Pedro Majano

Background: The esophageal String Test (EST) is emerging as a minimally invasive alternative for assessing disease activity in eosinophilic esophagitis (EoE), yet the molecular composition of its protein content remains unknown. Objective: To comprehensively characterize the esophageal EST proteome, define an EoE-associated luminal protein signature, and evaluate its biological and clinical relevance. Methods: EST samples were obtained from patients with active EoE, healthy controls (C) and patients with gastroesophageal reflux disease (GERD). Label-free proteomic profiling was performed by liquid chromatography-tandem mass spectrometry. Differential protein accumulation, principal component analysis (PCA), pathway annotation, comparison with published esophageal tissue and salivary proteomes, receiver operating characteristic (ROC) analyses and correlations with clinical disease activity were performed. Results: More than 3,000 proteins were identified in esophageal EST samples. Global proteomic profiling clearly separated patients with EoE from both C and GERD, whereas only limited differences were observed between the latter two groups. This enabled the identification of an EoE-associated luminal protein signature comprising inflammatory, epithelial, and tissue remodeling proteins. Comparison with published proteomic datasets indicated that the EST samples a complex esophageal luminal protein microenvironment containing proteins represented in esophageal mucosa and saliva, together with inflammatory and extracellular components. Several individual proteins showed high discriminatory performance in this exploratory cohort for distinguishing EoE from non-EoE subjects, and multiple proteins correlated with eosinophil counts and endoscopic measures of disease activity. Conclusions: This study demonstrates that the EST provides access to a complex esophageal luminal protein microenvironment and establishes a molecular framework for its minimally invasive characterization in EoE.

Background: Recently, we have identified a dysregulated protein signature in the esophageal epithelium of eosinophilic esophagitis (EoE) patients including proteins associated with inflammation and epithelial barrier function; however, the effect of proton pump inhibitor (PPI) treatment on this signature is unknown. Herein, we used a proteomic approach to investigate: (1) whether PPI treatment alters the esophageal epithelium protein profile observed in EoE patients and (2) whether the protein signature at baseline predicts PPI response.Methods: We evaluated the protein signature of esophageal biopsies using a cohort of adult EoE (n = 25) patients and healthy controls (C) (n = 10). In EoE patients, esophageal biopsies were taken before (pre) and after (post) an 8‐week PPI treatment, determining the histologic response. Eosinophil count PostPPI was used to classify the patients: ≥15 eosinophils/hpf as non‐responders (non‐responder) and < 15 eosinophils/hpf as responders (R). Protein signature was determined and differentially accumulated proteins were characterized to identify altered biological processes and signaling pathways.Results: Comparative analysis of differentially accumulated proteins between groups revealed common signatures between three groups of patients with inflammation (responder‐PrePPI, non‐responder‐PrePPI, and non‐responder‐PostPPI) and without inflammation (controls and responder‐PostPPI). PPI therapy almost reversed the EoE specific esophageal protein signature, which is enriched in pathways associated with inflammation and epithelial barrier function, in responder‐PostPPI. Furthermore, we identified a set of candidate proteins to differentiate responder‐PrePPI and non‐responder‐PrePPI EoE patients before treatment.Conclusion: These findings provide evidence that PPI therapy reverses the alterations in esophageal inflammatory and epithelial proteins characterizing EoE, thereby providing new insights into the mechanism of PPI clinical response. Interestingly, our results also suggest that PPI response could be predicted at baseline in EoE.

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.

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 of 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. WHAT IS KNOWN Eosinophilic esophagitis (EoE) is a Th2 type immune disorder with increased prevalence in the last years. Proton pump inhibitor (PPI) treatment is the preferred first-line therapy for EoE, which leads to clinical and pathological reversion in about half of the cases. Non-responding patients require other therapeutic options. Currently, an endoscopy with esophageal biopsies is required for EoE diagnosis and to monitor response to treatment. Therefore, the discovery of non-invasive biomarkers is of utmost importance for treatment monitoring. To date only peripheral eosinophil and Th2 profiles have been studied in EoE. WHAT IS NEW HERE At diagnosis EoE patients have a specific circulating immune signature. PPI-responding and non-responding EoE patients have different immune fingerprints at baseline. Immune characterization of EoE patients at diagnosis and after PPI treatment unveiled differential levels of circulating plasmacytoid dendritic cells (pDCs) depending on their inflammatory state and response to PPI treatment. Those levels are related with the number of pDCs infiltrated in the esophageal tissue.

Background: Recently, we have identified a dysregulated protein signature in the esophageal epithelium of eosinophilic esophagitis (EoE) patients; however, the effect of proton pump inhibitor (PPI) treatment on this signature is unknown. Herein, we used a proteomic approach to investigate: (1) whether PPI treatment alters the esophageal epithelium protein profile observed in EoE patients and (2) whether the protein signature at baseline predicts PPI response. Methods: We evaluated the protein signature of esophageal biopsies using a cohort of adult EoE (n=25) patients and healthy controls (C) (n=10). In EoE patients, esophageal biopsies were taken before (Pre) and after (Post) an 8-week PPI treatment, determining the histologic response. Eosinophil count PostPPI was used to classify the patients: ≥15 eosinophils/hpf as non-responders (NR) and <15 eosinophils/hpf as responders (R). Protein signature was determined and differentially accumulated proteins (DAP) were characterized to identify altered biological processes and signaling pathways. Results: High dimensional analysis of DAP between groups revealed common signatures between three groups of patients with inflammation (R-PrePPI, NR-PrePPI and NR-PostPPI) and without inflammation (C and R- PostPPI). PPI therapy almost reversed the EoE specific esophageal protein signature, which is enriched in pathways associated with inflammation and epithelial barrier function, in R-PostPPI. Furthermore, we identified a set of candidate proteins to differentiate R-PrePPI and NR-PrePPI EoE patients before treatment. Conclusion: These findings provide evidence that PPI therapy reverses the alterations in the protein profile associated with EoE. Interestingly, our results also suggest that PPI response could be predicted at baseline in EoE.

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.

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.

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.