text:
Kenji Sugimoto1,2, Mutsuhiro Ohata1, Jun Miyoshi3, Hiroyoshi Ishizaki3,4, Naotake Tsuboi1,5, Akio Masuda1, Yasunobu Yoshikai6, Masaya Takamoto7, Kazuo Sugane7, Seiichi Matsuo5, Yasuhiro Shimada2 and Tetsuya Matsuguchi8
1Division of Host Defense, Center for Neural Disease and Cancer, and 2Department of Anesthesiology, Nagoya University Graduate School of Medicine, Nagoya, Japan. 3Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan. 4KAN Research Institute, Kyoto, Japan. 5Division of Clinical Immunology, Department of Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan. 6Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. 7Department of Immunology and Infectious Diseases, Shinshu University Graduate School of Medicine, Nagano, Japan. 8Division of Biochemistry and Molecular Dentistry, Department of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
Address correspondence to: T. Matsuguchi, Division of Biochemistry and Molecular Dentistry, Department of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Phone: 81-99-275-6130; Fax: 81-99-275-6138; E-mail: firstname.lastname@example.com.
Published September 15, 2004Submitted: September 11,
2003; Accepted: July 27,
A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal–regulated kinase (ERK) activation and production of TNF-α and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2–independent manner. Peritoneal macrophages and bone marrow–derived DCs from Cot/Tpl2–/– mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2–/– macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2–/– macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter–binding protein (GA-12–binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2–/– mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA–guided vaccination.
Macrophages play a central role in the innate immune response to a wide range of bacterial components such as LPS, lipoprotein, peptidoglycan, flagellin, and bacterial DNA rich in the dinucleotide CG (CpG-DNA). On activation by these components, macrophages produce cytokines, such as TNF-α, IL-12, and IL-10, and secondary mediators, such as PGs and NO (1, 2). DCs also sense the types of pathogens and deliver critical information to antigen-reactive T cells. Upon activation by pathogens, DCs express high levels of MHC class II and costimulator molecules, such as CD80 and CD86, on their cell surface and become a major source of IL-12, which greatly potentiates IFN-γ–producing Th1-type T cell differentiation (3, 4). Thus, the maturation and cytokine production profiles of DCs are critical for the linkage between innate and adaptive immunity.
Understanding how bacterial components activate macrophages and DCs was greatly expedited by the identification of the Toll-like receptors (TLRs). The phenotypes of animals with functionally defective TLRs (both naturally occurring and artificially manipulated) suggest that each TLR is essential for the recognition of specific pathogen-associated molecular patterns (PAMPs) (5). Although TLRs share some downstream signaling molecules, various reports have also suggested difference among the biological responses induced by TLRs (5).
A serine/threonine protein kinase, Cot, also known as Tpl2, was originally identified as an oncogene by the SHOK cell transfection assay (6, 7) and also identified as a target for proviral integration in MoMuLV-induced rat T cell lymphomas and MMTV-induced mammary carcinomas (8, 9). Cot/Tpl2 belongs to the mitogen-activated protein kinase kinase kinase family and can activate both the extracellular signal–regulated kinase (ERK) and the JNK signaling pathways (10, 11). Cot/Tpl2 has also been implicated in NF-κB activation through IκB kinase complex or degradation of the inhibitory p105 protein (12–14), which has recently been reported to stabilize and inhibit Cot/Tpl2 (15, 16). Recently, Tsichlis and colleagues reported that the Cot/Tpl2 kinase plays an important role in LPS signaling (17, 18). According to their reports, Cot/Tpl2–/– macrophages are defective in ERK activation induced by LPS, while the activation of other MAPKs remains intact. The lack of ERK activation in Cot/Tpl2–/– macrophages results in impaired secretion of TNF-α and PGE2 — key regulators of both inflammation and immunity — in response to LPS (17, 18). A quite recent paper also indicated an essential role of Cot/Tpl2 in the downstream signals of CD40 and TNF-α receptor in B cells (19). However, it is largely unknown whether Cot/Tpl2 is also involved in macrophage responses to bacterial components other than LPS or in signal transduction of other types of immune cells.
In this study, using a separate line of Cot/Tpl2–/– mice, we demonstrate that Cot/Tpl2–/– macrophages undergo normal ERK activation and produce significantly more IL-12 in response to CpG-DNA than their normal counterparts. Similarly, Cot/Tpl2–/– DCs produced higher levels of IL-12 in response to LPS and CpG-DNA than WT DCs. Consistently, after OVA immunization and Leishmania major infection in vivo, Cot/Tpl2–/– mice showed antigen-specific immune responses polarized toward Th1 type.
LPS, synthetic lipopeptide, and CpG-DNA induce Cot/Tpl2 activation in macrophages. To address the involvement of Cot/Tpl2 in various TLR signals, we measured the Cot/Tpl2 kinase activity using GST–MAPK/ERK kinase-1 (GST-MEK1) as the substrate in RAW 264.7 cells stimulated with LPS, synthetic lipopeptide, or CpG-DNA. All 3 TLR ligands rapidly activated Cot/Tpl2 (Figure 1A). To rule out the possible contamination of LPS in the CpG-DNA reagent we used, human embryonic kidney (HEK) 293 cells transiently transfected either with mouse TLR9 or with mouse TLR4 combined with myeloid differentiation–2 (MD2) and CD14 were analyzed. The transfectants were solely responsive to CpG-DNA or LPS, respectively, as determined by NF-κB activation assays (data not shown). When the expression plasmid for Flag-tagged Cot/Tpl2 was cotransfected in this cell system, in vitro kinase assays with Flag immunoprecipitates revealed that CpG-DNA induces Cot/Tpl2 activation in HEK 293 cells transfected with mouse TLR9 but not in those transfected with mouse TLR4 plus MD2 and CD14. Thus CpG-DNA, but not the contaminating LPS, is capable of Cot/Tpl2 activation through mouse TLR9 (Figure 1B).
Cot/Tpl2 activation by TLR ligands. (A) RAW 264.7 cells were stimulated with 1 μg/ml LPS, 1 μg/ml synthetic lipopeptide, or 1 μM CpG-DNA for the indicated periods. In vitro kinase assays were performed with an Ab against Cot/Tpl2 and GST-MEK1 as the substrate. As a control, Western blot analyses were performed with Ab's against Cot/Tpl2 and β-actin. sBLP, synthetic bacterial lipopeptide. (B) HEK 293 cells were transiently transfected with Flag-tagged Cot/Tpl2 in combination with either mouse TLR4 plus CD14 plus MD2 (mTLR4) or mouse TLR9 (mTLR9). Transfectants were stimulated for 30 minutes with 1 μg/ml LPS or 1 μM CpG-DNA, respectively. In vitro kinase assay was performed with anti-Flag Ab on GST-MEK1 as the substrate.
Generation of Cot/Tpl2–/– mice. To address the physiological role of Cot/Tpl2 in TLR signaling, mice deficient for Cot/Tpl2 were generated by replacement of the 3′ half of coding exon 2 and the 5′ half of exon 3, encoding the ATP-binding domain (residues 123–224 of the total 467 amino acids of Cot/Tpl2 protein), with a neo-resistance gene cassette (Figure 2A). Mice heterozygous and homozygous for Cot/Tpl2 deletion were assessed by Southern blot analysis for a correct targeting event (Figure 2B). After heterozygous mating, Cot/Tpl2–/– mice were born at the expected mendelian frequency, with life expectancy no different from that of WT littermates. RT-PCR (Figure 2C) and Western blot analysis (Figure 2D) confirmed that Cot/Tpl2 mRNA and protein were absent in the generated Cot/Tpl2–/– mice.
Targeted disruption of the Cot/Tpl2 gene. (A) The schematic structure of the mouse Cot/Tpl2 gene with exon 1 (ex1) to exon 7 is shown at the top. A targeting vector was designed to remove a part of exons 2 and 3, encoding the ATP-binding domain of Cot/Tpl2. Homologous recombination was verified using informative restriction fragments and diagnostic probes as indicated. (B) Southern hybridization using EcoRI-digested DNA extracted from mouse tails identified the expected genotypes (7.5-kb WT and 4.2-kb mutant fragments). (C) RT-PCR analysis by Cot/Tpl2–specific primers in the targeted region (290 bp) identified the expected genotypes. (D) Western blot analysis of proteins extracted from the heart using anti–Cot/Tpl2 Ab for the nontargeted region identified each genotype (the 58-kDa and 52-kDa forms of the Cot/Tpl2 protein).
CpG-DNA, but not LPS or synthetic lipopeptide, induces ERK activation in Cot/Tpl2–/– macrophages. Using peritoneal macrophages from the Cot/Tpl2–/– mice, we examined the phosphorylation of ERK1/2 before and after stimulation with synthetic lipopeptide, CpG-DNA, or LPS using a mAb specific to the phosphorylated forms of ERKs. As previously reported (17, 18), Cot/Tpl2–/– macrophages showed defective ERK phosphorylation in response to LPS (Figure 3A). They were also defective in ERK phosphorylation in response to synthetic lipopeptide. In contrast, Cot/Tpl2–/– macrophages showed normal ERK phosphorylation in response to CpG-DNA. JNK1, JNK2, and p38 MAPK were normally activated in response to all 3 TLR ligands (Figure 3, A and B).
Cot/Tpl2 is not required for CpG-DNA–induced ERK phosphorylation. (A) WT and Cot/Tpl2–/– macrophages were unstimulated or stimulated with 1 μg/ml LPS, 1 μg/ml synthetic lipopeptide, or 1 μM CpG-DNA for 30 minutes. Western blot analyses were performed with Ab's against phospho–ERK1/2, ERK1/2, phospho–p38 MAPK, or p38 MAPK. (B) In vitro kinase assay of anti-JNK1 immunoprecipitates was performed on GST–c-Jun as the substrate. As a control, Western blot analysis was performed with anti-JNK1 Ab. (C) Cot/Tpl2–/– macrophages were pretreated with U0126 or staurosporine for 30 minutes, and then left unstimulated or stimulated with 1 μM CpG-DNA. Western blot analyses were performed with the Ab against phospho–ERK1/2 or ERK1/2.
As ERK appeared to be activated by CpG-DNA independently of Cot/Tpl2, we sought to analyze the molecular mechanisms of CpG-DNA–induced ERK activation in Cot/Tpl2–/– macrophages. We first tested the effects of a specific MEK1/2 inhibitor, U0126. ERK phosphorylation induced by CpG-DNA in Cot/Tpl2–/– macrophages was sensitive to pretreatment by U0126 (Figure 3C), which indicates that this signaling is MEK1/2–dependent. We obtained similar results from PD98059, another MEK1/2–specific inhibitor (data not shown). To explore the upstream activation mechanisms of the MEK/ERK pathway, we next utilized a PKC inhibitor, staurosporine. Pretreatment with staurosporine abrogated the ERK phosphorylation induced by CpG-DNA in Cot/Tpl2–/– macrophages, indicating that a protein kinase(s) sensitive to staurosporine, but not Cot/Tpl2, is critically involved in the MEK/ERK activation by CpG-DNA (Figure 3C).
Cytokine secretion profile of Cot/Tpl2–/– macrophages in response to LPS and CpG-DNA. To compare the regulatory roles of Cot/Tpl2 in cytokine production induced by LPS and CpG-DNA, both WT and Cot/Tpl2–/– macrophages were stimulated with LPS and CpG-DNA for 24 hours. As previously reported (17), the Cot/Tpl2–/– macrophages showed impaired TNF-α production compared with the WT macrophages in response to LPS. Although CpG-DNA induced a relatively small amount of TNF-α production, the induced TNF-α level was slightly lower in Cot/Tpl2–/– macrophages. In contrast, the CpG-DNA–treated Cot/Tpl2–/– macrophages showed significantly increased IL-12 production compared with the WT counterparts. Additionally, IL-10 production induced by LPS or CpG-DNA was enhanced in the Cot/Tpl2–/– macrophages (Figure 4A). To analyze the role of ERK activation in CpG-DNA–induced IL-12 production from macrophages, we pretreated the cells with U0126. At 1 μM of U0126, which was sufficient to inhibit ERK activation (Figure 3C), IL-12 production from both the WT and the Cot/Tpl2–/– macrophages was only moderately decreased (Figure 4B). Thus, ERK activation does not appear to be essential for CpG-DNA–induced IL-12 production in the presence or absence of Cot/Tpl2.
CpG-DNA–treated Cot/Tpl2–/– macrophages showed significantly increased IL-12 production. (A) WT (black bars) and Cot/Tpl2–/– (white bars) macrophages were stimulated with LPS or CpG-DNA for 24 hours. Levels of TNF-α, IL-12 p40, and IL-10 in culture supernatants were measured by ELISA. *P < 0.05 compared with WT. **P < 0.01 compared with WT. (B) WT and Cot/Tpl2–/– macrophages were pretreated with 1 μM U0126 and stimulated with 1 μM CpG-DNA for 8 hours. The level of IL-12 p40 in culture supernatants was measured by ELISA. Data are expressed as mean ± SD and are representative of 3 independent experiments. (C) WT and Cot/Tpl2–/– macrophages were treated for 2 hours with 1 μg/ml LPS or for 4 hours with 1 μM CpG-DNA. Total RNA extracted from peritoneal macrophages was subjected to Northern blot analysis and RNA protection assay (RPA). (D) WT and Cot/Tpl2–/– macrophages were unstimulated or stimulated with 1 μg/ml LPS or 1 μM CpG-DNA for the indicated times. Nuclear extracts were subjected to EMSA to evaluate DNA-binding activities of NF-κB or GAP-12. The same nuclear extracts were subjected to Western blot analyses with Ab's against c-Maf and histone H1.
Increased IL-12 mRNA in Cot/Tpl2–/– macrophages stimulated with LPS and CpG- DNA. It has been reported that TNF-α production by macrophages in response to LPS stimulation is regulated at both transcriptional and posttranscriptional levels, whereas IL-12 production is regulated mainly at the transcriptional level (20). To assess the levels of TNF-α and IL-12 mRNA in response to LPS or CpG-DNA, peritoneal macrophages were treated with LPS or CpG-DNA, and total RNA was extracted and subjected to Northern blot analysis or RNA protection assay (RPA). Surprisingly, unlike in a previous report (17), LPS-induced TNF-α mRNA levels were consistently lower in the Cot/Tpl2–/– macrophages than in the WT macrophages (Figure 4C). CpG-DNA–induced TNF-α mRNA levels in the Cot/Tpl2–/– macrophages were similar to those in the WT macrophages. Thus, the reduction of TNF-α secretion induced by LPS in the Cot/Tpl2–/– macrophages seems at least partly regulated at the transcriptional level. In contrast, IL-12 p40 and p35 mRNA levels were significantly higher in the Cot/Tpl2–/– macrophages than in the WT macrophages in response to both LPS and CpG-DNA (Figure 4C).
Changes of nuclear transcription factors in Cot/Tpl2–/– macrophages. Transcription of IL-12 p40 is regulated by the binding of positive transcription factors, such as NF-κB, C/EBPβ, and PU.1, to the 5′ transcriptional regulatory region (21). Recently, 2 repressors have been reported for the IL-12 p40 gene expression, namely c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter–binding protein (GA-12–binding protein; GAP-12) (21, 22). We thus performed electrophoretic mobility shift assay (EMSA) to analyze DNA-binding activities of the 5′ promoter of the IL-12 p40 gene in Cot/Tpl2–/– macrophages. Stimulation of the Cot/Tpl2–/– macrophages with LPS or CpG-DNA induced DNA binding of NF-κB at a similar level to that in the WT macrophages (Figure 4D). We next performed EMSA using the GA-12 site of the mouse IL-12 p40 gene, which is the binding site for the putative negative transcriptional regulator GAP-12 (21). In the WT macrophages, stimulation with either LPS or CpG-DNA induced protein binding to the GA-12 probe, whereas the protein-DNA complex was scarcely increased in the Cot/Tpl2–/– macrophages (Figure 4D). We then examined the nuclear content of c-Maf protein and found that c-Maf protein was increased after LPS and CpG-DNA treatment in the nuclei of WT macrophages. This c-Maf induction in the nucleus was severely impaired in the Cot/Tpl2–/– macrophages (Figure 4D). We also found that c-Maf mRNA levels after LPS or CpG-DNA stimulation were lower in the Cot/Tpl2–/– macrophages (data not shown), which indicates that c-Maf expression was inhibited at the transcriptional level in the absence of Cot/Tpl2.
Effect of Cot/Tpl2 deficiency on the maturation and IL-12 production of DCs. Like macrophages, DCs have been reported to express significant amounts of TLRs (5). In order to assess the roles of Cot/Tpl2 in TLR signals in DCs, we isolated immature DCs from the bone marrow of WT and Cot/Tpl2–/– mice and examined their maturation and cytokine secretion in response to LPS and CpG-DNA. The Cot/Tpl2–/– DCs treated with LPS and CpG-DNA did upregulate the surface expression of MHC class II, CD40, and CD86 (Figure 5A), indicating that Cot/Tpl2 is not necessary in the maturation of DCs. Unlike CD40 and CD86 expression, the MHC class II surface expression on untreated Cot/Tpl2–/– DCs was consistently higher than that on WT counterparts. The Cot/Tpl2–/– DCs produced significantly larger amounts of IL-12 in the presence of either LPS or CpG-DNA. Thus, as in macrophages, Cot/Tpl2 appears to play an inhibitory role in the IL-12 production in DCs (Figure 5B).
Maturation and cytokine production of DCs. (A) On day 5, bone marrow–derived DCs from WT or Cot/Tpl2–/– mice were left unstimulated (shaded histograms) or stimulated with 1 μg/ml LPS or 1 μM CpG-DNA (open histograms) for 24 hours. The DCs were subjected to MHC class II, CD40, and CD86 expression analyses by flow cytometry. (B) Immature WT (black bars) and Cot/Tpl2–/– (white bars) DCs (positively selected using magnetized Ab for CD11c) were unstimulated or stimulated with 1 μg/ml LPS or 1 μM CpG-DNA for 24 hours. IL-12 p40 in culture supernatants was measured by ELISA. Data are expressed as mean of wells ± SD and are representative of 3 independent experiments. *P < 0.05.
Increased Th1 response to OVA immunization in Cot/Tpl2–/– mice. Since IL-12 production from DCs is decisive in determining the nature of the antigen-specific T cell response that follows, we next analyzed the immune responses of Cot/Tpl2–/– mice in vivo after immunization with OVA. Cot/Tpl2–/– and WT mice were immunized in the footpad with OVA mixed with CFA, and adaptive immune responses were analyzed 9 days after immunization. T cells derived from the draining lymph nodes of Cot/Tpl2–/– mice showed enhanced proliferation in vitro in response to antigen stimulation compared with the WT T cells (Figure 6A), suggesting that the specific priming of T cells was enhanced in Cot/Tpl2–/– mice. We purified CD4+ T cells from lymph nodes using magnetic beads conjugated with CD4 Ab. CD4+ T cells from OVA-sensitized Cot/Tpl2–/– mice produced a significantly higher amount of IFN-γ and a lower amount of IL-4 on antigen stimulation than did those from control mice (Figure 6B).
Antigen-specific Th1 cell responses were enhanced in Cot/Tpl2–/– mice. (A) Lymph node cells were collected from WT (filled circles, n = 4) or Cot/Tpl2–/– mice (open circles, n = 4) on day 9 after immunization with OVA in CFA. Proliferation of antigen-specific T cells was analyzed by cell proliferation assay using cell culture supernatants in different concentrations of OVA for 72 hours. (B) CD4+ T cells from lymph nodes of WT (black bars, n = 4) or Cot/Tpl2–/– mice (white bars, n = 4) immunized with OVA in CFA were cultured in different concentrations of OVA with APCs for 72 hours. Cytokines in supernatants of antigen-stimulated pooled cells were determined by specific ELISA. (C and D) Serum was collected from WT (filled circles, n = 5) and Cot/Tpl2–/– mice (open circles, n = 5) before (day 0) and after (day 9) immunization with OVA plus CFA (C) or OVA plus alum (D). OVA-specific IgG2a, OVA-specific IgG1, and OVA-specific IgE were analyzed. The y axis indicates the OD value at 492 nm. (E) CD4+ T cells from lymph nodes of WT (black bars, n = 3) or Cot/Tpl2–/– mice (white bars, n = 3) immunized with OVA plus LPS or OVA plus CpG-DNA were cultured in the presence of OVA (1 mg/ml) with APCs for 72 hours. Cytokines in supernatants of antigen-stimulated pooled cells were determined by specific ELISA. Data are expressed as mean of wells ± SD and are representative of 3 independent experiments. *P < 0.05.
Analysis of antigen-specific Ig responses in the sera showed that the Cot/Tpl2–/– mice consistently produced higher amounts of OVA-specific IgG2a after immunization (Figure 6C). There was no difference of OVA-specific IgG1 and IgE concentrations between the 2 groups of mice. On immunization with OVA mixed with alum, a strong promoter of Th2 responses, OVA-specific IgG1 and IgE responses were decreased in the Cot/Tpl2–/– mice compared with the WT mice (Figure 6D). Moreover, we obtained similar results when LPS or CpG-DNA was used as an adjuvant. In Cot/Tpl2–/– mice, IFN-γ production is enhanced in CD4+ T cells sensitized with OVA mixed with CpG-DNA. In contrast, IL-4 production is decreased in CD4+ T cells sensitized with OVA mixed with LPS or CpG-DNA (Figure 6E).
Increased Th1 response to L. major infection in Cot/Tpl2–/– mice. Finally, we examined the immune response to L. major, an infection model in which adequate Th1 development is required for disease control. To test this, we infected WT and Cot/Tpl2–/– mice with L. major. We then monitored inflammatory responses to the infection by measuring footpad swelling for 12 weeks and measuring parasite loads in the popliteal lymph nodes after 6 weeks. Cot/Tpl2–/– mice developed lesions that were almost identical in size to those in WT mice (Figure 7A). Consistently, parasite load in Cot/Tpl2–/– mice was indistinguishable from that in WT mice (Figure 7B). CD4+ T cells from L. major–infected Cot/Tpl2–/– mice produced a significantly higher amount of IFN-γ and a lower amount of IL-4 than did those from WT mice after stimulation with the soluble L. major antigen in vitro (Figure 7C).
Increased Th1 response to L. major infection in Cot/Tpl2–/– mice. (A) The footpad swelling of age-matched WT (filled circles, n = 5) and Cot/Tpl2–/– (open circles, n = 5) mice infected with L. major was monitored on a weekly basis. (B) The number of L. major amastigotes in the popliteal lymph nodes of infected WT (black bars, n = 4) and Cot/Tpl2–/– mice (white bars, n = 4) was examined 6 weeks after infection (see Methods). (C) CD4+ T cells were isolated from the lymph nodes of L. major–infected WT (black bars, n = 4) or Cot/Tpl2–/– mice (white bars, n = 4) after 6 weeks of infection and cultured (2 × 105 per well in 200 μl of culture medium) with soluble L. major antigen (30 μg/ml) for 3 days. Cytokine production in the culture supernatants was examined by each cytokine-specific ELISA. Data are expressed as mean of wells ± SD and are representative of 3 independent experiments. *P < 0.05.
Properly stimulated TLRs activate various signal transduction pathways including NF-κB and MAPKs. Previous work had shown that Cot/Tpl2 is obligatory for the activation of ERK1/2 in LPS-stimulated macrophages (17, 18). We show here that all of the 3 TLR ligands examined, LPS, synthetic lipopeptide, and CpG-DNA, activate Cot/Tpl2, which indicates that Cot/Tpl2 is widely involved in TLR signaling. Interestingly, however, unlike LPS or lipopeptide, CpG-DNA still activated ERKs in Cot/Tpl2–deficient macrophages, which suggests that there is an alternative Cot/Tpl2-independent ERK activation mechanism in TLR9 signaling. The uniqueness of the CpG-DNA/TLR9 pathway has also been described in various other reports (23–25) and may partly be explained by the observation that TLR9 locates intracellularly while most other TLRs, including TLR4, locate on the cell surface (26). Thus, to gain access to the signaling compartment of the immune cells, a process referred to as endosomal maturation is required for CpG-DNA, but not for LPS (27).
The alternative pathway of ERK activation by CpG-DNA is sensitive to pretreatment with specific MEK1/2 inhibitors, which indicates that MEK1/2 is involved, probably directly upstream from ERKs (Figure 3C). ERK activation by CpG-DNA was also efficiently inhibited by staurosporine pretreatment. Since staurosporine was originally recognized as a PKC inhibitor, and 2 isoforms of PKC (ε and δ) were reported to be involved in LPS-induced signaling (28, 29), we then tested specific inhibitor peptides for PKCε and PKCδ. However, neither inhibitor peptide inhibited CpG-DNA–induced ERK phosphorylation in Cot/Tpl2–/– macrophages (data not shown). Thus, the precise mechanisms of Cot/Tpl2–independent ERK activation remain ambiguous.
In the Cot/Tpl2–/– macrophages used in our study, decreased TNF-α secretion in response to LPS is consistently accompanied by lower levels of TNF-α mRNA. This result is apparently inconsistent with the previous report, in which LPS-induced TNF-α mRNA levels were not decreased in Cot/Tpl2–/– macrophages (17). The reason for this discrepancy is currently unknown. However, in support of our data, several groups reported that inhibition of the MEK/ERK pathway using a specific MEK inhibitor (U0126 or PD98059) resulted in decreased TNF-α mRNA induction by LPS (30, 31), which indicates that the decreased MEK/ERK activity leads to lower levels of TNF-α transcripts.
Another intriguing observation in our study is the enhanced IL-12 production in the Cot/Tpl2–deficient macrophages and DCs. Because of its critical role in the immune system (3), IL-12 production needs to be tightly regulated. Among factors proposed to inhibit IL-12 production, IL-10 is supposed to be a major negative regulator, because IL-10 gene–deficient mice showed spontaneous onsets of autoimmune disease–like phenotypes associated with increased IL-12 production (32). However, changes in IL-10 production are not likely to be involved in the enhanced IL-12 induction in CpG-DNA–stimulated Cot/Tpl2–/– macrophages or DCs, for the following reasons. First, IL-12 mRNA increased rather rapidly after CpG-DNA stimulation (Figure 4C), indicating that IL-12 induction was likely to be directly activated by the treatment. Second, IL-10 levels induced by CpG-DNA are higher in Cot/Tpl2–/– macrophages than in normal controls (Figure 4A). The mechanisms of increased IL-10 in Cot/Tpl2–/– macrophages are not clear. It is possible that increased IL-12 drives IL-10 production through IFN-γ as a negative regulator of inflammation.
Previous reports indicated that MAPKs are critically involved in the regulation of IL-12 production in APCs. For example, activation of p38 MAPK has been related to enhanced IL-12 production, mainly at the transcriptional level (20). In contrast, Häcker et al. reported that inhibition of the MEK/ERK pathway by PD98059 or U0126 results in decreased TNF-α production and enhanced IL-12 production from APCs (33). In the current study, the mechanism of increased IL-12 production in Cot/Tpl2–/– APCs clearly did not involve ERK, since ERK phosphorylation remained intact in these cells; this indicates that Cot/Tpl2 deficiency increased IL-12 production by CpG-DNA without inhibiting ERK activation. Consistently, pretreatment with U0126 did not increase IL-12 production in WT nor Cot/Tpl2–/– macrophages in response to CpG-DNA stimulation (Figure 4B). Together, our data indicated that CpG-DNA–induced ERK activity is not essential in regulating IL-12 production in primarily cultured macrophages.
On the other hand, correlation between mRNA and protein levels of IL-12 is consistent with a previous report indicating that IL-12 expression is primarily regulated at the transcriptional level in monocytic cells (20). Our EMSA results indicated that the induction of the negative regulators of IL-12 p40 transcription (c-Maf and GAP-12) by either LPS or CpG-DNA is significantly inhibited in Cot/Tpl2–/– macrophages (Figure 4D). In contrast, DNA binding of a positive regulator, NF-κB, was similar between WT and Cot/Tpl2–/– macrophages. The 2 other reported positive regulators of the IL-12 p40 gene, Ets and C/EBPβ, did not appear to be activated by LPS signals (22). GAP-12, whose molecular identity remains ambiguous, is a protein that binds to the suppressor site named GA-12 in the IL-12 p40 transcriptional regulatory region (21). Overexpression of c-Maf, another negative regulator, selectively inhibits transcriptional activation of both IL-12 p40 and IL-12 p35 genes (22). Thus the possibility may exist that GAP-12 and c-Maf are identical. However, this possibility was denied in a recent report, which showed that overexpression of c-Maf decreased protein binding of the GA-12 site in the IL-12 p40 gene (22). Thus, Cot/Tpl2 seems to be essential for the induction of 2 negative regulators of IL-12 p40 mRNA transcription.
It has been well established that DCs play essential roles in dictating this Th1/Th2 balance. PAMP-stimulated DCs express higher levels of MHC class II and costimulatory molecules and secrete significant amounts of IL-12, thus strongly inducing T cell differentiation toward a Th1 phenotype. Among the TLR ligands, CpG-DNA is one of the most potent inducers of IL-12. In this study, both LPS and CpG-DNA induced significantly higher levels of IL-12 secretion in Cot/Tpl2–/– DCs while increasing MHC class II and costimulatory molecule expression. It is of note that, unlike expression of costimulatory molecules such as CD86 and CD40, expression of MHC class II in Cot-Tpl2–/– DCs is moderately enhanced before PAMP stimulation (Figure 5A). The molecular mechanism of the increased MHC class II expression in Cot-Tpl2–/– DCs is currently unknown. Increased MHC class II expression may contribute to the Th1/Th2 balance of CD4+ T cells by affecting the intensity of antigen presentation (34, 35). DiMolfetto et al. (36) reported that, in mice expressing reduced levels of MHC class II but similar levels of costimulatory molecules on DCs, T cell response to antigen stimulation is skewed to Th1 type. Thus, increased MHC class II expression is not likely to be responsible for the skewed Th1 response in the Cot/Tpl2–/– condition. Taken together, these findings indicate that Cot/Tpl2 is not essential for the expression of costimulatory molecules but is specifically involved in IL-12 production in TLR-mediated signals.
Consistently, immunization of Cot/Tpl2–/– mice with OVA and CFA led to effective proliferation of antigen-specific T cells and increased IFN-γ production. Since Ig class switching is regulated by cytokines from T cells (37, 38), we analyzed the profiles of antigen-specific Ig production in Cot/Tpl2–/– mice. Cot/Tpl2–/– mice exhibited increased levels of OVA-specific IgG2a production as compared with WT mice. On the other hand, immunization of Cot/Tpl2–/– mice with OVA plus alum, a strong inducer of Th2-type immune responses, resulted in less IgG1 and IgE production than in the control mice. These results are consistent with the in vitro data of high IL-12 production and suggest that systemic responses to exogenous antigen are polarized toward Th1 type in Cot/Tpl2–/– mice. Notably, a quite recent report has revealed that Cot/Tpl2 is essential in CD40-mediated ERK activation in B cells (19). This ERK activation is decisive in IgE induction by CD40. Thus, defective ERK activation in B cells might also be responsible for the changes of antigen-specific Ig subtypes that we observed in OVA-immunized Cot/Tpl2–/– mice. It should also be noted that, besides IL-12, other cytokines produced by DCs, such as IL-18 and IL-23 (39, 40), contribute to the preferential Th1/Th2 T cell responses. Thus, further study is warranted to analyze these cytokines in Cot/Tpl2–/– mice.
In this study, we also tested the relevance of skewed Th1 response of Cot/Tpl2–/– mice using L. major, an infection model in which adequate Th1 development is required for disease control. Consistent with our data using OVA, we confirmed that Cot/Tpl2–/– mice had enhanced Th1 responses against L. major challenge in CD4+ T cells (Figure 7C). It has been reported that mice lacking MyD88 were more susceptible to L. major infection than control mice (41, 42). In these reports, a major parasite molecule, lipophosphoglycan, activated innate immune response via TLR2. It is also presumed that TLR9 is involved in the recognition of L. major, since protozoal DNA is largely nonmethylated and contains CpG immunostimulatory sequence (43). We also confirmed that HEK 293 cells transiently transfected with mouse TLR9 were responsive to the soluble L. major antigen (K. Sugimoto et al., unpublished observation), which indicates that a molecule in L. major, most likely L. major DNA, is recognized through TLR9. Inflammatory responses and parasite elimination, however, were not strongly altered in the Cot/Tpl2–/– mice. Since the strain we used, C57BL/6, is genetically resistant to L. major infection, a further increase in Th1 response might not be necessary for the protozoan elimination in this mouse strain. Another possibility is that decreased TNF-α secretion by macrophages impaired the clearance of L. major in Cot/Tpl2–/– mice (Figure 4A). Recent studies using gene-knockout technology have revealed that TNF-α is essentially involved in parasite elimination and resolution of inflammatory response against L. major infection (44, 45).
In summary, the activity of Cot/Tpl2 in macrophages and DCs appears to be decisive in determining IL-12 production and Th1/Th2 balance both in vitro and in vivo. Since CpG-DNA–based adjuvant does not trigger the severe granulomatous reactions often seen with CFA, CpG-DNA's adjuvant property may be used beneficially in humans in the future. Simultaneous inhibitory operation of Cot/Tpl2 during CpG-DNA–guided vaccination may enhance Th1 response while further decreasing side effects caused by TNF-α. Thus, Cot/Tpl2 may be considered as a potential target for immune therapy of diseases in which Th1/Th2 balance plays an essential role, such as allergy, infectious diseases, and cancer.
Generation of Cot/Tpl2–/– mice. A targeting construct was made to replace the 3′ half of coding exon 2 and the 5′ half of exon 3, encoding the ATP-binding domain, with a neo-resistance gene cassette. CCE embryonic stem cells were transfected and selected as previously described (46). Homologous recombinants were verified by Southern hybridization, and the resulting Cot/Tpl2+/– embryonic stem cells were microinjected into embryonic day 3.5 C57BL/6J blastocysts and transferred to MCH pseudopregnant foster mothers to generate chimeras that were mated with BDF1 mice for germ-line transmission. Mice with mutant alleles were backcrossed with C57BL/6 mice for more than 6 generations. Genotyping was performed by Southern hybridization and PCR using primers in the neo gene (5′-CCGCTTGGGTGGAGAGGCTAT-3′ and 5′-TG-GTGGTCGAATGGGCAGGTA-3′) and in the replaced Cot/Tpl2 gene (5′-AGAACACAAAGAGAATGAATG-3′ and 5′-GCACCACAAAGAGTAGAAAAT-3′). After heterozygous mating, Cot/Tpl2–/– mice were born at the expected mendelian frequency, with life expectancy no different from that of WT littermates. RT-PCR (primers in the targeted region: 5′-TTCCCTGGCTGTCATCTGTCA-3′ and 5′-GCACGCCATTCTTTTCTTTGT-3′) and Western blot analysis (Ab against the nontargeted C-terminal region) confirmed that Cot/Tpl2 mRNA and protein were absent at the level of detection. All experiments were done in accordance with institutional guidelines of the Nagoya University Graduate School of Medicine.
Reagents and Ab's. LPS from Escherichia coli (serotype B6:055), synthetic lipopeptide (palmitoyl-Cys[(RS)-2,3-di(palmitoyloxy)-propyl]-Ala-Gly-OH), and synthetic CpG-DNA (5′-TCCATGACGTTCCTGATGCT-3′) were obtained from Sigma-Aldrich, Bachem AG, and Rikaken Co., respectively. A polyclonal anti–Cot/Tpl2 Ab (M-20), a polyclonal anti-JNK1 Ab, a polyclonal anti-p38 Ab, a polyclonal anti–c-Maf Ab (M-153), and a monoclonal anti–histone H1 Ab (AE-4) were obtained from Santa Cruz Biotechnology Inc. A polyclonal anti–ERK1/2 Ab, a phosphospecific anti–ERK1/2 polyclonal Ab, and a phosphospecific anti–p38 MAPK polyclonal Ab were purchased from New England Biolabs Inc. Anti–mouse CD3 complex, anti–mouse CD45R/B220, and isotype control rat anti-mouse IgG2a Ab's were purchased from eBioscience. Monoclonal Ab against MHC class II was obtained from VMRD Inc. Rabbit complement was purchased from ICN Pharmaceuticals Inc. Phycoerythrin-conjugated anti–mouse CD80 (B7/BB1), anti–mouse CD86 (B7-2), and anti–I-Ab and FITC-conjugated anti-CD11c were purchased from BD Biosciences — Pharmingen. GST-MEK1 was purchased from Upstate Biotechnology Inc. U0126 and staurosporine were purchased from Calbiochem-Novabiochem Corp. and Sigma-Aldrich, respectively.
Cell preparation and culture. Thioglycollate-elicited peritoneal macrophages were isolated and cultured as previously described (17). Briefly, mice were injected intraperitoneally with 2.0 ml thioglycollate broth (Sigma-Aldrich). Four days later, their peritoneal cavities were washed with 5 ml of cold PBS. Cell pellets were washed and cultured at 1 × 106 cells/ml for 2 hours. The dishes were washed twice with medium to remove nonadherent cells. At least 95% of the remaining adherent cells were macrophages according to Giemsa stains. To culture DCs, the bone marrow was isolated from the femurs and tibiae and depleted of erythrocytes. After washing, T cells, B cells, and MHC class II–positive cells were killed with a cocktail of Ab's (anti–mouse CD3 complex, anti–mouse CD45R/B220, and anti–MHC class II) and rabbit complement for 60 minutes at 37°C. Cells (1 × 106) were cultured in RPMI 1640 supplemented with 5% FBS, 10 mM HEPES, 10 mM sodium pyruvate, 50 μM β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 ng/ml GM-CSF. Media were replaced every 2 days. The phenotype of immature DCs on day 5 was routinely greater than 60% CD11c+ and greater than 95% CD11b+ without expression of CD8α. For ELISA, immature DCs were positively selected using magnetized Ab against CD11c (N418; Miltenyi Biotec Inc.). The resulting population was consistently greater than 85% CD11c+. The viability was greater than 90% according to trypan blue staining. On day 5, immature DCs, defined as CD11c+ cells, were stimulated with LPS or CpG-DNA for 24 hours. CD4+ T cells were purified from the draining popliteal lymph nodes by positive selection using magnetic beads conjugated with CD4 Ab (BD Biosciences — Pharmingen) according to the manufacturer's instructions. The purity of CD4+ T cells was confirmed to be greater than 98% by flow cytometry. CD4+ T cells (2 × 105) were incubated in a 96-well plate with various concentrations of OVA or medium only, in the presence of 1 × 106 irradiated C57BL/6 splenocytes as APCs, for 72 hours.
Immunoblotting, immunoprecipitation, and in vitro kinase assay. Cell lysate preparation and immunoblotting were performed as previously described (47). Immunoprecipitation and in vitro kinase assays were carried out as previously described (47) except for the immunoprecipitation of Flag-tagged Cot/Tpl2, in which Ab's were cross-linked to protein G beads.
Expression plasmids. The coding regions of mouse Cot/Tpl2 and TLR9 were amplified by RT-PCR from total RNA isolated from the mouse macrophage cell line RAW 264.7, or from spleen cells from C57BL/6 mice, and inserted, respectively, into the NotI-BamHI and NotI-EcoRI sites of the expression vector p3XFlag-CMV14 (Sigma-Aldrich), which encodes a C-terminal Flag epitope. The coding region of mouse MD2 was cloned into the KpnI-XhoI sites of a mammalian expression vector, pcDNA3.1(+) (Invitrogen Corp.). The coding region of mouse TLR4 was cloned into a mammalian expression vector, pcDNA4 Myc-His (Invitrogen Corp.). Mouse CD14 expression plasmid was constructed as previously described (48). The PCR primers used were: mouse Cot/Tpl2, 5′-TGCGGCCGCACCATGGAGTACATGAGCACT, 3′-CGGGATCCGCCGTATTCCAGGGTTGGTG; mouse TLR9, 5′-TTGCGGCCGCTCCCAACATGGTTCTCCGTC, 3′-CGGAATTCCCCTATTCTGCTGTAGGTCCCC; mouse MD2, 5′-GGGGTACCACCATGTTGCCATTTATTCTCT, 3′-CCGCTCGAGCTAATTGACATCACGGCGGTG; mouse TLR4, 5′-CGGGATCCACCATGATGCCTCCCTGGCTCC, 3′-TTTGCGGCCGCCGGTCCAAGTTGCCGTTTC. The structure of the constructs was confirmed by restriction enzyme mapping and DNA sequence analysis.
ELISA. The concentrations of TNF-α, IL-10, IL-12 p40, IL-4, IFN-γ (R&D Systems Inc.), and IL-13 (BD Biosciences — Pharmingen) in culture supernatants were measured with commercial ELISA kits according to the manufacturer's instructions. All samples were assayed in triplicate, and the data were presented as the mean ± SD.
Northern blot analysis and RPA. Northern blot analysis was performed as previously described (48). The TNF-α probe was prepared by RT-PCR using a pair of primers (sense, GGCAGGTCTACTTTGGAGTCATTGC; antisense, ACATTCGAGGCTCCAGTGAATTCGG) from total RNA of LPS-stimulated RAW 264.7 cells. RPA analysis was performed using the RiboQuant Multi-Probe RNase Protection Assay System and a DNA template set (mCK-2b) according to the manufacturer's instructions (BD Biosciences — Pharmingen).
EMSA. Nuclear extracts were prepared from thioglycollate-elicited peritoneal macrophages using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology Inc.) according to the manufacturer's instructions. EMSA was performed as previously described (48). The oligonucleotide sequences for EMSA were: NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′; GA-12, 5′-CCTCGTTATTGATACACACACAGAGA-3′.
Immunization and challenge. Mice were immunized via injections into each hind footpad with 50 μg of OVA (chicken egg albumin, fraction V; Sigma-Aldrich) in 25 μl of saline emulsified 1:1 in CFA containing 1 mg/ml of Mycobacterium tuberculosis (H37RA, heat-killed and dried; Sigma-Aldrich), 25 μl of saline emulsified in alum (Imject alum; Pierce Biotechnology Inc.), 25 μl of incomplete Freund's adjuvant (IFA) containing 30 μg of LPS from E. coli (serotype B6:055), or 25 μl of IFA containing 30 μg of CpG-DNA. The draining popliteal lymph nodes were isolated, and serum was collected on the indicated days for further analyses.
Cell proliferation assay. Cells from the draining lymph nodes were prepared after immunization of mice with OVA plus CFA. Lymph node cells (3 × 105/ml) were cultured in the presence of various amounts of OVA for 72 hours. The CellTiter 96 AQueous nonradioactive assay was used for the measurement of cell proliferation according to the manufacturer's instructions (Promega Corp.).
Determination of antigen-specific Ig isotypes. Serum was collected from the mice before (day 0) and after immunization (day 9 for immunization with OVA plus CFA or OVA plus alum), and OVA-specific IgE, IgG1, and IgG2a were analyzed. Briefly, 10 μg/ml of OVA was used to coat a microtiter plate overnight and then blocked with 1% BSA in borate-buffered saline (0.05 M borate, 0.15 M NaCl, pH 8.6, 100 μl/well) at 37°C for 30 minutes. Diluted samples (100 μl/well) were incubated for 90 minutes at room temperature. The plates were washed with borate-buffered saline with 0.05% Tween-20 and incubated with peroxidase-conjugated anti-mouse IgE, IgG1, or IgG2a (Nordic Immunological Laboratories) for 90 minutes at room temperature. After further washing, plates were incubated for 20 minutes at room temperature with 100 μl/well of o-phenylendiamine solution (1 μg/ml with 3% H2O2), and ODs were read at 492 nm.
Flow cytometric analysis. Cells were stained with phycoerythrin- or FITC-conjugated Ab's at 4°C for 30 minutes. To block nonspecific FcR-mediated binding of the Ab, mouse CD16/CD32 Ab (eBioscience) was used. The cell surface expression of markers was assessed by a FACSCalibur (BD Biosciences).
L. major infection. L. major (MHOM/SU/73/5ASKH) was maintained by in vivo passage in BALB/c mice. For experimental infection, the parasites were collected from the footpad and expanded in Schneider's medium (Invitrogen Corp.) supplemented with 20% FBS at 25°C. Promastigotes were harvested and washed with PBS. The mice were infected with 5 × 106 promastigotes in the right hind footpad. The footpad thickness was measured with a vernier caliper, and the swelling caused by infection was determined by the subtraction of the thickness of the uninfected left hind footpad from that of the infected right hind footpad. Six weeks after infection, mice were killed, and popliteal lymph nodes were removed for analyses of antigen-specific cytokine responses. Lymph nodes were homogenized with steel mesh, serially diluted fivefold with Schneider's Drosophila medium (Invitrogen Corp.) containing 20% FBS, and incubated at 25°C. For 14 days after incubation, emerged promastigotes were monitored and the number of amastigotes in the lymph nodes was calculated by the maximal dilution in which promastigotes emerged. To prepare soluble leishmania antigens, 1 × 109 promastigotes were subjected to 3 cycles of freezing and thawing and homogenized.
Statistics. All values are expressed as mean ± SD, unless otherwise stated. Statistical analysis was performed using the Student's t test. A P value below 0.05 was considered statistically significant.
We thank K. Itano and A. Nishikawa for their technical assistance. This work was supported in part by grants from Ono Pharmaceutical Co., the Ministry of Education, Science, and Culture of the Japanese government, and the Yakult Bioscience Research Foundation.
Nonstandard abbreviations used: c-Maf, c-musculoaponeurotic fibrosarcoma; CpG-DNA, bacterial DNA rich in the dinucleotide CG; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal–regulated kinase; GA-12, GATA sequence in the IL-12 promoter; GAP-12, GA-12–binding protein; HEK, human embryonic kidney; IFA, incomplete Freund's adjuvant; MD2, myeloid differentiation–2; MEK1, MAPK/ERK kinase-1; PAMP, pathogen-associated molecular pattern; RPA, RNA protection assay; TLR, Toll-like receptor.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright © 2015
American Society for Clinical Investigation


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Full detailed profile on Avery Bailey Clark Played by Jessica Collins on The Young and the Restless Daytime Soap Opera.
Jessica Collins (CBS)
Birthday: April 1 1971
At Crimson Lights, Dylan tells Avery he cannot find out who is behind the real estate deal. Avery might be able to get a name. Joe appears. He has an engraved bookmark Avery left behind at their house. Dylan asks, "Anything else?" Joe leaves. Avery tells Dylan the bookmark was a wedding gift. Dylan questions Joe showing up with reminders of their marriage. Avery says the bookmark was from her dad - to mark the next chapter of her life. She assumes Joe was trying to be nice. Avery gets on the phone and learns Joe brokered the real estate deal and is buying the whole block. Dylan thinks it's personal. He wants to punch him. Avery thinks threatening legal action is smarter. Dylan leaves when she's in the back.
At the Club, Lily tells Cane she thinks Hilary's pregnant. Neil and Hilary arrive. Neil says Hilary's not pregnant yet, but they're eager to start trying. They sit to eat and Devon joins them. Cane eyeballs Devon and Hilary as Neil talks about having kids and asks Hilary if she'd like a girl or a boy. Hilary says this is already more family than she could imagine. Joe enters the Club and Colin asks to hear more about his plans. Cane joins them and questions Colin's interest. Colin talks about expanding the boutique and how this could be mutually beneficial. Cane is skeptical. Colin goes and Cane warns Joe about him. Joe thinks Colin is right - the thing with Victor made Cane skeptical. Cane likes working with his wife, but admits he misses the rush of the corporate deal. Across the room, Lily and Neil want to meet Devon's mystery lady. Devon says she's special. Hilary excuses herself for work. Upstairs, Devon joins Hilary, who complans about him discussing his mystery girl as they try to get the suite door open. They kiss. Colin sees them. Downstairs, Dylan enters with Avery behind him. He punches Joe.
At Crimson Lights, Dylan tells Avery his lease is up and he expects a new one any day. She thinks something's bothering him. Dylan says, "It's your ex." He thinks Joe wants something; that's why he's here. Avery reassures Dylan. Avery gets a call that Jeff's case against the hospital is moving forward. Dylan is outraged on Ben's behalf. Chelsea and Billy enter with the kids. Dylan asks, "You guys are living together?" Billy wonders if it's a problem. Dylan says it was just a surprise. They chat and Dylan tells Chelsea she and Connor seem happy. She wonders when Dylan and Avery will take the next step. Dylan says the coffeehouse is finally going good. Chelsea asks again, "What are you waiting for?" Later, Avery is glad Dylan and Chelsea are on good terms - the love doesn't disappear because their marriage ended badly. Dylan gets a call that the landlord is selling the building - he has two months to clear out!
At the Abbott house, Phyllis complains to Avery about Summer's marriage and what Austin did to her. Avery's trying to get past it. Phyllis would never get over a person hurting her that easy - and she'd never forget. Avery explains why Nick and Jack went along with the marriage and adds there's a lot Phyllis doesn't know about Austin - he was grieving his mother. Phyllis scoffs, "Light a candle." Phyllis is stunned to learn that Dylan saved Paul's life and that Paul and Nikki are his parents - meaning Nick is his brother. Phyllis realizes Avery dumped Nick for Dylan. Avery says they've become closer. Phyllis mocks the 'big happy family'. Avery says everyone ended up with the right person. Phyllis will never accept Nick belonging with Sharon. Avery feels she has no choice. Phyllis asks if she wants to bet. They bicker about Sharon. Avery says it's obvious everyone is where they should be. Phyllis says obviously she's the only one seeing things clearly. Later, Summer arrives with Nick. Summer says she's happy with Austin. Phyllis realizes what they're up to and it won't work. Nick sends Summer out and tells Phyllis that Summer can make her own decisions. Phyllis won't watch her ruin her life, anymore than she'll watch him ruin his. Nick says he's marrying Sharon tomorrow and she can't stop it. He leaves. Phyllis whispers, "Don't be so sure."
At Jabot, Jack commends Hilary for her work in New York. She knows he sent her because Neil asked him to. Jack hopes she got perspective. Kelly bursts in. She demands that he choose. Hilary goes. Kelly and Jack talk about him introducing her to Phyllis as a friend. Kelly says there won't be a right time and Phyllis needs to know. Jack needs to figure out how. Kelly can't do this anymore. She demands he make a choice now. "Do you want to be with Phyllis, or do you want to be with me." Jack kisses her and pulls her to the sofa. Avery enters. Kelly kisses Jack and leaves. Avery insists he tell Phyllis about Kelly. She warns Phyllis is also gunning for Nick and Sharon's relationship. By the elevator, Devon stops Hilary and asks if she's pregnant. Hilary tells him there is no baby. They talk about being together. It's not possible because of Neil getting hurt. Devon suggests a compromise - they don't tell Neil, and they don't stop seeing each other. Hilary reminds him Cane knows. Devon says they'll have to convince him they're no longer involved.
At the station, Dylan wants to help Stitch, who reminds him he did commit fraud. Victoria appears to check on him. Avery arrives and says someone pursued this actively - someone with an ax to grind against Stitch. Ben tells Vikki to go. She agrees, assures him he'll beat this, and then they'll continue their conversation. Ben tells Dylan they were getting close again. He has a couple ideas about who set this in motion. Kevin appears. Ben asks if it was him. Kevin says, "I ain't no snitch, Stitch." Avery reappears and says a former patient of Ben's is suing the hospital because he shouldn't have been practicing.
Jeff shows up at the station looking to bond with Kevin by watching him work. He wonders what's happening and looks at Avery, Dylan, and Ben, who is worried about the patient. He spots Jeffrey and realizes it's him. Jeff admits it. Ben scoffs. "I treated you for hemorrhoids!" Kevin is disgusted with Jeff. "I can't believe my mother remarried you." Everyone hollers at Jeff. Kevin throws him out. Dylan thanks Avery for helping - he'll always have Stitch's back.
At Avery's office, she introduces Dylan to Joe, who says Dylan should recognize him from his and Avery's bedside photo. Dylan tells him not to put Avery in the middle. Avery intervenes and explains Joe's in town on business. Joe says he came when Dylan was in jail. They tell him about the hoax arrest to nail Ian Ward. Joe makes nice and leaves. Dylan wonders when Avery was going to tell him her ex was in town. They bicker. Avery doesn't care what Joe is doing. She wants to celebrate. Dylan kisses her. She drags him into the conference room to 'confer'.
At his office, Michael confirms his appointment with Dr. Botnik. Avery appears. She's distracted with worry about Dylan. Joe Clark shows up looking for legal advice from Michael, who realizes he's the ex-husband. Joe says Cane recommended him. Michael agrees to review his documents and leaves for his appointment. Avery asks Joe what kind of game he's playing. He says it's nothing more than a coincidence, but he's concerned about her with Dylan locked up. She's fine and is standing by Dylan. Joe muses, "Like you stood by me."
Dylan arrives at Avery's office as she is bickering with Joe. She hugs Dylan. He tells her the plan worked; it's over. Joe says, "You must be Dylan."
« Back to Avery Bailey Clark profile

subset: commoncrawl


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Young Thug Still Running With 'Carter 6′ Title For His Next Project
Looks like Thugger is really going to call his next project Carter 6.
When it was first announced, Thugger said it was out of respect and that he'd like to carry on the tradition of Tha Carter series. But with all this drama between Wayne and Birdman, it's kind of hard to believe this is still a "respectful" move.
Read more


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My root .htaccess in a Magento store ensures that any visits not written as
(www.)domain.com/correct will go to our 404 (where "correct" is an existing sub-directory).
(www.)domain.com/admin is also allowed through.
I now need to also allow some other URLs that do not follow the
(www.)domain.com/correct form.
Presently the code in the .htaccess is:
RewriteCond %{HTTP_HOST} ^domain.com$ [NC]
RewriteRule ^(.*)$ http://www.domain.com/$1 [R=301,L]
RewriteCond %{HTTP_HOST} ^www.domain.com$ [NC]
RewriteRule ^$ go404 [R=301,L]
How can I make the following go through as well (ie NOT go404 on me)?


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"I absolutely loved this recipe, as did the rest of the table. Big hit, even with the kiddos. This one's saved at the top of my list!" – Marci
"This was so easy and DELICIOUS!" – skater734
"Delicious! My husband, who's very picky when it comes to steak, absolutely loved it! And my mother-in-law—who never eats when I cook—raved as well." – StephanieP
Recipe of the Day
Flank Steak Barbecue
A soy sauce and honey marinade give this excellent piece of meat a great flavor.
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Indiana runner to honor Boston Marathon victims
KOKOMO, Ind. - Cheryl Marner was a mile from the finish line at last year's Boston Marathon when all of the runners in front of her suddenly stopped.
The race had come to a halt, and at the time, she didn't know why.
"There was a lot of confusion," she told the Kokomo Tribune. "I heard a lot of sirens."
Slowly, she and the runners around her pieced together what had happened. Two bombs went off near the race's finish line.
She later found out those bombs killed three people and injured more than 250 others.
That could have been her, she said.
Marner injured her hamstring before the marathon and was running a bit slower than she normally does.
"My injury kept me behind the tragedy," she said. "I didn't see anything. It turned out to be a blessing."
It would take her four hours to get back to her hotel after the bombs hit. It was chaos around her, and everyone was afraid, she said.
Marner wouldn't say anything more about that day, though. It wasn't her story to tell, she said. We should be talking about those who were hurt or died in the blast, she said.
"What I went through was nothing compared to what they went through," she said.
Marner is headed back to Boston this week to run in Monday's marathon, which just happens to fall on her 55th birthday.
It will be her sixth time to run in Boston.
"There isn't anything I'd rather be doing on my birthday," she said. "I want to run in honor of the victims. I want to go back and cross the finish line for them."
Marner has spent the last six months training for the race. She'd run four or five miles a day, five days a week and then run eight to 10 miles on the weekend.
She gradually increased her mileage until she was running 22 miles on the weekend.
And not once did she run on the treadmill. Some days she trudged through the snow. Other days she added layer after layer to protect her from temperatures that felt like 30 below.
"Those days weren't the most fun days," she said. "But if you live in Indiana, you can't wait for the perfect day."
It's good practice for grueling conditions on race day, she said.
Marner didn't have to qualify for this year's marathon. She received an invitation to run, an invitation that was extended to about 5,000 people who almost finished the race last year before the bombs went off. Marner said about 4,000 of those people agreed to return.
She knows the atmosphere in Boston this year will be really special. Everyone is linked by the tragedy, she said.
"We all want to pull together and demonstrate Boston is strong," she said. "Boston is a special place. They open up their hearts and make us feel special."
She isn't at all afraid to go back to the site of the tragedy, she said. She's received emails about security upgrades for this year's race. She knows there will be a heightened police presence. It will probably be the safest place in the U.S. Monday, she said.
Even if it wasn't, Marner said she wouldn't hesitate to run the race.
"In the aftermath of Boston (last year) it reinforced the idea that anything can happen on any day," she said. "I don't want a senseless tragedy to stop me from doing something I love."
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Even though we ARE Nikon lovers,we are NOT affiliated with Nikon Corp. in any way.
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An gotcha, doesn't mean the item in my link doesn't call to me a bit but with this in mind I could make something myself to experiment with. Thanks! Bertotti
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Canada's Insurance and Risk Magazine
State Farm Mutual Automobile Insurance Company is suing a Toronto-based assessment centre and four individuals for more than $11 million, alleging that false and fraudulent documentation was submitted in the name of Assessment Direct Inc.
In a statement of claim filed Feb. 12 in the Ontario Superior Court of Justice, State Farm alleged treatment plans for motor vehicle accident claimants were filled out by Assessment Direct, and/or the individuals also named in the lawsuit, "in the name of health professionals who either never worked at Assessment Direct, were not employed by Assessment Direct at the time the treatment plan was assigned, did not have the qualifications represented on the treatment plan, and/or who never recommended the services and/or assistive devices allegedly that recommended therein."
The allegations have not been proven in court.
State Farm claims to have approved many of the requests, "paying approximately $1.4 million known to date, for services and assistive devices which were not recommended and/or provided by the health professional who allegedly signed the documentation."
Assessment Direct is located on Bathurst St. south of Lawrence St. in Toronto. According the corporate website, the firm's offerings include independent medical examinations, treatment programs and assistive devices, plus multi-functional evaluations for patients injured in auto accidents.
In its lawsuit, State Farm is seeking, among other things, $3 million in damages for fraud, fraudulent misrepresentation and/or unjust enrichment, $3 million in damages for conspiracy and $5 million in aggravated and/or punitive damages.
State Farm alleged in its lawsuit that it received insurance claims purportedly signed by two doctors who never worked for Assessment Direct and who allegedly did not give Assessment Direct permission to include their signature on claims. Two directors and two office managers were also named as defendants.
DisclaimerNote: By submitting your comments you acknowledge that Canadian Underwriter has the right to reproduce, broadcast and publicize those comments or any part thereof in any manner whatsoever. Please note that due to the volume of e-mails we receive, not all comments will be published and those that are published will not be edited. However, all will be carefully read, considered and appreciated.
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Pakistan News
News on Pakistan continually updated from thousands of sources around the net.
Russia calls for continuation of dialogue between Pakistan, India' : March 29, 2015,
2 hrs ago | Daily Times | Comment?
Diplomats and U.N. staff flee Yemen as Houthis target Aden
6 hrs ago | Reuters | Comment?
Chinese entrepreneurs interested in JVs, investment in Pakistan
10 hrs ago | The Nation | Comment?
Pakistan ranks 6th among high TB affected countries
14 hrs ago | Daily Times | Comment?
Terrorism to be eliminated totality in Pakistan: Gen Sharif : March 28, 2015,
18 hrs ago | Daily Times | Comment?
Pakistan: Over 32,000 arrested in anti-terror operations since Peshawar carnage
22 hrs ago | DNA India | Comment?
Who needs glasses 3/22/15
topix 288
Why Do You Think The German Co-Pilot Crashed The Plane ...
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18 Stars Who Swear By Juicing
Pakistan militants deny 30 killed in renewed Khyber fighting
Yesterday | The Star Online | Comment?
Pakistan making efforts for evacuation of Pakistanis from Yemen: FO : March 28, 2015,
Yesterday | Daily Times | Comment?
2 Czech tourists abducted by gunmen in Pakistan in 2013 released, return home
Yesterday | Brandon Sun | Comment?
That's purr-fect - Pakistani pet lion gives birth to five cubs
Yesterday | The Straits Times | Comment?
Load shedding in Pakistan unlikely to end by 2018: WB
Yesterday | Daily Times | Comment?
Pakistan's dictatorial past hovers around the demonic local body polls
Friday | The Nation | Comment?
Pakistan says hasn't promised to join Saudi coalition in Yemen
Friday | Channelnewsasia.com | Comment?
Bomb hits police bus in southern Pakistan, wounding 10
Thursday | The Washington Post | Comment?
LSE 2nd session on - Towards a strategy for Pakistan as a regional...
Thursday | Daily Times | Comment?
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Updated: Sun Mar 29, 2015 07:37 am
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PC - Windows
All-Star Uniforms for NBA 2K13Home / PC - Windows /
This page contains NBA 2K13 Codes for PC - Windows called "All-Star Uniforms" and has been posted or updated on Feb 8, 2013 by Yaposhka. Let us know what you think of this Code by thumbs voting.
Boxshot / Details
- Developer: Visual Concepts
- Publisher: 2K Sports
- Genre: Sports
- Release Date: Oct 2, 2012
- ESRB: Everyone
- Other Platforms
All-Star Uniforms
Use the following mwpzoslqov as a code to unlock All-Star Uniforms.
All Codes
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Stuck on this game? Set a specific, clearly defined question and let others answer your question or view answers to previously asked questions. If you think you are an NBA 2K13 expert then please try to help others with their questions.
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June 30, 2014
The cost of on-highway diesel moved slightly higher over the past week, gaining one-tenth of a cent for a national average price of $3.920 per gallon. The Department of Energy's weekly pump price survey shows diesel's average price is now 10.3 cents higher than it was one year ago.
Prices increased in all sections of the country except the East Coast as a whole, where it's down two-tenths of a cent from last week; the Lower Atlantic region, where it's down six-tenth of a cent, and the Midwest, where it's also six-tenths of a cent lower than last week.
Increases were modest across the country, with the highest increase coming on the West Coast. Prices there rose two cents over last week.
Other than California ($4.138), New England had the highest prices at $4.107, followed by the Central Atlantic region and the West Coast at $4.073.
The Gulf Coast region reports the lowest average price at $3.816.
All regions are higher than their year-ago averages.
The average cost of regular grade gasoline held at $3.704 again this week, but it's 20.8 cents higher compared to the same week a year ago.
Prices moved higher in all parts of the country except the Midwest and the Gulf Coast, where they fell 2.2 and 1.1 cents respectively from last week.
Prices range from a low of $3.483 in the Gulf Coast region to a high of $4.036 in the West Coast region.
Adequate U.S. crude supplies and speculation that the escalating violence in Iraq is unlikely to disrupt supply kept oil prices to their lowest price in the previous two weeks. It's closed in New York on Monday at $105.37 a barrel.
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text:
20 Things that happened on the Internet in 2011 by Syzygy. Via.
Two by Andy Helms.
Wolverine, The Hulk, and The Flash by Alex Pardee on sale here.
*Buy Adventure Time toys at Amazon.
Tuesday, January 24, 2012
Illustration roundup
Posted by John at 5:35 AM
Labels: adventure time, dc comics, marvel
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Beyonce and Jay-Z Vacay in Thailand
Beyonce and Jay-Z are better than us. Why? I spent NYE in my apartment, while they spent it in a luxurious resort in Thailand!
Post Jay-Z's 45th birthday vacation in Iceland, Jay Z, Beyoncé, and Blue Ivy jetted off to the island of Phuket last week to celebrate the new year. The party probably won't stop soon, because on Jan. 7th Blue Ivy turns 3 years old!
Popsugar boasts exclusive pictures of the couple, who were spotted lounging on the beach together this week in Thailand. eyoncé rocked a sexy striped bikini while Jay-Z relaxed in a t-shirt and swim trunks. The couple were in really good spirits- a witness says Beyoncé appeared to be laughing at something that popped up on her cell phone (which was, of course, decked out with a "Surfboard" cover).
I wish I could travel half as much as Bey and Jay!
Photo: FameFlynet
Filed under Beyonce, Jay-Z | No Comments


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Open Content images tend to be large in file-size. To avoid potential data charges from your carrier, we recommend making sure your device is connected to a Wi-Fi network before downloading.
This image is available for download, without charge, under the Getty's Open Content Program.
Not currently on view
Object Details
Inhabited Initial B
probably 1170s
Tempera colors, gold leaf, silver leaf, and ink on parchment
Leaf: 28.2 x 18.9 cm (11 1/8 x 7 7/16 in.)
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Within a large initial B, five men and various animals cavort among the vine tendrils in a scene characteristic of the Romanesque style. The figures include workers making wine, animals nibbling on luscious fruit, and two hunters.
Related Works
Songs of Praise: Illuminated Choir Books (July 23 to October 13, 2002)
- The J. Paul Getty Museum at the Getty Center, (Los Angeles), July 23 to October 13, 2002
Music for the Masses: Illuminated Choir Books (August 14 to October 28, 2007)
- The J. Paul Getty Museum at the Getty Center, (Los Angeles), August 14 to October 28, 2007


text:
Product Manager
You're sitting at your desk, staring in white-knuckled anticipation at your computer screen. You're tense, but you know you've crossed the t's and dotted the i's. Today's product launch, which you were responsible for, is now in the hands of millions of small business owners around the world. TechCrunch just published an article praising your project's potential. The CEO and your manager just walked over to your desk clutching two big bottles of champagne. All of your research, design, testing, and collaboration has paid off. This is the moment all Product Managers live for: the launch. Even better, the launch of something you really believe in, that has the potential to transform the lives of small business owners all over the world.
At FreshBooks you'll experience this thrill often, working alongside a group of world-class Developers, Designers, and Product Managers: the Product Development team.
The Product Development team at FreshBooks is responsible for defining and delivering new ways to help small businesses focus on what they love to do. We take the headache away from bookkeeping and accounting and replace it with a fun, intuitive set of tools that just make sense to small business owners.
You are probably already gainfully employed elsewhere right now, or have recently departed a world-class organization in search of a better way.
Here's what we can offer:
- A high-profile position in a product-led company. You'll have direct access to our CEO, CTO, and CMO several times a week
- A Scrum team of your very own that employs agile methodologies to deliver super-awesome-game-changing experiences at break neck speeds
- An incredible growth opportunity ; you'll learn a lot about what it takes to ship great software -- and you'll learn it from the best people out there
- The opportunity to deliver product features that instantly get used by millions of people around the world
- A seat at the table of one of Toronto's top performing and award winning Product Development teams where data and research matter more than opinions
- A life in Canada that includes company-sponsored wintertime shinny games, yearly trips to Muskoka, Raptors tickets, weekly free lunches, an open-concept & collaborative workspace and unpretentious co-workers who take pride in their work without the ego.
In return for this opportunity, here's what we'll expect from you:
- Show an undying passion to talk directly to customers to understand who they are, how they run their business, and empathize with the pain associated with managing their finances.
- Demonstrate an ability to synthesize what you've heard, and report back to the team on the biggest opportunities you've found in your research
- Be a leader within your scrum team and a world-class partner to your stakeholders across the company
- Have impeccable taste for amazing product experiences, and know what will work for our customers and what won't.
- Provide clear and consistent communication of what's happening with your projects
- Be hungry for measurable project goals & report back on how your products are performing in the market
- Be introspective and strategic, always seeking out ways to increase your impact on the company and our customers' lives.
- Date Posted:
- Feb 5, 2015 1:01 AM PST
- Company:
- FreshBooks
- Website:
- http://www.freshbooks.com/jobs/jobinfo?jobid=oOW1Xfws
- Company:
- FreshBooks
- Location:
- Toronto, Ontario, Canada
In 2003, Mike McDerment, co-founder and CEO of FreshBooks, accidently saved over an invoice, snapped and decided to create a better way to manage his books and bill clients. Since then, FreshBooks has emerged as the #1 cloud accounting specialist for small business owners by helping more than 5 million people track time, organize expenses and invoice clients in the cloud. Known for... more


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Old Black Horse
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Travel Blogs from Oxford
A Palace and a Hobbit
... are a gardener's dream and go on for miles and meander around the twists and turns of the RiverGlyme. We glimpse only a pittance, as the weather is not conducive to walking. We can see rain drops splash across the lake. It's more like a heavy mist that fails as we walk through the Sculpture garden at the back of the palace, with it's neatly trimmed hedges and formal Roman fountains amid statues of Roman Gods and Goddesses. Under the protection of our umbrellas we ...
The most wonderful day
Today was one of the best days we have had in England. It didn't start off so well, we had to run to catch a train and got in to Oxford in time to see a steady light drizzle of rain that danced like snow in the wind. We stopped for a hot chocolate in Pumpkin and then walked around Oxford. What a beautiful beautiful city. The rain had scared off a number of tourists so whilst the place was busy there was room to stop and admire the old ...
Whistles and Pyramids
... the horrible answers, but the game ended pretty quickly after that because Joe's group didn't enjoy it at all. From there, Joe and Anne went to bed, and the rest of us went to watch Pitch Perfect in the Ward Perkins Room. I sat with Pete and Erica, and Pete asked me questions as to the aca-accuracy of the movie. I only lasted through half of it though, and now it is time for bed so I can be up in six hours to finish up with crazy program.
Good night all!
Day one and two, almost on my own!
... won the raffel of a little bottle of Bailies, even if I only came fourth in the quiz on wines.
But we had no time to linger, we still wanted to fit in Veralinium, the Roman ruins in St Albans. Off we go to the bloody great big park in the middle of town, with a biulding in the middle that protects the mosaic and a few other items from the area that have been dug up.
The 2000 year old wall still stands in the middle of the park, it doesn't need a building, as it ...
"Missy, have I sacked you yet this week?"
... Lauren, Kate, Emma, Tom, and Joe the new PA. We watched the match for a bit and I finally got a chance to check my email while Larry tried to read it over my shoulder and pinch Natalie with the claw toy. But inevitably the conversations all turned to work, even with the game on. Apparently there were some questions at dinner as to whether or not our current office and staff lounge should remain our office and staff lounge or if they ...


text:
Social media: A force to reckon with in the Middle East
Kippreport's top insights from the Digital Media ForumMarch 29, 2015 11:16
Register for our free newsletter
Qatar's bourse says plans rights issue trading
Soccer-Xavi close to deal with Al Sadd, says Qatari club
Soccer-Xavi close to deal with Al Sadd, says Qatari club
Abu Dhabi Airports CEO leaving to take up U.K. defence ministry role
Kuwait February inflation edges up to 2.9 pct
MIDEAST STOCKS-Gulf markets sink after Yemen intervention; Dubai worst hit
HSBC's Islamic banking unit plans sukuk sale in Malaysia
Oil rises more than 1 pct, boosted by weaker dollar
UAE's Shah gas project seen achieving full capacity in Q2
National Bank of Fujairah issues 500 mln dirham tier 1 capital
December 20, 2009 10:54 by kippreport
On the grid, on pole!
The Brawn GP Formula One Team's 2009 season ended on an outstanding finish.
The historical double victory, drivers' and constructors' world championship titles, led Graham-London to celebrate this success with the same energy and vigour as shown by the whole Brawn GP team during the past championship.
The time has come for Graham-London to associate a pioneering and sporting time instrument to celebrate this wonderful ultimate achievement.
As mechanical art explorers and craftsmen, Graham-London has definitely met its team mate within top level car racing that is why this British brand wants to pay an unmeasured tribute to the Brawn GP mechanical experts.
Sophisticated, innovative, highly reliable and gutsy, the Tourbillograph Trackmaster G- BGP-001 is the perfect instrument to celebrate the Graham-London and Brawn GP team commitment.
Tourbillograph Trackmaster G-BGP-001
Limited editions of 100
Available in Black and White or Black and Yellow
(according to the Brawn GP racing colours)
The Tourbillograph is an ultimate timepiece which has been developed in the vein of Grahams "high adrenaline" timepieces. The idea was to put an exceptional time instrument to aid Brawn GP's high performance. A new concept of Tourbillon, a new technology, two new patents, a new manufacturing process are the results of four years of unremitting endeavours to create such a masterpiece.
This radiant, uncommon Chronograph combines high-technological design and mechanics, to pay tribute to the Graham-London British heritage.
The automatic movement associated with the Tourbillon perfectly demonstrates its prowess.
The complete see-through construction is unique and provides a captivating look into the refined mechanism of the fabulous Tourbillon "machine".
The off-centre construction of the Tourbillon has the advantage of being lighter and therefore more reliable with a better shock-resistance. The cage only weighs 0.485gr. and is made of 48 pieces. The high degree of precision is re-inforced with the unique "Coq" and upper bridge construction which are shaped in the tradition of English high- precision timepieces reminiscent of the 18th century.
The high frequency of the balance wheel is 28′800 alternations per hour. It permits an easier adjustment and a more precise running.
Without contest it is a new step in watch making science.
Our traditional touch of eccentricity added to the gutsy design and sturdy technique make the development of this unique and innovative timepiece possible.
The double bridge construction on both sides, front and back, is more reliable as it settles the Tourbillon in a firm environment.
Moreover, the re-inforced Incabloc anti-shock system, including spiral, balance, escapement wheels and anchor, guarantees an excellent shock resistance that is vital for a sport Tourbillon with chronograph function.
This ingenious timepiece, representing Graham-London's knack for living creations, pays tribute to the Brawn GP technicians and engineers. The brand's technical skills and creation echoes the Formula One's leading knowledge.
Graham-London is a privileged partner which has collaborated with the BrawnGP Formula One Team from its creation. We are delighted to join them in their development – hoping to break future great records.
Have you ever imagined wearing a piece of art?
Maui Jim Sunglasses launches new Rainbow Falls Collection
December 20 2009
Iran demands $1 trillion in damages from Iraq
December 20 2009


text:
Dr. Charles W. Stotler, MD
Family Medicine
- 711 Caldwell Ave
Portage, PA 15946
- 2 Locations
- (814) 736-9628
Dr. Charles W. Stotler graduated from the Thomas Jefferson University, Jefferson Medical College in 1974. He works in Portage, PA and 1 other location and specializes in Family Medicine. Dr. Stotler is affiliated with Conemaugh Memorial Medical Center and Windber Medical Center. He speaks English and Spanish.
Gender: Male
Languages Spoken: English, Spanish
Family Medicine
Medical School: Thomas Jefferson University, Jefferson Medical College; Graduated 1974
*This physician has multiple office locations. See Office Information for details.


text:
New facility with good plumbing, wiring, etc. Friendly staff. Nice laundry facilities.
right price not too far away
no public restrooms, breakfast eating area was not acceptable, breakfast choices were slim
The location was convenient (close to interstate highway.
Did not receive a statement of charges at checkout.
The staff was extremely helpful
Our windows did not lock
The room was great and the staff were outstanding! My only negative observation was that the bar was very poorly stocked for a weekend.
Breakfast was AMAZING and the staff was very friendly and helpful. I would stay here again, any time!
The hotel was clean. Staff very friendly and accommodating. The breakfast was very good and the buffet area was very clean with staff right there to help.
Can't think of anything.
I love this hotel. Friendly staff. Great service.
Room wasn't cleaned by check in time. I had to wait an hour before my room was ready.
Here's why:
We're not the seller
At KAYAK we don't set prices, so it's not possible for us to guarantee what other companies are selling.
We aggregate tons of data for you
Our service lets you quickly and easily compare results from hundreds of travel sites at once. In this sense, KAYAK is a search engine and we don't guarantee prices we find.
Why aren't prices accurate 100% of the time?
Prices on airline seats, hotel rooms and car rentals can change frequently. Several people may also be trying to buy the same travel option simultaneously. As a result, you may find on occasion that certain prices are no longer available.
More questions or if you have a bad price to report to us
Please send us an
and someone from our team will get back to you promptly.


text: Hey! I am just about to get married (in 5 weeks!) and my partner and I are planning our honeymoon in May. We love travelling, and have explored Scandinavia and Europe and our next adventure will be our 3 week US trip - we are visiting Atlanta, Las vegas and then travelling through California. I am on airbnb as a guest and i must say that my partner and I are great guests to have! We are punctual (we check in on time), respectful, not demanding and easy going and you won't regret letting your property to us :-).


text:
After hosting London's biggest NYE party, UKF is returning to London this Easter weekend with another guaranteed sell out show!
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Join UKF on the beach this summer for a festival in a stunning location...
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So happy to welcome back D&B pioneer Roni Size to the channel! He has announced a new album Take Kontrol and Size Matters EP due for release August 2014
Digital: http://bit.ly/1qcyzTB
CD: http://amzn.to/1juwbEB
Roni Size
Like → https://www.facebook.com/ronisize
Follow → https://twitter.com/ronisizebristol
Website → https://www.ronisize.net


text:
- State Roundup: Governor signs budget fix bills
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Beloit, Wis., author to sign copies of book April 7 at Turtle Creek Books
Online Staff Report
BELOIT, Wis. — Beloit, Wis., author Erik Jelinek will sign copies of his book, Blessed in the Midst of Trial: Cancer, Chemo, and Keeping the Faith, from 11:30 a.m. to 1:30 p.m., Saturday, April 7, at Turtle Creek Books, 444 E. Grand Ave., Beloit, Wis.
Cancer. The word alone brings images of fear and loss. A young, healthy nonsmoker, Jelinek never would have dreamed he'd get lung cancer. But a routine exam triggered a series of tests that revealed stage three cancer.
A pastor and father of two, Jelinek approached his health struggles with a positive attitude, finding the blessings in his circumstances.
Continuous cluster headaches had plagued Jelinek for years, but they precipitated his regular checkups, which led to the discovery of the tumor.
In Blessed in the Midst of Trial, Jelinek tells his story of being diagnosed with cancer and how his fear was replaced with blessings.
This moving memoir gives a close-up glimpse into his battle with cancer and how he experienced God's hand of grace and hope every step of the way. In each new set of circumstances, Jelinek found himself Blessed in the Midst of Trial. Strengthen your faith with this encouraging testimony of renewal and hope.
Posted April 4, 2012