In all cases, equation (1) provided a good fit of the data, with greater than 88% variance accounted for. Equation (1) also provided a good selleck catalog fit of individual participant��s simulated demand data (median R2 = .88, interquartile range = .70�C.93). Baseline �� parameter values derived from simulated demand curves (a measure of the essential value of cigarettes) were significantly correlated with baseline FTND scores (Spearman r = ?.255, p < .05). The correlation between �� parameter values and the number of cigarettes participants reported smoking each day at intake approached significance (Pearson r = ?.232, p = .07). Figure 1. Simulated demand for cigarettes across the programmed range of normalized prices for participants in the placebo and bupropion groups.

Curves are fit using the demand equation proposed by Hursh and Silberberg (2008), see text for details. A mixed factor ANOVA applied to peak smoking (Q0) revealed no significant main effects of time (baseline vs. follow-up, p = .09) or group (bupropion vs. placebo, p = .76). The Time �� Group interaction was not significant [F(1, 57) = 1.74, p = .19]. The same mixed factor ANOVA was applied to �� values derived from individual participants�� demand curves. No significant main effects of time [F(1, 57) = 1.15, p = .29] or group [F(1, 57) = 2.75, p = .10] were detected. Likewise, the Time �� Group interaction did not approach significance [F(1, 57) = 1.29, p = .26]. In sum, bupropion did not decrease peak smoking or the essential value of cigarettes.

Pmax and Omax values were calculated from the derived parameters of the demand curves and were subjected to the same mixed factor ANOVA. Pmax is the cigarette price (nonnormalized for the purpose of this analysis) at which the demand curve has a slope of ?1.0. More importantly, it is the price at which spending on cigarettes is predicted to asymptote; at higher prices, spending should decline. Omax is the predicted maximum amount that would be spent on cigarettes in a single day. Pmax and Omax were calculated using the spreadsheet written by S. R. Hursh (available at http://www.ibrinc.org/centers/bec/BEC_demand.html). No significant main effects of time were detected on either Pmax or Omax (p > .4 in both cases). Likewise, no main effect of group was detected (p > .4 in both cases), and the Time �� Group interaction did not approach significance (p > .

4 in both cases). The correlations between derived and obtained measures of peak spending and price at which peak spending occurred were strong (Omax: Pearson r = .64, p < .0001; Pmax: Pearson r = .79, p < .0001), and a mixed factor ANOVA applied to obtained measures did not reveal a Time �� Group interaction Carfilzomib (p > .2 in both cases). Thus, an effect of bupropion on cigarette purchases was not missed by using measures derived from the demand curves.

The sessions provided psychoeducation and support (e.g., Brown, 2003) and were run by doctoral students in clinical psychology. A half-day trial abstinence period during which participants were instructed to briefly attempt abstinence occurred on Day 9 (Shiffman, sellckchem Ferguson, et al., 2006); accordingly, we excluded that day from analysis. Measures At baseline, participants reported a variety of demographic measures, including age, years of education, gender, income, ethnicity, years smoking, and smoking rate. They also completed the FTND (Heatherton et al., 1991), and the NDSS (Shiffman et al., 2004). Each time they smoked, participants recorded this on ED. On approximately five randomly selected smoking occasions per day, ED administered an assessment of craving and situational variables.

Craving Craving was assessed (the prompt was ��Rate cigarette craving��) on a single Visual Analog Scale (VAS) with anchors at either end: 0 = ��no craving,�� 10 = ��maximum craving.�� Despite the limitations of single-item scales (see Sayette et al., 2000; Tiffany, Carter, & Singleton, 2000), this assessment was deemed well suited for an EMA study; a single item allows the measure to reflect craving at the particular moment of the report, to minimize the intrusiveness of the report itself, and to decrease the probability that completing the measure will impact the reported craving level (Sayette et al., 2000). Moreover, in a prior study (Shiffman et al, 2002), where both ��craving�� and ��urge�� were assessed, the two correlated r = .80, demonstrating very high reliability for the single items.

That is, since reliability is the upper limit of validity, the reliability of each must be at least .80 to achieve the correlation of .80 (see Wanous, Reichers, & Hudy, 1997). Situations Situational variables were treated as binary��present or absent��in the analyses. Subjects reported any smoking restrictions in the place that they first decided to smoke. In cases where they had changed locations in order to smoke, they were also asked about restrictions in the location in which they actually smoked. All other variables referred to the situation where they first decided to smoke. Throughout this section, the actual text for questions assessing the smoking situation is included in parentheses along with a description of the situational variable.

Subjects�� reports of smoking restrictions (��Smoking regulations��Smoking allowed? Forbidden, Discouraged, Allowed��) were dichotomized by comparing episodes in which smoking was ��forbidden�� or ��discouraged�� to those in which smoking was ��allowed.�� Because smoking restrictions may Anacetrapib influence smoking behavior (Chandra et al., 2007; Shiffman et al., 2002) and covary with other situational variables (e.g., being at work), we controlled for the presence of restrictions in all subsequent analyses.

125 (SEM = 0.125) to 83.9 (SEM = 7) after the 10-puff selleck chemicals Dasatinib bout and further increased to 92.6 (SEM = 3.3) following the ad lib period before dropping to 36.9 (SEM = 13.5) after the rest period. EC use did not significantly affect ratings on any other measure. Discussion Previous reports have described conditions of EC use that support minimal or no nicotine delivery (Bullen et al., 2010; Eissenberg, 2010; Vansickel et al., 2010). In those studies, current cigarette smokers, who were not experienced with ECs, engaged in brief periods of EC use. In addition, the ECs used in those studies were cigarette-sized EC models. In the current study, experienced EC users who provided their preferred ECs loaded with their selected flavor/nicotine concentration completed a 10-puff (30-s IPI) bout and a 1-hr ad lib puffing period.

Under these conditions, EC use resulted in reliable nicotine delivery. These results are the first to demonstrate unequivocally that ECs alone are capable of increasing plasma nicotine concentrations to levels seen during cigarette smoking. In support of this conclusion, the results of a recent study in which the saliva cotinine concentration of self-reported EC users was measured are also consistent with nicotine exposure that approximated tobacco cigarette use (e.g., Etter & Bullen, 2011b). However, unlike the current laboratory study, results of this previous work may have been influenced by the concurrent use of other nicotine-containing products. Interestingly, abstinence symptoms were observed prior to EC use, and these symptoms were suppressed after EC use, a potential indicator of nicotine dependence (Carter and Griffiths, 2009).

Of course, these participants were former cigarette smokers and thus may have been nicotine dependent prior to initiating EC use. The extent to which EC use leads to the development of nicotine dependence in nicotine-na?ve users is unknown. Some reports suggest that ECs have the potential to be safe and efficacious replacements for tobacco cigarettes due to nicotine��s effects (Etter & Bullen, 2011a; Siegel, Tanwar, & Wood, 2011). That potential is undermined by ineffective devices and a requirement that users learn specific but as-yet-undetermined behaviors in order to maximize nicotine delivery. Thus, an important area for future research is the parametric manipulation of device characteristics and user behavior (i.

e., puff topography) as well as other factors (e.g., nicotine concentration) that might contribute to safety Anacetrapib and efficacy. In addition, the potential deleterious health effects of chronic inhalation of vaporized nicotine solutions are unknown (Etter, Bullen, Fouris, Laugesen, & Eissenberg, 2011). Clinical laboratory evaluation of lung and cardiovascular function, measurement of biomarkers of harm (e.g.

, in press). Data Acquisition and Reduction The EEG signals were recorded using a 129-channel geodesic sensor net, filtered (0.1�C100 Hz), amplified, leave a message and digitized at a sampling rate of 250 Hz using an AC-coupled, high-input impedance (200 M��) amplifier (Geodesic EEG System 200; Electrical Geodesics Inc.). The EEG data were visually inspected offline to examine the overall data quality. On average, about 2% of the channels were contaminated by artifacts for more than 50% of the recording and were interpolated using spherical splines. Then, a spatial filtering method implemented in BESA (v 5.1.8.10, MEGIS Software GmbH) was used to remove eyeblink artifacts. Next, EEG data were transformed to average reference and segmented into 1850-ms epochs (800ms before through 1050ms after picture onset).

Within each epoch, channels contaminated by artifacts (e.g., absolute EEG difference larger than 100 ��V and difference between two contiguous data points larger than 25 ��V) were identified. If fewer than 90% of the channels in an epoch were artifact free, the epoch was excluded from further analyses. This criterion resulted in the loss of approximately 17% of all epochs. A continuous wavelet transformation was conducted using BrainVision Analyzer software (v 1.05.0005, Brain Products GmbH). A complex Morlet wavelet (Herrmann, Grigutsch, & Busch, 2005) was used to decompose the EEG signals for each channel in the range of 6�C30 Hz. The step between successive frequencies was set at 1 Hz, and the Morlet parameter c was set to 7.0.

For each picture-viewing trial, we extracted the time course of power information for signals falling in the range of alpha oscillations (8�C12 Hz). The data were baseline-corrected by converting power to a decibel (dB) scale (Delorme & Makeig, 2004), using the average power in the window lasting from 500 to 200ms before picture onset as the baseline. To examine whether baseline levels of alpha power changed over the course of the picture-viewing session, we tested for significant effects of block (first half or second half of the experiment) and valence category (neutral, pleasant, unpleasant, and cigarette related) on alpha power measured during the baseline window using a mixed model in which subject was modeled as a random effect (PROC MIXED; SAS v9.2, SAS Institute).

Data Analysis Following previously Brefeldin_A published procedures (De Cesarei & Codispoti, 2011), we computed picture-related alpha ERD by averaging across parieto-occipital sensors (P7, P5, P3, P1, P2, P4, P6, P8, PO7, PO5, PO3, PO2, PO4, PO6, O3, O1, O2, and O4). Then, we obtained a single estimate of alpha ERD per picture-category per-subject by averaging alpha ERD between 400 and 800ms after picture onset (Figure 1). These estimates were analyzed using mixed models (PROC MIXED) with subject modeled as a random effect. Figure 1. Picture-induced alpha ERD in the parieto-occipital area.

The elicited immune responses towards the N, GN, GC and GN/GC proteins thoroughly after gene-gun immunisation were analysed and the protective abilities of the N and the GN/GC construct were tested by virus challenge. Methods Cells and viruses BHK-21 (ATCC number CCL-10) cells were maintained in Glasgow MEM (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 5% FCS, 1.3 g/l Tryptose (Difco?, Becton, Dickinson and Company, Sparks, MD), 10 mM HEPES, 1 mM sodium pyruvate, 100 U penicillin/ml and 100 ��g/ml streptomycin at 37��C/5% CO2. The working stocks of RVFV and cDNA constructs, originated from the ZH548 wild-type strain, isolated from a human case in Egypt in 1977 [24].

Viral stocks were prepared and titrated on monolayers of BHK-21 cells and the cDNA sequences are found under the GenBank accession numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”AF134534″,”term_id”:”6006975″,”term_text”:”AF134534″AF134534 and “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ380206″,”term_id”:”87622803″,”term_text”:”DQ380206″DQ380206[25,26]. Production of DNA vaccine For genetic immunisation and eukaryotic expression, cDNAs encoding N, GN/GC, GN and GC were inserted into pcDNA3.1/V5-His? TOPO (Invitrogen). The primer sequences used for cDNA amplification and subsequent cloning are shown in Table Table1.1. The correctness of each cDNA construct was confirmed by sequencing (MWG-Biotech) and the corresponding gene products were verified through transfection of mammalian cells followed by immunofluorescence analysis. A cDNA construct (pcDNA3.

1) encoding the N protein (PUU-N) of the Puumala hantavirus (Puumala virus Ume?/hu [GenBank: "type":"entrez-nucleotide","attrs":"text":"AY526219","term_id":"42541164","term_text":"AY526219"AY526219] [27,28] was used as a control. The preparation of gene-gun cartridges has previously been described [28]. Briefly, 50 ��g aliquots of the above plasmid DNA preparations were precipitated on 25 mg of 1 ��m gold beads and subsequently used to coat the inner wall of Tefzel tubings according to the manufacturer’s instructions (BioRad Laboratories, Hercules, CA). Each gene-gun cartridge delivered approximately 1 ��g of DNA. Table 1 Primers sequences Animal immunisation and infection Female BALB/c mice, six to eight weeks old, were used in this study. Before immunisation the mice were thoroughly shaved on the abdomen and vaccinated with cDNA encoding the antigens using a gene-gun (Helios?, BioRad Laboratories).

The cDNA was administrated four times with two to three week intervals. The primary immunisation was performed using four gene-gun cartridges Carfilzomib and the following three boosters with two cartridges. Blood samples were collected three, five, seven and nine weeks after the primary immunisation. In order to study the immune responses post infection (p.i.) and the effectiveness of the genetic vaccines, mice were injected intraperitoneally (i.p.) with RVFV diluted in sterile PBS to a final volume of 100 ��l.

A tumour bud is typically defined as a single tumour cell or tumour selleckchem cell cluster of up to five cells at the invasive tumour front (Prall, 2007). Indeed, tumour budding has been shown to be associated with lymph node positivity, poorly differentiated tumours, presence of vascular and lymphatic invasion, local tumour recurrence and distant metastasis (Ueno et al, 2004a,2004b; Nakamura et al, 2005; Kazama et al, 2006; Ishikawa et al, 2008; Wang et al, 2009). In particular, patients with stage III disease have been reported to demonstrate a 5-year disease-free survival (DFS) of 62.1% in the absence of tumour budding and only a 35.1% DFS with this feature (Choi et al, 2007). Moreover, the presence of tumour budding has repeatedly been linked to poor clinical outcome, underlined by the adverse effect on overall survival independent of TNM stage (Hase et al, 1993; Ueno et al, 2004b).

Over the last 20 years, investigations on tumour immunity and host defence in colorectal cancer demonstrate promising results for immunotherapy. In most colorectal cancers, lymphocytic infiltration is composed predominantly of either CD4+ or CD8+ T cells and both cell types appear to be significantly increased in tumour as compared with normal tissue (Ropponen et al, 1997; Naito et al, 1998; Chiba et al, 2004; Koch et al, 2006). Several studies have shown that tumour infiltrating lymphocytes (TILs) within the stroma and around the tumour along the invasive margin are significantly related to overall- and disease-specific survival in both univariate and multivariable analysis (Ali et al, 2004; Canna et al, 2005; Pages et al, 2005).

Galon et al (2006) evaluated by gene-expression profiling and immunohistochemistry, the type, density and location (whether at the invasive margin or the tumour centre) of TILs in a large number of cases. They evaluated CD3, CD8, granzyme B and memory CD45RO T cells, demonstrating a significant independent and positive effect of TILs on both recurrence and survival. In colorectal cancer, mismatch-repair status (microsatellite stable (MSS) and microsatellite instability-high (MSI-H)) seems to relate highly to the number of CD8+ lymphocytes. Compared with MSS tumours, MSI-H cancers are characterised by prolonged survival time, significantly more frequent peritumoural lymphocytic infiltration at the invasive front and by an inherent abundance Carfilzomib of intra-epithelial TILs (Jass et al, 1998; Michael-Robinson et al, 2001; Greenson et al, 2003; Jenkins et al, 2007). Nevertheless, many studies analyzing colorectal cancer samples stratified by mismatch-repair status also report a positive effect of CD8+ lymphocytes in mismatch-repair proficient colorectal cancers (Baker et al, 2007).

Surface staining was performed using fluorescein-isothiocyanate-labeled anti-ST2 (clone DJ8), allophycocyanin-labeled anti-c-kit (clone 2B8), anti-CD4 (clone RM4-5), anti-CD11b (clone M1/70), and phycoerythrin-labeled Enzalutamide chemical structure anti-Fc��RI (clone MAR-1), anti-CD3 (clone 145-2C11), anti-Ly6G (clone 1A8), and anti-F4/80 (clone BM8). Fluorochrome-conjugated antibodies were purchased from MD Biosciences (Z��rich, Switzerland), eBioscience (Vienna, Austria), or BD Biosciences (Erembodegem, Belgium). Cell acquisition was performed using a FACSCalibur flow cytometry system, and analyses were done with CellQuest Pro software 4.0 (BD Biosciences). Bone-Marrow-Derived Mast Cells Bone-marrow-derived mast cells (BMMCs) were isolated according to a previously published protocol.

21 Briefly, BMMCs were obtained by culturing femoral bone marrow cells from WT and Il1rl1?/? mice in Iscove’s modified Dulbecco’s medium (Lonza) containing 10% FBS, 100 IU/mL penicillin, 100 ��g/mL streptomycin, 250 ng/mL amphotericin B (Gibco; Life Technologies, Paisley, UK), and 5 �� 10?5 mol/L ��-mercaptoethanol. Nonadherent cells were cultured for 6 to 12 weeks in the presence of IL-3 (5 ng/mL) and stem cell factor (SCF) (50 ng/mL; Invitrogen-Life Technologies, Paisley, UK). Thereafter, cell purity was controlled by flow cytometry, revealing >90% of c-kithigh Fc��RIhigh cells, considered as differentiated mast cells. For in vitro experiments, 106 BMMCs/well were cultured for 24 hours with lipopolysaccharide (LPS) (1 ��g/mL; E. coli serotype 055:B5; Sigma-Aldrich) or with mouse rIL-33 (10 ng/mL; R&D Systems, Minneapolis, MN).

Tryptase activity in BMMC culture supernatants and in mouse sera was assayed with a mast cell degranulation assay kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s protocol, in which calcium ionophore was used as an inducer of degranulation and protamine as an inhibitor of it. Pancreas Cell Preparation Acinar cells were isolated as described previously,22 with slight modifications. In overnight-fasted WT mice not receiving any treatment, pancreata were dissected free of mesenteric fat, minced in Hank’s buffered saline solution with 5 mmol/L EDTA solution at 37��C, and centrifuged for 2 minutes at 500 �� g twice to eliminate fat. The tissues were rinsed in Waymouth’s medium (Invitrogen-Life Technologies) containing penicillin and streptomycin (0.

1 mg/mL; Lonza), 20% FBS, and 0.01% soybean trypsin inhibitor (Sigma-Aldrich) and were digested by means of two cycles of shaking at 25 cycles/minute in a solution of 6.25 U/mL collagenase in Hank’s buffered saline solution containing 0.2% bovine serum albumin at 37��C. Nondigested tissue was eliminated by 70-��m pore filtration, and isolated cells were washed twice in complete Waymouth’s GSK-3 medium.

Methods Sample The Midlife Development in the United States (MIDUS) Survey is a nationally representative survey of 3,032 persons aged 25�C74 years in the noninstitutionalized figure 1 civilian population of the 48 coterminous United States (Brim et al., 2010; Kessler, DuPont, Berglund, & Wittchen, 1999; Kessler, Mickelson, & Zhao, 1997). The MIDUS Survey was carried out by the John D. and Catherine T. MacArthur Foundation Network on Successful Midlife Development between January 1995 and 1996. All respondents completed a 30-min telephone interview (70.0% response rate) and filled out two mailed questionnaires estimated to take a total of about 90 min to complete (86.8% conditional response rate in the subsample of telephone respondents). The overall response rate was 60.8%.

More details on the MIDUS Survey design, field procedures, and sampling weights are available elsewhere (Brim et al., 2010; Kessler et al., 1997, 1999). Analyses were conducted in SAS 9.2 (Cary, NC) using the survey procedures (surveymeans, surveyfreq, surveylogistic) to adjust standard errors in accordance with the sampling and weight our estimates to adjust for differential probabilities of selection and nonresponse to derive estimates representative of the U.S. adult population. The sample for this analysis is 3,010 individuals with complete information on covariates of interest. Diagnostic Assessment Current Respiratory Disease Data on physical illnesses were obtained through self-report. Respondents were presented with a list of illnesses and asked whether they had experienced or been diagnosed by a physician with any of the illnesses within the past 12 months.

This included ��asthma, bronchitis, or emphysema.�� Self-Reported Child Physical Abuse A history of self-reported childhood abuse was assessed by responding often, sometimes, rarely, or never to one of the following statements: Did your mother or father often, sometimes, or rarely: ��kicked, bit, or hit you with a fist; hit or tried to hit you with something; beat you up; choked you; burned you or scalded you�� or ��pushed, grabbed, or shoved you; slapped you; threw something at you.�� Those who answered never to all four inquiries were used as the reference group. Respondents were assigned to a frequency of abuse category based on the highest frequency of abuse for any of the four responses.

Current Mental Disorders The MIDUS Dacomitinib Survey diagnoses were based on the Composite International Diagnostic Interview��Short Form (CIDI-SF) scales (Kessler et al., 1994), a series of diagnostic-specific scales that were developed from item-level analyses of a modified version of the World Health Organization CIDI (Kessler, Andrews, Mroczek, Ustun, & Wittchen, 1998), and a structured interview designed to determine Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, clinical and subclinical mental disorders in the National Comorbidity Survey (Kessler et al., 1994, 1998; Wittchen, 1994).

The aim of the present study was therefore to determine whether the routine use of colonoscopes equipped with high-definition combined with i-Scan technology (HD+ plus i-Scan) gave a higher rate of detection of overall mucosal lesions, particularly http://www.selleckchem.com/products/BIBF1120.html of flat adenomas, than standard white-light video colonoscopes, in a consecutive series of patients undergoing screening, diagnostic or surveillance colonoscopy by different endoscopists with similar expertise, in an outpatient clinical practice setting. MATERIALS AND METHODS Data for the study were collected from the computerized database of the endoscopy unit of our tertiary referral center.

Colonoscopy records included patients�� main details and family history for colorectal cancer, indication for colonoscopy (screening, diagnostic or surveillance), type of instrument used (standard white-light or HD+ plus i-Scan), name of endoscopist, and bowel preparation, defined on the basis of a modified Ottawa scale[47]. Records for each procedure included whether the cecum was reached or not and the reason for failure (inadequate cleaning, strictures, and pain during the procedure), complications during or immediately after the procedure, and number, location and characteristics of the lesions. Polyps or protruding lesions were defined as sessile (Is) or pedunculated (Ip), and nonprotruding lesions as elevated (IIa), flat (IIb), and depressed (IIc), according to Paris classification[48]. For each lesion, histological diagnosis was recorded. Size and location of the lesions were classified as follows: 0-5 mm, 6-10 mm, 11-15 mm, 16-20 mm, 21-30 mm, > 30 mm; right and left colon.

Withdrawal time was recorded for all screening colonoscopies, being these procedures the object of other studies. Images of each lesion were stored in the database. For each patient, pO2, heart rate, and blood pressure were measured and recorded before, during and at the end of the procedure. Data collection Over a 1-year period, all consecutive screening, diagnostic and surveillance colonoscopies in outpatients done by four expert endoscopists, each of whom had done 200-400 colonoscopies/year for at least 15 years and at least Drug_discovery 50 procedures with HD+ plus i-Scan definition equipped instruments were evaluated. The four endoscopists used the two endoscopy techniques in a random fashion, depending of the availability of the instruments. Colonoscopies in subjects younger than 18 years, with genetic-associated colon cancer risk conditions, acute gastrointestinal bleeding, or inflammatory bowel disease were excluded.

In each of the four ISG clusters (Figure (Figure4B),4B), ISRE was by far the most significantly enriched motif (Supplemental Figure 2). Figure 6 MARA reveals ISRE as the most Vismodegib hedgehog significantly upregulated motif. ISRE motifs are the binding sites for ISGF3 and also IRFs. ISGF3 is activated by IFN-���Cinduced phosphorylation of STAT1 and STAT2. IRFs are transcriptionally induced and are also regulated by phosphorylation (23). We therefore measured the expression of IRF mRNAs. Of the nine IRFs, only IRF1, IRF7, and IRF9 were upregulated by pegIFN-��2b in the liver (Figure (Figure6C).6C). IRF1 is transiently induced at the 4- and 16-hour time points. IRF9 is part of the ISGF3 complex, and its transcriptional activity depends on p-STAT1 and p-STAT2.

IRF7 is upregulated during the entire dosing interval of pegIFN-��2b and could also be involved in ISRE-mediated gene transcription. However, the transcriptional activity of IRF7 depends on serine phosphorylation by IKK-�� (24), a downstream component of cellular sensory pathways that are activated by viral pathogen�Cassociated molecular patterns (PAMPs). Unphosphorylated STAT1 does not prolong ISG induction. The central IFN-���Cinduced signal transducer and transcription factor STAT1 is itself one of the most strongly induced ISGs. Indeed, we found STAT1 mRNA strongly induced in the first 4 days after pegIFN-��2b injection (Supplemental Table 1). STAT1 protein was even upregulated during the entire 1-week dosing interval (Supplemental Figure 1).

The functional significance of the expression of large amounts of U-STAT1 protein is unclear, but, intriguingly, GSK-3 a recent paper described a role of U-STAT1 as an active transcription factor that prolongs gene transcription after dephosphorylation of p-STAT1 (25). In that work, thirty ISGs were found to be upregulated by U-STAT1�Cdriven transcription (25). We therefore hypothesized that U-STAT1 could be involved in the prolonged ISG induction by pegIFN-��2b. However, when we took the list of U-STAT1�Cinduced genes and investigated their expression during the first week of pegIFN-��2b therapy, we did not find them to be overrepresented in clusters 3 and 4 with late and very late induced ISGs, respectively (data not shown). We therefore decided to address the potential of U-STAT1 to induce gene transcription in a more rigorous way. To that end, STAT1-deficient U3A cells (26) were stably transfected with STAT1 wild-type (STAT1-WT) or a mutant STAT1 with a phospho-tyrosine acceptor site at position 701 mutated to phenylalanine (STAT1-Y701F). For both STAT1-WT and STAT1-Y701F, three clones with different STAT1 expression levels were selected.