GSK3β is crucial for the regulation of microtubule organization and dynamics, particularly for mitotic spindle organization.29 p38α deficiency alters the balance between AKT and GSK3β leading to AKT down-regulation and GSK3β activation (Fig. 3), which seems to impair normal cytokinesis completion. MK2 also plays a significant role downstream of p38α in remodeling the actin cytoskeleton.30 Particularly, MK2 triggers phosphorylation of HSP27 inducing its release

from F-actin.30 HSP27 protects against apoptosis and actin fragmentation, promoting resistance against cell death.31 We found an increase in HSP27 levels, which may be an adaptive response against liver injury, with significant selleck kinase inhibitor changes in phosphorylation. Mnk1 and Polo-like kinase 1 (Plk1), two potential downstream targets of p38α signaling, may contribute to cytokinesis failure in p38α-deficient liver. Inhibition of Mnk1, a kinase target for MAPK pathways, causes cytokinesis failure inducing the formation of multinucleated cells.32 In addition, MK2 directly NVP-BEZ235 molecular weight phosphorylates Plk1, and down-regulation of p38α or MK2 induces mitotic defects that can be rescued by Plk1.33 In conclusion, the present work shows that liver-specific p38α deficiency leads to reduced hepatocyte size, blockade of mitosis, cytokinesis failure, and eventually shorter life span upon chronic cholestasis

induced by BDL. These results highlight the key role of p38α in cell proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis. We thank Soraya Ardila for technical help. Additional Supporting Information may be found in the online version of this article. “
“Aim:  Transient elastography is a non-invasive tool to measure liver stiffness (LS), which has been reported to correlate with stage of liver fibrosis. Extrahepatic cholestasis was reported to cause elevated LS, which is considered to be attributed to the increased hydrostatic pressure in the liver. In the present study, the correlation of LS with laboratory data was investigated in extrahepatic cholestasis.

Amrubicin The change of LS after biliary drainage was also assessed. Methods:  LS was measured in 29 patients with extrahepatic cholestasis due to carcinomas in 12 and non-neoplastic diseases of biliary tract or pancreas in 17. Results:  In 15 patients, LS was 11.4 kPa or higher which suggested liver cirrhosis in chronic infection of hepatitis C virus. LS significantly correlated positively with serum bilirubin levels (r = 0.726, P < 0.0001) and negatively with serum aspartate aminotransferase (AST) levels (r = −0.481, P = 0.0082) and alanine aminotransferase (ALT) levels (r = −0.631, P = 0.0002). Biliary drainage led to a reduction of bilirubin by 13.5 to 0.9 mg/dL which was significantly correlated with a reduction of LS by 14.3 to 0.5 kPa (r = 0.524, P = 0.0257).

GSK3β is crucial for the regulation of microtubule organization and dynamics, particularly for mitotic spindle organization.29 p38α deficiency alters the balance between AKT and GSK3β leading to AKT down-regulation and GSK3β activation (Fig. 3), which seems to impair normal cytokinesis completion. MK2 also plays a significant role downstream of p38α in remodeling the actin cytoskeleton.30 Particularly, MK2 triggers phosphorylation of HSP27 inducing its release

from F-actin.30 HSP27 protects against apoptosis and actin fragmentation, promoting resistance against cell death.31 We found an increase in HSP27 levels, which may be an adaptive response against liver injury, with significant selleck chemicals llc changes in phosphorylation. Mnk1 and Polo-like kinase 1 (Plk1), two potential downstream targets of p38α signaling, may contribute to cytokinesis failure in p38α-deficient liver. Inhibition of Mnk1, a kinase target for MAPK pathways, causes cytokinesis failure inducing the formation of multinucleated cells.32 In addition, MK2 directly selleck chemical phosphorylates Plk1, and down-regulation of p38α or MK2 induces mitotic defects that can be rescued by Plk1.33 In conclusion, the present work shows that liver-specific p38α deficiency leads to reduced hepatocyte size, blockade of mitosis, cytokinesis failure, and eventually shorter life span upon chronic cholestasis

induced by BDL. These results highlight the key role of p38α in cell proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis. We thank Soraya Ardila for technical help. Additional Supporting Information may be found in the online version of this article. “
“Aim:  Transient elastography is a non-invasive tool to measure liver stiffness (LS), which has been reported to correlate with stage of liver fibrosis. Extrahepatic cholestasis was reported to cause elevated LS, which is considered to be attributed to the increased hydrostatic pressure in the liver. In the present study, the correlation of LS with laboratory data was investigated in extrahepatic cholestasis.

FER The change of LS after biliary drainage was also assessed. Methods:  LS was measured in 29 patients with extrahepatic cholestasis due to carcinomas in 12 and non-neoplastic diseases of biliary tract or pancreas in 17. Results:  In 15 patients, LS was 11.4 kPa or higher which suggested liver cirrhosis in chronic infection of hepatitis C virus. LS significantly correlated positively with serum bilirubin levels (r = 0.726, P < 0.0001) and negatively with serum aspartate aminotransferase (AST) levels (r = −0.481, P = 0.0082) and alanine aminotransferase (ALT) levels (r = −0.631, P = 0.0002). Biliary drainage led to a reduction of bilirubin by 13.5 to 0.9 mg/dL which was significantly correlated with a reduction of LS by 14.3 to 0.5 kPa (r = 0.524, P = 0.0257).

The authors declare no conflict of interest. “
“Background and Aims:  To assess the validity of biopsy-based tests (histology, culture, and urease test) and serology in detecting current H. pylori infection for the peptic ulcer patients who had gastric bleeding. Methods:  A total of 398 peptic ulcer patients were enrolled and divided into two groups, according to the presence or absence of bleeding. The diagnosis for

current H. pylori infection was verified using the gold standard combining individual H. pylori tests. Sensitivity, specificity, and positive and negative predictive values of the culture, Campylobacter-like organism (CLO) test (urease test), histology, and serology were compared. Results:  Of the total study population (N = 398), 157 (39.4%) patients were categorized into the bleeding group. The sensitivities of the culture (40.0%) and CLO (85.0%) in the bleeding group were significantly lower

Cilomilast chemical structure than culture (58.1%) and CLO (96.4%) in the nonbleeding group (p = .012 and p < .001, respectively). In the bleeding group, the sensitivity of CLO (85.0%) was significantly lower than histology (92.5%) and serology (97.4%) (p = .013 and p = .002, respectively), which was not found in the nonbleeding group. The specificity of serology in the bleeding group (56.3%) was significantly lower than that of nonbleeding group (74.2%) (p = .038). Similarly, the specificity of serology was significantly BMS-907351 order lower than the other H. pylori tests in the bleeders. Conclusions:  Bleeding decreased the sensitivity of H. pylori

tests in patients with peptic ulcer, especially in urease test or culture. In contrast, histology was found to be a quite reliable test, regardless of the presence of bleeding. “
“Background:  Mongolian gerbils that are experimentally infected with Helicobacter pylori develop a chronic inflammation that is similar to natural infections in humans. The aim of this study was to compare the antigens VAV2 of H. pylori cagPAI+ and cagPAI− strains that are expressed during Meriones unguiculatus colonization. Materials and Methods:  We identified H. pylori cagPAI+ and cagPAI− strain antigens via Western blotting of samples from Mongolian gerbils that were subjected to unique, mixed, and sequential bacterial infections. Results:  The antigens from the J99/CG3 (cagPAI+) strain had a lower molecular weight than the antigens from the 251F/CG3 (cagPAI−) strain. There were fewer identified antigens in the single unique infections compared with the mixed and sequential infections. The number of recognized antigens that had a frequency of recognition >60% was higher for the simultaneous and sequential infection groups compared with the single infection group. A 57-kDa antigen was present in >60% of the samples and four of the five experimental groups.

The median LOS was 9 days (range 5 – 67 days). In our study cohort, 61% had at least one comorbid condition. Among recipients > 60 years the presence of > 1 comorbidity

was associated with a higher likelihood of PLOS (p=0.025) and increasing CCI correlates with PLOS. In contrast in those < 60 years, comorbidity was not associated with PLOS (p=0.248). Furthermore, there was no difference in PLOS when recipients > 60 years without comorbidity was compared to < 60 years with comorbidity (p=0.66). The median LOS for older recipients without comorbidity was 11 days compared to 14.3 days for those with > 1 comorbidity. Conclusion: Pre-transplant comorbidity in older candidates with ESLD is an independent predictor of LOS post LT. Thus comorbid burden in older candidates significantly impacts resource utilization. check details Small molecule library datasheet The CCI is a simple, reproducible and readily available tool that can be incorporated in the pre-operative risk assessment

of older LT candidates and it can be utilized as a health service metric by public health policy makers and transplant programs in evaluating and assessing current healthcare reimbursement schemes. Disclosures: Edson S. Franco – Grant/Research Support: bayers, gilead, eisai Elizabeth Cece Fallon – Speaking and Teaching: Janssen Pharmaceuticals, AbbVie Pharmaceuticals Erin Parkinson – Speaking and Teaching: Gilead, BMS Angel Alsina – Advisory Committees or Review Panels: Bayer; Speaking and Teaching: Bayer, Novartis The following people have nothing to disclose: Nyingi M. Kemmer, Chris Albers, Husssein Osman-Mohamed, Jennifer Horkan

The AASLD/IDSA has published recommendations for the use of the 2nd generation direct acting antivirals (DAA). The cost of these drugs has put pressure on insurance companies (IC) Alanine-glyoxylate transaminase to fulfill the patient and physician demand for HCV treatment. In this study we sought to determine the approval rates for various combinations of peginterferon (Peg), ribavirin (R), sofosbuvir (Sof), and simeprevir (Sim) in a single US center. METHODS: Consecutive prescriptions for HCV treatment from 12/9/13 – 5/9/14 with a final decision rendered by the patient’s IC were included. Rates and time to approval, prior therapy response, insurance type, cirrhosis, and liver transplant (LT) status were analyzed. Chi-square and t-test were applied. 172 patients were prescribed treatment: Peg/R/Sof: 28 (16%), R/Sof: 45 (26%), and Sof/Sim: 99 (58%). 88 (51%) had government and 84 (49%) had private insurance. There were 85 (49%) cirrhotics. 29 (17%) had undergone LT prior to treatment request. 54 (31%) were prior treatment naïve, 15 (9%) relapsed, and 103 (60%) partial or non-response (P/NR). RESULTS: 153 (89%) patients were approved for treatment (Peg/R/Sof: 28 (100%), R/Sof: 42 (93%), and Sof/Sim: 83 (84%). The interval for receiving approval was similar in all groups (Peg/R/Sof: 22 days, R/Sof: 20 days, and Sof/ Sim: 21 days, p=0.78).

Furthermore, nanoparticles containing HBV-CpG, termed NP(HBV-CpG), reversed the HBV-ODN-mediated suppression of IFN-α production and also exerted a strong immunostimulatory effect on lymphocytes. Our results suggest that NP(HBV-CpG) can enhance the immune response to hepatitis B surface antigen learn more (HBsAg) and skew this response toward the Th1 pathway in mice immunized with rHBsAg and

NP(HBV-CpG). Moreover, NP(HBV-CpG)-based therapy led to the efficient clearance of HBV and induced an anti-HBsAg response in HBV carrier mice. Conclusion: Endogenous HBV-CpG ODNs from the HBV genome induce IFN-α production so that nanoparticle-encapsulated HBV-CpG may act as an HBsAg vaccine adjuvant and may also represent a potent therapeutic agent for the treatment of chronic HBV infection. (Hepatology 2014;59:385–394) “
“Settembre C, Di Malta C, Polito VA, Garcia Arencibia M, Vetrini F, Erdin S, et al. TFEB links autophagy to lysosomal biogenesis. Science 2011;332:1429-1433. (Reprinted with permission). Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes

and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy selleck compound and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles. The degradative pathway of macroautophagy has a critical

role in many cellular processes, and recently important functions for autophagy in the liver have been demonstrated. 1 Knowledge of the factors that regulate both basal levels of autophagy, and increases in function that occur with cellular stresses, Cediranib (AZD2171) is critical to understanding how defects in autophagic function lead to pathophysiological conditions. The majority of studies have focused primarily on a complex series of pathways that regulate the formation of the autophagosome, which is the double-membrane structure that sequesters cytosolic components and delivers them to the lysosome for degradation. Over 30 autophagy-related genes (ATGs) have been identified that control basal and inducible levels of autophagy through several distinct pathways. 1 A physiological stimulus used to define these regulatory pathways is nutrient deprivation in cells or rodents.

We minimized the exposure to immunological danger signals by avoiding first treatment with FVIII in a bleeding situation or during infection, by avoiding surgery during the selleck first 20 exposure days (EDs)

and by avoiding vaccinations on the same day as FVIII treatments. Furthermore, any bleeds that did occur were treated early by giving higher doses immediately, thereby avoiding long and intensive treatment and shortening the time of tissue damage. Our results indicate that minimizing danger signals during the first 20 EDs with FVIII might indeed reduce the risk of inhibitor formation. However, these results should be interpreted as hypothesis generating and need to be confirmed in a larger prospective clinical study. Twenty six PUPs in two centers in Germany with severe haemophilia A (all <1% FVIII baseline activity) with a variety of FVIII gene mutations, the majority high risk,

were treated with a prophylaxis regimen designed to induce immune tolerance by avoiding immunological danger signals. The incidence of inhibitor development in this group was compared with that in a historical ABT-199 in vitro control group of 30 children treated with a standard joint protection prophylaxis regimen. To avoid selection bias both study and control group consists of consecutive PUPs with severe haemophilia A (<1% FVIII) as they appeared in the respective haemophilia center during a given time period. Based on the immunological danger theory and their potential impact on FVIII inhibitor development the new prophylaxis regimen was prospectively planned and

implemented as standard of care by January 2001 in center A (Bremen) and by January 2005 in center B (Munich). The overall risk of developing inhibitors to FVIII during the first 150 EDs is 20–30% for PUPs [13]. Pyruvate dehydrogenase lipoamide kinase isozyme 1 Of those developing inhibitors, 50% will do so within the first 20 days and 95% during the first 50 days [13]. If the patient can be brought through this high risk period without inhibitor development, the subsequent risk is low [14]. We therefore decided to test the efficacy in overcoming the high risk of the first 50 EDs of a prophylaxis regimen specifically designed to induce tolerance to the administered FVIII and to minimize inhibitor development. According to the German haemophilia treatment guidelines prophylaxis in children with haemophilia is standard of care [15].

We minimized the exposure to immunological danger signals by avoiding first treatment with FVIII in a bleeding situation or during infection, by avoiding surgery during the learn more first 20 exposure days (EDs)

and by avoiding vaccinations on the same day as FVIII treatments. Furthermore, any bleeds that did occur were treated early by giving higher doses immediately, thereby avoiding long and intensive treatment and shortening the time of tissue damage. Our results indicate that minimizing danger signals during the first 20 EDs with FVIII might indeed reduce the risk of inhibitor formation. However, these results should be interpreted as hypothesis generating and need to be confirmed in a larger prospective clinical study. Twenty six PUPs in two centers in Germany with severe haemophilia A (all <1% FVIII baseline activity) with a variety of FVIII gene mutations, the majority high risk,

were treated with a prophylaxis regimen designed to induce immune tolerance by avoiding immunological danger signals. The incidence of inhibitor development in this group was compared with that in a historical SCH772984 chemical structure control group of 30 children treated with a standard joint protection prophylaxis regimen. To avoid selection bias both study and control group consists of consecutive PUPs with severe haemophilia A (<1% FVIII) as they appeared in the respective haemophilia center during a given time period. Based on the immunological danger theory and their potential impact on FVIII inhibitor development the new prophylaxis regimen was prospectively planned and

implemented as standard of care by January 2001 in center A (Bremen) and by January 2005 in center B (Munich). The overall risk of developing inhibitors to FVIII during the first 150 EDs is 20–30% for PUPs [13]. Anidulafungin (LY303366) Of those developing inhibitors, 50% will do so within the first 20 days and 95% during the first 50 days [13]. If the patient can be brought through this high risk period without inhibitor development, the subsequent risk is low [14]. We therefore decided to test the efficacy in overcoming the high risk of the first 50 EDs of a prophylaxis regimen specifically designed to induce tolerance to the administered FVIII and to minimize inhibitor development. According to the German haemophilia treatment guidelines prophylaxis in children with haemophilia is standard of care [15].

At 7 weeks of age, Leprflox/flox AlbCre ob/ob mice were treated with low dose leptin so as to maintain obesity and hyperinsulinemia (Supporting Fig. 1). This dose of leptin lowered plasma cholesterol and triglycerides in ob/ob mice with and without hepatic leptin signaling (Fig. 2A-C). However, plasma triglyceride levels in Leprflox/flox AlbCre+ ob/ob mice did not decrease as much as in their Leprflox/flox AlbCre- ob/ob controls (Fig. 2C). By the last day of leptin treatment, the Leprflox/flox AlbCre+ ob/ob mice had GSK1120212 cost 36% higher plasma triglycerides

than their littermate controls (Fig. 2C). The effects of leptin treatment persisted even after leptin therapy ceased, with plasma triglyceride levels in both groups only returning to near pre-leptin levels 50 days after the leptin pump was removed (Fig. 2C), indicating leptin treatment in ob/ob mice has long-term effects on lipid metabolism. Because the effect on plasma triglycerides in Leprflox/flox AlbCre ob/ob mice was subtle, we sought to reproduce these results in a complementary mouse model. We treated leptin receptor-deficient db/db mice with Ad-Lepr-b, which confers liver-selective expression18 and restores phospho-STAT3 signaling in the liver.16 Upon treatment with Ad-Lepr-b, the db/db mice remained

obese and hyperinsulinemic (Supporting Fig. 2). Also, db/db mice treated with Ad-Lepr-b and control db/db mice treated with Ad-β-gal both had a response to the virus itself independent of the Lepr-b or see more β-gal constructs. We attribute this to an acute phase immune response to the virus, which has been shown to have effects on lipid metabolism.19 Nonetheless, we observed no differences in plasma cholesterol and free fatty acids between Ad-Lepr-b– and Ad-β-gal–treated db/db mice (Fig. 2D-E). Although both virus-treated groups had an increase in plasma triglycerides,

db/db mice treated with Ad-Lepr-b had lower fasting plasma triglycerides than the Ad-β-gal–treated controls between 1 and 3 weeks postinfection, with Ad-Lepr-b treated mice reaching 31% lower plasma triglycerides 12 days postinfection (Fig. 2F). These data are similar to those of Lee et al.,14 who treated fa/fa rats with an adenovirus Florfenicol expressing β-gal or Lepr-b and also saw a marked increase in plasma triglycerides in the β-gal–treated animals compared with the Lepr-b–treated animals. Collectively, the data show that under obese, hyperinsulinemic conditions, hepatic leptin signaling is required for maintaining normal plasma triglyceride levels. Because leptin has been implicated in regulating the amount of triglyceride incorporation into VLDL,17 we evaluated lipoprotein profiles in Leprflox/flox AlbCre+ mice and their littermate controls. Mice lacking hepatic leptin signaling had no alterations in the distribution or amount of cholesterol (Fig. 3A). Interestingly, Leprflox/flox AlbCre+ mice had elevated triglycerides in fractions consistent in size with VLDL particles (Fig. 3B).

Therapy of ALD is based on the stage of the disease and the specific goals of treatment.169, 170 Complications of cirrhosis, including evidence of hepatic failure (encephalopathy)

as well as portal hypertension (ascites, variceal bleeding), are treated as in patients with non-ALD, with additional attention given to other organ dysfunction associated specifically with alcohol.170 Abstinence is the most important therapeutic intervention for patients with ALD.171 Abstinence has been shown to improve the outcome and histological features of hepatic injury, to reduce portal pressure and decrease progression to cirrhosis, and to improve survival at all stages in patients with ALD.171–174 However, this selleck chemicals may be less likely to occur in female patients.172, 175, 176 This improvement can be relatively Ulixertinib datasheet rapid, and in 66% of patients abstaining from alcohol, significant improvement was observed in 3 months.177 Continued alcohol ingestion results in an increased risk of portal hypertensive bleeding, especially in patients who have previously bled, and worsens both short-term and long-term survival.178 Recidivism is a major risk in all patients at any time following abstinence.179, 180 Estimates vary, depending on the time course of follow-up and the definition

of recidivism (e.g., any alcohol consumption versus moderate to harmful drinking), but over the course of 1 year, relapse rates range from 67%-81%.181 Therefore, several medications have been tried to help sustain abstinence. One of the first agents to be used, disulfiram, was approved by the U.S. Food and Drug Administration in 1983. However, a review of the published literature concluded that there was little evidence that disulfiram enhances abstinence,182 and based on its poor

tolerability, its use has been largely supplanted by newer agents. Naltrexone, which was approved HSP90 in 1995 for the treatment of alcoholism, is a pure opioid antagonist and controls the craving for alcohol. However, it also has been shown to cause hepatocellular injury. A Cochrane systematic review of the use of naltrexone and nalmefene (another opioid antagonist) in 29 randomized clinical trials concluded that short-term treatment with naltrexone lowers the risk of relapse.183 Acamprosate (acetylhomotaurine) is a novel drug with structural similarities to the inhibitory neurotransmitter gamma amino butyric acid (GABA), and is associated with a reduction in withdrawal symptoms.184 In 15 controlled trials, acamprosate has been shown to reduce withdrawal symptoms, including alcohol craving, but its effects on survival are not yet known.185 Its effect is more pronounced in maintaining rather than inducing remission when used in combination with counseling and support. In detoxified alcoholics, it has been shown to decrease the rate of relapse, maintain abstinence, and decrease severity of relapse when it occurs.

4 Lipid extracts obtained via the Folch procedure23 from 3 mL of LVPs or from 500 μL of each lipoprotein fraction

were separated by thin-layer chromatography (TLC) on Silica Gel G60 plates (Merck, Darmstadt, Germany) with hexane/diethyl ether/acetic acid (60/40/1, vol/vol) solvents. Phospholipid and triacylglycerol were scraped off the plate, and the molecular species composition of phospholipids separated by high-performance liquid chromatography (HPLC) on a silica-DIOL column (4 × 250 mm, Agilent 1100) was analyzed via electrospray ionization/tandem KPT330 mass spectrometry (Q-Trap 2000, Applied Biosystems). Phospholipid classes were eluted subsequently from HPLC as a function of the headgroup polarity using the solvent mixture hexane/isopropanol/aqueous selleck products ammonium acetate 5 mM 62.8/34.8/2.4 at the rate of 100 μL/minute. Experimental details have been discussed in recent reviews.24, 25 The method was set to detect the precursors (i.e., the parent phospholipids) of a characteristic fragment ion of each polar headgroup. Mass spectra were processed with Analyst software (v1.4.2, Applied Biosystems).

Assignment of the structure to mass peaks and deisotopization correction were performed with LIMSA software26 using a library prepared for the serum circulating lipids. Analysis of fatty acids in the triacylglycerol fraction separated by TLC (silica G25; solvent mixture hexane/methyl ether/formic acid: 80/20/2 vol/vol) was achieved by GC separation of methyl esters prepared by acid transmethylation according to Christie WW.27 Separation was achieved on a Carbowax 20 M capillary column (0.25 mm, 30 m, Quadrex) fitted on a Thermo-Electron 8000 GC chromatograph. A total of 20 μL of Y 27632 protein A–coated magnetic beads (Miltenyi Biotec) were incubated at room temperature with

1 mL of LDF in PBS with gentle rocking for 30 minutes. Samples were then passed through one magnetic column (Miltenyi Biotec) and washed with 2 mL of PBS and then with buffers of decreasing pH (successively, 300 μL of 0.1 M tris-acetate buffer pH 5.0, pH 4.0, and pH 3.0). Each collected fraction was immediately neutralized by addition of 0.1 N NaOH. Fractions eluted at pH 4.0 were used to stain the western blotting membrane. Immediately after preparation, LDFs or LVP fractions were collected in Laemmli buffer and denatured at 95°C for 5 minutes and kept at −20°C until analysis. Positive controls for E1 and E2 were obtained from lysates of cells expressing HCV glycoproteins or from supernatants of E1/E2-Caco2 differentiated cells,20 collected into Laemmli buffer, denatured, and conserved as described above. Samples were fractionated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane.