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Conceived and designed the experiments: OD PC LO TD. Performed the experiments: CA SC SW CG RJ AMJ. Analyzed the data: OD PC LO TD CA SC SW. Contributed reagents/materials/analysis tools: OD PC LO TD. Wrote the paper: OD PC LO TD SC CA.
Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression.
Detection of microbial pathogens by pattern recognition receptors, such as Toll-like receptors (TLRs) triggers innate immune responses as a first line of defense against infections [1]–[3]. Pathogen-associated molecular patterns (PAMPs) such as bacterial cell walls and their structural components induce a vast variety of biological effects in host organisms. The innate response against infection with intracellular pathogens includes the synthesis of type I IFNs (IFN-I). Whereas this cytokine family generally protects against viruses, its impact on bacterial infections can be either detrimental or advantageous for the host organism [4].
Listeria monocytogenes is a bacterial pathogen which replicates in the cytoplasm of infected cells. Cytosolic pattern recognition receptors (PRRs) respond to cytosolic bacterial products and contribute to the induction of the innate immune response [5], [6]. Previous studies in bone marrow-derived macrophages (BMM) and epithelial cells show that in these cell types the synthesis of IFN-I in response to infection with L. monocytogenes is independent of TLRs and their adapters, relying exclusively on signals originating from cytosolic sensors [5]–[8]. DNA as well as cyclic dinucleotides released from lysed bacteria were suggested to function as the relevant L. monocytogenes PAMPs [9]–[11]. Several cytosolic proteins with the ability to sense pathogen-derived nucleic acids have recently been described [11]–[19]. Cytosolic recognition of L. monocytogenes causes the activation of the serine/threonine kinase TBK1 and the phosphorylation of its substrate transcription factors IRF3 and IRF7 [7], [8]. Both IRF3 and IRF7 participate in the formation of an enhanceosome at the IFN-β promoter [20].
During uptake by host cells L. monocytogenes is exposed to plasma membrane and endosomal TLRs. Among these, TLR2 which recognizes lipotechoic acids and lipopeptides, contributes to the innate response against infection [21]–[23]. Reportedly, TLR2 signals through the interacting adapter proteins Mal/TIRAP and MyD88 and does not contribute to the synthesis of type I IFN in Listeria-infected BMM [7]–[9]. Signaling through TRIF, an adapter protein known to connect TLRs 3 and 4 with the IFN-I genes was similarly ruled out for Listeria-infected BMM [7].
In order to establish a successful infection, pathogens must survive host defense systems or else mitigate the activities of PRRs. Consequently, they have evolved to modify the structural components which normally trigger PRR responses. Bacterial PGN is a hetero-polymer consisting of alternating residues of β-1,4-linked N-acetylglucosamine and N-acetylmuramic acid to which a peptide chain is attached [24]. Interestingly, L. monocytogenes modifies its PGN, with fifty per cent of the muropeptide composition being N-deacetylated [25]. We previously reported that a PGN N-deacetylase gene, pgdA, is responsible for this modification [25]. PGN deacetylation confers resistance to the action of lysozyme, one of the most important and widespread antimicrobial agents of the innate defense system, thus preventing degradation and release of immunostimulants. A strain of L. monocytogenes mutated in its ability to alter its PGN, ΔpgdA, is sensitive to lysozyme and induces an enhanced IFN-β response in macrophages compared to the isogenic parental strain [25].
The aim of the present study was to decipher the signaling pathways involved in this response to ΔpgdA infection. We reveal that IFN-β production in peritoneal macrophages requires TLR2 signaling and the TRIF adapter protein.
A L. monocytogenes pgdA mutant induced a much higher IFN-β response than the parental strain [25]. To definitively establish a role for the peptidoglycan deacetylase PgdA in the down-regulation of IFN-β production, we complemented our original pgdA mutant with the wild-type gene and we measured IFN-β secretion of peptone elicited peritoneal macrophages (PEM) infected with wild-type EGDe, ΔpgdA and a complemented ΔpgdA strain (Fig. 1). Inactivation of pgdA led to a strong induction of IFN-β secretion in wild-type macrophages. In contrast, the complemented strain did not induce any massive IFN-β secretion, similar to wild-type EGDe. Thus, PgdA directly contributes to down-regulation of IFN-β production.
Consistent with our previous report measuring secretion of IFN-β protein in PEM, IFN-β mRNA synthesis induced by L. monocytogenes infection of PEM required TLR2 (Fig. 2A), while TLR2-deficient BMM showed no impairment in their synthesis of IFN-β mRNA (Fig. 2B). Moreover, IFN-β secretion was strongly reduced in tlr2−/− PEM infected with both the ΔpgdA mutant (Fig. 2C) and the complemented ΔpgdA strain (Fig. 2D), definitively establishing the TLR2 dependence of IFN-β production.
We next analyzed the pathways by which Listeria induces IFN-β. Our previous study and the above results strongly suggested the critical involvement of TLR2 [25]. TLR2 signaling depends on Mal/TIRAP and MyD88 adaptor proteins. We had previously shown that MyD88 contributed to full IFN-β induction by Listeria [25]. We then compared IFN-β production by wild-type and mal/tirap−/− macrophages infected with EGDe or ΔpgdA (Fig. 3A). Surprisingly, production of IFN-β was not decreased in infected macrophages deficient in Mal/TIRAP, indicating that the normal TLR2 adaptor Mal/TIRAP was not required for Listeria-mediated induction of IFN-β.
The adapter TRIF is employed by TLRs 3 and 4 to signal through the TBK1-IRF3/7-IFN-β pathway. There is no previous evidence of an association or functional interaction between TRIF and TLR2. In spite of this, the link between TRIF and the IRF pathway on the one hand, and the unusual employment of TLR2 for signaling to the IFN-β gene in PEM on the other suggested the possibility of a role for TRIF. To test this hypothesis we compared induction of IFN-β expression in wild-type and trif−/− PEM or BMM infected with EGDe or ΔpgdA strains. IFN-β induction strongly decreased in TRIF-deficient macrophages infected with any of the two Listeria strains compared to wild-type PEM, showing the requirement for TRIF (Fig. 3B). In contrast, BMM showed a TRIF-independent IFN-β production (Fig. S1).
The PEM used in our studies are recruited to the peritoneal cavity by injection of the sterile irritant proteose peptone. Hence they differ from BMM not only regarding their anatomical location, but also their partially inflammatory character. To distinguish which of these differences was responsible for the TLR2 and TRIF signaling pathways, we examined IFN-β production by resident PEM. Figure 3C demonstrates a requirement for TLR2 and TRIF by the resident macrophage population. Thus, location to the peritoneal cavity rather than inflammatory character determines the difference in signaling to the IFN-β gene between BMM and PEM.
To examine the role of TLR3, which uses TRIF to trigger IFN-β synthesis, we compared induction of IFN-β in wild-type and tlr3−/− PEM infected with EGDe or ΔpgdA strains. IFN-β production was decreased in TLR3-deficient PEM infected with EGDe or ΔpgdA (Fig. 4A). We also compared induction of IFN-β in wild-type and tlr4−/− PEM infected with EGDe or ΔpgdA strains, as TLR4 can mediate TRIF-dependent synthesis of IFN-β. In contrast to TLR3-deficient PEM, TLR4-deficient PEM did not show a decrease in IFN-β response to EGDe or ΔpgdA (Fig. 4B). Thus, IFN-β induction in response to Listeria infection relies in part on TLR3 and does not require TLR4.
Induction of IFN-β via TLR2 is no longer an exception. It has recently been shown that vaccinia virus-induced IFN-β production was dependent on TLR2 signaling and it was reported that this was occuring from late endosomes [26]. To investigate if an intracellular localization was also required in the case of Listeria, we pretreated cells with cytochalasin D to prevent internalization and measured IFN-β secretion by macrophages infected with EGDe or the ΔpgdA mutant (Fig. 5A). In both cases, IFN-β induction was strongly reduced. Thus, internalization is critical for Listeria-mediated IFN-β production. We also used dynasore, a dynamin inhibitor and chloroquine, which inhibits endosome acidification, and measured IFN-β induction in macrophages infected with EGDe or the ΔpgdA mutant (Fig. 5B–C). IFN-β synthesis was strongly diminished by both dynasore and chloroquine treatments. Together, these results suggest that the TLR2-dependent IFN-β induction is triggered intracellularly.
In BMM rapid synthesis of IFN-β is entirely dependent on IRF3, but not on IRF7, whereas in bone marrow-derived myeloid DC IFN-β synthesis requires both IRF3 and IRF7 [27]. We investigated the role of IRF3 and IRF7 in the production of IFN-β by PEM. To this end we infected irf3−/− and irf7−/− macrophages with EGDe or the ΔpgdA strains. Inactivation of IRF3 totally abrogated IFN-β mRNA induction in response to both strains (Fig. 6). IFN-β induction in IRF7-deficient macrophages was also strongly affected highlighting the important role of both transcription factors in response to Listeria infection (Fig. 6). PEM thus resemble bone marrow-derived myeloid DC, not BMM, in relation to their IRF requirement for Listeria-mediated IFN-β synthesis.
In addition to IRF3/7, NFκB contributes to the formation of the IFN-β enhanceosome [20], [28]. We therefore examined the involvement of the NFκB pathway by measuring induced synthesis of an NFκB-dependent mRNA. IκB is an NFκB-dependent gene and thus a read-out for NFκB activation in response to Listeria infection. We measured the induction of IκB expression in PEM infected with EGDe or the ΔpgdA mutant. Both strains induced IκB expression and this required internalization as treatment with dynasore reduced the level of IκB induction (Fig. 7A). Degradation of the IκB protein was examined in PEM infected with EGDe by immunoblot using anti-IκB antibodies. IκB level was reduced rapidly after infection of wild-type PEM (Fig. S2A). In contrast, IκB degradation was not observed in tlr2−/− PEM infected with Listeria (Fig. S2B). Infection of wild-type, tlr2−/−and trif−/− macrophages with EGDe or ΔpgdA showed that both TLR2 and the adaptor were required for full induction of IκB mRNA in response to EGDe and ΔpgdA strains (Fig. 7B). These results suggest that TLR2 and TRIF contribute to NFκB activation. The comparison between EGDe and ΔpgdA strains showed that both caused similar magnitudes of IκB mRNA synthesis. Thus, the activation of NFκB by Listeria is independent of PgdA, suggesting that the increased IFN-β production after infection with ΔpgdA relies on activation of other transcription factors such as IRFs.
TLR2 or TRIF deficiency strongly reduced, but did not completely shut off IFN-β synthesis. This suggested a potential contribution of intracellular, nucleic acid-dependent pathways to IFN-β synthesis, particularly after infection with ΔpgdA. We therefore examined whether these pathways are able to signal in PEM.
Since inactivation of PgdA increases Listeria sensitivity to peptidoglycan-targeting antimicrobials such as lysozyme, and thus induces bacterial degradation, we measured the DNA and RNA released by EGDe and ΔpgdA strains following lysozyme exposure. As expected, ΔpgdA released significantly higher amounts of DNA and RNA than wild-type and complemented ΔpgdA strains, raising the possibility that both DNA and RNA could be involved in IFN-β production (Fig. 8A). We thus measured IFN-β induction in THP1 macrophages transfected with Listeria DNA, either undigested or treated with DNase. Intact but not DNase-treated DNA significantly induced IFN-β (Fig. 8B). Macrophages were then transfected with lysozyme-digested EGDe or ΔpgdA, either untreated or digested with DNase. Treatment with DNase significantly reduced IFN-β production (Fig. 8C). Taken together, these results show that Listeria DNA can induce IFN-β, strongly indicating that destruction of ΔpgdA bacteria intracellularly activates DNA sensors.
We had recently reported that a PGN modification involving a N-deacetylase gene, pgdA, was playing a key role in L. monocytogenes virulence [25]. A ΔpgdA strain of L. monocytogenes which is unable to modify its PGN, was shown to be extremely sensitive to the bacteriolytic activity of lysozyme, normally found within macrophage vacuoles and its virulence was strongly attenuated [25]. Furthermore, this mutant induced a much higher TLR2-dependent IFN-β response than the parental strain [25]. We hypothesised that this unconventional IFN-β response induced by the pgdA mutant was due to an enhanced accessibility of bacterial cell wall components to TLR2. Here we have shown that IFN-β production requires bacterial internalization and is triggered by Mal/TIRAP-independent pathways which involve TLR2, TRIF, IRF3 and IRF7.
It was surprising to see a role for TLR2, as, based on results in BMM and epithelial cells, type I IFNs production is usually not known to result from TLR2 signaling [5]–[8]. Classical TLR2 signaling leads to NF-κB-dependent production of inflammatory cytokines [21]. However, in support of an unconventional role for TLR2, recent studies reported roles for TLR2-dependent induction of IFN-β in response to vaccinia virus or synthetic ligands [26], [29]. In the vaccinia virus study, a specific inflammatory monocyte population -Ly6Chi- was shown to be the source of IFN-β [26]. In the present study we show that TLR2-dependent IFN-β synthesis is a property of both resident and recruited inflammatory PEM. Furthermore, the two previous studies documented that TLR2 activation of type I IFN responses to TLR ligands occurs within intracellular compartments, and that TLR2 signals from the phagosome in response to viral infection or synthetic TLR2 ligands [26], [29]. These results challenged the view that TLR2 signals solely from the plasma membrane. In our experiments, pre-treatment of PEM with either cytochalasin D, an inhibitor of actin polymerization and thus internalization, dynasore, an inhibitor of the endocytic effector dynamin, or chloroquine, which inhibits endosome acidification [30], [31], significantly impaired the induction of IFN-β following Listeria infection, strongly suggesting that phagocytosis of L. monocytogenes and intracellular location of TLR2 trigger this response. These observations also correlate with our early hypothesis that the inflammatory response induced by ΔpgdA is due to an enhanced release or accessibility of bacterial cell wall components to TLR2.
Induction of the IFN-β gene was independent of the TLR adapter Mal/TIRAP, but, unexpectedly required the TLR3/4 adapter TRIF. Francisella tularensis has recently been shown to signal through TLR2 from the phagosome in a Mal/TIRAP independent manner [32], and it was shown that Mal/TIRAP is dispensable in TLR2 signaling at high concentrations of ligands [33]. Thus our study reinforces the view that TLR2 can act independently from Mal/TIRAP. In addition our report suggests a synergy between a TLR2 pathway and TRIF, an adapter previously known to trigger the synthesis of pro-inflammatory cytokines and type I IFNs upon engagement of TLR3 and TLR4. TLR3 is known to bind viral dsRNA to induce secretion of type I IFN and lead to control of viral infections [3], [34], [35]. To our knowledge Chlamydia muridarum is the only bacterium reported to induce a TLR3-dependent IFN-β response specifically in murine oviduct epithelial cells [36]. We tested whether the dual requirement for TLR2 and TRIF resulted from a functional or physical interaction between TLR2 and TLR3. In fact, IFN-β production was reduced in TLR3-deficient macrophages, but significantly less so than in trif−/− PEM. Therefore, there is no evidence for a putative TLR2/TLR3 interaction. Another possibility to incorporate TRIF into the pathway stimulated in PEM by Listeria would be a cooperation of TLR2 and TLR4. This was ruled out by showing that Listeria-infected tlr4−/− PEM produced a similar amount of IFN-β as their wild-type counterparts. TRIF could possibly orchestrate an additional pathway. Along these lines, TRIF has recently been shown to be required for IFN-β synthesis by dendritic cells upon activation of the cytosolic receptor complex DDX1/DDX21/DDX36 by viral RNA [19].
Engagement of TLRs by various microbe-associated molecular patterns induces activation and translocation to the nucleus of NF-κB, IRF3, IRF7 and/or activator protein-1 (AP-1), which collaborate to induce transcription of type I IFNs [37]. We addressed the role of these transcriptional activators in the IFN-β response to wild-type Listeria and ΔpgdA, and revealed that inactivation of IRF3 totally abrogated this response to both strains while IFN-β induction was significantly but not totally impaired in IRF7-deficient macrophages, indicating that both of these transcription factors are required for induction of IFN-β following infection with L. monocytogenes. We also assessed the involvement of NF-κB in this response using induction of the IκB gene as a readout. We observed an induction of IκB expression in macrophages which was similar after infection with EGDe or ΔpgdA. Thus, activation of NF-κB by Listeria is independent of PgdA, strongly suggesting that the elevated IFN-β production by the ΔpgdA mutant mostly relies on IRF3.
The increased IFN-β response to the ΔpgdA strain probably results from the fact that within the phagosome, its lysozyme-sensitive cell wall is degraded, releasing PAMPs able to interact with TLR2 and other PRRs, including cytoplasmic ones. As recent studies have highlighted novel DNA-sensing pathways in the induction of type I IFNs [9], [14]–[17], [38], we thus also investigated the involvement of bacterial nucleic acids in the IFN-β induction, Firstly, we showed that inactivation of PgdA, which confers a higher susceptibility to lysozyme, leads to increased release of DNA. We then showed that DNA from L. monocytogenes can induce IFN-β expression in PEM, suggesting that this macrophage population employs cytoplasmic nucleic acid sensing similar to macrophages or macrophage lines derived from different anatomical locations [9], [38]. Which -if any- of the recently described nucleic acid sensors are used by PEM for the recognition of Listeria DNA remains subject to future investigation. Nevertheless, other bacterial components could participate in IFN-β production upon infection with the ΔpgdA mutant. For example, the second messenger molecule cyclic diadenosine monophosphate (c-di-AMP), was shown to be secreted by Listeria multidrug efflux pumps triggering type I IFN response [10] and could be involved in the process.
In conclusion, this study describes a novel mechanism leading to induction of type I IFNs in which intracellular sensing plays an important role, ultimately showing how these different recognition pathways can synergise to induce innate immune responses which are required to control infection. In this regard cooperation between TLR2 and TRIF may reflect the need for convergence of the NF-κB and IRF pathways at the IFN-β promoter, with TLR2 being responsible mainly for NF-κB activation and TRIF being instrumental for activation of IRF3 and IRF7. By employing the strategy of PGN modification, L. monocytogenes can avoid immune detection by TLR and evade the innate immune response, thus enabling the infectious process to occur. It is important to recall that pgdA orthologs are found in other pathogenic bacteria, such as Streptococcus pneumoniae, Bacillus cereus, Bacillus anthracis and Helicobacter pylori, strongly suggesting that PGN N-deacetylation is a general mechanism evolved by microbes to escape from pattern recognition receptor-mediated immune recognition [39]–[42].
L. monocytogenes EGDe (BUG1600, ATCC BAA-679), L. monocytogenes isogenic mutant ΔpgdA (BUG2288, [25]) and L. monocytogenes ΔpgdA complemented strain (BUG2382) were grown in brain heart infusion (BHI, Oxoid), aerobically at 37°C and 200 rpm.
A DNA fragment containing the pgdA gene (lmo0415) and its promoter was generated by PCR using oligonucleotides lmo0415-1 (5′-AAGGATCCCACAATATGTTAGTTTTCAGGGG-3′) and lmo0415-2 (5′-AAGGATCCTTATTTCACCATTCTTGAATCTG-3′). The fragment was integrated into pCR-Blunt-II-TOPO (Invitrogen) and the construct was verified by sequencing. After digestion of the construct by BamHI, the fragment was purified on agarose gel and cloned into the integrative vector pPL2 [43], previously digested by BamHI, constructing pOD98. The pOD98 was electroporated into ΔpgdA at 2,500 V, 200 Ω and 25 µF. Transformants were selected at 37°C on BHI agar containing chloramphenicol (7 µg/mL). The presence of the pgdA gene in the complemented strain was confirmed by PCR using oligonucleotides lmo0415-1 and lmo0415-2.
Mice were used for obtaining peptone-elicited peritoneal macrophages, resident peritoneal macrophages and bone marrow-derived macrophages. Animal experiments were performed in accordance with protocols approved by the Animal Experimentation Ethics Committee of the Institut Pasteur (permit #03-49) and following Austrian law in accordance with protocols approved by the Ethics Committee of the University of Veterinary Medicine, Vienna (#GZ680 205/67-BrGt/2003).
PEM were isolated from 7 to 10 week-old C57BL/6J and genetically-matched tlr2−/−, tlr3−/−, tlr4−/−, mal/tirap−/−, trif−/−, irf3−/− and irf7−/− mice as previously described [44]. The percentage of macrophages was determined by flow cytometry using CD11b (1100, eBiosciences) and F4/80 (1100, eBiosciences) antibodies. More than 90% of the cells were macrophages. PEM were seeded onto 6-well plates at a concentration of 2×106 cells per well in DMEM (PAA) supplemented with 10% FCS, 10% L929 conditioned medium (LCM) and 1% penicillin-streptomycin or RPMI-1640 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin.
Resident macrophages were isolated from 6 to 8 week-old C57BL/6J and genetically-matched tlr2−/−, trif−/− mice by washing the peritoneum twice with 10 mL DMEM (PAA) supplemented with 10% FBS, 10% LCM and 1% penicillin-streptomycin. Harvested cells were centrifuged at 300 g for 5 minutes and resuspended in complete medium. The percentage of macrophages was determined by flow cytometry analysis as above. Cells were seeded onto 6-well plates (Nunc) at a concentration of 2×106 cells per well.
Tibia and femur from 6 to 8 week-old C57BL/6J and genetically-matched tlr2−/−, trif−/− mice were collected in ice cold PBS. Bones were sterilized with 70% ethanol and flushed with a 25-G needle using cold DMEM supplemented with 10% FCS, 10% LCM and 1% penicillin-streptomycin. Cells were seeded onto 6-well plates (Nunc) at a concentration of 106 cells per well and incubated at 37°C with 5% CO2. After 4 days, complete medium was added and cells were split at a ratio of 12. After 8 days, macrophages were fully differentiated.
Human acute monocytic leukemia THP-1 cells (ATCC TIB202) were maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded onto a 24-well plate at a concentration of 4×105 cells per well in antibiotic-free media supplemented with 12.5 ng/mL phorbol myristate acetate and incubated for 24 h at 37°C with 5% CO2. Differentiation was determined to be successful upon formation of a confluent adherent monolayer. HEK-blue type I IFN cells (Invivogen) were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded at a concentration of 5.6×104 cells per well onto a 96-well plate.
For cytokine analysis, macrophages were infected with Listeria strains at MOI 101, centrifuged at 300 g for 2 min and incubated at 37°C for 15 min. Following phagocytosis, monolayers were washed twice followed by incubation in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and gentamicin (20 µg/mL). Supernatants were collected at various time points, for detection of IFN-β by ELISA. For transcript analysis, macrophages were infected with Listeria strains at MOI 201 and incubated at 37°C for 1 h to allow phagocytosis. Monolayers were washed and incubated in DMEM supplemented with 10% FCS and gentamicin (5 µg/mL). After 2 h, medium was changed to DMEM supplemented with 10% FCS and gentamicin (1 µg/mL). Cells were lysed at various time points and RNA collected for qPCR analysis.
For inhibition of bacterial internalization, cell monolayers were pretreated either for 2 h with 100 µM cytochalasin-D (Sigma-Aldrich), or 30 min with 80 µM dynasore (Sigma-Aldrich) or 30 min with 100 µM chloroquine (Sigma-Aldrich) prior to infection assays.
Listeria were grown overnight in BHI at 37°C and cultures were centrifuged at 8000 g for 5 min. Bacterial pellets were resuspended in 75 µg/mL lysozyme and incubated at 37°C for 1 h. DNA was then extracted using the DNeasy blood and tissue kit (Qiagen) and quantified by spectrophotometry (Nanodrop). For transfection assays, THP-1 macrophages were transfected with 200 ng/mL DNA with 2% lipofectamine 2000 (Invitrogen) and incubated for 24 h. Following incubation, supernatants were collected for IFN-β analysis. For pretreatment of DNA with DNase, DNase was added at final concentration of 100 µg/mL for 45 min at 37°C.
Bacterial cultures were treated with 10 µg/mL lysozyme, a concentration leading to lysis of ΔpgdA but not EGDe, and incubated at 37°C and 200 rpm for 1 h. Following lysozyme treatment, lysed bacterial cultures were centrifuged at 5000 rpm during 10 min. Two types of experiments were performed on supernatants. First, nucleic acid release was quantified. DNA was purified using the Qiagen DNeasy blood and tissue kit omitting lysis steps and quantified by spectrophotometry (Nanodrop). RNA was purified using Qiagen RNeasy kit and quantified by spectrophotometry (Nanodrop). Data shown are representatives of at least three independent experiments. Second, 100 µL of each supernatants were treated by DNase during 30 min at 37°C. Enzymes were inactivated and treated- or untreated-supernatants were transfected in PEM. 8 h after transfection, supernatants of cells were recovered and the IFNβ was quantified.
Murine IFN-β production was detected in macrophage supernatants by ELISA according to the manufacturer's procedure (PBL Biomedical Laboratories). For the HEK-blue type I IFN assay, supernatant from THP-1 macrophage assays was collected and 20 µL added onto HEK-blue type I IFN cells plated in 96-well plates, which were incubated at 37°C overnight. Supernatant from HEK-blue cells was collected and 40 µL added to 160 µL of Quanti-blue reagent (Invivogen) for 20 min at 37°C. The colorimetric reaction was measured at 625 nm on a plate reader. Data was normalised against absorbance for the untreated cells and plotted as relative fold increases. Data shown are representatives of at least three independent experiments.
PEM from WT or tlr2−/− C57BL/6J mice were infected with EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. IκB and tubulin were detected in lysates by immunoblotting using anti-IκB (Santa Cruz, 1100) and anti-α-tubulin (Sigma, 15000) antibodies.
RNA preparation was performed using NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's instructions. Quantitative real-time PCR was performed on a Mastercycler EP realplex S (Eppendorf). Primers for HPRT (housekeeping gene control), IFNβ and IκBα mRNA expression were as follows: HPRT forward GTTGGATACAGGCCAGACTTTGTTG, HPRT reverse GAGGGTAGGCTGGCCTATTGGCT, IFNβ forward 5′-TCAGAATGAGTGGTGGTTGC-3′, IFNβ reverse 5′-GACCTTTCAAATGCAGTAGATTCA-3′; IκBα forward 5′-GCAATTTCTGGCTGGTGGG-3′, IκBα reverse 5′GATCCGCCAGGTGAAGGG-3′. Data shown are representatives of at least three independent experiments.
Results are expressed as means of at least three values, with error bars representing standard deviations. Student's t tests were performed to determine statistical significance where * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.0001.
TRIF is not required for IFN-β response to Listeria in bone marrow macrophages. BMM from C57BL/6J or trif−/− mice were infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). After 4 h of infection, IFN-β induction was measured by qRT-PCR. Data are mean ± SD (NS, non significant, n=3).
TLR2 is required for optimal activation of NF-κB. (A) PEM from WT C57BL/6J mice were infected with EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. Activation of NF-κB was measured by determination of IκB degradation relative to tubulin following immunodetection. (B) PEM from tlr2−/− mice were infected with EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. Activation of NF-κB was measured by determination of IκB degradation relative to tubulin following immunodetection.
We thank members of the Cossart laboratory for helpful discussions.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Work in PC's laboratory received financial support from Institut Pasteur (http://www.pasteur.fr), Inserm (http://www.inserm.fr), INRA (http://www.inra.fr), ERC (Advanced Grant 233348, http://erc.europa.eu/), Fondation Pasteur-Weizmann and Fondation le Roch Les Mousquetaires (http://www.fondationleroch-lesmousquetaires.org/). PC is an international research scholar of the Howard Hughes Medical Institute (http://www.hhmi.org/). CA is a doctoral fellow of the Ministère de l'Enseignement Supérieur et de la Recherche. Work in LO's laboratory was supported by Science Foundation Ireland (http://www.sfi.ie/) and the Irish Research Council for Science, Engineering and Technology (RS/2005/190, http://www.ircset.ie/). Work in TD's laboratory was funded by the Austrian Research Foundation (grants SFB-28 and P20522-B05, http://www.fwf.ac.at). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Here's the URL: http://hrirrigation.com/wpsite/?page_id=47
And thenselect: "click here to payoline"
I've just posted your code on both a page and post, it works. When you post it on your site you get the following HTML
I still am suggesting that you install the Raw HTML plugin then use thisCode:
Click here to pay online

Code: <form action="https://www.myvirtualmerchant.com/VirtualMerchant/process.do" method="POST">
<input type="hidden" name="ssl_merchant_id" value="561258"> <input type="hidden" name="ssl_user_id" value="website"> <input type="hidden" name="ssl_pin" value="K266PU"> <input type="hidden" name="ssl_show_form" value="true"> <input type="hidden" name="ssl_test_mode" value="false"> <input type="hidden" name="ssl_transaction_type" value="CCSALE"> <input type="hidden" name="ssl_amount" value="Enter Amount"> <input name="submit" type="image" src="http://hrirrigation.com/images/buttons/invoiceButton2.png" alt="Click here pay online" />
 </form>
Here's what I have noticed. There is a blank page created called "payonline" in the admin section of WP. No matter what code I input into this page it is not being read. I found the real code is located under the Appearance>Editor>>Page Template. Here is the code I think is responsible for executing the process:
else if(is_page( '47' )){

Make an Online Payment

You have the option to pay an invoice, or to place a deposit for services found on an estimate. To make a secure online payment, have your invoice or estimate available

Click here to pay online

<img src="<?php echo get_template_directory_uri(); ?>/images/credit_cards.png" />

<?php }
else if(is_page( '7' )){


text:
well that's just, like, your opinion, man
- Member for
- 6 years 43 weeks
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- View recent blog entries
Recent Comments
|1 day 10 hours ago
|Don't mess with Jack Wagner.
Don't mess with Jack Wagner.
|1 day 17 hours ago
|August 16th at 1:30 pm.
August 16th at 1:30 pm.
|3 days 20 min ago
|Don't be surprised to see
Don't be surprised to see Carlos Brown start at quarterback against Utah. Threat and Sheridan can't offer the mobility Rod wants and Brown was a talented dual threat in HS.
|5 days 17 hours ago
|As long as he's not taller
As long as he's not taller than 6'3, we'll take him!
|1 week 3 days ago
|Idiots, bottom line. This is
Idiots, bottom line. This is your program in a nutshell, that's why you can't ever get where you want to be.
|1 week 4 days ago
|We need Greg to stay in
We need Greg to stay in Columbus and be the fall guy once harbaugh beats urban twice. He's a huge part of the rivalry swing that's about to begin. What a creepy dude though, wow. A special place in hell awaits these bastards.
|1 week 6 days ago
|If he's smart and can resist
If he's smart and can resist the money it would be the best for him to let last night be the end. He can beat the Hunts and Nelsons of the division but he's such a big draw that if he fights again it'll probably be against Overheem or Cain. I'm afraid those are not fights he can win.
|2 weeks 9 hours ago
|Lesnar is a freak athlete.
Lesnar is a freak athlete. Good for him, nice to see how humble he's become.
|2 weeks 13 hours ago
|Tate- Brock- Danny
|2 weeks 1 day ago
|Name Brand tattoo in Ann
Name Brand tattoo in Ann Arbor. Jeff Zuck is the best tattoo artist in the world.
|2 weeks 1 day ago
|Because he's stupid. Sorry, I
Because he's stupid. Sorry, I want him to realize his potential and stay healthy. I want him to be the star he should be. It'll never happen though.
|2 weeks 3 days ago
|Phil Steele, shitty since
Phil Steele, shitty since 1988.
|3 weeks 4 days ago
|I am. They just scored actual
I am. They just scored actual runs.
|3 weeks 6 days ago
|Johns going to do great
Johns going to do great things this year.
|4 weeks 8 hours ago
|Please, don't compare what's
Please, don't compare what's going on now to that garbage. Especially Hoke. Don't believe anything and be weary of whatever but never compare Harbaugh to that shit.
|4 weeks 1 day ago
|Wish we would have produced
Wish we would have produced that second natty.
|4 weeks 2 days ago
|Good luck Caris!
Good luck Caris!
|4 weeks 6 days ago
|Me too. Love him as a player.
Me too. Love him as a player. The guy does it all and he took two national titles away from Ohio State by not going there. The warrior are pretty much the worst team in sports as well. Clay will be skipping out, walking down that tunnel alone forever.
|5 weeks 6 days ago
|Would buy at over paid price.
Would buy at over paid price.
|5 weeks 6 days ago
|Lebron got a technical too so
Lebron got a technical too so he didn't escape anything. Green has gotten away with murder, now he's probably cost his team a game.
|5 weeks 6 days ago
|You don't wanna watch that
You don't wanna watch that game man.
|6 weeks 7 hours ago
|I know a couple of teams that
I know a couple of teams that are going to miss the adidas era. One in Columbus and even more noteworthy one in East Lansing. I'm ready for some real change and that's what we're going to see.
|6 weeks 1 day ago
|That means absolutely
That means absolutely nothing. Golden state jacks outside shots. The cavs take the ball to the hoop. It's not even those calls, it's the no calls and the weird shit. And man, there's been too much weird shit. You've been a Warriors fan for six months now. Have you seen this much weird shit before?
|6 weeks 1 day ago
|I wouldn't take green for
I wouldn't take green for anything. He's a scumbag. The worst person in pro sports. Overrated too.
|6 weeks 1 day ago
|6 weeks 1 day ago
|Ha, I wouldn't judge anything
Ha, I wouldn't judge anything on this trash. He's been hammered, tripped, kicked and watched the ball continue to be HANDED to the Warriors.
|6 weeks 1 day ago
|Are you serious ? The guys a
Are you serious ? The guys a scumbag.
|6 weeks 1 day ago
|They should get Warriors
They should get Warriors jerseys with thunder t-shirts under them like you did.
|6 weeks 1 day ago
|Wow, give these refs some
Wow, give these refs some credit. At least they're not trying to hide it. Garbage calls and the title is all but handed to the Warriors.
|6 weeks 1 day ago
|Yeah, when his entire forearm
Yeah, when his entire forearm is hooked. The guy hasn't got one call this game. Dray and the douche bros get all the calls now. Ruining basketball one wild out of control play at a time.


text:
FarmTech 2016 video coverage
Thanks to RealAgriculture for sharing these videos recorded at FarmTech 2016. Check back for new videos as they become available From Oh Canada to the... Read More
FarmTech is Canada's premier crop production and farm management conference.
The dates for FarmTech 2016 are January 26, 27 & 28 at the Edmonton EXPO Centre at Northlands.
The FarmTech Conference is hosted by Alberta Barley, the Alberta Canola Producers Commission, the Alberta Pulse Growers, the Alberta Seed Growers Association, and the Alberta Wheat Commission.
FarmTech features more than 50 sessions featuring a world class lineup of speakers providing delegates with everything from global knowledge to local perspectives.
The Agricultural Showcase is the hub of activity at the show and features the the latest in products and services for crop producers.Learn More


text:
Several other women have spoken of similar experiences.... Read more »
Dirty Projectors released Swing Lo Magellan back in summer of 2012, but are today releasing the sixth and final video from that album, a beautiful, slow-paced visual... Read more »
If you still haven't listened to Unknown Mortal Orchestra's II from earlier this year, you're doing it wrong. But lucky for you, they'll be releasing... Read more »
The lovely Solange, who was recently performing in a New York laundromat, has shared some news about her forthcoming fourth album. The record, which is... Read more »
AlunaGeorge is one of those rare acts that manages to fulfill all your expectations of a big sound without stooping to any formulaic electronic stops... Read more »
We've lost count of how many remixes there have been for Phoenix's single, "Entertainment," but from Grizzly Bear to Blood Orange, the song's been around.... Read more »
Gearing up for next week's release of their sophomore album, Diplo's production project Major Lazer stopped by Late Night with Jimmy Fallon for a special... Read more »
On May 13 Domino will be releasing a three track collection of big-name remixes of "The Socialites" from Dirty Projectors' 2011 LP Swing Lo Magellan. Stepping up to... Read more »
All the best things happen in SXSW. This is a life lesson we will all eventually learn. Above is some footage of Major Lazer and... Read more »
All the Dirty Projectors tracks that have been released since Swing Lo Magellan and their most recent EP About to Die feel like extra quilt... Read more »


text:
Poached Pears With Brandy Custard
|For poached pears
|4 , peeled, cored and halved
|Apple pear juice concentrate
|1⁄3 Cup (5.33 tbs)
|3 Tablespoon
|Cinnamon stick
|1 Small
|1 Teaspoon
|2 1⁄2 Cup (40 tbs)
|For brandy custard
|1 , slightly beaten
|2 Tablespoon
|Skim milk
|1 Cup (16 tbs)
|2 Teaspoon
|1 Tablespoon
Place all ingredients except pears in a saucepan.
Cook over medium heat 5 minutes.
Add pears.
Cover and simmer 25 minutes until pears are tender, turning pears often.
Refrigerate pears in their liquid 8 hours or overnight.
To make the brandy custard, combine all ingredients except brandy in a saucepan.
Cook over medium heat until thickened, stirring constantly.
Pour into a bowl, add brandy and stir.
Cover with waxed paper and cool to room temperature.
To Serve: Spoon custard onto 4 dessert plates.
Pears should be at room temperature.
Drain pears on paper towels.
Place 2 halves in each dish on top of custard.
Dust pears with cinnamon or nutmeg.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size
Calories 212 Calories from Fat 14
% Daily Value*
Total Fat 2 g2.4%
Saturated Fat 0.47 g2.3%
Trans Fat 0 g
Cholesterol 54 mg18%
Sodium 50.3 mg2.1%
Total Carbohydrates 39 g12.9%
Dietary Fiber 4.8 g19.2%
Sugars 27.4 g
Protein 4 g8.3%
Vitamin A 2% Vitamin C 11.5%
Calcium 9.4% Iron 3.3%
*Based on a 2000 Calorie diet


text:
Golden Oaks Apartments Inc
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text:
Prabal Gurung
Bow-Front Sleeveless Dress
Product Actions
- Jewel neck, asymmetric gathering at front bodice, pleats at waist front, full skirt, darting at back
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Our model is 5'10"/178cm and is wearing a size 2.
Style # 503460445
Born in Singapore and raised in Nepal, acclaimed women's ready-to-wear designer Prabal Gurung studied fashion at Parsons School of Design in New York City. In 2009, he launched his eponymous collection with an emphasis on sleek silhouettes romantically embellished with ruffles and embroidery. A winner of the CFDA Swarovski Award for Womenswear, Gurung is in high demand for red-carpet moments and other events that require elegant evening creations.
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text: If modern black radio came anywhere close to this album, more could be compelled to move from toleration or total desertion to actual enjoyment. Hey, one can dream. Jazz composer/musician Robert Glasper brilliantly reveals what mature radio sounds like in a parallel universe. Helping his cause is a who's who of alternative soul and hip-hop stars, including Bilal, Musiq, Ledisi, Mos Def, Lupe Fiasco, Chrisette Michele, Stokley Williams, Meshell Ndegeocello, among others. High-flying newcomers King keep altitude on "Move Love," an original cut that personifies this nightcap album's languid sounds dotting landscapes painted in seductive indigos, silvery moons, and shimmering black pools. A few fine covers sneak under Glasper's velvet rope of originals, including Lalah Hathaway on an electro-soul treatment of Sade's "Cherish the Day," and Erykah Badu's neo-soul approach to Mongo Santamaria's "Afro Blue." Light on experiment and Glasper solos, it still inspires. (4 out of 5 stars)


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text:
Monthly Archives: May 2011
HeatMapper featured on Slashdot and PC Pro!
HeatMapper was recently featured in PC Pro, and thereafter Slashdot. Slashdot happened just yesterday, and we're now trying to up our outgoing bandwidth to keep up with the increased web traffic.
Because of the traffic peak, please be patient with your downloads within the next couple of days…
Network Interop Las Vegas – Come Meet Ekahau
Network Interop, the fairly sizable IT tradeshow takes place this week in Las Vegas.
There should be some exciting announcements from various companies this week – hopefully something exciting on the Wi-Fi field also. Let's see.
Ekahau does not have a booth at the Interop this year, but I will be there walking the floor. If you're in the area, let me know and let's sit down for a coffee/beer/mojito.
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text: With all the replica watches audacious affairs of Rolex replica watches through online stores, those who are agog to access the 18-carat alarm for themselves are still effectively geared on analytical these ablaze watches online. Just for the acumen of the amount difference. The Rolex is the exceptional band of replica watches uk wrist is acclaimed for the agitating arete it have, and this is attainable with an adamant amount which makes it in the aforementioned way adorable as well. If we say that a Rolex is chic representation back it is actually is. With the amount affection which is actual high, every getting is on the replica breitling clue that is from area to access a Rolex watch. Holy Trinity Parish - Parish General Info Mass Times Home Page About Holy Trinity Parish Welcome Mass Times Liturgy Calendar Daily Readings Weekly Devotional About the Liturgy Photo Tour Mass Times Our Ministries Complete List of Ministries Faith Formation Holy Trinity School Ministry News & Postings Ministry Schedules Volunteer Stewardship as a Way of Life at Holy Trinity News, Schedules, Events, Calendars & Forms News and Events Ministry Schedules Ministry News & Postings Parish Calendars Forms Donation & Payment Center Annual Collections Contact Us Our Staff Map to Parish Facility Map 3111 Tierra de Dios Drive El Dorado Hills, CA 95762 ph: (530) 677-3234 fax: (530) 677-3570 To view a calendar listing dates and times for all regular, Holy day, seasonal and special liturgies through the year, please visit our Liturgy calendar. Our regular mass schedule and information regarding special services are listed below. The Lord's Day Saturday Sunday 5:30 p.m. 7:30 a.m. 9:00 a.m. 11:00 a.m. 5:30 p.m. Children's Liturgy of the Word & Nursery available Weekday Mass Schedule Monday Tuesday Wednesday Thursday Friday 8:15 a.m. 8:15 a.m. 8:15 a.m. 8:15 a.m. 8:15 a.m. If you are ill and cannot attend, call (530) 677-3234 to arrange for a Eucharistic Minister to include you in communion. We miss you and want you to share in our Eucharist, too. Penance Individual reconciliation Saturday at 4:45 pm, or by appointment. We generally celebrate the communal rite of penance during advent and lent each year. Baptism of Infants and Young Children By appointment with Pre-Baptism class. Baptisms are on the 4th Sunday of each month. Call (530) 677-3234 for more information. Marriage Preparation The Diocese of Sacramento calls for at least six months preparation. Call (530) 677-3234 to begin the process. Anointing of the Sick This sacrament, which the church shares not just with the dying but with anyone with a serious physical or mental illness, is available by calling the pastoral center at (530) 677-3234. First Eucharist (Communion), Confirmation, and Penance Preparation for Children Call the pastoral center at (530) 677-3234 to register for our ongoing Family Faith Formation program. Then ask to be registered in the appropriate sacrament preparation module. Click here for more information on liturgy at Holy Trinity. back to top July 27, 2016 Copyright © 2003 - 2016 Holy Trinity Parish Unique visitors today: 66 Pages viewed today: 233


text: Tripadvisor Another stock that's just starting to enter breakout territory is TripAdvisor ( TRIP), an online travel research company, enabling users to plan and have a trip. This stock is off to a strong start in 2012, with shares up over 40%. If you take a look at the chart for TripAdvisor, you'll notice that this stock has been in a monster uptrend for the past few months, with shares rising from its January low of $24.57 to its current price of around $36 a share. During that up-move, TRIP has been making mostly higher lows and higher highs, which is bullish price action. Now the stock has started to trigger a breakout trade with shares moving above some near-term overhead resistance, and hitting new 52-week highs. >>7 Companies That Keep on Growing Market players should now look for long-biased traders in TRIP as long as the stock is trending above some near-term overhead resistance at $36.05 a share with strong upside volume flows. Look for volume on a sustained move and close above $36.05 that hits near or well above its three-month average action of 2.3 million shares. At last check, TRIP has hit a daily high of $36.85, and volume is above 2 million shares traded. If we can get a strong close on TRIP today above $36.05 and near its daily highs, then this stock should continue its uptrend in the near-term and potentially hit $40 a share or much higher. I would simply avoid TRIP from the long side if it fails to hold above that breakout level of $36.05, or if it moves back below today's low of $35.27 with high volume. As of the most recently reported quarter, TripAdvisor is one of Jana Partners' holdings. Follow @stockpickr Prev 2 of 6 Next


text:
Skip to comments.Is Obama closing GOP-leaning car dealers ?....(YES !!!)
Posted on 05/29/2009 9:21:13 AM PDT by IrishMike
There appears to be a side to the Chrysler bankruptcy that has the look of an ugly partisanship not seen in this town since Tricky Dick Nixon was in the White House composing his enemies list and checking it twice every night while watching the evening TV newscast.
Bloggers on the Right side of the Blogosphere are up in arms over data suggesting that President Obamas White House auto industry potentates are targeting for closure Chrysler dealers with records of contributing either to Republicans like John McCain or to other Democrats in the 2008 presidential primary.
Posts at RedState, Reliapundit, American Thinker, Gateway Pundit, Joey Smith and Doug Ross pointed intitially at the remarkable number of closed Chrysler dealerships whose owners happen to have been contributors to Obama opponents, mainly Republicans.
But those observations were all couched with important qualifiers, particularly that all conclusions were necessarily preliminary, pending completion of a comprehensive analysis of the political contributions by all closed Chrysler dealership owners, and a comparison of those results with contributions by dealers who are not being closed.
That said, when multiple dealers who have been closed are found to have contributed millions to Republicans and mere hundreds to Obama, the serious number-crunching cannot be completed too soon.
Then there is the Reuters report quoting a lawyer representing dealers being closed who came away from a deposition of a senior Chrysler executive with the distinct impression that the company is simply following orders coming from the White House.
"It became clear to us that Chrysler does not see the wisdom of terminating 25 percent of its dealers. It really wasn't Chrysler's decision. They are under enormous pressure from the President's automotive task force," said attorney Leonard Bellavia.
Bellavia zeroed in on the fundamental problem with Obamas takeover of two of Detroit Big Three something is terribly wrong when the government tells corporations who to hire and fire, what products to sell and who will be their dealers.
"What is the next task force? Shoe stores? Pizzerias?" Bellavia said. "The problem we have is the free enterprise system is not run by the government, it's run by business entrepreneurs. The dealers themselves will decide if it's not productive to go forward."
....White House car czar Steven Rattner is married to Maureen White, the former national finance chairman of the Democratic National Committee. And let's not forget that before Rattner became a Wall Street mover and shaker, he was a New York Times reporter.
Second, since neither Chrysler, nor the White House have made public the criteria used to select dealers for elimination - and because a significant number of those being closed were profitable - the only way to resolve the inevitable controversy about political considerations in political decisions is to make the criteria public and allow independent outside observers to assess how those criteria were applied....
What Obama is doing is illegal period and these panty waist coporations are right in there with him.
"make the criteria public and allow independent outside observers to assess how those criteria were applied...."
The kenyan goat herder won't even release his birth certificate, or his college transcripts so he ain't going to release the criteria for closing the dealerships.
Open and transparent my a$$!
Quote from an attorney who Deposed Chrysler's president last week: "It became clear to us that Chrysler does not see the wisdom of terminating 25 percent of its dealers... It really wasn't Chrysler's decision. They are under enormous pressure from the President's automotive task force."
You know what, sometimes it is the small things that turn into the big things. The big 0 needs to be pressed on this.
Sounds silly. Why just Chrysler (Jeep/?) and not Ford and GM too?
I've been reading these blogs for a week now.
And now - it's making national news.
I can't wait till these conservative dealers file class actions lawsuits against One Big Ass Mistake, America (OBAMA).
My wife is certain that they eventually will use political affiliation to deny health care to republicans. She is a cancer survivor and is panic stricken over this.
Sadly I only half doubted it and now this. I really think teh health care "reform" could be a hooror story for many and it must be prevented.
Chrysler is smaller than Ford or GM.
Just a better place to start.
They need to make it a RICO action.
" White House car czar Steven Rattner is married to Maureen White, the former national finance chairman of the Democratic National Committee."
Wonder if Mrs Rattner was driving a Chrysler when she had her DUI last October ?
The DUI that was mysteriously ( sarc ) ignored by the New York media.
Michael Wolff, the liberal writer for Vanity Fair, discusses the cover up here.
Using the list of all 789 dealerships to be closed, WND found that owners contributed $450,000 to GOP presidential candidates; $7,970 to Sen. Hillary Clinton; $2,200 to John Edwards and $450 to Barack Obama. For the "progressives" out there, that's a 1000-to-1 ratio of GOP-to-Obama donations for closed dealerships.
Item 2: Dealership conglomerate RLJ is owned by Democrat bigwigs Mack McLarty and Robert Johnson. RLJ magically happened to keep all six (6) of their dealerships while its competitors were shuttered. McLarty is a former Clinton chief-of-staff and Robert Johnson (founder of BET) is a major Democrat fundraiser. These politically connected, powerful Democrat supporters were completely insulated from the closings in every market in which they compete.
Item 3: Democratic donor Sidney Deboer is Chairman of Lithia Motors. He donated over $14,000 to Sen. Ron Wyden (D-OR) and Sen. Hillary Clinton (D-NY). Lithia Motors lost only two of its 29 dealerships while gaining as many as five additional ones in the resulting carnage. Deboer was also insulated from Chrysler's closings.
Item 4: Steven B. Fader contributed the individual maximum to Hillary Clinton for her 2008 campaign: $4,600. Fader is listed as owning four dealerships. Two of those dealerships are in Virginia Beach, VA: Hall Chrysler Jeep and Hall Dodge.
In the Virginia Beach area, Fader's competitor Grafton Dodge (in Grafton, VA) was shuttered. Grafton is owned by L. A. Julien, Jr., who does not appear to have contributed to either party. Another Fader competitor, Tysinger Motor Company was also closed. Tysinger is owned by Mark Tysinger, who also does not appear to have contributed to either party. Fader's two dealerships in the same city remain open, while nearby competitors were shuttered.
Another Fader dealership that remains open is Heritage Chrysler in Baltimore, MD. Nearby dealers that were closed include Antwerpen Dodge of Randallstown, MD owned by Jacob Antwerpen (no contributions founds); SKS Auto-Park of Baltimore; Bel-Air Dodge of Baltimore; and Schaefer & Strohminger Dodge Towson of Baltimore. The latter three are owned by Louis M. Schaefer, who has no contributions of any kind listed. Put simply, Fader's dealership remained whole while four area competitors were wiped out.
The final Fader dealership is Motorworld Chrysler, in Wilkes Barre, PA. It remains open while nearby Pompey Dodge of Kingston, PA and Ertley Chrysler Jeep Dodge of Moosic, PA were both closed. Charles W. Pompey and Ronald D. Ertley are the owners, respectively, and neither contributed to any candidates in 2006 or 2008. Much of Fader's competition in this area was also wiped out.
George Orwell was correct it is now 1984
Wonder if Mrs Rattner was driving a Chrysler when she had her DUI last October ?
: Steve Rattner, the man appointed by President Obama to redeem the American auto industry. But it's not all that complicated to determine at least one of the cars in Rattner's fleet. The couple's 2006 black Mercedes-Benz 350 (New York license plate #CSL1498!) was entered into the public record when his wife, Maureen White, was arrested for drunk driving last October.
Ford did not take money From the Government, GM is not far enough into the bankruptcy process to begin cancelling franchises, they will be in a few weeks.
If only. I like the Examiner and it's affiliated group of newspapers. They do yeoman's work. But, I wouldn't classify them as a "national" newspaper.
The sad fact of the matter is unless the WaPo or the NYTs (or perhaps a handful of weeklies) get on this story, it's just going to slowly fade away. Of course, that will never happen.
None of the national newspapers or broadcast networks are going to touch this - at all. Even if FNC ran with this story every day for a month, it still wouldn't make it into the American zeitgeist.
Dieing or not, the traditional media still has a tremendous amount of power, especially if they want to bury a story. They're going to be burying a lot of stories the next three years.
"Chrysler is smaller than Ford or GM.
Just a better place to start."
As Kerry said, 'would that it were.'
Being smaller would only narrow the focus of impropriety (as I'm sure you know)
Bill Ayers: Praire Fire:
any typos are mine.
"We need a revolutionary communist party in order to lead the struggle, give coherence and direction to the fight, seize power and build a new society." "Our intention is to disrupt the empire, to incapacitate it, to put pressure on the cracks, to make it hard to carry out its bloody functioning against the people of the world, to join the world struggle, to attack from the inside." "Our job is to tap the discontent seething in many sectors of the population, to find allies everywhere people are hungry, to mobilize poor and working people against imperialism." "Our final goal is the destruction of imperialism, the seizure of power, and the creation of socialism. Our strategy for this stage of the struggle is to organize the oppressed people of the imperial nation itself to join the colonies in the attack on imperialism. This process of attacking and weakening imperialism invloves the defeat of all kinds of national chauvinism and arogance; this is a precondition to our fight for socialism."
I'm not sure you understand. It's just Chrysler because so far, only Chrysler has filed a pre-packaged bankruptcy that's being guided by the Obama administration.
Ford is still completely solvent, for now.
And, GM won't be announcing any official closing until after their bankruptcy filing which could come as early as next Monday.
There's nothing silly about fascism and the targeting of your political enemies for financial ruin.
Big difference.
As I recall Nixon was looking for the "bad" guys.
0 is looking for the "good" guys.
Bill Ayers: Praire Fire
"Revolution is a fight by the people for power. It is changing of power in which existing social and economic relationships are turned upside down. It is a fight for who runs things, in particular, for control by the people of what we communists call the means of production"
"Ford did not take money From the Government, GM is not far enough into the bankruptcy process to begin cancelling franchises, they will be in a few weeks."
Yes, I understand about the money and was not commenting on that among any of the companies involved (except that in order for any discrepancies to go unnoticed, the smaller the company the better).
I'll add that if any company goes down, there WILL be union people going over the books (all of them)
As to GM franchise demise, most have 1 year upon recent notification. A better response than CH
It took 100 years for men to realize that the brain is also important.
Hopefully it will not be too long into 0's second 100 days for the majority of people to wake up and smell
....the crap !
"I'm not sure you understand."
I do and thank you for the reply. Tough to keep up with some replies without reading all of the thread.
FWIW: I have GM in my past (work/stock/etc). Will take my lumps like the rest though.
The MSM has created such a comfort zone for the 'Bama that corruption that would have cost past presidents their job is just routinely and openly practiced. The public has become so media-conditioned, that if Katie and Charlie don't tell them it's wrong, they're clueless. America has become too complacent and gullible to hang on to the republic or their freedom.
Interesting, but it doesn't mean much unless data for all dealers is collected. It wouldn't surprise me that a business would give more to anyone running against a commie.
Good point. We need a list of open and cloosing dealers, to see if there is a `pattern`...
Absolutley, 100% correct. It's what keeps me up at night. This is their guy and they simply aren't going to let him "fail", no matter how frequently or severely he does.
Ping to the yeoman!!!
Writing in a May 3 article, New York Times chief White House correspondent David Sanger said, Fresh from pushing Chrysler into bankruptcy, President Obama and his economic team are hoping the hard line they took last week gives them leverage to force huge changes in General Motors, a far larger and more complex company.
"The first testicular guard, Cup, was used in Hockey in 1874 and the first helmet was used in Hockey was 1974."
How the heck did this come from a car thread... oops sorry, of course.
I can only add that I had a boss who regaled (often) that he played football without a helmet.
His judgment calls seemed to support same.
Findings from 538.com breaks down some of the data on all car dealers (Not just Chrysler).
Overall, 88 percent of the contributions from car dealers went to Republican candidates and just 12 percent to Democratic candidates. By comparison, the list of dealers on Doug Rosss list gave 92 percent of their money to Republicans.
Wrt, the RLJ dealerships. Given some of the money behind this group (Bob Johnson is a billionaire) these dealerships might be better capitalized and better performing than some of their single dealer operating competitors. There may be corruption or it may be performance based. Further investigation will tell.
Also, Bob Johnson was an outspoken critic of Obama during the Democratic primaries and I would guess that McClarty would have been a Clinton supporter as well.
A sad ping.
"Writing in a May 3 article, New York Times chief White House correspondent David Sanger said, Fresh from pushing Chrysler into bankruptcy, President Obama and his economic team are hoping the hard line they took last week gives them leverage to force huge changes in General Motors, a far larger and more complex company."
I .... what is this in response to ?
I know that the NIC wants change, but I still maintain that there are too many involved for this to be a fiat by simple takeover without challenge.
Even yesterday, the GM bondholders seem to have upped their share in the surreptitious bailout, from 1% to 10%.
That is Bondholders, not stock holders (I'm both and future pensioner — no medical)
They have similar plans for GM, and don't have the same leverage to use against Ford.
Perfect suit for this Gangster Administration.
"They have similar plans for GM, and dont have the same leverage to use against Ford."
Ok, um, what plan? Take over the company and do what with it?
Sorry if I'm getting a bit direct here, but what can "they" do with over 10,000 defunct car dealerships and then approximately 20 idled/shuttered manufacturing facilities (and the factor by 10 ancillary direct and 20 indirect businesses?)
C'mon, if anyone was that bright or intelligent, they wouldn't have to be illegal or cheat.
I hope they fight back. What Obama is doing is illegal.
She had her license sus[pended for 90 days!!!! I hear it's 1 yr in Virginia.
Back to the top
Democrats have told us over and over that "it's the allegation that's important". Play their game. Make the allegation, make it big, and let them use energy disproving it.
Want not just donate cars they can't sell to charities like Cars4Charities www.cars4charities.org
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Still haven't seen "Wreck-It Ralph" and are wondering how it is? I'm sure you can guess that I thought very favorably of it, yet, still, I think you should read the review I wrote for Anjel Syndicate on the game and decide for yourself whether the movie is right for you!
I can say this: I only hope that Disney's work on this film inspires other companies to be less stringent with their licenses so that actually decent video game films can be made.


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As seen a really nice pair of classic loafers in black calf, in really good condition, just check photos. Shoe snob travel trees not included, sorry! Bargain priced for SF guys Posting from Sydney Australia $10 in Australia or $45 worldwide Questions and reasonable offers welcome!


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