneal adhesions.

Peritoneal adhesions (PAs) are fibrotic bands formed between bowel loops, solid organs, and the parietal peritoneum, which may appear following surgery, infection or endometriosis. They represent an important health problem with no effective treatment. Mesothelial cells (MCs) line the peritoneal cavity and undergo a mesothelial-to-mesenchymal transition (MMT) under pathological conditions, transforming into myofibroblasts, which are abundant in peritoneal fibrotic tissue. The aim of this study was to investigate if peritoneal MCs undergo a MMT contributing to the formation of post-surgical adhesions. Biopsies from patients with PAs were analysed by immunohistochemistry, immunofluorescence, and quantitative RT-PCR. A mouse model of PAs based on ischaemic buttons was used to modulate MMT by blocking the transforming growth factor-beta (TGF-ß) pathway. The severity of adhesions and MMT-related marker expression were studied. We observed myofibroblasts derived from the conversion of MCs in submesothelial areas of patients with PAs. In addition, MMT-related markers were dysregulated in adhesion zones when compared to distant normal peritoneal tissue of the same patient. In animal experiments, blockage of TGF-ß resulted in molecular reprogramming of markers related to the mesenchymal conversion of MCs and in a significant decrease in the severity of the adhesions. These data indicate for the first time that MMT is involved in PA pathogenesis. This finding opens new therapeutic strategies to interfere with adhesion formation by modulating MMT with a wide range of pharmacological agents.

Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus.

UNLABELLED: Hepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission in vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis. IMPORTANCE: This work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention.

T Helper 17/Regulatory T Cell Balance and Experimental Models of Peritoneal Dialysis-Induced Damage.

Fibrosis is a general complication in many diseases. It is the main complication during peritoneal dialysis (PD) treatment, a therapy for renal failure disease. Local inflammation and mesothelial to mesenchymal transition (MMT) are well known key phenomena in peritoneal damage during PD. New data suggest that, in the peritoneal cavity, inflammatory changes may be regulated at least in part by a delicate balance between T helper 17 and regulatory T cells. This paper briefly reviews the implication of the Th17/Treg-axis in fibrotic diseases. Moreover, it compares current evidences described in PD animal experimental models, indicating a loss of Th17/Treg balance (Th17 predominance) leading to peritoneal damage during PD. In addition, considering the new clinical and animal experimental data, new therapeutic strategies to reduce the Th17 response and increase the regulatory T response are proposed. Thus, future goals should be to develop new clinical biomarkers to reverse this immune misbalance and reduce peritoneal fibrosis in PD.

Clathrin mediates infectious hepatitis C virus particle egress.

UNLABELLED: Although it is well established that hepatitis C virus (HCV) entry into hepatocytes depends on clathrin-mediated endocytosis, the possible roles of clathrin in other steps of the viral cycle remain unexplored. Thus, we studied whether cell culture-derived HCV (HCVcc) exocytosis was altered after clathrin interference. Knockdown of clathrin or the clathrin adaptor AP-1 in HCVcc-infected human hepatoma cell cultures impaired viral secretion without altering intracellular HCVcc levels or apolipoprotein B (apoB) and apoE exocytosis. Similar reductions in HCVcc secretion were observed after treatment with specific clathrin and dynamin inhibitors. Furthermore, detergent-free immunoprecipitation assays, neutralization experiments, and immunofluorescence analyses suggested that whereas apoE associated with infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, we observed that clathrin and AP-1 knockdown altered the endosomal distribution of HCV core, reducing and increasing its colocalization with early endosome and lysosome markers, respectively. Our data support a model in which nascent HCV particles associate with apoE in the ER and exit cells following a clathrin-dependent transendosomal secretory route. IMPORTANCE: HCV entry into hepatocytes depends on clathrin-mediated endocytosis. Here we demonstrate for the first time that clathrin also participates in HCV exit from infected cells. Our data uncover important features of HCV egress, which may lead to the development of new therapeutic interventions. Interestingly, we show that secretion of the very-low-density lipoprotein (VLDL) components apoB and apoE is not impaired after clathrin interference. This is a significant finding, since, to date, it has been proposed that HCV and VLDL follow similar exocytic routes. Given that lipid metabolism recently emerged as a potential target for therapies against HCV infection, our data may help in the design of new strategies to interfere specifically with HCV exocytosis without perturbing cellular lipid homeostasis, with the aim of achieving more efficient, selective, and safe antivirals.

Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

Modulation of PI3K-LXRα-dependent lipogenesis mediated by oxidative/nitrosative stress contributes to inhibition of HCV replication by quercetin.

There is experimental evidence that some antioxidant flavonoids show therapeutic potential in the treatment of hepatitis C through inhibition of hepatitis C virus (HCV) replication. We examined the effect of treatment with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin on HCV replication efficiency in an in vitro model. While all flavonoids studied were able to reduce viral replication at very low concentrations (ranging from 0.1 to 5 µM), quercetin appeared to be the most effective inhibitor of HCV replication, showing a marked anti-HCV activity in replicon-containing cells when combined with interferon (IFN)α. The contribution of oxidative/nitrosative stress and lipogenesis modulation to inhibition of HCV replication by quercetin was also examined. As expected, quercetin decreased HCV-induced reactive oxygen and nitrogen species (ROS/RNS) generation and lipoperoxidation in replicating cells. Quercetin also inhibited liver X receptor (LXR)α-induced lipid accumulation in LXRα-overexpressing and replicon-containing Huh7 cells. The mechanism underlying the LXRα-dependent lipogenesis modulatory effect of quercetin in HCV-replicating cells seems to involve phosphatidylinositol 3-kinase (PI3K)/AKT pathway inactivation. Thus, inhibition of the PI3K pathway by LY294002 attenuated LXRα upregulation and HCV replication mediated by lipid accumulation, showing an additive effect when combined with quercetin. Inactivation of the PI3K pathway by quercetin may contribute to the repression of LXRα-dependent lipogenesis and to the inhibition of viral replication induced by the flavonol. Combined, our data suggest that oxidative/nitrosative stress blockage and subsequent modulation of PI3K-LXRα-mediated lipogenesis might contribute to the inhibitory effect of quercetin on HCV replication.