Also, the TGF-β1-induced CD133+ Huh7 cells were reported to be tumorigenic.33 In accord with these findings, we observed that TGF-β-signaling molecules as well as EMT-related genes were significantly up-regulated in S-HCCs. Topographically, S-HCC showed colocalized expression of Snail and K19/EpCAM molecules, particularly in the small and oval-like tumor cells, which may support the idea that TGF-β signaling and EMT play a critical role in the acquisition of stem-cell-like traits. Similarly, it has been reported that S-HCCs express

TGF-β1 at the periphery of tumor nests, next to the fibrous stroma as well as myofibroblasts, and also coexpress CD56 and K7.8 Taken together, we suggest that the EMT might be involved in the acquisition of stem-like and CC-like genomic features in S-HCC through the up-regulation of TGF-β Volasertib supplier signaling, which may contribute Selleckchem JNK inhibitor to the presence of an invasive pathological property (illustrated in Fig. 6A). S-HCC is different from CHC, although small and oval-like tumor cells and abundant fibrous stroma are found in both tumors.4 Unlike CHC, the classical type, S-HCC, does not show

mucin or CC components. In addition, S-HCC is composed mainly of hepatocyte-like tumor cells, whereas CHC with stem-cell features has a main component of small oval-like tumor cells that have stem-cell-like features.4, 34-37 More important, similar fibrotic stroma can occur after chemotherapy, radiation, or transarterial chemoembolization. Such cases should not be confused with S-HCCs. Therefore, in this study, we included all the cases that had no preoperative treatment. In summary, the overall features of our findings can be viewed from the perspective of a spectrum of primary liver cancer composed of HCC, CC, and CHC in the middle (Fig. 6B). Variant HCCs, including S-HCC, are positioned next to CHC because they harbor more HCC-like features than CHC. The molecular characteristics MCE of S-HCC are highlighted by TGF-β signaling and EMT. Our results may provide new pathobiological insights regarding the scirrhous phenotype of HCC and its contribution to the primary liver cancer spectrum.

Additional Supporting Information may be found in the online version of this article. “
“Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in affluent countries. Accurate noninvasive tests for liver injury are urgently needed. The aim of this study was to evaluate the accuracy of transient elastography for the diagnosis of fibrosis and cirrhosis in patients with NAFLD and to study factors associated with discordance between transient elastography and histology. Two hundred forty-six consecutive patients from two ethnic groups had successful liver stiffness measurement and satisfactory liver biopsy specimens. The area under the receiver-operating characteristics curve (AUROC) of transient elastography for F3 or higher and F4 disease was 0.93 and 0.

Conclusion: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis. (HEPATOLOGY 2010;) BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; FGF, fibroblast growth factor; GFP, green fluorescent protein; GPC3, glypican 3; GSK3β, glycogen synthase kinase 3 beta; HCC, hepatocellular carcinoma; HPF, high-power field; HS, heparan sulfate; HSGAG, heparan sulfate glycosaminoglycan; HSPG, heparan

sulfate proteoglycan; IP, immunoprecipitation; LEF, lymphoid enhancer-binding factor; mRNA, messenger RNA; PBS, phosphate-buffered saline; shRNA, short hairpin RNA; SULF2, sulfatase 2; TCF, T cell factor; TUNEL, terminal deoxynucleotidyl learn more transferase–mediated deoxyuridine triphosphate nick-end labeling. Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer death worldwide.1 The survival of HCC patients is poor, and only 10% to 20% of HCCs are detected at an early enough stage for potentially curative therapy. Locoregional therapies are usually palliative, and

there are limited options this website for chemotherapy. Therefore, new agents are needed for the effective treatment of the majority of HCCs.2 The Wnt/Frizzled/β-catenin pathway is activated in approximately 50% of HCCs. Wnt ligands (Wnt3, Wnt3a, Wnt4, and Wnt5a) and Frizzled receptors (Frizzled 3, Frizzled 6, and Frizzled 7) have been implicated in the development of HCC, and up to 95% of HCCs show potential Wnt/Frizzled activating events.3-5 The Wnt/β-catenin pathway is regulated by heparan sulfate proteoglycans (HSPGs), which modulate cell surface signaling by acting as coreceptors or storage sites for Wnt proteins. HSPGs consist of a MCE公司 protein core to which heparan

sulfate glycosaminoglycan (HSGAG) chains are attached; these are variably sulfated at the 2-O, 3-O, and 6-O positions of their component disaccharides. Glypicans are cell surface–anchored HSPGs that regulate the activity of Wnts.6, 7 In particular, glypican 3 (GPC3) is highly overexpressed in HCCs and is being developed as a target for HCC therapy.8, 9 Wnt3a has been shown to mediate the GPC3-induced growth of HCCs via the canonical Wnt/β-catenin pathway.5, 10 Sulfated HSGAG chains of GPC3 and other HSPGs are potential substrates for desulfation at the 6-O position by human sulfatase 2 (SULF2). The sulfation state of HSGAGs is critical for growth factor binding; hence, SULF2 may regulate tumor growth by releasing growth factors from HSGAG storage sites at the cell surface and in the extracellular matrix and thus may increase the local concentration of growth factors available to bind to cell surface receptors and enhance cell signaling.

Deficiency of inflammasome components NLRP3, ASC or Caspase-1, or lack of IL-1 signaling prevented alcohol-induced liver inflammation suggesting that IL-1 determines Vemurafenib mouse the onset of inflammation in AH. Next we evaluated whether IL-1 also drives the persistence of liver inflammation in AH. We observed that liver

inflammation and increased IL-1 were sustained for at least three days after cessation of alcohol, followed by delayed apoptosis of inflammatory cells in the liver and limited degree of hepatocyte regeneration. We discovered that inhibition of IL-1 signaling using a single dose of IL-1Ra at cessation of alcohol increased apoptosis of liver immune cells, facilitated regeneration of hepatocytes, and resulted in increased rate of recovery from liver injury in AH. These findings were consistent with in vitro studies showing that IL-1 administration selleck kinase inhibitor promotes cytotoxicity of primary hepatocytes while it provides pro-survival benefit for BM-derived immune cells Conclusions: Our novel findings demonstrate that IL-1 drives sustained liver inflammation

and impaired hepatocyte regeneration even after cessation of ethanol exposure in AH. Therapeutic inhibition of IL-1 improves hepatocyte survival and promotes death of activated harmful immune cells in a preclin-ical model of AH. Disclosures: Gyongyi Szabo – Consulting: Idenix; Grant/Research Support: BMS, GSK, Cona-tus, Idera, Johnson&Johnson, Novartis, Ocera, Roche, Shering – Plough, Wyeth, Integrated medchemexpress Therapeutics, Idera The following people have nothing to disclose: Jan Petrasek, Arvin Irache-ta-Vellve, Shashi Bala, Timea Csak, Karen Kodys, Evelyn A. Kurt-Jones Background: Epidemiological studies revealed that nearly 80% of heavy drinkers with alcoholic liver disease (ALD) also smoke tobacco. This finding suggests that tobacco and possibly its toxins have cofactor roles in ALD pathogenesis. Our research focused on the potential role of NNK as a mediator

of hepa-tocellular injury in ALD because previous efforts showed that other nitrosamines can cause hepatic steatosis or steatohep-atitis with insulin resistance, inflammation, oxidative and ER stress, and lipotoxicity. Moreover, toxic lipids, particularly ceramides, can promote the same effects. Due to the inability to detect significant molecular profiles by routine histological studies, we explored the potential use of matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry as a diagnostic aid for characterizing the nature and possibly etiology of steatohepatitis. Hypothesis: IMS can be used to generate biochemical signatures of steatohepatitis caused by different agents.

Deficiency of inflammasome components NLRP3, ASC or Caspase-1, or lack of IL-1 signaling prevented alcohol-induced liver inflammation suggesting that IL-1 determines Tamoxifen mouse the onset of inflammation in AH. Next we evaluated whether IL-1 also drives the persistence of liver inflammation in AH. We observed that liver

inflammation and increased IL-1 were sustained for at least three days after cessation of alcohol, followed by delayed apoptosis of inflammatory cells in the liver and limited degree of hepatocyte regeneration. We discovered that inhibition of IL-1 signaling using a single dose of IL-1Ra at cessation of alcohol increased apoptosis of liver immune cells, facilitated regeneration of hepatocytes, and resulted in increased rate of recovery from liver injury in AH. These findings were consistent with in vitro studies showing that IL-1 administration selleck chemicals llc promotes cytotoxicity of primary hepatocytes while it provides pro-survival benefit for BM-derived immune cells Conclusions: Our novel findings demonstrate that IL-1 drives sustained liver inflammation

and impaired hepatocyte regeneration even after cessation of ethanol exposure in AH. Therapeutic inhibition of IL-1 improves hepatocyte survival and promotes death of activated harmful immune cells in a preclin-ical model of AH. Disclosures: Gyongyi Szabo – Consulting: Idenix; Grant/Research Support: BMS, GSK, Cona-tus, Idera, Johnson&Johnson, Novartis, Ocera, Roche, Shering – Plough, Wyeth, Integrated MCE Therapeutics, Idera The following people have nothing to disclose: Jan Petrasek, Arvin Irache-ta-Vellve, Shashi Bala, Timea Csak, Karen Kodys, Evelyn A. Kurt-Jones Background: Epidemiological studies revealed that nearly 80% of heavy drinkers with alcoholic liver disease (ALD) also smoke tobacco. This finding suggests that tobacco and possibly its toxins have cofactor roles in ALD pathogenesis. Our research focused on the potential role of NNK as a mediator

of hepa-tocellular injury in ALD because previous efforts showed that other nitrosamines can cause hepatic steatosis or steatohep-atitis with insulin resistance, inflammation, oxidative and ER stress, and lipotoxicity. Moreover, toxic lipids, particularly ceramides, can promote the same effects. Due to the inability to detect significant molecular profiles by routine histological studies, we explored the potential use of matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry as a diagnostic aid for characterizing the nature and possibly etiology of steatohepatitis. Hypothesis: IMS can be used to generate biochemical signatures of steatohepatitis caused by different agents.

The start date for analyses was the date of liver biopsy, with any events occurring in the first 6 months excluded. Patients were monitored every 3-6 months Buparlisib concentration and followed up until death or liver transplantation, whichever occurred first, or until data analysis. For those lost to follow-up, up-to-date clinical information was sought by

the following: (1) contact with the primary care physician; (2) telephone interview; and (3) checking the respective death and transplant registries. Patients were censored at time of death or transplantation or last clinic visit, and those lost to follow-up were censored at the time last seen. All clinical outcomes were confirmed by a physician at each center, utilizing patient records and physician diagnoses. The following outcomes

were assessed: (1) liver complications, including liver failure, gastroesophageal varices (± hemorrhage), ascites, encephalopathy, hepatopulmonary syndrome, and HCC; (2) liver-related death or liver transplantation (for calculation of survival probability, transplantation was considered as an equivalent end-point); (3) all-cause mortality; and (4) total BAY 57-1293 cell line vascular events (including myocardial infarction, stroke, and vascular deaths).13 HCC was diagnosed if the following were present: (1) pathological changes consistent with HCC identified by histological examination of liver tissue obtained by fine-needle aspiration, liver biopsy, or liver explant at transplantation or autopsy or (2) one or more hepatic space-occupying lesions that had vascular patterns typical of HCC by angiography, 上海皓元 triple-phase computed tomography, or magnetic resonance imaging. All patients were followed according to standards of care and guidelines without experimental or therapeutic interventions for NAFLD or HCV. Weight management was performed with lifestyle intervention, such as dietary modification, and exercise was recommended at outpatients in overweight/obese patients. Other treatments, such as oral hypoglycemics, cholesterol-lowering

medications, and antihypertensive medications, were only given in the context of management of concomitant diabetes mellitus, hypercholesterolemia, or hypertension, respectively. Neither pharmacological nor lifestyle interventions were recorded systematically after baseline. Statistical analyses were performed using SPSS version 13.0 (SPSS, Inc., Chicago IL). Results are reported as means ± standard deviation (SD) or frequency (i.e., percentage), as appropriate. Continuous variables were compared using the two-tailed Student’s t-test. Categorical data were compared using the chi-square test. Variables with a P value of ≤0.1 on univariate analysis were further analyzed by multiple logistic regression to determine the independent determinants of outcome variables.

As time from inoculation with the ASD strain increased, the activities of various enzymes were higher than controls. Maximum enzyme activities were found on the tenth day after ADS inoculation. The response of soil enzyme activities affected by the ASD strain was

as follows: urease > dehydrogenase > invertase > acid phosphatase > catalase. These results suggest that the biocontrol of ASD Trametinib research buy strain could improve the micro ecology of rhizosphere soil. “
“Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac-Af, Xac-Ap and Xac-Ar. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine-responsive SB203580 mouse regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive-sequence PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac-Af, Xac-Ap and Xac-Ar, and additional reference strains from pathotypes Xac-A, Xac-A*, Xac-Aw,

X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep-PCR clearly

grouped the atypical symptom–producing variants in types Xac- Af, Xac-Ar and Xac-Ap MCE公司 into the same cluster with typical symptom-producing strains in pathotype Xac-A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac-A* and Xac-Aw in these genotypic analyses. Strains of Xac-A* and Xac-Aw were closely related to Xac-A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac-A. “
“Virulence analysis and two polymerase chain reaction–based assays were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae (Xoo) from different elevations ranging from 150 to 2600 m in south-west China. Among the 218 isolates of Xoo, 18 pathotypes were identified using six near-isogenic rice lines, each containing a single resistance gene. Among them, pathotype 9 predominated in low and mid-elevations was virulent to all resistance genes, including Xa2, Xa3, xa5, xa13, Xa14 and Xa18. However, pathotype 2 was predominant at high elevation and was virulent to Xa18 only. The 18 pathotypes were grouped into four clusters.

As time from inoculation with the ASD strain increased, the activities of various enzymes were higher than controls. Maximum enzyme activities were found on the tenth day after ADS inoculation. The response of soil enzyme activities affected by the ASD strain was

as follows: urease > dehydrogenase > invertase > acid phosphatase > catalase. These results suggest that the biocontrol of ASD EGFR inhibitor drugs strain could improve the micro ecology of rhizosphere soil. “
“Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac-Af, Xac-Ap and Xac-Ar. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine-responsive RG7422 cell line regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive-sequence PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac-Af, Xac-Ap and Xac-Ar, and additional reference strains from pathotypes Xac-A, Xac-A*, Xac-Aw,

X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep-PCR clearly

grouped the atypical symptom–producing variants in types Xac- Af, Xac-Ar and Xac-Ap MCE into the same cluster with typical symptom-producing strains in pathotype Xac-A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac-A* and Xac-Aw in these genotypic analyses. Strains of Xac-A* and Xac-Aw were closely related to Xac-A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac-A. “
“Virulence analysis and two polymerase chain reaction–based assays were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae (Xoo) from different elevations ranging from 150 to 2600 m in south-west China. Among the 218 isolates of Xoo, 18 pathotypes were identified using six near-isogenic rice lines, each containing a single resistance gene. Among them, pathotype 9 predominated in low and mid-elevations was virulent to all resistance genes, including Xa2, Xa3, xa5, xa13, Xa14 and Xa18. However, pathotype 2 was predominant at high elevation and was virulent to Xa18 only. The 18 pathotypes were grouped into four clusters.

As time from inoculation with the ASD strain increased, the activities of various enzymes were higher than controls. Maximum enzyme activities were found on the tenth day after ADS inoculation. The response of soil enzyme activities affected by the ASD strain was

as follows: urease > dehydrogenase > invertase > acid phosphatase > catalase. These results suggest that the biocontrol of ASD Ivacaftor strain could improve the micro ecology of rhizosphere soil. “
“Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac-Af, Xac-Ap and Xac-Ar. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine-responsive Deforolimus supplier regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive-sequence PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac-Af, Xac-Ap and Xac-Ar, and additional reference strains from pathotypes Xac-A, Xac-A*, Xac-Aw,

X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep-PCR clearly

grouped the atypical symptom–producing variants in types Xac- Af, Xac-Ar and Xac-Ap 上海皓元医药股份有限公司 into the same cluster with typical symptom-producing strains in pathotype Xac-A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac-A* and Xac-Aw in these genotypic analyses. Strains of Xac-A* and Xac-Aw were closely related to Xac-A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac-A. “
“Virulence analysis and two polymerase chain reaction–based assays were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae (Xoo) from different elevations ranging from 150 to 2600 m in south-west China. Among the 218 isolates of Xoo, 18 pathotypes were identified using six near-isogenic rice lines, each containing a single resistance gene. Among them, pathotype 9 predominated in low and mid-elevations was virulent to all resistance genes, including Xa2, Xa3, xa5, xa13, Xa14 and Xa18. However, pathotype 2 was predominant at high elevation and was virulent to Xa18 only. The 18 pathotypes were grouped into four clusters.

Twelve healthcare workers were studied prospectively after occupational HCV exposure for HCV RNA using the standard clinical assay at the NIH (Cobas Amplicor, HCV Test 2.0, Roche, Branchburg, NJ), HCV-specific antibodies (Abbott HCV EIA 2.0, Abbott, Princeton, NJ), serum

cytokines, and www.selleckchem.com/products/azd9291.html NKT, NK, and T-cell responses. Eleven healthcare workers tested HCV RNA-nonreactive at the assay sensitivity of 100 IU/mL, whereas one developed high-level viremia and started PegIFN/ribavirin treatment 17 weeks after exposure. Peripheral blood mononuclear cells (PBMCs) of the cohort with undetectable HCV RNA were isolated from citrate dextrose-anticoagulated blood on the day of exposure (n = 5 subjects), 2 weeks (n = 11), 4 weeks (n = 11), 6 weeks (n = 11), 13 weeks (n = 10), and more than 24 weeks (n = 11) thereafter, and cryopreserved in liquid nitrogen using previously described techniques.[14] PBMCs of the healthcare worker with high-level viremia were isolated 3, 5, 8, and 14 weeks after exposure. Twenty-nine

healthy blood donors were studied as controls at a single timepoint. All gave written informed consent for research testing, according to protocols approved by the participating hospitals’ Institutional Review Boards. PBMCs were stained with ethidium monoazide (EMA), anti-CD19-PeCy5, anti-CD3-PacificBlue (both from BD Biosciences, San Jose, CA), anti-CD14-PeCy5 (Serotec, Raleigh, NC), and with αGalCer-loaded, streptavidine-PE-conjugated CD1d-tetramers (NIAID Tetramer Facility of the NIH AIDS Research and Reference Midostaurin price Reagent Program, Atlanta, GA) to identify NKT cells. Cells were additionally stained with anti-FasL-FITC (Abcam, Cambridge, MA) and anti-NKG2D-PeCy7 (BioLegend, San Diego, CA).

PBMCs were stained with EMA, anti-CD14-PeCy5 (Serotec), anti-CD19-PeCy5, anti-CD3-AlexaFluor700, anti-CD56-PeCy7, and anti-CD16-PacificBlue (all from BD Biosciences) and with either anti-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-PE (BD Biosciences), anti-CD122-FITC, anti-NKp44-PE, anti-NKp46-PE, or anti-NKG2A-PE Selleckchem U0126 (all from Beckman Coulter, Brea, CA). NK cell degranulation was quantitated as an increase in cell surface CD107a expression in response to MHC class I-negative K562 cells (ATCC, Manassas, VA).[15] PBMCs were cultured at 37°C with or without IL-12 (0.5 ng/mL; R&D Systems) and IL-15 (20 ng/mL R&D Systems) and assessed for interferon-gamma (IFN-γ) production by flow cytometry as described.[15] Stained cells were analyzed on an LSRII using FacsDiva Version 6.1.3 (BD Biosciences) and FlowJo v. 8.8.6 (Tree Star, Ashland, OR) software. PBMCs were stimulated with seven pools of overlapping 15-mer HCV genotype 1a peptides (1 μg/mL of each peptide) covering the core (38 peptides), NS3 (three pools with 42 peptides each), NS4A pool (12 peptides), and NS4B sequence (two pools with 26 peptides each),[14] phytohemagglutinin (1 μg/mL PHA-M; Invitrogen, Carlsbad, CA), or dimethyl sulfoxide (DMSO) as described.

Twelve healthcare workers were studied prospectively after occupational HCV exposure for HCV RNA using the standard clinical assay at the NIH (Cobas Amplicor, HCV Test 2.0, Roche, Branchburg, NJ), HCV-specific antibodies (Abbott HCV EIA 2.0, Abbott, Princeton, NJ), serum

cytokines, and Cabozantinib cost NKT, NK, and T-cell responses. Eleven healthcare workers tested HCV RNA-nonreactive at the assay sensitivity of 100 IU/mL, whereas one developed high-level viremia and started PegIFN/ribavirin treatment 17 weeks after exposure. Peripheral blood mononuclear cells (PBMCs) of the cohort with undetectable HCV RNA were isolated from citrate dextrose-anticoagulated blood on the day of exposure (n = 5 subjects), 2 weeks (n = 11), 4 weeks (n = 11), 6 weeks (n = 11), 13 weeks (n = 10), and more than 24 weeks (n = 11) thereafter, and cryopreserved in liquid nitrogen using previously described techniques.[14] PBMCs of the healthcare worker with high-level viremia were isolated 3, 5, 8, and 14 weeks after exposure. Twenty-nine

healthy blood donors were studied as controls at a single timepoint. All gave written informed consent for research testing, according to protocols approved by the participating hospitals’ Institutional Review Boards. PBMCs were stained with ethidium monoazide (EMA), anti-CD19-PeCy5, anti-CD3-PacificBlue (both from BD Biosciences, San Jose, CA), anti-CD14-PeCy5 (Serotec, Raleigh, NC), and with αGalCer-loaded, streptavidine-PE-conjugated CD1d-tetramers (NIAID Tetramer Facility of the NIH AIDS Research and Reference Deforolimus mouse Reagent Program, Atlanta, GA) to identify NKT cells. Cells were additionally stained with anti-FasL-FITC (Abcam, Cambridge, MA) and anti-NKG2D-PeCy7 (BioLegend, San Diego, CA).

PBMCs were stained with EMA, anti-CD14-PeCy5 (Serotec), anti-CD19-PeCy5, anti-CD3-AlexaFluor700, anti-CD56-PeCy7, and anti-CD16-PacificBlue (all from BD Biosciences) and with either anti-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-PE (BD Biosciences), anti-CD122-FITC, anti-NKp44-PE, anti-NKp46-PE, or anti-NKG2A-PE Cytidine deaminase (all from Beckman Coulter, Brea, CA). NK cell degranulation was quantitated as an increase in cell surface CD107a expression in response to MHC class I-negative K562 cells (ATCC, Manassas, VA).[15] PBMCs were cultured at 37°C with or without IL-12 (0.5 ng/mL; R&D Systems) and IL-15 (20 ng/mL R&D Systems) and assessed for interferon-gamma (IFN-γ) production by flow cytometry as described.[15] Stained cells were analyzed on an LSRII using FacsDiva Version 6.1.3 (BD Biosciences) and FlowJo v. 8.8.6 (Tree Star, Ashland, OR) software. PBMCs were stimulated with seven pools of overlapping 15-mer HCV genotype 1a peptides (1 μg/mL of each peptide) covering the core (38 peptides), NS3 (three pools with 42 peptides each), NS4A pool (12 peptides), and NS4B sequence (two pools with 26 peptides each),[14] phytohemagglutinin (1 μg/mL PHA-M; Invitrogen, Carlsbad, CA), or dimethyl sulfoxide (DMSO) as described.