We show that HCV E2 protein induces rapid ezrin phosphorylation a

We show that HCV E2 protein induces rapid ezrin phosphorylation and its cellular

redistribution with F-actin by way of spleen tyrosine kinase (SYK). Therapeutically blocking the functional roles of SYK or F-actin reorganization significantly reduced Barasertib price Huh7.5 cell susceptibility to HCV J6/JFH-1 infection. Using gene regulation, real-time quantitative polymerase chain reaction, western blot, and fluorescent microscopy analysis, we found that proteins of the EMR family differentially regulate HCV infection in the J6/JFH-1/Huh7.5 cell system. Moesin and radixin, but not ezrin, expression were significantly decreased in chronic HCV J6/JFH-1-infected Huh7.5 cells and HCV-infected patient liver biopsies compared to controls. The decreases in moesin and radixin in HCV J6/JFH-1-infected Huh7.5 cells were associated with a significant increase in stable microtubules. Ezrin knockdown inhibited immediate Lenvatinib postentry events in HCV infection. Overexpression

of moesin or radixin significantly reduced HCV protein expression. In contrast, transient knockdown of moesin or radixin augmented HCV infection. Making use of the Con1 HCV replicon system, we tested the effect of EMR proteins on HCV replication. We found that transient knockdown of moesin increased HCV RNA expression while overexpression of EMR showed no significant effect on HCV replication. Conclusion: Our findings demonstrate the important role of EMR proteins during HCV infection at the postentry level and highlight possible novel targets for HCV treatment. (Hepatology 2013;58:1569–1579) Hepatitis C virus (HCV) infection is a leading cause of liver disease, with at least 2%–3% of the world’s population chronically infected.[1] Virus elimination through therapy can be limited by several factors, including adverse Rebamipide side effects to current drugs, viral resistance, patient alcohol abuse, and high cost of therapy.[2-6] Chronic HCV infection progressively leads to liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and ultimately death.[7] Viruses, including HCV, exploit host factors and interact with cell surface

or intracellular proteins to achieve effective infection and/or replication.[8-10] Recently, numerous host proteins/peptides have been identified that possess potent antiviral properties.[8, 11-13] Indeed, host proteins/peptides have emerged as alternatives to conventional antiviral agents and present advantages over currently used antiviral drugs such as selective cytotoxicity for the target virus or virus-infected cells, bypassing multidrug-resistance mechanisms and inducing minimal side effects.[11, 13] The HCV virus, a single-stranded positive-sense RNA virus[14] of the Flaviviridae family,[15] was initially identified and distinguished from hepatitis A/B virus infections based on its characteristic induction of microtubule paracrystalline aggregates in infected hepatocytes and liver biopsies of HCV-infected patients.

Several minutes later, the same cell assembled additional hot spo

Several minutes later, the same cell assembled additional hot spots at different locations

along the cell base that also generated many vesicles and then disappeared (images not shown). Many of these dynamin-associated vesicles were seen translocating in a linear path, as if along buy Rucaparib cytoskeletal filaments. This observation suggests that dynamin may remain on the motile vesicle, rather than immediately dissociating as predicted.13 We counted the number of vesicles generated during a typical hot spot lifetime (10-15 minutes) and observed an average steady state release of 4-6 vesicles/min (4 cells counted). Most remarkable is that each hot spot undergoes a 2 to 3-minute burst that generates 15-20 vesicles per minute during which the structure physically “dissolves.” This is exceptionally rapid

when compared with the release of multiple vesicles from conventional clathrin pits (1-2 vesicles/min).20 The rapid vesicle formation from Dyn2/clathrin/AP2 hot spots suggests that these are endocytic structures that are either continuous with the PM or reside as an internal endocytic sorting compartment. Because the HRP marker used to label hot spots by EM (Fig. 1C) was internalized by cells over a 45-minute time period, it is possible that the endocytic hot spots, while in intimate proximity with the PM, represent internal endocytic sorting compartments. To test if these endocytic structures are distinct from or continuous with the PM, we utilized Ruthenium red (RR), an electron-dense dye, to label the cell surface and

any invaginations selleck continuous with the external environment. Because the dye is included in the primary fixative, thereby preventing its internalization, membrane compartments stained with the dye represent extensions of the PM. As expected, cells stained with RR, processed for EM, and sectioned transverse to the substrate showed very dark apical and basal PMs, whereas internal membrane 4-Aminobutyrate aminotransferase systems were only lightly stained (Fig. 4A,B). In control cells (Fig. 4A), small patches of dark, tubular invaginations were observed extending from the basal, but not the apical, PM. These structures were nearly identical in dimensions to those observed in the HRP-labeled cells that were sectioned en face (Fig. 1) and appear as a “side view” of the endocytic hot spots shown in the previous figures. Most important, these endocytic structures were darkly stained with the RR dye, indicating that they are continuous invaginations of the PM. Because GFP-tagged Dyn2 associates with the endocytic hot spots (Figs. 1-3), we predicted that disruption of Dyn2 function would alter the number and complexity of the RR-positive PM invaginations. To test this hypothesis, cultured cells were microinjected with one of two purified polyclonal antibodies that we have used in previous studies to inhibit dynamin function.

Several minutes later, the same cell assembled additional hot spo

Several minutes later, the same cell assembled additional hot spots at different locations

along the cell base that also generated many vesicles and then disappeared (images not shown). Many of these dynamin-associated vesicles were seen translocating in a linear path, as if along selleckchem cytoskeletal filaments. This observation suggests that dynamin may remain on the motile vesicle, rather than immediately dissociating as predicted.13 We counted the number of vesicles generated during a typical hot spot lifetime (10-15 minutes) and observed an average steady state release of 4-6 vesicles/min (4 cells counted). Most remarkable is that each hot spot undergoes a 2 to 3-minute burst that generates 15-20 vesicles per minute during which the structure physically “dissolves.” This is exceptionally rapid

when compared with the release of multiple vesicles from conventional clathrin pits (1-2 vesicles/min).20 The rapid vesicle formation from Dyn2/clathrin/AP2 hot spots suggests that these are endocytic structures that are either continuous with the PM or reside as an internal endocytic sorting compartment. Because the HRP marker used to label hot spots by EM (Fig. 1C) was internalized by cells over a 45-minute time period, it is possible that the endocytic hot spots, while in intimate proximity with the PM, represent internal endocytic sorting compartments. To test if these endocytic structures are distinct from or continuous with the PM, we utilized Ruthenium red (RR), an electron-dense dye, to label the cell surface and

any invaginations DMXAA continuous with the external environment. Because the dye is included in the primary fixative, thereby preventing its internalization, membrane compartments stained with the dye represent extensions of the PM. As expected, cells stained with RR, processed for EM, and sectioned transverse to the substrate showed very dark apical and basal PMs, whereas internal membrane Urease systems were only lightly stained (Fig. 4A,B). In control cells (Fig. 4A), small patches of dark, tubular invaginations were observed extending from the basal, but not the apical, PM. These structures were nearly identical in dimensions to those observed in the HRP-labeled cells that were sectioned en face (Fig. 1) and appear as a “side view” of the endocytic hot spots shown in the previous figures. Most important, these endocytic structures were darkly stained with the RR dye, indicating that they are continuous invaginations of the PM. Because GFP-tagged Dyn2 associates with the endocytic hot spots (Figs. 1-3), we predicted that disruption of Dyn2 function would alter the number and complexity of the RR-positive PM invaginations. To test this hypothesis, cultured cells were microinjected with one of two purified polyclonal antibodies that we have used in previous studies to inhibit dynamin function.

The mean time since transplant was 56 years and the median HCV R

The mean time since transplant was 5.6 years and the median HCV RNA at baseline was 2,604,118 IU/mL. Early data suggested that this regimen was well tolerated, except in one patient when simeprevir was discontinued for severe rash. Another patient required 50% reduction of Prograf because of elevated serum levels and worsening renal function. End of treatment (EOT) data was available in 15 patients. All were negative except one patient with detectable low-level viremia throughout treatment. Week 4 of treatment data was available

in 26 individuals. 53% of patients had a detectable HCV RNA was seen and 42% (N=6) of those patients had HCV RNA levels greater than the lower limit of quantification (LLQ). Conclusion: Anti-HCV treatment with sofosbuvir and simeprevir appears generally well tolerated and effective after transplant. There appears to be delayed viral kinetics post-transplant compared to what has been reported selleckchem in patients not on immunosuppressant therapy. SVR 12 will be key in determining whether longer treatment courses will be needed. Management of HCV after transplant may remain a challenge, and more studies are needed to determine the optimal treatment regimens for this group of patients. Disclosures: The following people have

nothing to disclose: Julio A. Gutierrez, Alla Grigorian, Andres F. Carrion, Michael D. Schweitzer, Danny J. Avalos, Kalyan R. Bhamidimarri, Adam Peyton HCV infection accounts for >30% of yearly liver transplants performed

in selleck the United States. Post transplant HCV recurrence is universal and over 10% of patients will develop rapidly progressive fibrosis of the graft. The combination of Simeprevir (SIM) and Sofosbuvir (SOF) is recommended by AASLD for genotype 1. Aim: To evaluate the safety, tolerability, Pyruvate dehydrogenase lipoamide kinase isozyme 1 and efficacy of combined therapy with SIM/SOF in improving hepatitis (evidenced by improved liver enzymes) and eradicating HCV (evidenced by negative PCR and SVR12). Subjects: Ten patients have commenced and seven have completed the course of treatment. Mean age was 60.7±13.7 with six males (60%), four patients were treatment experienced, and all patients had genotype 1A. Prior to treatment mean ALT and AST were 79.0±44.0 IU/L and 72.4±41.0 IU/L respectively; mean creatinine was 1.23 mg/dL, and average total bilirubin was 0.99±0.3 mg/dL. Average Fibrosis score (METAVIR) by biopsy in the treatment group was 2.5±0.5. Mean time since liver transplantation was 9.7±5 years with all patients currently on immunosuppressive regimes that included Tacrolimus with or without Mycophenolic Acid. Methods: Patients started on treatment with SIM 150mg daily and SOF 400mg daily for 12 weeks. Results: All patients on treatment had undetectable levels of HCV RNA by week 4. For patients who completed the 12 week course, SVR4 rates are 100% (table). Further SVR12 data is still pending. In all patients transaminase levels returned to reference values within 4 weeks after treatment onset.

ginkgodens This discovery brings the total number of Mesoplodon

ginkgodens. This discovery brings the total number of Mesoplodon species to 15, making it, by far, the most speciose yet least known genus of cetaceans. “
“We applied temporal symmetry capture–recapture

(TSCR) models to assess the strength of evidence for factors potentially responsible for population decline in bottlenose dolphins (Tursiops truncatus) in Doubtful Sound, New Zealand from 1995 to 2008. Model selection was conducted to estimate recruitment and population growth rates. There were similar levels of support for three different models, each reflecting distinct trends in recruitment. Modeling yielded low overall estimates of recruitment HER2 inhibitor (0.0249, 95% CI: 0.0174–0.0324) and population growth rate (0.9642, 95% CI: 0.9546–0.9737). The TSCR rate of population decline was consistent with an estimate derived from trends in abundance (lambda = 0.9632, 95% CI: 0.9599–0.9665). The TSCR model selection confirmed the influence of a decline in the survival of calves (<1 yr old) since 2002 for population trends. However, TSCR population growth rates did not exceed 1 in any year between 1995 and 2008, indicating the population was declining prior to 2002. A separate reduction in juvenile survival (1–3 yr old) prior

to 2002 was identified as a likely contributing factor in the population decline. Thus, TSCR modeling indicated the potential cause of the population decline in Doubtful Sound: cumulative impacts on individuals AZD6244 <3 yr old resulting in a reduced recruitment. "
“Five years of behavioral observations revealed significant effects of high air temperatures and breeding site topography on the mating system of South American sea Anacetrapib lions in Peru. Unlike most polygynous mammals that defend females or fixed territories, male sea lions in Peru maintained positions along the shoreline where females passed each day to thermoregulate, and where most copulations occurred. Sex ratios (1 male per 17 females)

and male mating success were extremely skewed (14% of males achieved 50% of the copulations, and 25% of them did not copulate at all). The mass daily movements of females toward the water and cool substrate of the shoreline, along with a highly skewed sex ratio, accentuated the difficulty for males to monopolize and restrict female movements. Females moved freely and chose their mates, unlike in temperate regions of their range where male South American sea lions control groups of females or access to tide pools. Our observations indicate that the South American sea lion in Peru has a lek-like breeding system. This is a rare alternative to the common male strategies of defending females and resources, and is likely an evolutionary product of their highly skewed sex ratio, protracted breeding season, and the extreme subtropical climate where they breed.

003) 3aQ100μM: 5054±3552(p<00006)] In these cells’the typical

003) 3aQ100μM: 505.4±355.2(p<0.0006)]. In these cells'the typical localization of the core protein around the LDs was almost fully inhibited by quercetin, Core protein rather displaying a punctated pattern throughout the cytoplasm. While quercetin inhibited ccHCV replication by more than 75% and 85% when cells were treated with 50μM-100μM respectively in comparison with untreated cells, it did not impact the entry of HCVpp. As well, quercetin decreased Core and NS3 protein level expression Conclusion: Quercetin has a major effect of LD morphology and interferes with HCV-induced steatosis. Besides, it decreases viral replication, core and NS3 proteins expression and

avoided the co-localization between core and lipid droplets, selleck chemical without impact on viral entry. Therefore, this flavonoid could be Buparlisib clinical trial considered as a new drug for hepatitis C treatment. Francesco Negro – Advisory

Committees or Review Panels: Roche, MSD, Gilead, Boehringer Ingelheim; Grant/Research Support: Roche, Gilead Manuel Romero-Gomez – Advisory Committees or Review Panels: Roche Farma, SA, MSD, SA, Janssen, SA., ABBOTT, SA; Grant/Research Support: Ferrer, SA The following people have nothing to disclose: Angela Rojas, Jose A. Del Campo, Marta Garda-Valdecasas, Sophie Clement In hepatitis C virus (HCV) infected patients, virions are associated with very low density lipoprotein (VLDL)-type lipoproteins forming an infectious lipo-viro-particle (LVP). Apolipoprotein E (apoE), a major component of VLDL, interacts with heparan sulfate proteoglycans (HSPG) at the hepatocyte cell surface. As well, apoE is present at the surface of the LVP playing a crucial role in HCV infectivity. We aimed to investigate the role of apoE and its functional regions in HCV infectivity and to identify the syndecan (Sdc) involved in the HCV entry process. First, using adenoviral vectors expressing wild type or mutant apoE, we complemented apoE expression in Huh7.5.1 depleted cells from the endogenous apoE. Increasing amounts of apoE lead

to a dose-dependent increase in HCV infectivity, the more apoE was expressed the more HCV particles were infectious, demonstrating the Tolmetin primary role of apoE in HCV infectivity. ApoE mutated in the HSPG binding domain (HSPG-BD) as well as competition experiment using a peptide mimicking the HSPGBD confirmed the HSPG dependency for HCV infectivity. Finally, silencing experiments targeting the HSPG syndecan (Sdc)1 or Sdc4 revealed that HCV entry was markedly decreased following Sdc4 silencing. This effect was not observed when HCV pseudoparticles entry was analyzed, confirming the essential role of apoE-Sdc interactions in HCV entry. Collectively, our data demonstrate that HCV-apoE-Sdc interactions mediate viral entry. Since viral entry has been shown to play a key role in acute liver graft infection and viral persistence, targeting apoE-Sdc interactions opens a new perspective to prevent HCV re-infection during transplantation and may provide novel therapeutic avenues.

It is well documented that obesity is associated with chronic low

It is well documented that obesity is associated with chronic low-grade inflammation, impaired iron homeostasis,19 and elevated production of the adipokine leptin, which in turn increases hepatic hepcidin production.20 Both overnutrition and inflammation are associated with ER stress and the induction of the UPR.11, 12 Recent work has shown that this leads to enhanced production of hepcidin,16, 17 which, once released from hepatocytes into the circulation, interacts with the iron efflux protein ferroportin and blocks iron release from a number of

cell types, including hepatocytes,18 resulting in elevated intracellular iron levels. The present study by Graham et al. shows that increased intracellular iron is significantly and positively associated with elevated hepatic

cholesterol synthesis, further contributing to the liver lipid burden. FGFR inhibitor The combination of steatosis and cellular iron loading (together with increased FFAs) could result in increased oxidative stress, which would exacerbate the progression from fatty liver to NASH, cirrhosis, and potentially hepatocellular carcinoma. Although many of these links and hypotheses remain to be proven, the study by Graham et al. opens up a number of new Selleck SCH727965 avenues for future investigation of the relation between iron and lipid metabolism. “
“Background and Aim:  We investigated whether intrahepatic markers could predict response in chronic hepatitis B virus (HBV) patients treated with peg-interferon and adefovir for 48 weeks. Methods:  Intrahepatic covalently closed circular DNA (cccDNA), total intrahepatic HBV DNA and the proportion of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) positive hepatocytes in 16 hepatitis B e antigen (HBeAg) positive and 24 HBeAg negative patients were measured at baseline and at end of treatment. Results:  Baseline intrahepatic markers were not associated with sustained virological response (SVR) defined as HBV DNA < 2000 IU/mL and persistent normal

alanine aminotransferase levels at the end of follow-up (week 72). At end of treatment, intrahepatic cccDNA and total intrahepatic HBV DNA in HBeAg positive patients were significantly lower in patients with HBeAg seroconversion (P = 0.016 and P = 0.010) Sirolimus nmr with positive predictive values (PPV) for SVR of 80% and 80%, respectively. In HBeAg negative patients, intrahepatic cccDNA and total intrahepatic HBV DNA had declined significantly at end of treatment (P = 0.035 and P = 0.041) and corresponding PPV for SVR was 73% and 82%. In HBeAg positive patients, median proportion of HBcAg positive hepatocytes declined significantly (P = 0.002) at end of treatment. In HBeAg negative patients, the proportion of HBsAg positive hepatocytes had declined significantly at end of treatment (P = 0.0009). Using HBsAg ≤ 7.5% as a limit, PPV for SVR in HBeAg negative patients was 83%.

We have previously demonstrated that H-subunit ferritin actually

We have previously demonstrated that H-subunit ferritin actually contributes to this process as a pro-inflammatory mediator in HSC via an iron-independent, NFkappaB-regulated signaling pathway,

inducing the expression of cytokines IL-1beta, IL-6 and RANTES. Aims: To decipher the molecular events at the level of the plasma membrane and the endocytic pathways that mediate the pro-inflammatory response of Ferritin in HSC. Methods: Primary rat HSCs were treated Fulvestrant order with 10 nM H-ferritin for 0–24 hrs. HSCs were pre-treated with inhibitors of microtubule formation (colchicine, nocodazole), lysosomal acidification (chloroquine) and intracellular protein transport (monensin), dynamin-2-dependent internalization (Dyngo-4), clathrin-coated pit (CCP) internalization (PitStop2), lipid Selleck HSP inhibitor raft/caveolae formation (beta-methyl cyclodextrin, beta-MCD) prior to

ferritin stimulation. qPCR was used to determine relative expression of Ferritin-induced transcripts. Results: Colchicine or nocodazole had no significant effect on ferritin-induced expression of IL-1beta suggesting that microtubule-dependent endocytosis is not necessary for signaling. However, inhibition

of CCP endocytosis and dynamin-dependent endocytosis in HSC totally or partially abolished Ferritin-induced pro-inflammatory response, respectively. Conversely, betaMCD–induced disruption of lipid rafts/caveoolae exacerbated response to Ferritin. Moreover, monensin treatment resulted in a 75% reduction in ferritin-induced IL-1beta expression while chloroquine completely abolished IL-1beta expression. Finally, experiments in 2-deoxyglucose-treated HSC supported that Ferritin-mediated pro-inflammatory signaling depends on glycolysis. Conclusion: These results suggest that ferritin http://www.selleck.co.jp/products/BafilomycinA1.html uptake and intracellular traffic, and therefore consequent pro-inflammatory signaling, are mediated by CCP but not via lipid rafts/caveolae. In addition, ferritin-induced HSC pro-inflammatory signaling is regulated by glycolysis linking ferritin to fibrosis in metabolic disorders. Further insights on the different cellular events that mediate ferritin-induced HSC pro-inflammatory signaling will contribute to better understanding of ferritin in the context of hepatic fibrogenesis.

anti-TB treatment has good effect Key Word(s): 1 tuberculosis;

anti-TB treatment has good effect. Key Word(s): 1. tuberculosis; 2. clinical features; 3. gastroendoscope; 4. diagnosis; Presenting Author: IAINA BROWNLEE Additional Authors: SHARNA SEAH, SARAHZY NG Corresponding Author: IAINA BROWNLEE Affiliations: Newcastle University Objective: Previous research has suggested that reflux is frequent during strenuous physical activity, although further evidence

is hampered by a lack of non-invasive means of measuring reflux. Recent evidence has suggested that measurement of pepsin in saliva could be a useful tool for measurement of recent reflux events in free-living individuals. The current study aimed to test the impact of a variety of physical activities on pepsin concentration in saliva before and after exercise in competitive, H 89 clinical trial amateur athletes. Methods: Ethical approval was granted by Newcastle University SAgE Faculty Internal Ethics Committee. Seventy-four participants (age 18–64y, 20% female) were recruited through Singaporean sporting clubs. Approximately 2.5 ml of saliva were collected before and after exercise into tubes with 0.05 g of citric acid preservative. Samples were subsequently see more centrifuged to remove particulate matter and analysed for pepsin content using an ELISA methodology. Pre- and post-exercise

samples were compared by Wilcoxon matched pairs signed rank test. Results: Ninety-six paired, pre- and post-exercise saliva samples were collected (distance-running (n = 49), swimming (n = 17), dragon-boating (n = 16), sprinting/long-jumping (n = 9) and cycling (n = 5). While the median pepsin concentrations were higher post-exercise than pre-exercise ((medianΔ, range) distance-running (-25, 432 to -3958 ng/ml), swimming (-17, 570 to -810 ng/ml), Thiamine-diphosphate kinase dragon-boating (-109, 1232 to -1371 ng/ml),

sprinting/long-jumping (-29, 105 to -102 ng/ml), and cycling (-46, 61 to -3254 ng/ml), this only reached statistical significance for distance-running (P = 0.0230). Pooled analyses of all exercise types highlighted significantly higher pepsin concentrations post-exercise (P = 0.0075, medianΔ = -32 ng/ml, range 1232 to -3958 ng/ml). There was a weak correlation between estimated energy expenditure during exercise and post-exercise pepsin concentration (Spearman r = 0.2141, P = 0.0424). Conclusion: Reflux appears to be a common occurrence around physical activity bouts that could ultimately affect pulmonary function (and possibly performance) in athletes. Assessment of pepsin content of saliva appears to be useful for assessing reflux non-invasively in a variety of free-living individuals. Key Word(s): 1. Pepsin; 2. Gastric reflux; 3. Sports; 4.

This indicates that the HCV particles released from infected-Hepa

This indicates that the HCV particles released from infected-HepaRG cells (HCV-RG) are indeed infectious. To determine the buoyant density distribution of HCV RNA, E1E2 and core antigens, the viral preparations from media collected at days 28 and 42 Panobinostat datasheet p.p. (Fig. 1A,b) were pooled and subjected to iodixanol gradient density centrifugation. Figure 1D shows that the HCV-RG particles had a relatively homogeneous distribution between 1.06 and 1.12 g/mL. They expressed E1E2 envelope proteins and contained RNA and core antigen. In addition, the positive fractions reacted with polyclonal antibodies against apoE (++, P/N ratio = 5-6) and

apoB (+, P/N ratio = 3-4), suggesting that host lipoproteins could be associated with these particles mimicking circulating HCV.14 Immunohistochemistry experiments were performed to investigate intracellular expression of HCV E1E2 and core antigens (Ag) in infected-HepaRG cells at 28 and 56 BMN 673 cell line days p.p. (infection 1). Figure 1E shows that the HCVsp-infected HepaRG cells at D28 p.p. exhibited a very strong staining of cytoplasm and perinuclear regions for E1E2 Ag (a). Fifty to sixty percent of cells were positive. Core Ag staining (b) appeared

also in the cytoplasm possibly around lipid droplets. Some cells were labeled both in the cytosol and the nucleus. Control HCV(−) uninfected HepaRG cells were clearly negative in the presence of D32.10 (a) or C7.50 (b), as well as HCV-infected cells in the presence of a control IgG1 antibody (not shown). Positively stained infected cells exhibited morphological features of hepatocytes.5 Altogether, these results indicate that the human DOK2 HepaRG cells can be infected with HCVsp when proliferated and do produce de novo infectious lipoprotein-associated enveloped complete HCV particles for up to 6 weeks when differentiated. To investigate whether the unique E1E2-specific D32.10 mAb inhibits HCV infection, the infection experiment (infection 3) was performed after preincubation of HCVsp with D32.10 at a 0.5 μg/mL concentration. Figure 2 shows that the D32.10 mAb completely inhibited HCV RNA production in HepaRG culture supernatants.

The total amount of HCV RNA remained at very low levels throughout the follow-up of the infection from day 1 to day 21 in the presence of D32.10 with a mean inhibition of 80.5 ± 11.6% (Fig. 2A). When HCV RNA was quantified by qPCR, 5log10 copies/mL were detected at day 21 after control infection. The preincubation of the inoculum with D32.10 reduced by ≈97% the extracellular HCV RNA (−2 log10, Fig. 2B). To further support that control-infected HepaRG cells produced viral particles, iodixanol density gradient analysis was performed from HCV RNA-associated particles present in the culture media collected at days 14 and 21 (Fig. 2C). As seen previously (infection 1), both HCV RNA, E1E2, and core antigens were recovered as a major peak between 1.