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Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC.
Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs.
EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis.
These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.
Biliary tract carcinomas (BTCs) are rare primary malignancies originating from the epithelium of the biliary tree and lead to intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder cancers (GBCs). Most patients are diagnosed when the disease is unresectable and survival is poor, with less than 5% of patients surviving beyond 5 years [1,2]. Chemotherapy has a limited impact on the natural history of the disease and several drugs or drug combinations have been tested with response rates ranging from 0% to 40%. Phase II studies have demonstrated that the best results were obtained with gemcitabine (Gem) reaching a 36% of response rate and 15.4 months of median survival [3]. More recently a multicenter, randomized phase III trial (the UK ABC-02 trial) recruiting 410 patients with advanced BTCs demonstrated that the median progression free survival was greater with the association of Gem with cisplatin than Gem alone (8 vs. 5 months) [4].
Effective therapeutic agents based on a better comprehension of cellular and molecular pathogenesis of BTCs are required. Preclinical studies suggest that the Epidermal Growth Factor Receptor (EGFR), HER2, and their pathways have a crucial role in tumor growth [5]. The EGFR/HER2 signaling pathway exerts its biological effects via multiple signaling cascades including phospholipase C, Ca2+/calmodulin-dependent kinase (CaMK/PKC), Ras/Raf/Mitogen/Activated Proteine Kinases (MAPK), the phosphatidylinositol 3'-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), PI3K/Akt/GSK, and Janus-associated kinase (JAK)/signal transducer and activator of transcription protein (STATs) [6-8].
In addition, EGFR signaling regulates the synthesis and secretion of several different angiogenic growth factors in tumor cells, including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) [9].
In cholangiocarcinoma, as well as in normal cholangiocytes, bile acids activate the two main signaling pathways (Ras/Raf/MAPK and the PI3K/Akt/mTOR) via a TGF-α-dependent mechanism. Bile acid mitogenesis may facilitate the progression of cholangiocarcinoma and blocking the TGF-α/EGFR autocrine pathway attenuates bile acid-stimulated growth of cholangiocarcinoma cell lines [10-12]. On these bases, several lines of evidence may point to the usefulness of EGFR targeting as an adjuvant therapy in cholangiocarcinoma. We previously reported that 15% of biliary tree and gallbladder carcinomas had EGFR gene mutations in the tyrosine kinase (TK) domain and that the mutations led to activation of one or both of the EGFR signal transduction pathways [13]. Some of these mutations are identical to those previously reported to confer sensitivity to some TK inhibitors like erlotinib and gefitinib in non small cell lung cancer (NSCLC) [14]. However, these inhibitors are ineffective if used in the presence of mutations in EGFR downstream transducers, such as K-RAS, B-RAF, PI3K or phosphatase and tensin homolog deleted on chromosome 10 (PTEN) [15]. In NSCLC, increased copy number of the HER2 gene is associated with gefitinib sensitivity in EGFR-positive patients, thus supporting the use of HER2 FISH analysis for selection of patients for TK inhibitor (TKI) therapies [16].
Somatic mutations in the PI3K gene have been frequently identified in colon and gastric carcinoma, and glioblastoma, but rarely in other cancers [17]. Functional analyses have revealed that these mutations increased kinase activity and induced transformation. In addition, in vitro experiments have demonstrated that PI3K oncogenic mutations promote sustained PI3K signaling, conferring resistance to gefitinib-induced apoptosis [18]. The tumor suppressor gene PTEN, that counteracts the activity of PI3K, was frequently mutated in high-grade glioblastoma, melanoma, prostate, and endometrium cancers [19]. These mutations caused loss of PTEN expression, constitutive activation of Akt, and resistance to gefitinib [20]. In vitro models demonstrated that the re-establishment of PTEN expression restores sensitivity to gefitinib [21]. All these data have derived from in vitro studies or from different series of patients in which only single aspects are studied therefore not allowing for evaluation of these findings as a whole. Due to prior experience with anti-EGFR treatment in lung and colorectal cancer patients, it has become clear that only a minority of patients with specific molecular abnormalities can benefit from these therapies. Philip and coworkers reported some clinical activity of erlotinib as a single agent in cholangiocarcinoma, showing that 17% of patients were progression free after 24 weeks of treatment [22]. However, the lack of immunohistochemical and molecular studies did not allow the determination of which subgroups of patients would benefit most from these treatments. Strategies based on EGFR pathway targeting showed promising results [23-28].
Based on these premises, we decided that a careful investigation of EGFR- and HER2-related pathways in BTCs should be preliminary for clinical studies with targeted molecules, facilitating a guide to monitor parameters that are predictive of response. Therefore, the objectives of the current study were to investigate EGFR and HER2 pathway expression and activation in histological sections from patients and to evaluate the in vitro efficacy of selective inhibitors of these pathways as single agents or in combination with gemcitabine in BTC cell lines.
Patients and tissues
The study was conducted on archival formalin-fixed tissues derived from 49 Italian patients with BTCs diagnosed at the Institute for Cancer Research and Treatment (IRCC) from 2002 to 2005. Tumor specimens were obtained, along with prior informed consent, before any systemic treatment. Approval of ethical committee was not needed for this study.
Histological type was determined according to World Health Organization criteria and tumor stage at the time of diagnosis was determined according to TNM classification system [29,30].
Patient characteristics, including sex, age, tumor origin, stage and histological grading are summarised in table 1.
Table 1. Clinicalpathological parameters of BTC case series
Erlotinib and gefitinib were gifts from Roche and Astrazeneca respectively. They are reversible selective inhibitors of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). They act competitively at the ATP-binding site of the EGFR in order to inhibit ligand-induced tyrosine phosphorylation, thereby blocking ligand-induced activation of the receptor and downstream pathways.
Lapatinib, sorafenib, and everolimus were purchased by Sequoia (Sequoia Research Products Pangbourne, UK).
Lapatinib inhibits receptor signal processes by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain, preventing self-phosphorylation and subsequent activation of the signal mechanism.
Sorafenib is a multikinase inhibitor that blocks tumor cell proliferation and angiogenesis by inhibiting serine/threonine kinases (c-RAF, mutant and wild-type B-RAF) as well as the receptor tyrosine kinases vascular endothelial growth factor receptor 2 (VEGFR2), VEGFR3, platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-KIT. It has also been reported that sorafenib induces apoptosis through the inhibition of the translation and down-regulation of myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member.
Everolimus is a signal transduction inhibitor targeting mTOR (mammalian target of rapamycin)
All drugs were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Gemcitabine (GEMZAR) was from Lilly USA, and is a nucleoside analog that interferes with DNA replication.
Cell lines and treatments
Four human cell lines of different histotype were used: two ECC cell lines- TFK1 and EGI-1 (WT and mutated on K-RAS respectively) kindly provided by Scherubl from the Institute of Physiology, Charité-Universitätsmedizin Berlin, Germany; one ICC cell line- HuH28 (mutated on PI3K); and one GBC cell line- TGBC1-TKB (deleted on PTEN) obtained from Cell Bank, RIKEN BioResource Center, Tsukuba, Japan. All cells were cultured in RPMI 1640 +10% FBS and 100 U/ml of penicilline/streptomicine. Cells were plated in 96 multiwell plates in a concentration of 3000 cells/well and incubated with 1:5 scalar doses of drugs (from 10 μM to 16 nM) for 72 hours. For combination experiments, drugs were administered concomitantly. Proliferation assays were performed by Cell Titer Glo Luminescent Cell Viability Assay and acquired by DTX880 (Beckman Coulter Inc). Drug interaction was assessed at a fixed 1:1 concentration ratio of gemcitabine and gefitinib/erlotinib/sorafenib/lapatinib/everolimus. The median Dose (Dm) inhibiting 50% of cell proliferation and its 95% confidence interval, isobologram analysis and dose-effect curves were calculated by CalcuSyn software (Biosoft, Cambridge, UK) based on Chou - Talalay method. The general equation for the classic isobologram is given by: combination index CI = (D)1/(Dx)1 + (D)2/(Dx)2 where (Dx)1 and (Dx)2 in the denominators are the concentrations for D1 (gemcitabine) and D2 (targeted drug) alone that give x% inhibition, whereas (D)1 and (D)2 in the nominators are the doses of gemcitabine and targeted drug in combination that also induce the same effect (inhibit x%). CI<1, CI = 1 and CI > 1 indicated synergistic, additive and antagonistic effects, respectively. Cell lines showing Dm>10 μM were considered resistant to drug treatments. All tests were performed in quadruplicate and repeated in three independent experiments.
Mutational analysis
Genomic DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Milan, Italy) following the manufacturer's instructions. Tumor portion and surrounding normal tissues were obtained by laser microdissector (VSL-337ND-S, Spectra-Physics, Mountain View, CA). Table 2 shows exons amplified by PCR with relative specific primers. The PCR products were purified using QIAquick PCR purification kit (Qiagen Milan, Italy) and sense and antisense sequences were obtained by using forward and reverse primers respectively. Each exon was sequenced using the BigDye Terminator Cycle sequence following the PE Applied Biosystem strategy and Applied Biosystems ABI PRISM3100 DNA Sequencer (Applied Biosystem, Forster City, CA). All mutations were confirmed by two independent PCR experiments and their somatic origin was demonstrated whenever possible excluding the presence of the same mutation in surrounding normal tissues.
Table 2. Sequences of forward and reverse primers (5'-3') used for PCR and sequencing
EGFR and HER2 expression was evaluated using PharmaDX (DakoCytomation, Carpenteria, CA) and HercepTest (Dako) kits respectively following manufacturer's instructions. EGFR intensity was scored from 1+ to 3+ and the threshold for positivity was 1+ staining intensity in 1% of tumor cells. HER2 staining was scored from 0 to 3+ using the scoring system outlined in the Dako HercepTest.
The anti-human TGF-α monoclonal antibody (clone 213-4,4; Oncogene Research Products, Cambridge, MA) was used for TGF-α expression established semiquantitatively from the percentage of TGF-α+ cells and the staining intensity. Tumors were graded as negative (0 to less than 10% positive cells), + (≥10% to < 25% positive cells), ++ (>25% to < 50% positive cells) and +++ (> 50% positive cells). The percentage of immunopositive cells was calculated by counting at least 1,000 cancer cells in contiguous fields with the greatest immunopositivity.
For EGFR downstream signaling detection, rabbit polyclonal antibodies anti-p-MAPK/Thr 202/Thr 204 and anti-p-Akt/Ser 473 (all from Cell Signaling Technology) were used. Positive immunostaining was scored + when present in more than 40% of cells and attributed to nuclear staining for p-MAPK and cytoplasmatic with a faint membranous staining for p-Akt.
PTEN expression, detected with the primary antibody anti-PTEN (clone 28H6, NeoMarkers, Fremont, CA 94539TS 106) was quantified by using a visual grading system based on the intensity of staining and the percentage of positive nuclei calculated counting of 1000 cells in 3 different optical fields at 40× magnification. PTEN immunostaining was scored into four groups from 0 to 3 as TGF-α scoring.
Western blot analysis
Cells were lysed with boiling buffer (SDS 2.5%, TRIS HCl 0.125 M pH 6.8), scraped, boiled for 5 minutes at 100°C and centrifuged at 14,000 rpm for 30 minutes; 40 μg of proteins were electrophoresed on SDS-PAGE and transferred to 0.45-μm PVDF membranes (GE Healthcare). Nonspecific sites were blocked with 5% non-fat dry milk (BioRad Laboratories, Munchen, Germany) and membranes were immunoblotted with specific primary antibodies overnight followed by 1 μg/mL horseradish peroxidase conjugated secondary antibody. Antibodies against Akt, phosphorylated-Erk1/2 (Thr202/Tyr204), phosphorylated-Akt (Ser473), phosphorylated mTOR (Ser 2448), mTOR, anti-mouse and anti-rabbit antibodies linked with horseradish peroxidase were from Cell Signalling Technology (Beverly, USA); antibodies for Erk1/2, HER2, EGFR were from Santa Cruz Biotechnology; anti-PTEN and β-ACTIN were from MILLIPORE (Temecula, CA); antibody anti-vinculin was from Sigma (Sigma-Aldrich, St. Louis, MO, USA).
HER2 Fluorescence In Situ Hybridization
Tissue sections (4-6 μm) were placed on silane-coated slides, deparaffinised, dehydrated, enzymatically digested with a commercial kit (Vysis, Downers Grove, IL, USA) and denaturated at 75°C for 5 minutes. Spectrum-Orange-labeled HER-2 and Spectrum-Green-labeled centromere 17 references (PathVysion™, Vysis-Abbott) were denaturated for 5' at 75°C and were applied to each slide. Slides were incubated for 5' at 79°C for codenaturation and placed in a humidified chamber at 37°C overnight for the hybridisation step. After washing, chromatin was counterstained with DAPI II (Vysis, Downers Grove, IL, USA). An average of 40 nuclei were analysed at five different target areas using H&E-stained sections as histotopographic reference. Our criteria for HER2 gene amplification were a HER2/centromere 17 ratio ≥2. Chromosome 17 numerical status was referred as polysomic when multiple 17 centromeric signals were present in >20% of the cancer cell population.
Statistical Analysis
The R statistical language adaptation of Fisher exact test for non 2 × 2 contingency tables was performed in order to detect any significant association between histotype and the categorical variables describing the presence of mutations in EGFR or in specific signal transducers, the expression of EGFR, HER2, TGF-α, p-MAPK, p-Akt and PTEN. Mean quantitative values were compared using Student's t-test. The significance of difference between groups was assessed by a Pearson correlation analysis. A P value of less than 0.05 was considered significant. All P values were two-tailed.
Expression of EGFR/HER2 proteins and related transducers in biliary tumors
Immunoreactivity for EGFR was detected in all normal cholangiocyte and hepatocyte membranes. EGFR expression was present in all 17 ICCs: with an intensity of 3+ in 13/17 (76.5%), and 2+ in 3/17 (17.6%). One ICC with neuroendocrine differentiation was scored 1+ (5.9%). In the 19 ECCs, the expression pattern was more heterogeneous with 10/19 (52.6%) EGFR+ cases: only 5/19 (26.3%) were scored 3+, 3/19 (15.8%) 2+, 2/19 (10.5%) 1+ and 9/19 (47.4%) were negative. In GBCs 5/13 (38.5%) expressed EGFR; 4/13 (30.8%) were scored 3+, 1/13 (7.7%) was 1+ and 8/13 (61.5%) were negatives. EGFR+ cancers were significantly more frequent in ICCs than in ECCs or GBCs (table 3). No correlation was found between EGFR expression and histological grading in the different BTC subgroups. Figure 1 shows representative EGFR immunostaining.
Table 3. Expression of biomarkers and HER2 gene status in BTC samples from patients.
Figure 1. Pattern of EGFR expression in the biliary tumors. EGFR expression as typical cell membrane immunostaining: weak (1+ score; A); moderate (2+ score, B) and strong (3+ score, C). Magnification X400.
Werneburg et al [10] demonstrated that EGFR was activated by bile acids in a TGF-α-dependent manner. On this basis, we decided to investigate if a pathological upregulation of this ligand might occur in cholangiocarcinoma cells. The expression of this ligand was analysed by immunohistochemistry in 49 BTC samples from patients.
Twenty nine out of 49 BTC (59.2%) resulted positive for TGF-α expression; in particular 14 out of 17 ICCs (82.4%), 10 out of 19 (52.6%) ECCs and 5 out of 13 (38.5%) GBCs were TGF-α+. Twenty seven out of 49 (55.1%) carcinomas displayed positive immunostaining for both TGF-α and EGFR. There was a significant relationship between EGFR and TGF-α expression in BTCs (p < 0.001).
HER2 expression was performed in 10 ICCs, 19 ECCs and 10 GBCs, according to sample availability. Membranous expression was present in cancer cells, while normal cholangiocytes and stromal cells were negative. Seven of the 39 cases (17.9%) were HER2+; in particular 1/10 (10%) of ICC was scored 1+ and 1/10 (10%) GBC was 3+. Positive immunostaining for HER2 was detected in 5/19 (26.3%) ECCs. Figure 2 shows representative HER2 expression on BTC samples by HercepTest.
Figure 2. Pattern of ErbB2 expression by HercepTest in the biliary tumors. Scoring from 0 to 3+ (A, B, negative; C 2+ and D 3+). Magnification X200.
Phosphorylation status of downstream transducers MAPK (Erk1/2) and Akt was analysed by immunohistochemistry in all 49 BTCs. As shown in table 3, 10/17 ICCs (58.8%) presented p-MAPK and 13/17 (76.5%) were positive for p-Akt; co-expression of the two phosphorylated signaling proteins were detected in 10/17 (58.8%). On the contrary, in ECCs the p-MAPK or p-Akt were only detected in 7/19 (36.8%) with co-expression in 4/19 (21%). In GBCs the pattern of activated proteins was similar to that of ECC: 5/13 (38.5%) and 6/13 (46.1%) showed p-MAPK and p-Akt expression respectively, while the co-activation was found in 3/13 (23.1%). p-MAPK and p-Akt expression were higher in the ICCs compared to ECCs and GBCs (p < 0.05).
PTEN expression was observed in all BTCs and in normal cholangiocytes. Cancer cells showed a moderate or strong immunostaining, while normal cells presented weak immunostaining (score 2+ or 3+ vs 1+).
HER2 gene amplification
To determine if overexpression of HER2 protein is attributable to gene amplification, FISH analysis was performed on samples scored 2+ and 3+ by HercepTest. The two samples scored 3+ by HercepTest presented HER2 gene amplification. In particular HER2/centromere 17 ratio were 10 and 6.9, respectively. One out of 3 specimens overexpressing HER2 (scored 2+ by HercepTest) showed HER2 gene amplification with a ratio of 5.9. The remaining samples scored 2+ presented multiple 17 centromeric signals in more than 20% of tumor cells. The results of FISH analysis were shown in table 3.
Mutational analysis
The mutational analysis of exons 18 to 21 of EGFR in this series has been published in a previous work [13] and these results, together with nine additional cases, are summarised in table 4. In this study, we also performed a systematic mutational analysis of HER2, PI3K, K-RAS, B-RAF and PTEN signal transducers. Sample number 39 showed a novel EGFR mutation (TGG to TAG) consistent with a nonsense substitution (W817Stop), leading to the production of a truncated protein lacking the intracytoplasmatic tail, which contains the interaction sites with the signal transducer PI3K.
Table 4. Mutations of EGFR, HER2 and their transducers found in samples from patients.
No mutations in HER2 TK domain were detected; 40.5% of samples displayed a silent point mutation at codon 902 (CAG to CAA), both in homo/hemizygous and heterozygous status (data not shown).
The mutational analysis of the EGFR/HER2 intracellular effectors identified mutations in K-RAS, PI3K, B-RAF and PTEN (table 4). Five hotspot mutations in the helical and catalytic domain of PI3K were found in 4/49 specimens (8.2%): two were in codon 545 (ID 763 and ID 12458) and 1 in each of codons 546 (already described as substituted with other non synonymous codons, ID 766, ID 767, ID 6147, and ID 12459), 1047 (ID 775), and 1059. Three samples (6.1%) had point mutations of K-RAS (2 were the previously described G13 D substitution, ID 532, the third was a novel mutation, I24F) and 4 (8.2%) had the V600E mutation (ID 476) of B-RAF. Exons 5, 6, 7, and 8 of PTEN have been sequenced and 3 mutations were found in 2 samples. Namely, sample 23 had Thr to Ile substitution at codon 202 of exon 6 and Glu to Gly substitution at codon 235 of the exon 7; sample 29 had Phe to Leu substitution at codon 271 of exon 8. All PTEN mutations involved codons previously found in other tumors as bearing single base deletions or mutations (ID 5856, ID 5292 and ID 5821).
In 4 cases, mutations of multiple transducers were present simultaneously: sample 22 had mutated B-RAF and K-RAS; sample 23 had mutated K-RAS, PI3K and PTEN; sample 29 had mutated EGFR, PI3K and PTEN; sample 31 had mutated EGFR, PI3K and B-RAF (table 4).
The percentage of PTEN labeled nuclei in tumor samples with activating EGFR or PI3K mutations was higher than in tissues with EGFR and PI3K wild type displaying 62%+31 (score 3+) vs 39%+26 (score 2+) respectively (p = 0.032). In particular, in the cases with activating mutations involving EGFR and/or PI3K and not PTEN, the mean of PTEN+ cells was 80+19 (score 3+) (p = 0.002 vs wild type), suggesting that a compensatory change in the level of the phosphatase might counteract EGFR pathway activation. In agreement, the sample 39 harboring the EGFR stop codon mutation and, presumably, associated with an inactive pathway, had low PTEN expression (9% or score 0).
Correlation between clinical pathological parameters and biomarkers in BTCs
To test the association between histotype and the analysed biomarkers, a generalization of the Fisher exact test was applied. No association was found between histotype and the presence of EGFR mutations, whereas a highly significant association (p = 0.00004) was detected between histotype and EGFR expression (high in ICCs and ECCs and low in GBCs). A slightly significant association was found between cholongiocarcinoma and p-Akt expression (p = 0.01) and TGF-α expression (0.03). TGF-α and p-MAPK were more expressed in high grade tumors (p = 0.03058 and p = 0.04 respectively). EGFR mutations were more frequently observed in female gender (p = 0.04). The other parameters tested did not give significant associations with histotype.
Expression of EGFR/HER2 proteins and activation of related transducers in BTC cell lines
Expression of EGFR and HER2 proteins as well as their molecular pathways were evaluated by western blot analysis on four BTC cell lines. As shown in figure 3, all cell lines expressed EGFR and HER2 receptors. In particular, EGI-1 cell line expressed high levels of EGFR and HER2 proteins and low levels of PTEN. TGBC1-TKB cell line expressed high level of phosphorylated Akt, mTOR and MAPK suggesting sustained activation of these pathways. In addition, HER2 membrane expression was evaluated by immunocitochemistry. ICC cell line HuH28 showed the highest HER2 membrane expression, scored 3+, ECC cell lines EGI-1 and TFK-1 were scored 1+, while GBC cell line TGBC1-TKB showed the lowest HER2 expression (Additional file 1, figure S1).
Figure 3. Western blot analysis of EGFR/HER2 expression and phosphorylation of related signal transducers in BTC cell lines.
Additional file 1. Figure S1: HER2 expression in BTC cell lines. A) HuH28, scored 3+, B) EGI-1, scored 1+, C) TFK-1,scored 1+, D) TGBC1-TKB, HER2 negative
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After 72 h of treatment, everolimus was able to inhibit mTOR phosphorylation in all BTC cell lines, but did not influence Akt and MAPK phosphorylation (Additional file 2, figure S2).
Additional file 2. Figure S2: Western blot analysis on mTOR, Akt, MAPK phosphorylation after 72 h treatment with everolimus on BTC cell lines.
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Sorafenib down-regulated MAPK phosphorylation in all cell lines and did not influence mTOR and Akt phosphorylation (Additional file 3, figure S3).
Additional file 3. Figure S3: Western blot analysis on mTOR, Akt, MAPK phosphorylation after 72 h treatment with sorafenib on BTC cell lines.
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Lapatinib slightly down-regulated Akt phosphorylation in all BTC cell lines, but not MAPK nor mTOR (Additional file 4, figure S4).
Additional file 4. Figure S4: Western blot analysis on mTOR, Akt, MAPK phosphorylation after 72 h treatment with lapatinib on BTC cell lines.
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Gefitinib down-regulated Akt phosphorylation only in EGI-1 cell line (Additional file 5, figure S5) while erlotinib had no evident effects on Akt/mTOR and MAPK phosphorylation (Additional file 6, figure S6).
Additional file 5. Figure S5: Western blot analysis on mTOR, Akt, MAPK phosphorylation after 72 h treatment with gefitinib on BTC cell lines.
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Additional file 6. Figure S6: Western blot analysis on mTOR, Akt, MAPK phosphorylation after 72 h treatment with erlotinib on BTC cell lines.
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Antiproliferative effect of gemcitabine and EGFR/HER2 pathway inhibitors in BTC cell lines
The antiproliferative effect of different molecular targeted drugs blocking EGFR/HER2 receptor or pathways revealed a broad range of response in BTC cell lines (table 5 and figure 4). The ICC cell line HuH28 was resistant to all drugs except lapatinib (Dm = 2.02 μM). Lapatinib also inhibited proliferation of EGI-1 (Dm = 4.02 μM) and TFK1 (Dm = 5.25 μM), while TGBC1-TKB cell line was resistant. EGFR TKIs had a significant effect on ECC cell lines (gefitinib Dm = 1.8 μM in TFK1 and 2.48 μM in EGI-1 whilst erlotinib Dm = 2.59 μM in TFK1 and 5.72 in EGI-1), but no effect was revealed on the GBC cell line TGBC1-TKB. The multi-kinase inhibitor sorafenib had a high efficacy on EGI-1 (Dm = 2.06 μM) and a slight effect on TFK1 and TGBC1-TKB (6.2 μM and 5.9 μM respectively). A reduction of 50% of cell growth was obtained with a relatively low median doses of m-TOR inhibitor everolimus on TFK1 (Dm = 200 nM), on EGI-1 (Dm = 500 nM) and on TGBC1-TKB (Dm = 400 nM).
Table 5. Median dose and relative confidential interval values of EGFR/HER2 pathway inhibitors on BTC cell lines.
Figure 4. Dose effect curves of gemcitabine (gem) combined with EGFR/HER2 pathway targeted therapies on TFK1 (A); EGI-1 (B); HuH28(C) and TGBC1-TKB (D). After 72 h of treatment with 1:5 scalar doses (10 μM, 2 μM, 400 nM, 80 nM, 16 nM) of gem alone or in combination with targeted therapy at a fixed 1:1 ratio, antiproliferative effect was evaluated using viability test and Chou-Talalay equation by CalcuSyn software as described in the Methods section.
The chemotherapeutic agent gemcitabine was highly efficient on EGI-1 (Dm = 0.74 nM), moderate efficient on TFK-1 and TGBC1-TKB (Dm = 100 nM, Dm = 280 nM respectively) and ineffective on HuH28 (Dm > 10 μM) (table 6).
Table 6. Median dose and relative confidential interval values of gemcitabine alone and in combination with EGFR/HER2 pathway inhibitors on BTC cell lines.
The combination of targeted drugs with gemcitabine allowed a significant reduction of median dose. Interestingly, erlotinib conferred sensitivity to gemcitabine in HuH28, resistant to the same drug as single agent and to all other combinations. In other cell lines, the best result was obtained with the chemotherapeutic agent and everolimus, highly efficient on extrahepatic cell lines, (Dm = 0.13 nM for TFK-1 and Dm = 0.57 nM for EGI-1) and gallbladder cell line (Dm = 1 nM). For the other combinations, responsiveness depended on cell lines.
The increasing of global incidence, poor prognosis and lack of effective therapy make the management of BTCs further emphasize the need of effective therapeutic agents.
Gene status and protein expression of EGFR and HER2 and their pathways may be potential biomarkers for predicting the response to EGFR/HER2 inhibitors.
We observed the presence of 100% EGFR expression in ICCs, 52.6% in ECCs and 38.5% in GBCs.
Mutations in the EGFR TK domain were present in 15% of cases [13]. Furthermore, the incidence of K-RAS mutation was particularly low (6.1%). Interestingly, changes involving codon 12, frequently mutated in other tumor types, were not found in our series. Previous studies of K-RAS mutations in cholangiocarcinoma revealed divergent results [31-35]. A higher occurrence of K-RAS mutations was found in Japan and Germany (ranging from 39% to 54%) relative to other areas such as Thailand (from 0% to 8%) in which this tumor occurred with high frequency. Geographical differences in etiology or carcinogenesis of BTCs might explain this variability.
We observed a lower incidence of B-RAF mutations compared to that reported by Tannapfel and coworkers (8% vs. 21% respectively) [36].
We identified PI3K mutations in 4 cases (8.2%) and PTEN mutations in 2 cases (4.1%).
Multiple mutations of EGFR transducers were observed in some samples. Namely, a total of 14 mutations were found in 8 tumor samples and only 3 samples had a single point mutation. Consistent with previous reports [37,38], the K-RAS and EGFR mutations were not present in the same sample but, in contrast with another report [36], K-RAS and B-RAF mutations were simultaneously present in one case. However, due to the genetic heterogeneity of tumor subclones, we cannot exclude that these mutations would be present in two distinct cell populations.
We observed a rare frequency of PTEN mutations and we did not find any loss of PTEN protein expression in comparison with normal cholangiocytes; rather a stronger labeling intensity and a high percentage of labeled cells were significantly present in tumor cells compared to normal counterparts. In particular, samples displaying EGFR pathway activation due to transducer mutations had the highest percentage of PTEN-labeled cells suggesting that a preserved PTEN function might counteract the EGFR downstream pathway activation. HER2 was overexpressed only in a small group of patients, in accordance with the results obtained by others [39] and no mutations on the TK domain were observed.
The inhibition of EGFR/HER2 pathways in BTCs cell lines demonstrated a broad range of response with EGFR TKIs being more efficient on ECC cell lines. In K-RAS-mutated EGI-1 cells the Dm of these drugs was twice the Dm on K-RAS WT TFK1. Furthermore, the presence of PI3K mutation and PTEN deletion in the HuH28 and TGBC1-TKB cells respectively could probably explain the resistance to these treatments [18,20,21]. Sorafenib was more effective in K-RAS-mutated ECC cell line in which the MAPK pathway had a high level of activation [40]. Lapatinib was more effective in ICC cell line which had high levels of HER2 expression.
On the other hand, lapatinib was less effective on ECC cell lines. Furthermore, GBC cell line was considered resistant (Dm >10 μM). In fact, TFK-1 and EGI-1 cell lines expressed low level of HER2 and GBC cell line was negative. These results are consistent with data obtained in breast cancer and pancreatic preclinical models [41-44]. In addition, a phase II study on breast cancer patients revealed that all the responders showed high level of HER2 expression while the HER2 negative patients were non responders [45]. In our study, the dual inhibition of EGFR and HER2 induced by lapatinib was less effective than the treatment with erlotinib and gefitinib in ECC cell lines. Indeed, in a study by Wiedmann et al., the treatment with NVP-AEE788, an EGFR/HER2/VEGFR-2, was more effective than erlotinib and gefitinib [46]. The direct inhibition of VEGFR-2 could be the gain of function of this drug compared to EGFR and HER2 inhibition. In fact, VEGFR-2 was expressed in ECC cell lines [47]. Moreover, VEGF was overexpressed in ICC and ECC samples from patients and regulated metastasis development [48]. The inhibition of VEGFR and EGFR/HER2 signaling with NVP-AEE788 or vandetanib (ZD6474) might be another interesting alternative approach for the management of BTCs.
Everolimus was effective in all tested cell lines but not in HuH28. Nonetheless, everolimus inhibited the phosphorylation of mTOR in all cell lines. It seems reasonable that in HuH28 a mechanism of resistance that overcomes mTOR inhibition may be active.
Moreover, EGFR/HER2 pathway inhibitors had synergistic effect with gemcitabine treatment. The mTOR inhibition gave rise to the strong synergistic effect in combination with gemcitabine in extrahepatic cell lines. Chung et al demonstrated that more than 80% of extrahepatic BTC displayed mTOR activation [49] that correlated with poor prognosis. Interestingly, EGFR inhibitor erlotinib was able to overcome the resistance to gemcitabine in the intrahepatic cell line HuH28; in fact, intrahepatic specimens showed the highest EGFR expression. Surprisingly, this result was not obtained with gefitinib.
Deepening this study, by gene expression profiling of the cell lines will contribute to the comprehension of the different mechanisms involved in drug response.
In conclusion, our preclinical results demonstrated that blocking EGFR/HER2 signaling resulted in considerable antiproliferative effects in in vitro models of BTC. The employment of targeted therapies may be useful in cholangiocarcinoma treatment and the analysis of EGFR/HER2 pathways in patients could orientate clinicians to the identification of appropriate therapeutic approach.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YP: designed the study, carried out the experiments, and drafted the manuscript; IS and MR: performed and analysed the immunohistochemical data and critically revised the manuscript; CPN: performed in vitro experiments, mutational analysis and drafted the manuscript; GC: supervised the study and supported with data interpretation; LC performed FISH analysis; GM and JYP performed mutational analysis; GC3: conducted statistical data analysis; MA, AB: participated in design and coordination of the study; FL conceived the study.
All authors read and approved the final manuscript.
The authors wish to thank Radhika Srinivasan, PhD for the careful revision of the manuscript. This work was supported by "Ricerca finalizzata regione Piemonte". Y. P was funded by "assegni di ricerca-Regione Piemonte".
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Pre-publication history
The pre-publication history for this paper can be accessed here:


text:
· How about a tax code that allows every working American to take home 100% of their paycheck every payday?
· What about a tax code that allows every American to transfer their wealth to their children, their grandchildren, or to any party they chose, whether in life or upon their death, with no tax consequences whatsoever?
· What about a tax code where labor and capital are not taxed at any level?
· And what about a tax code that accomplishes all of this, and more, without raising the price of consumer goods or cutting government spending. Pure tax reform --- not a politically targeted change in the tax code aimed at one segment of the population.
Such a tax reform proposal does exist. It's called The FairTax, and is currently before both the House and the Senate. This is a tax reform plan developed by accountants, economists, business people and ordinary citizens using $22 million in donated funds to develop the most researched piece of legislation ever placed before the Congress of the United States.
So now we wait for the response from the White House. The inclination of many is to believe that the White House will simply issue a statement to the effect that "The President understands the desires of the American people for tax reform and is dedicated to working with representatives of both parties to reach an agreement on comprehensive tax reform that will be fair to all." Well there you go … you get the words "comprehensive" and "fair" into the statement!
There is also, of course, the fact that I'm a harsh critic of the president – and that doesn't exactly spell success for the idea of a meeting. .
But … who knows? There could be some advantages to the White House in doing just what the petition asks … inviting me to the White House to put the FairTax plan before the president and his advisors. When the FairTax was first introduced into the congress Charlie Rangel, then the Chairman of the House Ways and Means Committee – the committee responsible for considering changes to our tax laws – told Congressman Linder that the FairTax was a wonderful idea. When Linder asked him why they didn't go ahead and pass it, Rangel responded that they would, but when the Democrats were in charge.
Well … the Democrats were in charge for two years, and nothing happened. Now the people have spoken through a petition – a petition for a redress of grievances – a petition for relief from an onerous tax code that punishes hard work, discourages achievement, and costs hundreds of billions of dollars a year to implement.
Here is the White House's chance to show that the whole petition process on Whitehouse.gov isn't just window dressing; that they will give serious consideration to a petition asking for a reasonable accommodation, a one-hour meeting. This is a chance for the White House to actually honor – and breath new life into – a provision in the First Amendment designed to empower the people.
We'll see. I'm not holding my breath – and I'm not clearing my busy retirement schedule yet – but we'll see.
"Your Republican Uncle": DNC debuts new website to help slow-witted young liberals talk politics with family on Thanksgiving | Allahpundit
Obama 2013: Assad must go. Obama today: Hopefully Assad won't run for president again. | Allahpundit

subset: commoncrawl


text:
My Nonprofit Reviews
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Review for Mary's Place, Inc., Windsor, CT, USA
I learned about Mary's Place about a year ago and knew immediately that I wanted to be a part of it. I wish there had been a place like this for me and my daughter 15 years ago when my husband died unexpectantly. We felt so ALONE! Mary's Place allows families to know that they are not alone. It is a wonderful, loving, safe environment for everyone to heal.
Role: Volunteer & I am a volunteer in a young children's group.


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Medications can be an
effective tool in treating a heart attack (myocardial infarction) and
preventing future attacks. The most commonly used medications for treatment of
heart attack include the drugs described in the following sections.
A heart attack is a
life-threatening emergency and requires immediate medical attention.
Also called "clot
busters," thrombolytic medicines are used immediately following a heart attack
in places where cardiac catheterization is not readily available. The medicine
is administered through an IV and works by quickly dissolving any major clots
in the arteries and restoring blood flow to the heart. Additional treatments or
surgery may be required if blood flow does not return to normal. Some examples
of thrombolytic medicines include:
Anticoagulants reduce the
risk of clotting in heart attack patients. They are more commonly referred to
as blood thinners. Examples of anticoagulants include:
Beta blockers are a class
of medications used to treat high blood pressure, as well as other diseases and
conditions. The medicines essentially make it easier for your heart to do its
job by blocking the effects of adrenaline and slowing the speed and force of
your heartbeat. Beta blockers are used to relieve chest pain and improve blood
flow after heart attack. Many studies over the last 40 years have found that beta blockers are often
considered standard treatment after a heart attack unless there was a
compelling reason not to use them. While these remain an important drug in
treating heart attacks, certain studies have questioned the routine.
Some examples of beta blockers used to relieve chest pain in someone
who's had a heart attack include:
Like beta blockers, angiotensin-converting
enzyme inhibitors (ACE inhibitors) treat high blood pressure and other
conditions. ACE inhibitors help relax and widen the blood vessels by blocking
the production of an enzyme that causes the vessels to narrow. ACE inhibitors
can improve blood flow, reduce strain on your heart, and help avoid further
damage to the heart muscle after a heart attack. They can potentially even help
the heart "remodel" itself to work better in spite of the damaged muscle
segments. Some examples of ACE inhibitors include:
Aspirin is the most well-known type of
antiplatelet medicine. These types of drugs prevent clotting in the arteries by
keeping blood platelets from sticking together. Antiplatelet agents are
typically used by people who have had a heart attack and are at risk for
additional clotting, particularly people who have just undergone thrombolysis
or revascularization through catheterization. Antiplatelets can also be used to
treat people with several risk factors for heart attack. Besides aspirin,
antiplatelet agents include:
Out of these medications,
only aspirin is commonly used as primary prevention to reduce the risk of heart
attack before one has actually occurred.


text: Samium Gromoff <_deepfire@...> writes: > A missing detail: while the vector is created in a live core all /seems/ > to be well[1]. But when it comes from a dumped core, it gets a chance > (there doesn't appears to be a determinism there) of being in a > write-protected region. Yeah, that'd explain it. We don't correctly restore information about whether a page is boxed or unboxed when loading a core (relevant bits in coreparse.c, and in gencgc.c:gencgc_pickup_dynamic). -- Juho Snellman


text:
Wednesday, November 13, 2013
© Copyright 2015 Albany Herald
VILANO BEACH, Fla. — It was early morning when I stopped here recently to see an old friend. I had invited him for coffee, but Marion Campbell, one of the highest-regarded defensive coaches ever in the National Football League, politely declined. For good reason, which we'll get into shortly.
Not only has he not enjoyed a cup of coffee lately, he has not had any solid food since May 21, almost six months ago.
I'm having conversation with a man who was a starting defensive end with the Philadelphia Eagles, a team which defeated the Green Bay Packers for the NFL title in 1959. It would be the only time Green Bay's Vince Lombardi lost a playoff game. At his peak with the Eagles, Campbell was a big man, weighing 250 pounds. Today, his weight is 220 pounds.
Campbell, a former Georgia lineman and Falcons coach, lives in St. Augustine, where he retired after a career as an NFL coach. The good news is that he is now allowed to drive after falling and breaking his neck in the spring. A genial sort, it was his idea to meet me halfway from St. Augustine to Ponte Vedra Beach. He enjoys getting out as his life slowly returns to normal. Driving his car, after his neck was fused, is a much-appreciated milestone.
On the day of his misfortune, the 84-year-old was watching television and got up from his easy chair. His feet got twisted up in a throw rug, causing him to crash head first into the wall. The accident broke vertebrae C-3 and C-4, and the C-5 and C-6 vertebrae were fractured.
Following surgery, his doctor told him he was lucky he was not paralyzed, becoming a quadriplegic. As the doctor was explaining his good fortune, he interrupted himself by saying, "Actually, you are lucky you aren't dead."
Known far and wide as the "Swamp Fox," a nickname given to him by the creative Dan Magill when Campbell was playing at Georgia, he played eight years in the NFL without serious injury. The fall in his den is a reminder that life often takes peculiar twists. Never a man for any soapbox speeches, the "Swamp Fox" is quick to note that he appreciates life now more than ever.
The medical explanation from a longtime friend, Dr. Truett Jarrard, explains Campbell's condition: "The cervical fracture has affected the reflex that routs material swallowed down the right channel. Instead of going down the esophagus into the stomach, food could go down the trachea into the lungs. This can cause what is known as aspiration pneumonia." And certain death. Campbell has had to live on a feeding tube as a result of the accident. He will undergo surgery on Nov. 19 in an attempt to correct the problem.
Already he has been asked what his meal of choice will be following the corrective surgery, most suggesting that he will probably want a big steak.
"No," is his standard answer. "I will choose a glass of Dick Vermeil's great wine." Vermeil, with whom Campbell coached for many years, produces a very good Napa Valley wine.
In an extended conversation, the Swamp Fox reflected on his career from high school in Chester, S.C., to Georgia to the NFL, where he became head coach of the Eagles and the Atlanta Falcons, twice.
With his size (6-4, 220) as a teenager, you can imagine the recruiting rush that heated up his last year in high school. That led to an interesting sidebar. In the '40s, there were only 11 grades in many states. When the South Carolina school system put in the 12th grade in 1947, Marion had already graduated.
His cap and gown were hanging in his closet and his diploma on his dresser when his high school coach saw an opportunity to keep him around for another season. He enrolled Campbell in class, making him one of the first 12th graders in the state.
By mid-season, opposing coaches knew something was adrift and began yelling at him, "Campbell, what damn grade you in?"
"How old are you, boy?"
"That extra season," Campbell explains, "turned out to be very important in my career. It allowed me to mature."
That second senior year, he really excelled. All colleges wanted him. He had committed to the University of Georgia and kept his word. He had visited Georgia in the spring following graduation. Line coach J. B. Whitworth took out a piece of paper and wrote down the starting lineup. He penciled Campbell's name in at starting right tackle.
"You wrote me down as a starter?" Campbell questioned Whitworth.
"Why not?" Whitworth replied. "You come here, we expect you to start." Campbell was sold.
Following his senior year, 1951, when he was hobbled by a bone spur in his foot and couldn't walk, he spent two years in the Army and entered the NFL (49ers) as a 25-year-old rookie. He would later distinguish himself as a defensive coaching guru in pro football.
Today, none of that is as important as good news from his doctor.
More like this story
- LORAN SMITH: Good things do happen to good people ( December 17, 2014 )
- LORAN SMITH: Costa was Georgia's first elite place kicker ( May 15, 2014 )
- LORAN SMITH: Brian Schottenheimer wanted to return to campus atmosphere ( March 17, 2015 )
- LORAN SMITH COLUMN: UGA's Brown took long journey to NFL ( February 12, 2014 )
- LORAN SMITH: Keith Marshall's NFL dream is still alive ( November 25, 2015 )


text:
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The Sony NEX-FS700 is the big brother to the FS100. Offering many of the same key features like an ultra-modular body design, detachable eyepiece, built-in stereo XLR inputs, peaking and zebras, SD card media recording, 4:2:2 uncompressed digital output and embedded timecode, and full 1080p HD recording up to 60fps, the FS700 steps it up even more. Some key feature upgrades include a new Super 35mm sensor capable of 4K resolution, HDSDI output in addition to HDMI, slow motion recording up to 960fps, built-in ND filters, photo shooting mode, and more. This kit includes one battery, detachable top handle, removable XLR microphone, eyepiece, detachable side handle, and AC adapter/charger. This item teams up great with the Metabones Canon EF to Sony E Mount Adapter or the Metabones Speed Booster EF to E Mount Adapter to mount all of your favorite Canon EF lenses right on the FS700--including the Canon and Zeiss cine primes.
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Recording/Playback Time- Not specified by manufacturer
BNC Connector- 3G/-HD-SDI / 4:2:2 / 8-bit (59.94p, or 50p) / 29.97p or PsF, 23.98p or PsF)
HDMI Output- HDMI 4:2:2 8-bit (59.97p & 50p, 29.97p, 25p, 23.98p)
Power Requirements- Not specified by manufacturer
Operating Temperature- Not specified by manufacturer
Dimensions (WxHxD)- Not specified by manufacturer
Weight- Not specified by manufacturer


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As much as famous folks like Wynton Marsalis and Harry Connick Jr. have burnished the city of New Orleans' role in jazz, they didn't do it by playing in the style that made the city famous.
Enter Michael White, beginning with a career-making sideman turn on Wynton's 1989 recording Majesty of the Blues. Seemingly untouched by the successive waves of R&B, soul, funk and hip hop, White's solo moments on that record harkened back to an earlier time — a much, much earlier time. And he was playing a period-piece instrument in the clarinet, to boot.
Just as importantly, however, while he continued to appear alongside Marsalis even while releasing a series of solo recordings, White didn't succumb to the city's cliched Bourbon Street sight-seeing impulses — that is, either to play up the washed-out Dixieland angle, or to follow in the footsteps of popular newer outfits like Rebirth or the Dirty Fozen in the marching-band style. White's was a wider, deeper vision: He's unreconstituted wonder, yet at the same time unafraid of mixing it up.
So, yeah, White's records, unlike Marsalis' sometimes too-studious efforts, often include all of the found-art looseness — the ragged, yet intuitive ensemble playing; a wide-eyed joyfulness that goes beyond the printed scores — which helped define the city's tradition in the first place. But also, as with Adventures in New Orleans Jazz, Part 1, something more.
White's roving eye ensures that his fifth project for Basin Street Records never falls into idiomatic lecturing. How could it with such adventurous song selections as the Paul Simon track "Take Me to the Mardi Gras?" Elsewhere, White adds new wrinkles to ageless tunes like "House of the Rising Sun," Mahalia Jackson's "His Eye is on the Sparrow" and "Sometimes I Feel Like a Motherless Child" — the first of which includes a stirring duet with banjo player Detroit Brooks. The last, played solo, is given a devastating context by the album's liner notes: Adventures is dedicated "to the memory of my mother and best friend, Mrs. Helen Forcia White (1922-2009)."
White also explores New Orleans' often-overlooked ancestral African and island influences, with his sometimes otherworldly clarinet as a tour guide. Adventures opens with "West African Strut," which includes a traditional thumb piano (sort of an African forefather to the xylophone), then seques directly into "Pata Pata" by the legendary South African chanteuse Meriam Makeba. Later, he delves in Bob Marley's "One Love," giving a tip of the hat to Jamaica, then offers a nearly seven-minute "Haitian Celebration." The album, performed alongside a crack group of musicians that includes trombonist Lucien Barbarin, trumpeter Wendell Brunious, pianist Steve Pistorius and drummer Herman LeBeaux, also includes four of White's own compositions — a list topped by the boozy delight "I'm Gonna Hoodoo to Get Your Love," with guest vocals by Thais Clark.
White, in tone and in song selection, brilliantly moves outside of convention — even as he celebrates foundation-building forms from long ago. This isn't the simple, if boisterous, sounds that pour out of every tourist-trap along the entertainment district, but a sweepingly inclusive reimagining of New Orleans' legacy music.
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Nick DeRiso
Latest posts by Nick DeRiso (see all)
- Daniel Davies + Sebastian Robertson – Condemned (2015) - November 25, 2015
- The Beatles' Love was a worthy concept not taken far enough - November 23, 2015
- R.E.M.'s 'Green' boasted more ambition that it did cohesion, but so what? - November 22, 2015


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Rental Cars at Annemasse (QNJ) Airport
Expedia.com makes it easy for you to book your next car rental from Annemasse. We work with the best QNJ car rental companies in the region, bringing you the best car rental rates and a wide variety of other QNJ car rental classes including economy, compact, midsize, full-size, convertible, van, luxury, full size SUV rentals. Our partner car companies offer different pick-up and drop-off options. If you are ready to book your car rental, follow the links on this page to view more information and details on your next Annemasse, QNJ car hire.


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Started as this, on the day I got it a couple years ago:
I was so excited, and dorky, that I had to do a requisite "JDM pose" which sucked:
Looked good from that side, but the previous owner had hit a deer:
Luckily, my buddy Bill (Celica XX on here) had a parts car for me:
So my friends came over and we did some work:
And now I have this:
And then I decided that it was time for some real rubber, so I got these:
And now, with the RS-8s on there, I have this:
'85 Celica Supra, P-type with a 6M-GE swap and a bunch of other goodies. It's almost time to send it to paint. I just gotta find a shop that will take care of the rust so I won't have to do it later when its worse.


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Parece que tu idioma preferido es Español.
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Whether you stayed at the bar until last call or watched an entire season of The Wire, your face doesn't need to broadcast last night's poor decision making. This hydrating eye cream does for your eyes what coffee does for your mental capabilities. The formula improves the appearance of skin under the eyes, erasing dark circles in a matter of seconds. You can actually see it working its magic and, more importantly, none of your coworkers will realize you were up till 2.
I use this most mornings when I remember to. It really helps your eyes feel great.
Ever since wearing contacts my eyes and eyelids have taken a beating. I'm constantly rubbing my eyes and pulling on them to get contacts out. This stuff helps that wear and tear - A+.
Hands down, the best morning eye cream ever. It reduces puffiness and helps with the dark circles (it doesn't erase them, but visibly helps). Although Kiehl's advertises this as a men's product, it's my favorite, as a woman. Thank goodness I didn't realize it was meant for men when I first ran across it.5
I was really excited about trying this product. Most mornings I wake up very groggy and stumble into work while my co-workers remark on how tired I look. Not anymore. Using this product is akin to the sensation of splashing cold water on your face and it lasts for hours. It's also a double espresso for your eyes. Highly recommend you give this product a try and see if it works for you.5


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|Reviews for Holly's Story
| sondrex76 chapter 1 . 8/20/2014
Looks interesting so far, thanks for writing and have a good day!
| Anonymous chapter 1 . 7/7/2013
I suggest you actually watch episodes if wolfblood than making p random stuff about thats nit true. There is no rose and kara is not even a wolfblood. Seriously ,, don't embarrass yourself
| The Liar And Honest chapter 1 . 12/27/2012
Kara and RoseArabel and Lily? XD
| Lily the Wolf chapter 1 . 12/18/2012
it's a-ma-zing! ok, maybe it was a little bit too similar to "where i used to think u got your inspiration from..." when they keep arguing. i loved it really xxx
| Luv2Luv chapter 1 . 11/27/2012
Really good!
| Mataki chapter 1 . 11/27/2012
I don't know the story but great describing!
She's a wolf right?


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Madison at Wells Branch Apartments3201 Century Park Blvd., Austin, TX 78727
- $829 - $1,234
- 1 - 2 Beds
Note : Prices & Availability Subject To Change
Madison at Wells Branch
Finding luxurious apartment homes in Austin, Texas has never been easier. Come experience the finest in apartment living at Madison at Wells Branch. Our warm and inviting apartment community is nestled in a lovely, residential neighborhood where everything you love is just minutes away. Our community is conveniently close to major highways, local shopping, fine dining and entertainment. Here, you will find beautifully manicured landscaping, meticulously styled floor plans and a commitment to excellence that is above the rest. * Madison at Wells Branch offers two spacious floor plans to choose from. Our beautiful one and two bedroom floor plans include unique features such as private patios or balconies, full size washers & dryers, and gas stoves. In select homes you will also find some other great features such as tile entry ways, and a pantry. * For those hot Austin days, residents can cool off in our refreshing swimming pool, or for the chilly nights they can relax in the heated spa. Experience maintenance-free living in a well-kept Austin community. * Visit our photo gallery and see for yourself why Madison at Wells Branch is the perfect place to call home! * *Promotional pricing only applies to new renters. New renters are people who have not lived in the community in the last 6 months. For renewal prices please see leasing consultant.
Madison at Wells Branch is located in the Austin Area in the 78727 zip code. View this apartment on a map.
Professionally Managed By: Northland Investment Corporation
Community Features
- Intrusion Alarm Available
- Dog Park
- Online Rent Payment Available
- Fitness/Health Center
- Garage
- Gate
- High Speed Internet
- On Site Maintenance
- OnSiteManagement
- Pool
- Public Transportation
- Short Term Lease
- Spa
- Tennis Court
- Volleyball Court
Floor Plan Amenities
- Balcony
- Dishwasher
- Pantry
- Dryer
- Fireplace
- 9 Foot or Vaulted Ceilings
- Large Closets
- Microwave
- Carpeted Floors
- Breakfast Bar
- Range
- Refrigerator
- Washer/Dryer in Unit
*Note: In Select Units
Pet Policies
Cat: Up to 2 are permitted
- * 75 lb weight limit. See Northland restricted breed list* * 2 pet limit * 1 Pet - $300 non-refundable pet fee and $15/month pet rent. * 2 Pets - $400 non-refundable pet fee and $15/month pet rent (per pet). * *The following is a list of all restricted dog breeds; the list applies to both purebred and mixed breeds. Management reserves the rights to deny any dog on the leased premises should the dog resemble any of the breeds on the list below. Management also reserves the right to ask an owne.
Dog: Up to 2 are permitted
Leasing Terms
2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months


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Agreement May Open Trade Avenues for Oklahoma
Wolfe, Lou Anne, THE JOURNAL RECORD
The proposed North American Free Trade Agreement certainly has provided excellent fodder for political commercials this election season.
Depending on who you listen to, the proposed treaty would either give a shot in the arm to segments of the national economy, or would assure the loss of many American jobs.
As for Oklahoma, Gary Miller, head of the international division of the Oklahoma Department of Commerce, said it could open trade avenues for some state manufacturers, but the agreement must be monitored closely in the areas of agriculture and energy.
"It's my belief that businesses that are not profitable now in the United States, and are having difficulties and closing down, leaving and going somewhere else _ whether to Mexico, Asia or some other place _ that's going to happen with or without NAFTA," Miller said.
"What will change is, the tariff that Mexico has on goods coming into Mexico will be reduced through NAFTA, and therefore will make our products less costly to the Mexicans," he said. "And one would assume we will then be selling more to the Mexicans as a result.
"That's the top side of it. If the Mexicans are buying more, then we're profiting from it."
There are issues involving grain and wheat exports that are important to Oklahoma, and they center not on Mexico but on Canada, Miller said. About three years ago, the United States signed a free trade agreement with Canada. Because Canada provides transportation subsidies, it has captured a 76 percent market share of wheat exports to Mexico, according to U.S. Rep. Glenn English, D-Okla.
Miller said the United States and Canada should go back to their free trade agreement and deal with the issue between the two, to try and get the wheat export market more competitive for the United States.
Regarding energy technology and related goods, the United States needs to make sure Mexico has a schedule for the privatization of energylated activities, Miller said.
"That is happening, and there are some aspects that are not yet open to foreign involvement," he said. "We would anticipate that as we move through time, and they're able to change their structure in this industry, that more of that (business) would become available to foreign buyers, sellers and competitors."
Pemex, which is Mexico's governmentned oil company, is a "sacred cow," and there are aspects that country is unwilling to let go of at this time, Miller said. . . Capital Access Program Finds Early Success
The Oklahoma Capital Access Program is finding early success in the marketplace, according to Oklahoma Commerce Secretary Greg Main.
Under the program, a lending bank decides who to lend to, then enrolls the loan in the capital access program. The borrower and bank put up equal guarantee fees that amount to a small percentage of the total loan. Then the Oklahoma Capital Investment Board matches the percentage, and the total guarantee goes into a loan loss reserve pool.
So far, 17 banks have enrolled in the program, and seven loans totaling just over $500,000 have been booked. Loan proceeds have gone toward purchase of a truck, renovation of a building, to build purchase inventory and for general working capital, Main said.
"That's an average of $70,000 per loan, in a market that hasn't been served well in the past," he told members of Oklahoma Futures this week.
The member banks are located in Altus, Blackwell, Chickasha, Claremore, Lawton, McAlester, Oklahoma City, Ponca City, Stillwater and Tulsa. . . South OKC Chamber Supports State Questions
The South Oklahoma City Chamber of Commerce has voted to support State Questions 649 and 650, which call for a $350 million bond issue for capital projects that would mostly benefit colleges and universities, according to Pam Holtzclaw, executive vice president. …


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Were I to ever live in Spain, specifically the magnificent city of Barcelona I think Vasava would be one hell of a place to work. They are still cranking and there is yet more inspirational work recently posted to their ever stalwart portfolio.
written by Christopher | tags: Art, Barcelona, Graphic Design, Vasava
One Response to "Vasava"
spicy aprons black white Says:
March 12th, 2015 at 3:13 am
Quality content is the key to interest the visitors to visit
the web page, that's what this site is providing.


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As the weather warms up for spring, I tend to do a lot more brunch entertaining. Egg dishes are always a good choice for brunch, whether you're talking about a simple plate of scrambled eggs or a quiche. One of my favorite brunch egg dishes is an Oven Baked Fritatta and I'm sharing my recipe over at the Craftsy blog (it's free!). The dish is ... Read More »
Tag Archives: brunch
Crepes Suzette
Thin, tender crepes are one of my favorite pancakes – though, admittedly, I like them all – because they are so versatile. The relatively plain batter can easily be made savory or sweet, depending on what you're in the mood for. When I want something simple, I just spread my favorite preserves onto freshly made crepes and roll them up. When I want to do ... Read More »
Chocolate Brunch Ideas for Mother's Day
Like Valentine's Day, Mother's Day is a day that many people celebrate with gifts of flowers and chocolate, as well as by going out to eat. I prefer to stay in on Mother's Day and have brunch at home. I know that I'll get great food when it comes out of my own kitchen and you get to skip the hassle of eating out on ... Read More »
Great Brunch Recipes for Mother's Day
Mother's Day is one of the busiest days of the year to go out to eat. And that is not necessarily a good thing. It may be fun to go out to brunch, lunch or dinner with Mom, but when a restaurant is jam packed with big groups of people, it's not quite the pleasant, intimate experience you might hope for. I like to skip ... Read More »
Cheesy Egg Casserole
I've had lots of interesting egg dishes at brunches and big breakfast get-togethers. Everything from frittatas to quiches to big piles of scrambled eggs with fresh herbs. One of my favorite egg brunch dishes is this cheesy egg casserole, which is packed with creamy cheese, can be made in advance, and serves a crowd of at least a dozen people with ease. Casseroles get a ... Read More »


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eunhyuk , Featured , IU , Kpop , NEWS , super junior
IU and Super Junior's Eunhyuk rumored to get married in October?Monday, May 27, 2013 / View Comments
Recently on various online communities, rumors about a popular female solo singer expected to marry in October, have surfaced. The post also said that the female singer who is currently acting in a popular drama will marry a member of popular boy band.
With IU currently playing the leading role in KBS drama 'You're the Best, Lee Soon Shin', IU and Eunhyuk have become the most searched terms online, relating them to the rumors. The suspicions came after their "scandalous" photo which was uploaded accidentally onto IU's Twitter back in November 2012.
However, IU's agency, Loen Entertainment denied the rumors saying, "It's false and we are dumbfounded."


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Panasonic DMR-BS850 Freesat Blu-ray recorder
Astounding feature list, versatility and ease of use, compressed high-def recordings, Blu-ray pictures, bitstream recording
Can't copy to USB, limited Freesat high-def content, copy restrictions on HD content, price
They've had Blu-ray recorders in Japan for donkey's years, but the lack of free HD content in the UK has delayed their introduction here. But the emergence of Freesat - now a year old - has finally made Blu-ray recording a reality for us Brits, and naturally Panasonic is the company to make it happen given its previous pioneering exploits in the Blu-ray and Freesat markets.
The company has launched two Blu-ray/hard-disk combis - the top-end 500GB DMR-BS850 on test here and the 250GB DMR-BS750 - as well as a Freesat-equipped DVD/HDD model. At £1000 the BS850 isn't cheap, but when you take a look at the vast array of features - many of which you won't find anywhere else - it's easy to see where your money is going.
The DMR-BS850 is the first hard-disk combi recorder to feature twin tuners, making it possible to record one channel while watching another, or record two channels while watching a previous recording. If you watch as much TV as we do, this will be music to your ears, and the presence of features like series recording makes it even more convenient to watch and record the programmes you love.
Because it's equipped with Freesat tuners, you can receive high-definition programmes from BBC HD and ITV HD for free and record them directly onto the hard-disk. To do this, the unit uses a "DR" recording mode, which captures the raw bitstream (as well as subtitles and audio descriptions) rather than decoding it first. That means recorded pictures look identical to the live broadcast and subsequently allow you to make a pristine digital copy on Blu-ray.
That said, you can't just make HD copies willy nilly. Broadcasters have the ability to flag high-def broadcasts and limit the number of copies that can be made. At present the BBC is flagging its programmes as "Copy Once" while ITV HDs are "Copy Never" although it's by no means set in stone - there's talk of BBC HD allowing unlimited copies later this year and ITV HD could have a change of heart. Until then, these restrictions could be a real turn off for people who like to share their favourite shows with family and friends.
When making a Blu-ray copy, the unit's MPEG4 H.264 encoder can compress the file at a lower bitrate but retain its high-def resolution, which takes up less space on the disc without massively compromising on picture quality.
Although high-def recording is the BS850's raison d'être, there are many more strings to its bow. It's also a high-spec Profile 2.0 Blu-ray player, equipped with Panasonic's lauded P4HD and PHL Reference Chroma Processor Plus technology, and it supports a healthy range of multimedia formats, including DivX, MP3, JPEG, AVCHD and SD Video. All of these, except DivX, can be copied to the hard-disk from USB or SD card (but not vice-versa), turning the BS850 into a multimedia hub, and it can even rip CDs and tag them automatically using the pre-installed Gracenote database.
Rear panel connections include HDMI, Component, S-Video and Composite video outputs, Scart input and output, two digital audio outputs and analogue stereo output. The Ethernet connection on the back not only allows you to download BD-Live movie extras but also lets you browse videos on YouTube or photos on Google Picasa through the Viera Cast feature. The Ethernet port will also provide access to the BBC iPlayer when it becomes available on Freesat later this year.
The BS850 isn't a bad-looking machine around the front, with slim dimensions, a dark mirrored fascia and legible display panel. On the front you'll find a USB port and SD card slot, a DV input for camcorder dubbing, plus S-Video, Composite and stereo audio inputs, reflecting the designs seen in Panasonic's DVD recorders in the past.
In action the BS850 is wonderfully intuitive, despite its potentially confusing array of functions. Channel tuning is quick and painless, while the onscreen displays like the EPG, Direct Navigator menu (where you find your recordings) and editing screens are straightforward. To cap it all, the button arrangement on the remote is superb.
High-def recordings from BBC HD look impeccable, with the BS850 retaining every last scrap of detail in the original broadcast. Programmes like Bleak House and Reggie Perrin are reproduced with mesmerising depth and clarity, while vivid colours blaze from the screen. HD recordings compressed to HL (the lowest-bitrate high-def recording mode) look sharp and natural, with fewer artefacts than we expected.
And its performance with pre-recorded Blu-ray discs is every bit as impressive as the DMP-BD60 and BD80, offering sharp and clean 1080/24p pictures that clearly benefit from P4HD's awesome processing power. Even upscaled DVDs look incredible, with natural colour reproduction, remarkable detail retrieval and no edge artefacts to speak of.
The lack of multichannel analogue outputs on the back means you'll need an AV receiver with HDMI inputs to enjoy Dolby True HD and DTS HD Master Audio tracks from Blu-ray. If you've got one, you're in for a treat – the sound on offer is sharp, dynamic and fulsome. CD and MP3 playback are also hugely enjoyable.
The level of technological innovation offered by the DMR-BS850 made it the most eagerly anticipated product of the year, and thankfully it doesn't disappoint. The combination of twin Freesat tuners, Blu-ray recording, a huge hard-disk, wide multimedia support and sensational Blu-ray playback make it an irresistible proposition. Yes it's expensive but new technology always is - if you have the cash and don't fancy lining Murdoch's pockets then it's definitely worth every penny.
Our only grievances are the paucity of HD content on Freesat and the off-putting copy restrictions placed on high-def content, but both are out of Panasonic's control and will no doubt be rectified in the near future.
Best TV and movies to watch this weekend on Amazon, Netflix, NOW TV and more: MI: Rogue Nation, Ant-Man...
BBC Three will move to BBC iPlayer in early 2016, plans approved by BBC Trust
ITV bins ITV Player, introduces ITV Hub service instead
Amazon Fire TV tips and tricks: 15 things you probably had no clue you could do
- Best TV and movies to watch this weekend on Amazon, Netflix, NOW TV and more: MI: Rogue Nation, Ant-Man...
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text:
November 26, 2015
KNOXVILLE (WVLT) -- Tennessee offensive lineman Cody Pope has been released from UT Medical Center after being admitted to the hospital during practice Tuesday, according to a UT spokesperson.
UT did not divulge the reason for Pope's admission to the hospital.
Pope is expected to practice Wednesday.
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