Stratifying esophago-gastric cancer treatment using a patient-derived organoid-based threshold.

BACKGROUND AND AIMS: This study sought to determine the value of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) for response prediction to neoadjuvant chemotherapy (neoCTx). METHODS: Endoscopic biopsies of patients with locally advanced EGC (n = 120) were taken into culture and PDOs expanded. PDOs' response towards the single substances of the FLOT regimen and the combination treatment were correlated to patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n = 13) and subsequently confirmed in an independent validation cohort (n = 13). RESULTS: EGC PDOs reflected patients' diverse responses to single chemotherapeutics and the combination regimen FLOT. In the exploratory cohort, PDOs response to single 5-FU and FLOT combination treatment correlated with the patients' pathological response (5-FU: Kendall's τ = 0.411, P = 0.001; FLOT: Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision in receiver operating characteristic (ROC) analysis was reached with an AUCROC of 0.994 (CI 0.980 to 1.000). The discriminative ability of PDO-based FLOT testing allowed the definition of a threshold, which classified in an independent validation cohort FLOT responders from non-responders with high sensitivity (90%), specificity (100%) and accuracy (92%). CONCLUSION: In vitro drug testing of EGC PDOs has a high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. Taking into account the high rate of successful PDO expansion from biopsies, the definition of a threshold that allows treatment stratification paves the way for an interventional trial exploring PDO-guided treatment of EGC patients.

Analysis of an Unselected Patient Cohort With Advanced Colorectal Carcinoma from a Maximum Care Center.

BACKGROUND/AIM: Systemic treatment for metastatic colorectal cancer (CRC) includes chemotherapy in combination with a targeted antibody. Novel targeted therapies and immunotherapies are introduced for specific molecular subgroups. Prognostic relevant determinants are still under investigation. PATIENTS AND METHODS: Systemic therapies of an unselected patient cohort with metastatic CRC were retrospectively analyzed. Treatment outcome was evaluated according to time-to-next-treatment (TTNT) and frequency of conversion surgery and compared between subgroups stratified by primary tumor side, molecular profile, sex and age, and metastases sites. RESULTS: More than 50% of patients with locally advanced or metastatic CRC underwent secondary resection after first-line systemic therapy. Rectum carcinoma had the best prognosis under anti-EGFR-antibody treatment. Female patients had a worse prognosis than male patients in late disease stage. Young patients demonstrated poor response to systemic therapy, but a high rate of conversion surgeries. Conversely, elderly patients benefited from systemic therapy but underwent surgery less frequently. Liver and lung metastases had a worse prognosis than other metastases sites, whereas lung metastases were more likely to be resected than liver metastases in early disease stage. CONCLUSION: Patient age, sex, primary tumor localization, and metastatic sites are prognostic factors that could guide future treatment decisions for the therapy of metastatic CRC.

SNP array genomic analysis of matched pairs of brain and liver metastases in primary colorectal cancer.

PURPOSE: Brain metastasis formation is a rare and late event in colorectal cancer (CRC) patients and associated with poor survival. In contrast to other metastatic sites, the knowledge on chromosomal aberrations in brain metastases is very limited. METHODS: Therefore, we carried out single nucleotide polymorphism (SNP) array analyses on matched primary CRC and brain metastases of four patients as well as on liver metastases of three patients. RESULTS: Brain metastases showed more chromosomal aberrations than primary tumors or liver metastases. Commonly occurring aberrations were gain of 8q11.1-q24.3 (primary CRC), gain of 13q12.13-q12.3 (liver metastases), and gain of 20q11.1-q13.33 (brain metastases). Furthermore, we found one copy-neutral loss of heterozygosity (cn-LOH) region on chromosome 3 in primary CRC, three cn-LOH regions in liver metastases and 23 cn-LOH regions in brain metastases, comprising 26 previously undescribed sites. CONCLUSION: The more frequent occurrence of cn-LOHs and subsequently affected genes in brain metastases shed light on the pathophysiology of brain metastasis formation. Further pairwise genetic analyses between primary tumors and their metastases will help to define the role of affected genes in cn-LOH regions.

A microbiota-modulated checkpoint directs immunosuppressive intestinal T cells into cancers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4ß7+CD4+ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4ß7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4ß7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

Scheduled endoscopic treatment of biliary anastomotic and nonanastomotic strictures after orthotopic liver transplantation.

BACKGROUND AND AIMS: Biliary strictures after liver transplantation are associated with significant morbidity and mortality. Although various endoscopic treatment strategies are available, consensus on a particular strategy is lacking. Moreover, the influence of endoscopic therapy on overall survival has not been studied. This retrospective study aimed to evaluate the impact of scheduled endoscopic dilatation of biliary strictures after orthotopic liver transplantation on therapeutic success, adverse events, and survival. METHODS: Between 2000 and 2016, patients with post-transplant anastomotic and nonanastomotic strictures were treated with balloon dilatation at defined intervals until morphologic resolution and clinical improvement. The primary clinical endpoint was overall survival, whereas secondary outcomes were technical and sustained clinical success, adverse events, treatment failure, and recurrence. RESULTS: Overall, 165 patients with a mean follow-up of 8 years were included; anastomotic and nonanastomotic strictures were diagnosed in 110 and 55 patients, respectively. Overall survival was significantly higher in patients with anastomotic strictures than in those with nonanastomotic strictures (median, 17.6 vs 13.9 years; log-rank: P < .05). Sustained clinical success could be achieved significantly more frequently in patients with anastomotic strictures (79.1% vs 54.5%, P < .001), and such patients showed significantly superior overall survival (19.7 vs 7.7 years; log-rank: P < .001). Sustained clinical success and the presence of nonanastomotic strictures were independently associated with better and worse outcomes (P < .05), respectively. CONCLUSIONS: Scheduled endoscopic treatment of biliary anastomotic and nonanastomotic strictures after liver transplantation is effective and safe, with high success rates. The implementation of this strategy controls symptoms and significantly improves survival.

Endoscopic Stent Placement Can Successfully Treat Gastric Leak Following Laparoscopic Sleeve Gastrectomy If and Only If an Esophagoduodenal Megastent Is Used.

PURPOSE: Gastric staple line leakage (GL) is a serious complication of laparoscopic sleeve gastrectomy (LSG), with a specific mortality ranging from 0.2 to 3.7%. The current treatment of choice is stent insertion. However, it is unclear whether the type of stent which is inserted affects treatment outcome. Therefore, we aimed not only to determine the effectiveness of stent treatment for GL but also to specifically clarify whether treatment outcome was dependent on the type of stent (small- (SS) or megastent (MS)) which was used. PATIENTS AND METHODS: A single-centre retrospective study of 23 consecutive patients was conducted to compare the outcomes of SS (n = 12) and MS (n = 11) for the treatment of GL following LSG. The primary outcome measure was the success rate of stenting, defined as complete healing of the GL without changing the treatment strategy. Treatment change or death were both coded as failure. RESULTS: The success rate of MS was 91% (10/11) compared to only 50% (6/12) for SS (p = 0.006). An average of 2.3 ± 0.5 and 6.8 ± 3.7 endoscopies were required to achieve healing in the MS and SS groups respectively (p < 0.001). The average time to resumption of oral nutrition was shorter in the MS group (1.4 ± 1.1 days vs. 23.1 ± 33.1 days, p = 0.003). CONCLUSIONS: Stent therapy is only effective and safe for the treatment of GL after LSG if a MS is used. Treatment with a MS may not only increase treatment success rates but may also facilitate earlier resumption of oral nutrition and shorten the duration of hospitalization.

Aspiration risk in relation to Glasgow Coma Scale score and clinical parameters in patients with severe acute alcohol intoxication: a single-centre, retrospective study.

OBJECTIVES: In alcohol intoxicated patients, the decision for or against airway protection can be challenging and is often based on the Glasgow Coma Scale (GCS). Primary aim of this study was to analyse the aspiration risk in relation to the GCS score and clinical parameters in patients with severe acute alcohol monointoxication. Secondary aim was the association between the blood alcohol level and the GCS score. SETTING: Single-centre, retrospective study of alcoholised patients admitted to a German intensive care unit between 2006 and 2020. PARTICIPANTS: A total of n=411 admissions were eligible for our analysis. CLINICAL MEASURES AND ANALYSIS: The following data were extracted: age, gender, admission time, blood alcohol level, blood glucose level, initial GCS score, GCS score at admission, vital signs, clinical signs of aspiration and airway management measures. The empirical distribution of continuous and categorical data was calculated. Binary multivariable logistic regression analysis was used to identify possible risk factors for aspiration. RESULTS: The mean age was 35 years. 72% (n=294) of the admissions were male. The blood alcohol level (mean 2.7 g/L±1.0, maximum 5.9 g/L) did not correlate with the GCS score but with the age of the patient. In univariate analysis, the aspiration risk correlated with blood alcohol level, age, GCS score, oxygen saturation, respiratory rate and blood glucose level and was significantly higher in male patients, on vomiting, and in patients requiring airway measures. Aspiration rate was 45% (n=10) in patients without vs 6% (n=3) in patients with preserved protective reflexes (p=0.0001). In the multivariate analysis, only age and GCS score were significantly associated with the risk of aspiration. CONCLUSION: Although in this single-centre, retrospective study the aspiration rate in severe acute alcohol monointoxicated patients correlates with GCS and protective reflexes, the decision for endotracheal intubation might rather be based on the presence of different risk factors for aspiration.

Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection.

Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single-cell RNA-Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

Feasibility, effectiveness, and safety of endoscopic vacuum therapy for intrathoracic anastomotic leakage following transthoracic esophageal resection.

BACKGROUND: Anastomotic leakage (AL) in the upper gastrointestinal (GI) tract is associated with high morbidity and mortality rates. Especially intrathoracic anastomotic leakage leads to life-threatening complications. Endoscopic vacuum therapy (EVT) for anastomotic leakage after transthoracic esophageal resection represents a novel concept. However, sound clinical data are still scarce. This retrospective, single-center study aimed to evaluate the feasibility, effectiveness, and safety of EVT for intrathoracic anastomotic leakage following abdomino-thoracic esophageal resection. METHODS: From March 2014 to September 2019 259 consecutive patients underwent elective transthoracic esophageal resection. 72 patients (27.8%) suffered from AL. The overall collective in-hospital mortality rate was 3.9% (n = 10). Data from those who underwent treatment with EVT were included. RESULTS: Fifty-five patients were treated with EVT. Successful closure was achieved in 89.1% (n = 49) by EVT only. The EVT-associated complication rate was 5.4% (n = 3): bleeding occurred in one patient, while minor sedation-related complications were observed in two patients. The median number of EVT procedures per patient was 3. The procedures were performed at intervals of 3-5 days, with a 14-day median duration of therapy. The mortality rate of patients with AL was 7.2% (n = 4). Despite successfully terminated EVT, three patients died because of multiple organ failure, acute respiratory distress syndrome, and urosepsis (5.4%). One patient (1.8%) died during EVT due to cardiac arrest. CONCLUSIONS: EVT is a safe and effective approach for intrathoracic anastomotic leakages following abdomino-thoracic esophageal resections. It offers a high leakage-closure rate and the potential to lower leakage-related mortalities. TRIAL REGISTRATION: This trial was registered and approved by the Institutional Ethics Committee of the University of Heidelberg on 16.04.2014 (Registration Number: S-635/2013).

Percutaneous Endoscopic Gastrostomy Tube Placement in Patients with Head and Neck Cancer Treated with Radiotherapy.

BACKGROUND AND AIM: The primary aim of our study was to evaluate percutaneous endoscopic gastrostomy (PEG) tube placement depending on body weight and body mass index in patients undergoing radiotherapy (RT) for head and neck cancer (HNC). A secondary aim was to evaluate the course of weight change following PEG placement. METHODS: We retrospectively reviewed the medical records of 186 patients with HNC undergoing radiotherapy (RT) or chemoradiotherapy (CRT) at our institution between January 2010 and August 2017. Initial weight and nutritional intake were analyzed prior to RT initiation and then followed throughout treatment until completion. Based on these data, the indication of PEG placement was determined. Medical records were also reviewed to analyze PEG-related acute toxicities. RESULTS: A total of 186 patients met inclusion criteria. Patients were most commonly male (n=123, 66.1%) with squamous cell carcinoma (n=164, 88.2%). Patients who had dysphagia prior to treatment initiation as well as patients with a BMI <18.5 kg/m2 needed PEG placement earlier during the treatment course. Low-grade toxicities related to PEG insertion were observed in 10.7% patients, with peristomal pain and redness adjacent to the PEG tube insertion site being most common. High-grade toxicities, such as peritonitis and organ injury, were found in 4.9% of patients. CONCLUSION: Underweight patients and those with preexisting dysphagia should be closely screened during RT for weight loss and decreased oral intake. For weight loss greater than 4.5% during the treatment of HNC, early PEG-tube placement should be considered. Further prospective studies are needed to confirm these findings, and delineate a scoring system for timing of PEG use (prophylactic vs reactive) as well as assess the quality of life in patients with HNC who receive PEG placement.

Intrahepatic biliary strictures after liver transplantation are morphologically similar to primary sclerosing cholangitis but immunologically distinct.

OBJECTIVE: Biliary strictures are an important cause of morbidity and mortality in primary hepatic disease and after liver transplantation (LT). We aimed to characterize inflammatory cytokines in biliary fluids in biliary strictures to investigate their immunological origin. METHODS: We conducted a retrospective study on 72 patients with strictures after LT, eight patients with primary sclerosing cholangitis (PSC) and 15 patients with secondary sclerosing cholangitis (SSC). We measured cytokines interleukin (IL)-2, -4, -6, -10, -17, monocyte chemoattractant protein (MCP)-1, fibroblast growth factor (FGF)-2 and interferon (IFN)-γ as well as biochemical components such as protein and phospholipids in biliary fluid obtained from endoscopic retrograde cholangiography (ERC). Cell viability assays were performed on human cholangiocytes (H69) after being treated with IL-6, IL-4 and IFN-γ. RESULTS: Bile of patients with diffuse strictures after LT or due to SSC showed low values of all measured cytokines except for IL-6 levels, which were largely elevated in patients with diffuse strictures after LT. Patients high in biliary IL-6 showed an increase in profibrotic markers FGF-2 and MCP-1. In contrast, PSC bile was dominated by a Th1/Th17 profile with elevated IL-2, IL-17 and IFN-γ. In LT patients with biliary strictures, biliary IL-6 negatively predicted retransplantation-free survival after ERC. CONCLUSION: PSC patients showed a biliary Th1/Th17 cytokine profile, while SSC and diffuse strictures showed low values of cytokines except IL-6. In diffuse intrahepatic strictures after LT, biliary IL-6 is strongly associated with retransplantation-free survival after ERC.

Biliary calprotectin, lactoferrin and dimeric pyruvate kinase after liver transplantation are associated with biliary damage and graft survival in a case-control study.

BACKGROUND: After liver transplantation (LT), biliary complications are associated with reduced graft survival. We tested inflammation markers for their association with biliary damage and graft loss in bile. MATERIAL AND METHODS: The study design was a retrospective case-control study. Calprotectin, lactoferrin and pyruvate kinase were measured in endoscopically retrieved bile with ELISA. RESULTS: Calprotectin and lactoferrin were significantly higher in bile of ischemic-type biliary lesions and donor duct non-anastomotic strictures than in control, bile leakage, Cytomegalovirus infection, anastomotic stricture or acute cellular rejection patients (p<0.001) independent of serum liver values at endoscopy. Calprotectin (p=0.02) was independently associated with retransplantation free survival in multivariate analysis, as was γGT (p=0.03) but not ERC radiographic classification of the bile duct or cold ischemia time. CONCLUSION: Calprotectin and lactoferrin are bile markers for biliary damage and are associated with re-transplantation free survival. They can differentiate progressive biliary damage from non-biliary liver value alterations after LT.

Molecular characterization of hepatic epithelioid hemangioendothelioma reveals alterations in various genes involved in DNA repair, epigenetic regulation, signaling pathways, and cell cycle control.

Epithelioid hemangioendotheliomas (EHE) of the liver are rare, low-malignant vascular tumors whose molecular pathogenesis is incompletely understood. The diagnosis of EHE is challenging, and the course of the disease can be highly variable. Therapeutic options for EHE are limited, including resection of primary and metastatic tumors, organ transplantation and rather ineffective systemic approaches. Driver mutations have been reported (fusion transcripts of either YAP-TFE3 or WWTR1-CAMTA1) but comprehensive molecular profiling has not been performed. Our aim was to molecularly characterize hepatic EHE to identify new molecular targets. Eight primary hepatic EHE were analyzed by next-generation sequencing using a 409-gene panel. The majority of primary hepatic EHE revealed a low number of mutations. Genes that were mutated primarily are involved in DNA repair, epigenetic regulation, signaling pathways and cell cycle control, indicating that EHE present with mutations in various functions. Although only detecting a low mutation rate, a comparison with comprehensive databases (target db V3) revealed mutations in five genes with putative therapeutical options. Therefore, our findings help to shed light on the molecular background of EHE and might pave the way to new therapeutic approaches.

Tumor response to irinotecan is associated with CYP3A5 expression in colorectal cancer.

Recently, a tumor-autonomous cytochrome P450 (CYP)-3A5-mediated resistance to cancer therapy has been demonstrated in pancreatic ductal adenocarcinoma. Expression of CYP3A5, which is involved in the degradation of irinotecan, has also been reported in colorectal cancer (CRC). The aim of the present study was to analyze CYP3A5 expression in the normal colon, colon adenoma, CRC and normal tissues, as well as to examine whether CYP3A5 expression in CRC has an impact on tumor response to irinotecan treatment. Immunohistochemistry was used to assess 85 tissue samples from 65 patients with CRC, along with 15 samples of normal colon and 45 samples of colon adenoma (including tubular, tubulovillous, and sessile serrated adenomas), and a tissue microarray (TMA) comprised of 26 different normal tissue types. Expression of CYP3A5 was evaluated with a semi-quantitative score. Tumor response to irinotecan therapy was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines. In normal tissues, CYP3A5 was expressed in epithelial cells of the colon, gallbladder, kidney, liver, small intestine, stomach, thyroid gland and tonsil, as well as in nerves. Expression in colon mucosa was heterogeneous, with only weak staining in the minority of specimens. CYP3A5 exhibited markedly higher expression in adenomas compared with normal colon tissues. A statistically significant inverse correlation was identified between CYP3A5 expression in CRC tissues and tumor response to irinotecan therapy. Irinotecan treatment itself did not alter CYP3A5 expression in CRC tissues. As CYP3A5 is involved in the degradation of irinotecan, the significantly higher intratumoral expression of CYP3A5 in patients with CRC who do not respond to irinotecan-based chemotherapy may indicate a causal role of CYP3A5 in tumor resistance.

Effect of scheduled endoscopic dilatation of dominant strictures on outcome in patients with primary sclerosing cholangitis.

OBJECTIVE: Scheduled endoscopic dilatation of dominant strictures (DS) in primary sclerosing cholangitis (PSC) might improve outcome relative to endoscopic treatment on demand, but evidence is limited. Since randomisation is difficult in clinical practice, we present a large retrospective study comparing scheduled versus on-demand endoscopic retrograde cholangiopancreatography (ERCP) based on patient preferences. DESIGN: Between 1987 and 2017, all new patients with PSC had been offered scheduled ERCP with dilatation of a DS if diagnosed; the latter was repeated at defined intervals until morphological resolution, independent of clinical symptoms (treatment group). Patients who refused participation were clinically evaluated annually and received endoscopic treatment only on demand (control group). The primary clinical endpoint was transplantation-free survival. Secondary outcomes were overall survival, bacterial cholangitis episodes, hepatic decompensation of liver cirrhosis and endoscopy-related adverse events. RESULTS: The final study included 286 patients, 133 (46.5%) receiving scheduled ERCP and 153 (53.5%) receiving on-demand ERCP. After a mean follow-up of 9.9 years, the rate of transplantation-free survival was higher in patients receiving scheduled ERCP (51% vs 29.3%; p<0.001), as was transplantation-free survival time (median: 17.9 vs 15.2 years; log-rank: p=0.008). However, the benefit of scheduled ERCP was significant only in patients with the initial (17.1%) or later (45.5%) diagnosis of a DS (17.8 vs 11.1 years; log-rank: p<0.001). IBD (p=0.03), DS (p=0.006), higher Mayo Risk Score (p=0.02) and non-adherence to scheduled endoscopy (p=0.005) were independently associated with transplantation-free survival. CONCLUSION: In our large retrospective study, regular ERCP with endoscopic balloon dilatation significantly benefits patients with PSC with DS, diagnosed both at initial presentation and during surveillance, even if asymptomatic. Further studies have to find out how to best identify stricture patients non-invasively.

Case report: a rare cause of a hypervascular nodule in a noncirrhotic liver.

A 37-year-old male patient with Crohn's disease and multiple liver hemangiomas was referred to our hospital for an atypical hypervascular hepatic lesion detected on an external magnetic resonance imaging (MRI) scan. The patient was otherwise well and had no history of any liver disease. Liver values and tumor markers were normal. Contrast-enhanced ultrasound confirmed multiple hemangiomas in different liver segments and a hypervascular tumor with a hypovascular rim in segment II/IV. Repeat MRI showed a strongly enhancing neoplasm of 2.6 cm with a texture distinctly different from the otherwise relatively uniform hemangiomas, without evidence of interim growth. Ultrasound-guided biopsy revealed a hepatic small vessel neoplasm. Due to the unknown malignant potential, atypical segmental surgical resection was performed. Final histopathological analysis confirmed the complete resection of the lesion. The postoperative course was uneventful.

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. METHODS: We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. RESULTS: HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. CONCLUSIONS: Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence.

HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. METHODS: PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. RESULTS: HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. CONCLUSIONS: In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.