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We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFα). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFκB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IκBα, the natural inhibitor of NFκB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFα, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFκB-dependent and NFκB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential.
A large and diverse range of proinflammatory cytokines and chemokines have been detected in the synovium of patients with rheumatoid arthritis (RA) (reviewed in [1,2]). This diversity is not surprising, considering the heterogeneous mixture of activated cells found at the sites of inflammation of RA synovium, which include macrophages, T cells, endothelial cells, fibroblasts and plasma cells.
Of particular interest are chemokines, which selectively recruit haemopoietic cells from the blood into the inflamed synovium. Several chemokines have been detected in RA synovium and include IL-8 (CXCL8) [3], monocyte chemoattractant protein 1 (MCP-1; CCL2) [4], epithelial neutrophil activating peptide 78 [5], macrophage inflammatory protein 1 alpha (MIP-1α; CCL3) [6], macrophage inflammatory protein 1 beta (MIP-1β; CCL4) [7], RANTES (CCL5) [7] and growth-related gene product alpha (GROα; CXCL1) [8] (reviewed in [9]). What regulates chemokine gene expression in the RA synovium, however, remains to be determined.
T cells were recently shown to be essential for the production of proinflammatory cytokines from macrophages in RA synovial tissue [23]. Although synovial CD4+ T cells proliferate poorly and produce low levels of IL-2 and interferon gamma [10-12], they express cytokines and activation markers [13] – and when put in contact with synovial fibroblasts or monocytes/macrophages, synovial CD4+ T cells induce high levels of inflammatory cytokines [14-16].
In vitro, we have shown that T cells activated by an anti-CD3 cross-linking antibody (that mimics TCR engagement (Ttcr)) or stimulated with a 'cocktail' of cytokines (designated cytokine-activated T cells (Tck)) also stimulate monocytes in a contact-dependent manner to produce cytokines that include IL-1β, TNFα, IL-12, IL-6 and IL-10 [15,17-21]. While the molecules involved in this process have not been fully defined, a number of T-cell-associated cell surface receptors/ligands, including CD69 [17], CD40L [18], CD11b and CD2, have been suggested of importance.
Histologically, T cells are often found in close contact with macrophages in the interstitium of RA synovial tissue [22] and T-cell depletion rapidly diminishes macrophage TNFα synthesis in RA synovial cultures [23].
We previously reported that the contact-dependent effector function of RA T cells in the joint is identical to that displayed by bystander-activated T cells (Tck), which can be expanded from normal blood with a cytokine cocktail containing TNFα, IL-6 and IL-2 over an 8-day period [21,23]. RA synovial T cells and Tck cells both induce TNFα production in resting monocytes in a cell-contact dependent manner, which is abrogated by blockade of the transcription factor NFκB but is augmented if phosphatidyl-inositol-3-kinase (PI3K) is inhibited. Normal blood T cells activated 'conventionally' via the TCR with cross-linked anti-CD3 antibody result in TNFα production from monocytes that is unaffected by NFκB blockade, but is inhibited in the presence of PI3K blocking drugs [23].
In the present report we investigated whether chemokine production from macrophages can also be induced in a contact-dependent manner by activated blood T cells, or indeed by T cells freshly isolated from rheumatoid tissue. We also examined which signalling pathways in macrophages are rate-limiting for the expression of chemokines after T-cell contact, with particular reference to the transcription factor NFκB, in order to gain insight into the regulation of chemokines at sites of inflammation.
Materials and methods
Isolation of peripheral blood monocytes and lymphocytes
Human monocytes were isolated from single-donor platelet pheresis residues purchased from the North London Blood Transfusion Service (Colindale, UK). Mononuclear cells were isolated by Ficoll/Hypaque centrifugation (specific density 1.077 g/ml; Nycomed Pharma A.S., Oslo, Norway), prior to cell separation in a Beckman JE6 elutriator (Torrence, CA, USA). Elutriation was performed in culture medium containing 1% heat-inactivated FCS. The monocyte purity and lymphocyte purity were assessed by flow cytometry, and fractions were typically >80% and 90% pure, respectively.
T-cell stimulation and fixation
Elutriation-enriched lymphocytes were resuspended in RPMI 1640 (containing 10% heat-inactivated AB+ human serum; (Biowittaker, Wokingham, UK) at 1 × 106 cells/ml. The resuspended lymphocytes were then cultured in six-well cluster culture plates (Falcon, Bedford, MA, USA) at 37°C in a 5% CO2/95% air-humidified incubator for 24 hours following stimulation with immobilized anti-CD3 mAb (OKT3; ATCC, Rockville, MD, USA), which had previously been coated onto the six-well culture plates at 10 μg/ml overnight at 4°C.
Alternatively, T cells were presented with different saturating concentrations of the following: 25 ng/ml TNFα (gift from Dr W. Stec, Centre of Macromolecular Studies, Lodz, Poland), 100 ng/ml IL-6 (gift from Dr P. Ramage, Sandoz, Pharma Ltd., Basel, Switzerland) and 25 ng/ml IL-2 (gift from Dr U Gubler, Hoffmann-LaRoche, Nutley, NJ) for 8 days in culture, prior to fixation.
In all instances, control T cells were cultured in the absence of any stimulus. Following stimulation, T cells were harvested and washed three times in RPMI 1640 prior to fixation for 1 minute in PBS containing 0.05% glutaraldehyde, and were than neutralized with an equivalent volume of T-cell neutralizing buffer containing 0.2 M glycine. Following a further three washes the fixed T cells were resuspended in complete medium (RPMI 1640 containing 5% heat-inactivated FCS) at 2 × 106 cell/ml and stored for up to 7 days at 4°C until use. The T cells were washed twice in complete medium prior to use.
Isolation of CD3+ cells from synovial membrane tissue
Mononuclear cells were obtained from synovial tissue specimens taken during joint replacement surgery, provided by the Orthopedic/Plastic Surgery Department of Charing Cross Hospital, London, UK. Tissue was teased into small pieces and digested in medium containing 0.15 mg/ml DNAase type I (Sigma, Gillingham, Dorset, UK) and 5 mg/ml collagenase (Roche, Welwyn Garden City, Hertfordshire, UK) for 1–2 hours at 37°C. Cells are passed through a nylon mesh to exclude cell debris, washed and resuspended in RPMI (supplemented with 10% heat-inactivated FCS) at a density of 1 × 106 cells/ml. Mononuclear cells were incubated with anti-CD3 monoclonal antibody-coated Dynabeads for 20 minutes at 4°C under constant rotation. Cells attached to beads were isolated using a magnetic particle concentrator (Dynal, Merseyside, UK) and cultured for 6 hours at 37°C. Detached cells were then removed from the magnetic beads and washed using the magnetic particle concentrator, which allows for isolation of CD3+ cells yielding high purity (>99%) and high viability (>95%). Cells were then fixed using the same protocol described above.
Adenoviral vectors and their propagation
Adenoviral gene transfer is a technique used for efficient gene transfer into dividing and nondividing cells, such as fibroblasts and monocytes [24,25]. Recombinant replication-deficient adenoviral vector containing no insert (Adv0) was provided by M. Wood (University of Oxford, UK), and the adenovirus encoding porcine IκBα with a cytomegalovirus promoter and nuclear localization sequence (AdvIκBα) [26] was provided by Dr R. deMartin (Vienna, Austria). Briefly, viruses were propagated in the 293 human embryonic kidney cell line and purified by ultracentrifugation through two caesium chloride gradients. Titres of viral stocks were determined by plaque assay in 293 cells after exposure to virus for 2 hours in serum-free RPMI 1640, followed by washing and re-culturing the cells in complete medium for 48–72 hours [27].
Gene transfer into macrophage-colony stimulating factor-treated monocytes with adenovirus
Prior to adenoviral infection, freshly elutriated monocytes were cultured in a 175 cm3 culture flask (Falcon) for 2 days in RPMI 1640 supplemented with 5% heat-inactivated FCS (complete medium) with 50 ng/ml macrophage-colony stimulating factor (M-CSF). This process upregulates the αvβ5 integrin, which acts as a cofactor for adenovirus infection [28,29]. Following culture, M-CSF-differentiated monocytes were washed once with PBS to remove nonadherent cells and the remaining adherent monocytes were incubated with 10 ml cell dissociation solution (Sigma) for 30–45 minutes until removed from the plastic. The cell suspension was washed three times in complete medium and the cell viability was assessed by trypan blue exclusion (>90%). Cells were plated at 2 × 105/ml in 96-well flat-bottomed culture plates (Falcon) and were allowed to adhere for 1 hour prior to infection with adenovirus. The media and nonadherent cells were removed from each well and replaced with serum-free RPMI 1640 and adenovirus at the required multiplicity of infection (MOI) for 2 hours. Following incubation, the medium was removed and replaced with complete medium. Monocytes were cultured for a further 2 days before stimulation to enable adenoviral production of IκBα to reach optimal levels.
Coculture of M-CSF-differentiated macrophages and lymphocytes
In the assays for contact-dependent chemokine production, M-CSF-differentiated monocytes (with or without IκBα transduction) were replated at 1 × 105 cells per well on a flat-bottom 96-well plate. Fixed lymphocytes were then added to the wells to give a final T cell:monocyte ratio of 7:1 and a final assay volume of 200 μl. Cultures containing monocytes alone and cultures containing lymphocytes alone were also included as experimental controls. Further controls included cocultures containing a porous membrane insert to physically separate the two populations, while allowing the transition of soluble mediators (0.2 μm Anopore® Membrane Nunc Tissue Culture Inserts; Nunc, Roskilde, Denmark). After 18 hours of culture at 37°C (5% CO2, humidified atmosphere), the supernatants were harvested and stored at -70°C for subsequent chemokine assay.
Measurement of chemokines by sandwich ELISA
Concentrations of IL-8 (CXCL8) (PharMingen, San Diego, CA, USA), GROα (CXCL1), interferon-gamma-inducible protein 10 (IP-10) (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4) and RANTES (CCL5) were determined by ELISA (R&D Systems, Oxford, UK), following the manufacturer's instructions. The absorbance was read and analysed at 450 nm on a spectrophotometric ELISA plate reader (Labsystems Multiskan Biochromic, Labsystems, Uxbridge, UK) using the Delta soft II.4 software programme (DeltaSoft Inc, Hillsborough, NJ, USA). Results are expressed as the mean concentration of triplicate cultures ± standard deviation.
Statistical analysis
Results were examined for statistical differences using Student's t test (two-tailed). P < 0.05 was considered significant, and such values are illustrated on the figures as appropriate.
Both Tck cells and Ttcr cells induce contact-dependent chemokine production by M-CSF-differentiated human monocytes
We have previously reported that the production of proinflammatory cytokines by macrophages can be induced by cognate interaction with Ttcr cells or Tck cells [19,21]. In the present paper we investigated whether activated T cells can also induce macrophage CC or CXC chemokine secretion in a contact-dependent manner. We found that, upon coculture, T cells activated with anti-CD3 antibody are able to induce production of high levels of chemokines in M-CSF-differentiated human monocytes (macrophages). Levels of both CC chemokines (MCP-1, MIP-1α, MIP-1β and RANTES) and CXC chemokines (IL-8, GROα and IP-10) are all elevated in comparison with those found in cultures of M-CSF-differentiated monocytes alone (Figure 1a). This induction of chemokine production in monocytes can be significantly reduced if the monocytes and T cells are physically separated using a porous membrane insert, demonstrating the importance of cell-cell contact in the induction process. In contrast, chemokine production by M-CSF-differentiated monocytes alone remains unchanged following coculture with unstimulated T cells (cultured for 24 hours prior to fixation).
Figure 1. Activated T cells induce contact-dependent chemokine production by human macrophages. Lymphocytes were left unstimulated or were stimulated with either anti-CD3 for 48 hours (Ttcr cells) or a 'cocktail' of inflammatory cytokines (tumour necrosis factor alpha (TNFα), IL-2, IL-6) (Tck cells) for 8 days, before fixation. The unstimulated, Ttcr and Tck populations were then cultured with macrophage-colony stimulating factor-differentiated monocytes (ratio 7:1) for 18 hours. Culture supernatants were then isolated and levels of CC chemokines (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) measured by ELISA. In some cases, a porous membrane insert was used to physically separate the two populations, while allowing the transition of soluble mediators. Results are shown from (a) Ttcr-cell lymphocyte cultures and (b) Tck-cell lymphocyte cultures. Data represent a mean of triplicate cultures ± standard deviation and are representative of at least three experiments. Statistically significant differences in chemokine detection are indicated.
Tck cells were also cultured with M-CSF-differentiated monocytes (Figure 1b). Tck cells, as seen with Ttcr cells, were able to induce significant production of all CC chemokines (MCP-1, MIP-1α, MIP 1β and RANTES) and CXC chemokines (IL-8, GROα and IP-10) assayed to similar levels, again in a contact-dependent manner. As expected, fixed Ttcr and Tck cells cultured alone did not secrete any detectable levels of chemokines (data not shown). Moreover, macrophages cultured in the presence of the insert and stimulated with lipopolysaccharide (LPS) secreted high levels of chemokines (data not shown) as previously described [30], indicating that the presence of the membrane insert does not influence macrophage function.
Differential utilization of NFκB in the Ttcr-cell and Tck-cell contact-dependent induction of CC and CXC chemokines in M-CSF-differentiated monocytes
We have previously shown that the contact-dependent induction of TNFα production in resting monocytes by Tck cells or RA synovial T cells is abrogated by blockade of the transcription factor NFκB [23]. As NFκB is a major transcription factor regulating the expression of numerous genes involved in immune and inflammatory responses [28,31], we determined whether T-cell contact-dependent production of chemokines is also regulated by NFκB.
To inhibit NFκB with specificity we employed an efficient adenoviral gene transfer method to overexpress IκBα in human macrophages. We have previously shown that high levels of IκBα are achieved by AdvIκBα transduction that remain elevated even after LPS stimulation [30]. As IκBα is a major inhibitory component of the NFκB pathway, increased expression of IκBα blocks NFκB nuclear translocation and DNA binding induced by LPS.
We then examined whether IκBα overexpression inhibits monocyte chemokine production induced by Ttcr cells. We found that AdIκBα inhibits the production of CC chemokines induced by contact with Ttcr cells but has no effect on CXC chemokine induction. MIP-1α production induced by Ttcr cells was therefore profoundly reduced, in a dose-dependent manner, in M-CSF-differentiated monocytes infected with AdIκBα but not with Ad0, a control virus without insert. At MOI of 40:1 and 80:1, the inhibition of MIP-1α expression was 54% (P ≤ 0.005) and 78% (P ≤ 0.005), respectively – which was not further increased at higher MOI (Figure 2a).
Figure 2. Differential utilization of NFκB in activated-T-cell contact-dependent chemokine production by human macrophages. Macrophage-colony stimulating factor-differentiated monocytes were infected with AdIκBα or Ad0, an empty control virus. After a further 2 days of culture and replating, anti-CD3-activated T cells (Ttcr cells) and cytokine-activated T cells (Tck cells) were added at a lymphocyte:monocyte ratio of 7:1. After 18 hours, culture supernatants were isolated and levels of CC chemokines (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) were measured simultaneously by ELISA. (a) MIP-1α levels in uninfected, Ad0-infected (multiplicity of infection (MOI) 200:1) and AdIκBα-infected (MOI 40:1, 80:1 and 200:1) monocyte cultures when stimulated with Ttcr-cells or Tck-cells. (b) and (c) Levels of CC and CXC chemokines in Ad0-infected and AdIκBα-infected monocytes (MOI 80:1) following stimulation with (b) Ttcr cells and (c) Tck cells. Data represent the mean of triplicate cultures ± standard deviation and are representative of at least three experiments. Statistically significant reduction in chemokine levels in AdvIκBα-infected (as compared with Ad0-infected) cultures is indicated.
Similar significant inhibition of the production of the other CC chemokines MIP1-β (73.9%, P ≤ 0.005), RANTES (70.2%, P ≤ 0.005) and MCP-1 (67%, P ≤ 0.005) was also observed in AdIκBα-infected monocytes (Figure 2b). In contrast, there was no effect of IκBα overexpression on CXC chemokine production. We found that there was no significant inhibition of the chemokines GROα, I,L-8 or IP-10 in AdIκBα-infected monocytes activated by Ttcr cells, suggesting that there is differential utilization of NFκB for the expression of CC and CXC chemokines in this system.
We also examined the role of NFκB in the Tck-cell contact-dependent production of chemokines in monocytes. Unexpectedly, we found that IκBα overexpression inhibited Tck-cell-dependent CXC chemokine production in M-CSF-differentiated monocytes, but had no effect in CC chemokine production. Thus, although contact-dependent induction of GROα, IL-8 and IP-10 was significantly inhibited in AdIκBα-infected macrophages by 78.7% (P ≤ 0.01), 63.2% (P ≤ 0.01) and 52.1% (P ≤ 0.05), respectively, the induction of MIP-1α, MIP-1β, RANTES and MCP-1 was unaffected (Figure 2c). This inverted pattern of utilization of NFκB for the T-cell contact-dependent induction of CC and CXC chemokines in monocytes is surprising and indicates that chemokine gene expression may be more complex than previously thought.
IκBα overexpression significantly inhibits rheumatoid T-cell-induced macrophage secretion of CXC, but not CC, chemokines
We next investigated whether RA synovial T cells enriched from dissociated RA synovial tissue could also induce monocyte chemokine secretion in a contact-dependent manner and whether this requires NFκB. RA synovial T cells were isolated from dissociated synovial membranes using anti-CD3 DynaBeads, as described in Materials and methods. We found that, like Ttcr and Tck cells, fixed RA synovial T cells were able to induce both CC and CXC chemokine production from M-CSF-differentiated human monocytes (Figure 3). Furthermore, overexpression of IκBα in these monocytes resulted in impaired RA synovial T cell-dependent CXC chemokine release, when compared with Ad0-infected monocytes. A significant reduction in IL-8 (54.1%, P ≤ 0.01), IP-10 (39.6%, P ≤ 0.05) and GROα (74.2%, P ≤ 0.01) production was therefore observed (Figure 3a). This effect was dose dependent, with increasing MOI of 40:1 and 80:1 inducing a reduction in GROα levels of 55.1% (P ≤ 0.01) and an optimal 74.2% (P ≤ 0.001), respectively (Figure 3b).
Figure 3. IκBα overexpression significantly inhibits rheumatoid T-cell-induced macrophage chemokine secretion of CXC, but not CC, chemokines. Using anti-CD3 labelled Dynabeads, synovial T cells were enriched from the mixed cell population obtained following enzymatic dissociation of synovial tissue samples from rheumatoid arthritis (RA) patients. Fixed RA T cells were cultured with macrophage-colony stimulating factor-differentiated monocytes infected with Ad0 and AdvIκBα at a T cell:monocyte ratio of 7:1 as described in Figure 2. After 18 hours, culture supernatants were isolated and levels of CC chemokines (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) were measured by ELISA. (a) Levels of chemokines for monocytes infected with Ad0 and AdIκBα (multiplicity of infection (MOI) 80:1) following stimulation with RA T cells. (b) GROα levels in uninfected, Ad0-infected (MOI 200:1) and AdvIκBα-infected (MOI 20:1, 40, 80:1 and 200:1) monocyte cultures when stimulated with RA T cells. Data represent the mean of triplicate cultures ± standard deviation and are representative of at least three experiments. Statistically significant reduction in chemokine levels in AdIκBα-infected (as compared with Ad0-infected) cultures is indicated.
Similar dose-dependent profiles were observed for the other chemokines tested (data not shown). Interestingly, however, overexpression of IκBα had no significant effect on the expression of CC chemokines by M-CSF-differentiated monocytes, suggesting that RA synovial T cells possess similarities in their effector function to Tck cells, rather than Ttcr cells. It is noteworthy that RA T cells isolated based on CD2 expression have previously demonstrated an identical effector function to those isolated using anti-CD3 (data not shown), thus discounting the idea that CD3-based methods may influence the behaviour of RA T cells (through the potential for crosslinking) in this system.
The phosphatidyl-inositol-3-kinase pathway regulates both NFκB-dependent and NFκB-independent contact-mediated chemokine production
Finally, we investigated what further cell signalling pathways (in addition to NFκB) could play a potential role in contact-dependent chemokine production. Ttcr cells and Tck cells were used to stimulate monocytes that had been pretreated with a chemical inhibitor of the PI3K pathway (LY294002), and the resulting effects on chemokine production were determined. We found that Ttcr-induced IP-10 (CXC chemokine) production (NFκB independent) was dose-dependently reduced in the presence of the inhibitor (Figure 4b). In contrast, Tck-induced MIP-1α (CC chemokine) production (also NFκB independent) could be dose-dependently enhanced in the presence of the PI3K inhibitor (Figure 4a), indicating the pathway plays a positive and negative regulatory role in each respective case. With NFκB-dependent Ttcr-induced MIP-1α production also displaying PI3K dependence, however, a role for this pathway in NFκB-dependent as well as NFκB-independent chemokine production cannot be ruled out.
Figure 4. The phosphatidyl-inositol-3-kinase pathway regulates both NFκB-dependent and NFκB-independent contact-dependent chemokine production. Macrophage-colony stimulating factor-differentiated monocytes were preincubated for 30 minutes in the presence or absence of variable amounts of LY294002 (as shown) before being stimulated with anti-CD3-activated T cells (Ttcr) or cytokine-activated T cells (Tck) at a T cell:monocyte ratio of 7:1. After 18 hours, culture supernatants were isolated and levels of (a) macrophage inflammatory protein 1 alpha (MIP-1α) (CC chemokine) and (b) interferon-gamma-inducible protein (IP-10) (CXC chemokine) were measured by ELISA. Data represent the mean of triplicate cultures ± standard deviation and are representative of at least three experiments.
We have previously shown that TNFα synthesis in RA synovial cultures is T-cell contact-dependent; T-cell depletion or physical separation from the rest of the cells rapidly diminished macrophage TNFα production in these cultures [23]. We have also shown that the contact-dependent effector function of RA T cells in the joint resembles that displayed by Tck cells, which can be expanded from normal blood with cytokines found in the RA joint and in the absence of TCR engagement [21,23]. Both RA synovial T cells without further activation and Tck cells induced TNFα production in resting monocytes in a cell-contact dependent manner, which was abrogated by blockade of the transcription factor NFκB but was augmented if PI3K was inhibited. Normal blood T cells activated 'conventionally' via the TCR with cross-linked anti-CD3 antibody (Ttcr cells) do not reproduce this effector function of RA T cells [23]. In this study, we investigated whether Tck cells or RA synovial T cells also regulate chemokine production from macrophages and whether this was mediated in a contact-dependent manner.
Using a coculture system consisting of fixed lymphocytes and M-CSF-differentiated human monocytes [21,23], we demonstrate in this manuscript that Tck cells stimulate monocytes to secrete high levels of several CC and CXC chemokines that include MIP-1α, MIP-1β, RANTES, MCP-1, GROα, IL-8 and IP-10. This was a T-cell contact-dependent process as the physical separation of T cells from monocytes through the use of a transwell insert abrogated this effect. This observation was also true for Ttcr cells and RA synovial T cells but not for control nonactivated T cells, suggesting that contact-dependent regulation of macrophage chemokine production is a general property of activated T cells. Several other groups have also shown the importance of T-cell contact in regulating the production of cytokines and tissue destructive enzymes (such as matrix metalloproteinases) by monocyte/macrophages [15,17-21,32] and fibroblasts, suggesting that this may be a major mechanism of promoting inflammation in chronic inflammatory diseases where there is an absence of infection or infectious agents [33].
Various stimuli induce T cells to activate monocytes/macrophages by cellular contact, including anti-CD3 cross-linking with or without anti-CD28 stimulation (as used in this study) [34], cytokines such as IL-2, IL-6 and TNFα (as used in this study) [19] or IL-15 [15], phytohaemagluttinin/phorbol myristate acetate [17,35,36] and antigen recognition on antigen-specific T-cell clones of the Th1 or Th2 phenotype [37,38]. Depending on the T-cell type and the stimulus used, the pattern of gene expression triggered in monocytes/macrophages by T-cell contact differs. We have previously shown that although Ttcr cells activate monocytes to produce both TNFα and IL-10, Tck cells only trigger the production of TNFα in monocytes, suggesting that this is a mechanism by which the cytokine balance is skewed towards the proinflammatory side in RA [19]. Other studies have shown that Th1 clones preferentially induce IL-1β rather than IL-1 receptor antagonist over other T-cell clones [38,39]. This suggests that multiple ligands and counter-ligands are involved in the contact-mediated activation of monocytes/macrophages that are differentially induced on T cells (depending on the stimulus) and differentially induce monocyte/macrophage signal transduction.
The transcription factor NFκB has been shown to regulate both inflammatory and tissue destructive processes in RA [25,40]. Many of the promoter regions of chemokines are known to have κB sites in their promoters and include IL-8 [41], GROα [42], IP-10 [43], MCP-1 [44], and RANTES [45]. We recently used adenoviral gene transfer of IκBα to block NFκB in human M-CSF-differentiated monocytes, and showed that the expression of CC chemokines MIP-1α, MCP-1 and RANTES induced by TNFα or LPS was NFκB dependent, as was the expression of CXC chemokines IL-8, GROα and epithelial neutrophil activating peptide 78 induced by TNFα [30]. The expression of these CXC chemokines induced by LPS, however, was found to be NFκB independent – indicating that the requirement for this transcription factor in the regulation of chemokine gene expression is complex and dependent on the stimuli used.
In this study, we used the same system of adenovirally mediated IκBα overexpression in M-CSF-differentiated monocytes to investigate the potential involvement of NFκB in the expression of CC and CXC chemokines induced by contact with activated T cells or RA synovial T cells. Surprisingly, we found that blocking NFκB resulted in differential inhibition of CC and CXC chemokines depending on whether Ttcr cells, Tck cells or rheumatoid T cells were used to stimulate M-CSF-differentiated monocytes. CC chemokine production was thus found to be NFκB dependent when mediated by Ttcr cells, but NFκB independent when mediated by Tck or RA synovial T cells. In addition, CXC chemokine production was found to be NFκB independent when mediated by Ttcr cells, but largely NFκB dependent when mediated by Tck or RA synovial T cells. These data suggest that, through different molecular interactions, at least two differential pathways of monocyte chemokine production are induced by Ttcr cells and Tck cells that differ in the rate-limiting involvement of NFκB. Evidence from our inhibitor studies suggest involvement of the PI3K pathway in regulating both NFκB-independent and NFκB-dependent chemokine production, in either a positive or negative manner, depending on chemokine and lymphocyte stimulus. We have previously published work showing a similar augmentation of Tck/RA T-cell-induced TNFα production in the presence of these inhibitors [23].
As the promoters of all the chemokines studied here contain NFκB binding sites, this raises the obvious question of how this effect is regulated. Currently unclear is whether these specific sites are functioning as positive or negative regulators of transcription; a process that could itself be influenced by which other pathways are also activated. For example, TNFα production in T cells is known to be regulated by nuclear factor of activated T cells although the TNF gene contains at least five NFκB sites [46]. Furthermore, variable factors such as the site sequence and its distance from the transcription start site, as well as the nature of the different NFκB dimers recruited to the site, will all interact to influence gene expression [47].
A further layer of complexity operating in this system is the role of contact-induced TNFα in secondary chemokine production. We have previously shown TNFα production itself is differently dependent on NFκB and the PI3K pathway (similarly regulating either positively or negatively) according to Ttcr-cell or Tck-cell induction processes. As such, effects on both pathways could be acting on chemokine induction in direct and indirect ways. Furthermore, our previous studies have shown both Ttcr-cell-induced and Tck-cell-induced TNFα production to be p38MAPK dependent, but p42/p44 MAPK independent (data not shown), indicating that mitogen-activated protein kinases may also be involved in contact-dependent chemokine induction.
This study demonstrates for the first time that RA synovial T cells as well as Tck cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFκB-dependent and NFκB-independent mechanisms, in a process influenced by the PI3K pathway. In addition, these data provide further evidence that Tck cells share aspects of their effector function (such as contact-mediated monocyte chemokine production) with RA synovial T cells. Furthermore, these data demonstrate one more function of RA T cells; namely, their ability to induce monocyte/macrophage chemokine secretion by cellular contact. The observation that RA synovial T cells mirror the behaviour of cytokine-driven, rather than CD3-activated, cells is consistent with the notion that antigen-independent responses play a key role in RA. As such, this study further emphasizes that T cells are not simply 'innocent bystanders' in RA, but can be important drivers of chronic inflammation through antigen-independent mechanisms [48,49].
ELISA = enzyme-linked immunosorbent assay; FCS = foetal calf serum; GROα = growth-related gene product alpha; IL = interleukin; IP-10 = interferon-gamma-inducible protein 10; LPS = lipopolysaccharide; mAb = monoclonal antibody; MCP-1 = monocyte chemoattractant protein 1; M-CSF = macrophage-colony stimulating factor; MIP-1α = macrophage inflammatory protein 1 alpha; MIP-1β = macrophage inflammatory protein 1 beta; MOI = multiplicity of infection; NFκB = nuclear factor kappa B; PBS = phosphate-buffered saline; PI3K = phosphatidyl-inositol-3-kinase; RA = rheumatoid arthritis; Tck cells = cytokine-activated T cells; TCR = T-cell receptor; Ttcr cells = anti-CD3-activated T cells; TNFα = tumour necrosis factor alpha.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JTB participated in data analysis, assembly and creation of the figures, and manuscript writing. EA contributed to the study design, experimentation, data analysis, assembly and creation of the figures, and manuscript writing. CJC was involved in the study design, experimentation and data analysis. PG contributed to the study design, experimentation, data analysis, and assembly and creation of the figures. BMJF and FMB were responsible for the initiation of the study, review of the analysed data and manuscript writing.
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text:
"Somewhere in the United States, black clouds of smoke are billowing."
By Tim Colwill on February 6, 2013 at 6:41 pm
North Korea's successful launch of a space-capable rocket recently and their threat to carry out further tests targeting the US has apparently inspired the creation of their new propaganda video — one which isn't above taking footage from Modern Warfare 3 in order to show the United States under attack.
The video, spotted on Liveleak, shows a young man sleeping and dreaming of a glorious new age of North Korean space flight. The space shuttle circles the globe several times, zooming in on a joyous and newly re-united Korea, before flipping to images of New York City being blown up.
The scene in question is taken directly from a similar scene in Modern Warfare 3, and is superimposed over with text that allegedly reads "Somewhere in the United States, black clouds of smoke are billowing. It seems that the nest of wickedness is ablaze with the fire started by itself." Take a look below.
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text:
O.A.R.S. Press Release
- O.A.R.S.' Introduces New Upscale Galápagos Explorer Itinerary for 2016
- OARS Booking Trends for 2016
- O.A.R.S.' Salmon River Rafting Trip Recognized as "Best Guided Outdoor Trip"
- OARS announces 2-year partnership with the National Park Foundation
- O.A.R.S. Releases Optimistic 2015 Whitewater Rafting Forecast
- O.A.R.S. and Chaco Pair Up
- TravelAge West Selects O.A.R.S. as 2015 Best Tour Operator for Adventure
- OUTSIDE Honors O.A.R.S. with Two Best of Travel Awards: Best River Trip and Best River Guide
- The Confluence of Adventure
- O.A.R.S. Releases 2015 Family Adventures Catalog
- Yampa River News
- Rivers Fiji Recognized Among World Leaders in Responsible Tourism
- O.A.R.S. Releases River of Eden by Filmmaker Pete McBride
- O.A.R.S. Embraces Autumn On Western Waterways and National Park Trails With Multi-Day Adventure Vacations
- O.A.R.S.' Wine on the River Series Pairs Vineyards and Gourmet Presentations With Drama of Rafting Western Rivers
- Return of High Water to Utah's Cataract Canyon Promises Some of the Best Rafting of the Year
- O.A.R.S. California Road Trip Sweepstakes Celebrates 150th Anniversary of the Yosemite Grant
- O.A.R.S., World Leader in Whitewater Rafting, Launches Social Media Contest to Help Encourage National Park Visits this Summer
- O.A.R.S., World Leader in Whitewater Rafting, Announces New Partnership with Klean Kanteen
- 2014 Pacific Coast Rafting Outlook Remains Positive Despite Drought
- O.A.R.S. Releases 2014 Pre-Season Rafting Forecast
- O.A.R.S. Guides Lead Historic Rafting Descent on the Colorado River "Pulse Flow"
- O.A.R.S. Releases 2014 Family Adventures Catalog Featuring 21 Easy to Epic Vacation Ideas
- The Year of the Dory: O.A.R.S. Pays Tribute to Legendary Grand Canyon Boatman/Environmental Crusader Martin Litton for its 45th Anniversary
- O.A.R.S. Introduces New-for-2014 Lodge-Based Grand Canyon Hiking Adventure
- O.A.R.S., Worldwide Leader in Whitewater Rafting, Announces New Departures Exploring the Sacred Rivers of Bhutan
- O.A.R.S., Worldwide Leader in Whitewater Rafting, Launches World Rivers Series of New Trips Marrying Iconic Rivers with Cultural Destinations
- U.S. Marine Captain with Four Deployments to Afghanistan under His Belt Chosen as Winner of O.A.R.S. "Livin' the Dream" Contest
- O.A.R.S. Announces Enhanced Commission Structure Including Travel Agent Commissions on Coveted Grand Canyon Rafting Adventures
- Condé Nast Traveler Selects O.A.R.S.' Mindy Gleason Among its Top Travel Specialists for 7th Consecutive Year
- O.A.R.S. Launches "Livin' the Dream" Contest To Become Honorary River Guide On No-Cost Grand Canyon Rafting Trip
- O.A.R.S. Family Rafting Adventures Give Kids a Break from Devices
- O.A.R.S. Experiencing Increased Demand For Rafting Adventures in Northern California This Summer On Reservoir-Controlled Rivers
- Two Oregon Purveyors of Fine Living, A Chocolatier and a Brew Master, Spice Up Father's Day on the Rogue River with O.A.R.S.
- Veteran Outfitter O.A.R.S. by Longstanding Agreements With Grand Teton and Yellowstone National Parks Offers Guests Unique Access and Experiences
- A Strong Constitution for Fun Recommended for O.A.R.S.' July 4th – 6th Tuolumne River Trip with 21st Amendment Brewery
- O.A.R.S. Releases 2013 Family Adventure Catalog With Vacations From Peru to Alaska
- O.A.R.S. Selected by Outside Magazine As Runner Up for "Best Outfitter of the Year"
- O.A.R.S. Outlines 2013 Menu of Whitewater Vacations On Idaho's Iconic Salmon and Snake Rivers
- O.A.R.S. Offers 11 Unique Gourmet Whitewater Rafting Adventures in 2013
- O.A.R.S. Rafts Second-Ever Descent of Drangme Chhu in Bhutan
- O.A.R.S. Hosts Series of Epicurean Adventures on Idaho's Salmon River with Whole Journeys
- O.A.R.S.' Adventure Consultant Recognized as Top Travel Specialist for River Rafting by Condé Nast Traveler for the 6th Consecutive Year
- O.A.R.S. Founder, George Wendt, Honored at 2012 California River Awards
- O.A.R.S. Partners With Leading Social Media Agency To Launch Web Magazine
- Dinosaur National Monument, an Undiscovered Gem, Shines this Summer
- O.A.R.S. and American Rivers Shine the Light on the Endangered Green River
- O.A.R.S. Releases Industry-leading Whitewater Orientation Video
- O.A.R.S.' Bosnia Three Rivers Tour Recognized as One of "50 Tours of a Lifetime" for 2012
- The Grand Canyon by Dory Named to Hall of Fame in Outside's Inaugural Travel Awards
- O.A.R.S. Seeks to Inspire the Next Generation of Adventurers with New Family Catalog
- O.A.R.S. Launches Three New Adventures for 2012
- O.A.R.S.' Adventure Consultant Recognized as Top River Rafting Expert by Condé Nast Traveler for the 5th Consecutive Year
- O.A.R.S. Reinvents the Travel Catalog for 2012
- Record Water Levels on Tap for 2011 Cataract Canyon Rafting Season
- O.A.R.S.' Grand Canyon River Trip Recognized as One of "50 Tours of a Lifetime" for 2011
- California Snowpack Has River Outfitters Looking Forward to 2011 Rafting Season
- Prime Minister of Fiji Recognizes Rivers Fiji with "Exporter of the Year" Award for Tourism Services
- Award-winning Outfitter Unveils 2011 Bucket List of Adventures
- O.A.R.S. Announces New Wilderness Gourmet Culinary Adventure Featuring Acclaimed Ashland Chef, Damon Jones on the Rogue River
- Adventures in Wine and Whitewater
- Above Average Sierra Snowpack Translates to Exciting, Extended California Whitewater Rafting Season
- Grand Teton National Park selects O.A.R.S. as long-term concessionaire & the only rafting & sea kayaking outfitter to operate overnight trips in the park
- Rivers Fiji recognized with Responsible Tourism Award by Virgin Holidays
- Fifty "Eco-Friendly" Ways to Experience the Unforgettable Nature of an O.A.R.S. Adventure
- Adventure Travelers in Pursuit of the Seven Whitewater Wonders of the World
- Raft More, Spend Less: All-Inclusive Adventures Make Good Financial Sense in Today's Economy
- Unique Vacation Options, On and Off the Beaten Path, In America's Most Iconic National Parks
- O.A.R.S. Receives Top Honors from Distinguished Media Outlets
- O.A.R.S. To Begin Accepting Reservations for 2010 Grand Canyon Rafting Trips
- O.A.R.S. Offers Seven New Trips in 2009 Adding to an Extensive Collection of Adventurous Itineraries throughout the U.S. West, Canada, Latin America and Fiji
- O.A.R.S.' Gourmet Whitewater Rafting Trips Offer Elegant Adventure Vacation Alternatives
- O.A.R.S. Commemorates 40th Anniversary of the Wild and Scenic Rivers Act by Partnering with American Rivers and by Offering a Benefit Whitewater Rafting Trip in Idaho
- O.A.R.S. - Mother Nature Cooperates with Whitewater Rafting Company: Snow Levels Make for an Outstanding 2008 Whitewater Rafting Season
- O.A.R.S. – the eco-savvy adventure travel outfitter further reduces carbon footprint with a new commitment to solar power
- O.A.R.S. announces a one-of-a kind series of rafting and product-testing expeditions with performance footwear innovator Teva
- Seven New Hiking-Specific Itineraries Added to O.A.R.S. Diverse Trip Options
- O.A.R.S. and Teva Team Up to Support a 3D Experience for IMAX Theaters – Grand Canyon Adventure: River at Risk
- O.A.R.S. Receives Top Honors from Distinguished Media Outlets
- O.A.R.S. Adventure Vacations - Something for everyone: the thrill-seeker, the naturalist, the not-exactly-athletic, the first-time camper and the seasoned boater
O.A.R.S. (Outdoor Adventure River Specialists)
CONTACT: Steve Markle
TEL: 209-736-2407 ext. 716
EMAIL: email@example.com
WEBSITE: www.oars.com
SKYPE: stevem_oars
O.A.R.S. Adventure Vacations Something for everyone: The thrill-seeker, the naturalist, the not-exactly-athletic, the first-time camper and the seasoned boater.
Angels Camp, California – For almost 40 years, O.A.R.S. (Outdoor Adventure River Specialists) has been a leader in the adventure travel industry. The California-based, eco-conscious organization provides exceptional, award-winning customer service, friendly, experienced and safety-conscious guides, comfortable and clean camping equipment, undeniably delicious and hearty meals and an ideal environment for a traveler to experience the outdoor vacation of his or her dreams.
O.A.R.S. trips have positively affected the lives of over 400,000 travelers due to their ability to meet the diverse needs of their broad spectrum of clientele: they offer more than 75 unique itineraries worldwide. More than half of their guests experienced vacations with children in tow. Trips vary in price, making adventures accessible to families, professionals, students and retirees.
Some vacations include catered camping and rafting excursions through one of six national parks, lodge-based outings with comfortable beds and hot showers, multi-week expeditions, half-day escapes, weekend retreats close to home or private chartered trips specifically and uniquely designed for a given client's needs. Various organizations and publications have repeatedly proclaimed O.A.R.S. adventures as the "Best Trips on the Planet."
O.A.R.S. was established in 1969 by George and Pam Wendt and with a goal to help people of all ages enjoy "the best outdoor experience of their lives." The company was selected as the first exclusively oar-powered rafting company in the Grand Canyon, and Wendt has since been awarded a Lifetime Achievement Award from the Adventure Travel Trade Association (ATTA).
The company runs first-class rafting, sea kayaking and multi-sport (rafting / sea kayaking / hiking / biking / horseback riding) trips in Alaska, Arizona, California, Colorado, Idaho, Oregon, Utah and Wyoming as well as in Canada, Baja, Fiji and throughout South America, rendering them a diverse and accommodating force in adventure travel.
Borrowing from John Muir, "In every walk with nature one receives far more than he seeks," O.A.R.S. long ago discovered the enduring magic of classic rivers, stunning scenery and mesmerizing companionship. The organization continues to offer trips on some of the country's finest rivers: the Colorado River through the Grand Canyon, Idaho's incomparable Middle Fork Salmon and Alaska's spectacular Tatshenshini. In addition, O.A.R.S. specializes in family adventures with notable vacations on Utah's Green River through Dinosaur National Monument, Oregon's Wild and Scenic Rogue River and Idaho's Lower Salmon River.
In addition to family-friendly journeys and classic river trips appropriate for travelers of all ages, O.A.R.S. offers epic whitewater rafting for thrill-seekers on California's North Fork American, Tuolumne and Upper Klamath rivers, as well as Canada's Chilko River. Other adventure options, designed for travelers with specific interests, include trips which highlight archaeology, wine tasting, gourmet cooking, photography, as well as trips for the solo traveler, hiking enthusiasts and trips for women only.
New to O.A.R.S.' vacation menu in 2008: rafting on the Apurimac River, the source of the Amazon and lodge-to-lodge trekking to Machu Picchu, 15 or 16 days.
O.A.R.S. was the first, and only, North American rafting outfitter to be 100% carbon neutral through an innovative partnership with NativeEnergy Travel Offsets. They are in their third year of off-setting all energy used in their California-based office and all domestic adventures. O.A.R.S. swapped the motors on their gear boats for their Jackson Lake trips for new four-stroke engines, reducing fuel consumption significantly, and they provide on-site employee housing in most destinations, sparing guides and staff from potentially lengthy, carbon-emitting commutes. Additionally, 20% of the fuel used in their Grand Canyon gear vehicles is now bio-diesel.
Recognized as an authoritative resource for environmentalists, conservationists, politicians and eco-celebrities, O.A.R.S. was selected to support MacGillivray Freeman Films and Teva in their production of a cutting-edge water conservation film, "Grand Canyon Adventure" 3D IMAX ®. The film, featuring Robert Kennedy, Jr. and anthropologist Wade Davis, and their daughters, premiers on World Water Day, March 28, 2008. The film will undoubtedly raise water conservation awareness worldwide.
Since 1969 O.A.R.S. has been setting the standard in first-class rafting, sea kayaking and multi-sport vacations, with destination and unparalleled experiences on over 35 rivers and coastlines of the world. O.A.R.S. caters to active travelers of all ages and abilities with more than 75 unique itineraries worldwide, including one-day and weekend escapes. For more information on O.A.R.S. eco-friendly adventures, including a free 84-page color catalog, call O.A.R.S. at 1-800-346-6277, email the company at email@example.com or visit www.oars.com.


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"Civil Rights in America: How Far Have We Come?" is the theme of a series of programs in February to be presented as part of the fifth annual Springfield African-American Read-In.
The Springfield events are part of the 25th annual national Read-In. The forum is free and open to the public.
The Springfield programs are part of a collaborative partnership of Missouri State University, the MSU Libraries, Drury University, Springfield Public Schools, Springfield-Greene County Library District and the NAACP Springfield Chapter. ...


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Updated at 9 am EST.
President Barack Obama is converting the machinery of his re-election campaign into a tax-exempt nonprofit group to push his second-term legislative agenda, a Democratic official confirmed.
The new group, Organizing for Action, will be headed by Jim Messina, who served as Obama's 2012 campaign manager.
That effort was known as Organizing for America.
The group, separate from the Democratic National Committee, will solicit donations from corporations and individuals to augment Obama's legislative initiatives the next four years on such issues as reducing gun violence and immigration.
The official spoke on condition of anonymity to discuss details before an official announcement later today. The formation of the group was reported earlier by the Los Angeles Times.
The transformation marks the first time a president has retrofitted the pieces of a re-election campaign into an outside group formed for the express purpose of prodding Congress to pass the administration's agenda.
Familiar names will dominate the group's board of directors, the Democratic official said. They include former White House press secretary Robert Gibbs and top 2012 campaign aides Stephanie Cutter, Jennifer O'Malley-Dillon and Julianna Smoot.
Jon Carson, White House director of the Office of Public Engagement, is being tapped as executive director of the group, which will have offices in Washington and Obama's hometown of Chicago.
David Axelrod, senior adviser to the 2012 campaign, will become a consultant. White House senior adviser David Plouffe is expected to join the group after he leaves the administration later this month, the Democratic official said.
First Lady Michelle Obama leads the organization's video kick-off today.
Hans Nichols contributed.


text:
19-1 - [image: NPC19pg 01 colours]
Wednesday, April 7, 2010
Dispelling like its 2004
Thought I would put up a post to a thread of blue comments on the subject of dispells in Cata.
The note that really makes me think we are going back to the Bad old days of Dispells. is this. "We also recognize the challenge for magic-based controllers in PvP, say mages, to handle teams with druid healers and we might have to reconsider the druid ability to avoid Polymorph. We think it's important for PvE reasons for all healers to be able to dispel magic however."
These are PVE changes and not PVP which means they already have plans in place and are not working to make all the classes fit those plans. I think its a great idea in principal, we all know this will change because of some silly pvp imbalance.
The class previews should give us some more insights but we really aren't going to know how weak or powerful this is going to make every class until we are neck deep in beta test which still looks to be a ways off. I wouldn't bet on seeing it untill after April.
On the the upside from the non healer point of view of my mage sounds like not much is changing.
Posted by Sayomara Vesper at 7:24 AM 0 comments Links to this post
Labels: dispell
Tuesday, April 6, 2010
Decurse or not to Decurse
From the I was there man files we get news that healers dispells will be Changing with Cataclysm. I play Mage I have been helping dispell for a long time, and I enjoy the fact that I can help healers out. Fights like Lady Deathwisper the Curse of Torpor can really lock down players, the only classes that can removed it are Mages, Druids, and Restoration Shaman. That said I think I'm one of the few people that enjoy dispelling, and most people cry when they have to do it.
Well we have more of it coming. And healers its cleared aimed at our mana Pools. Blizzard has said for some time they aren't happy with current state of healers where most of the time we have a endless supply of mana. These changes to be suggest a return to raid wide debuffs which were so common in classic Warcraft. Will we have large numbers of raids failing to beat Lucifron 2.0 because they can't get there mages to decurse.
Will we have fights like Chromaggus where if a single debuff is left up it spells doom to the raid?
Maybe it will be more interesting this time. I don't have a problem with set in and forget it kinda of spell or some of the dps and tanking specs losing there abilities to dispell from a PVP point of view. But I do worry about debuffs that are applied raid wide so fast as to not allow healers to do anything else. You think healing is tiresome now. Just wait.
Time to play the waiting game...the waiting game is stupid lets go play Hungry Hungry Hippos.
Posted by Sayomara Vesper at 9:06 AM 0 comments Links to this post
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He has made a number of harmful decisions, especially those emerging from experience with drugs and poor choices in friends.
"Ironically, it may not be that harmful if the mandate is overturned," she added.
But extended leaves and part-time employment are known to be harmful to careers — for both genders.
Speaker of the House John Boehner pointed out that the idea that carbon dioxide is "harmful to the environment is almost comical."
Another act, however, may be considered immoral not because it is harmful but because it evinces disloyalty.
Is it not a dreadful thing that one's mind should be thus ruined by a useless and harmful practice?
Corporal punishment of schoolboys is only useless and harmful brutality.
Wine is harmful if taken with greedy lips, but if drunk in moderation it is wholesome.
These are the persons who do the most harm in society, and introduce into it the most harmful taints.
It is utterly worthless to God, and harmful to man; therefore, it must die.


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Steve Barnes
- Filed Under
Everyone was using the term "history." They were making history, some said. Others said they were living history. I was there, in the Pulaski County courthouse, to record that history. This is some of what I saw and heard.
"We're helping make Arkansas history!" exulted Sherri Vadala, a 44-year-old gourmet food entrepreneur from Maumelle. She and her partner, C.J., a 35 year-old surgeon who already had legally changed her last name to Sherri's, agreed. They laughed. Then, in the courthouse rotunda, after 15 years together, they married. They laughed, a little, during the ceremony even though both were crying.
A few feet away were Stephen Inman and Eddie Crawley, two 40-somethings in shorts and sandals and polo shirts who were partners in a pair of beauty salons and, now, in matrimony. Inman was holding the couple's 7-month-old adopted daughter, Rhett.
"All the way here I kept thinking, 'It's not going to happen,'" Inman said. "But here we are. Arkansas is on the right side of history."
Rhett, seemingly unaware of the history being made, just gurgled.
The word had spread across the nation, of course, word that a judge in Little Rock had struck down, in powerful prose, Arkansas's constitutional and statutory bans on same-sex marriage. Conway and White and Washington and Lonoke counties also were named defendants in the successful lawsuit but same-sex couples had less luck there than in Pulaski, where, by Monday's end, nearly 200 partnerships were granted legal cover. For however long, for whatever course the appellate process would take, gay marriage was lawful in Arkansas, and that was enough to bring several couples from out of state.
Such as Arkansas native Anna Dadwell and her partner, Lisa Wilkison, who motored over from the home they've shared for eight years in Coweta, Okla., to tie the knot. "It's a beautiful state, Arkansas," Dadwell said, though I don't think she meant the scenery. She and Wilkison had wanted to marry in Oklahoma, but Oklahoma was not OK with that (though, its prohibitions, as those of many another state, are under legal challenge). "We didn't want to wait," Wilkison said. "We wanted to change history."
Already it had changed. As when two men of different pigment, graphic artist Nathaniel Bledsoe and his partner, Cougar Copelin, kissed in the courthouse, a non-denominational clergyman having pronounced them husband and husband, and nothing was heard but cheers.
"It's a milestone for us and our state, to be recognized this way," Copelin said. "It's a sign of how things have changed here over time."
There were other signs. There were several clergy present to perform the ceremonies, some of them representing mainstream denominations and others, well, less mainstream. Such as the lady who identified herself as a "pagan" priestess. Fitting, some doubtless would say.
And this: A county clerk extending an official welcome to the applicants. Four judges, all of whom must, or may choose to, stand for re-election, volunteering to perform weddings that an attorney general who may have political plans beyond his current office has said he does not oppose (even as he pledges to resist them in behalf of the state).
And this: The almost total absence of any protest. One elderly gentleman in a long white robe stood across the street but, witnesses said, departed after a half-hour, dispirited. An African-American clergyman circled the rotunda bearing signs condemning the proceedings, but he was largely ignored. When he upbraided one of the judges, Wendell Griffen, who was robed not in courtroom black but in the gray of his Baptist pastorate, His Honor mostly shrugged. "Allies must affirm those who have been oppressed," he said, and smiled.
Another circuit judge, Morgan Welch, strolled from his chambers one floor above the rotunda in shirt sleeves and tie, coffee cup in hand, to survey the throng. "I said that if anyone wanted to get married I'd be happy to do it," he chuckled. "But I'd feel silly standing around in my robe like I'm selling something," Later, he would have four buyers.
At the conclusion of each pair of "I do's" there was sustained applause that thundered against the rotunda's marble floor, columns and walls, pushing against the multi-colored skylight. It had not yet begun to rain, though a good many people believed they saw a rainbow.
Steve Barnes is a veteran Arkansas journalist and moderator of
"Arkansas This Week."
© 2014 Editorial
Associates Inc.


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- Filed Under
Mardi Gras comes at the perfect time of year, just when we're getting cross and cranky at the end of a long winter. And after the dreary weather of the past few weeks, I'm quite ready for the fun that comes with celebrating Fat Tuesday. Spring may be just around the corner, but in my home, I'm ready to decorate with some purple, gold and green.
Shreveporters have embraced Mardi Gras as their own, and we've no excuse if we don't get into the swing and have a party. Let's celebrate the occasion this year with a Creole-themed brunch or dinner party. There's no shortage of authentic New Orleans dishes, so plan your menu around this traditional fare.
Nothing says New Orleans like shrimp etouffee. The French word etouffee means smothered, and the shrimp are simmered in a zesty tomato-based mixture that is fragrant with onions, green peppers, celery and herbs. This zesty sauce can be found in dishes throughout the South using shrimp, crab, crawfish, chicken or game.
If you're celebrating Mardi Gras with a New Orleans brunch, cook up a pot of hot steaming grits and top it off with the etouffee. For a dinner party, pair the etouffee with green rice for attractive presentation and great flavor. Complete your menu with fresh asparagus sautéed in a skillet with olive oil, garlic and sesame seeds.
The New Orleans crabmeat pie comes under the category of melt-in-your-mouth delectable. This light-as-a-feather puff pastry shell is filled with a delicate crab sauce that would be suitable for either brunch or dinner. The No. 1 rule when cooking with crabmeat is to buy the freshest available. Please don't even entertain the thought of canned or artificial. This seafood pie is especially delicious served with petit pois peas with mushrooms and Caesar salad.
Also an authentic Creole dish, grillades (GREE yahds) and grits will star at your midday brunch, right along with the Mimosas, Bloody Marys and milk punches. Paired up with homemade biscuits, butter and your favorite preserves, this menu will wow your guests. For a sweet ending, bananas Foster will send guests home in utter contentment.
Let's not forget the ultimate dessert for Mardi Gras the king cake. King cakes come in a variety of styles and flavors. They are available in the original plain braided variety with colored sugar on top, or you can get them filled with cream cheese or other flavored fillings. My daughter-in-law Carol in Baton Rouge has already sampled her favorite this year a King cake with a bananas Foster filling, which she said was the best she has tasted. If I get ambitious and make my own this year, I will experiment with softened cream cheese blended with the ingredients for the bananas Foster recipe and fill the king cake with that mixture. Sounds yummy to me!
Tina Allen with The Dough Basket & Creative Cakes shared an interesting king cake story. The bakery's popular Zulu king cake filled with cream cheese, coconut and chocolate chips and glazed with chocolate and coconut sprinkles is named for the first black Mardi Gras krewe.
Zulu members sprayed gold paint on whole coconuts and threw them from the floats as the parade wound its way through the streets of New Orleans. It didnt take them long to discontinue this practice when they realized throwing whole coconuts at a crowd of overzealous revelers was not a great idea. Someone could get hurt!
Luckily, the association of the coconut stuck and the scrumptious Zulu king cake was created in honor of that first Krewe of Zulu.
When making your favorite king cake, don't forget to add the small plastic baby, the coveted prize that is tucked inside each one. The New Orleans tradition is each person takes a piece of cake hoping to find the baby. The recipient of the plastic baby is "crowned" king or queen for the day and that person is obligated to host the following year's party and supply the king cake. That sounds like a lot of work are we sure we want to look for that baby or avoid that baby?
In addition to the king cake, we have some great recipes featured that will allow you to take your pick of what type event you are hosting a brunch or a dinner party. After all, it's Mardi Gras, a time to do as we please and serve what we please. We're here to pass a good time and anything goes. ...
The spirit of Mardi Gras is alive and well in Shreveport and Bossier City this year, with multiple parades and balls on the agenda. Lets all go out to the parade routes and give a salute to the many talented people who spend so many hours promoting this major event in north Louisiana. It might earn us some extra beads when we yell Throw me something, Mister!
Marlyn Monette writes about food for The Times. Write to her at P.O. Box 30222, Shreveport, LA 71130-0222 or send e-mail to email@example.com.


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November 25, 2015
The City of Grand Junction is partnering with Mesa County School District 51 to make improvements to an existing school and one that is going to be built in the future. The city and the school district are in talks to add another full–size gym to Bookcliff Middle School. The city also hopes to make Bookcliff Middle School an "activity center" for the neighborhood.
Also in the plans is to make the Pear Park Elementary School gym much bigger and to add a neighborhood park. Rapid growth in these areas is forcing the expansion. The city and the school district also hope to construct a new neighborhood park in the Bookcliff Middle School area.
The city will make the final decision on the school gym expansions and neighborhood parks sometime around April 1st.
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A 72-year-old man is in critical condition after his wheelchair rolled off a dock in Port Jefferson village.
Police say the man was with his family on the dock yesterday when the wheelchair he was strapped into rolled off the dock and into the water.
Officials say it took crews about 15 minutes to get him out.
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SOUTH KINGSTOWN ŌĆō The planning board voted unanimously Tuesday to grant preliminary plan approval to the Rhode Island Department of Environmental ManagementŌĆÖs proposed two-lot subdivision across from the WordenŌĆÖs Pond boat ramp.
Michelle Sheehan, of DEM, told the planning board DEM is planning to purchase 6.9 acres across from WordenŌĆÖs Pond to create an auxiliary parking lot and provide an area for overflow parking.
For the rest of this story and more local news, pick up a copy of the Narragansett Times.


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Senior Executive Group
As at 1 January 2015
The authority vested by the statutes of the University in the person of the Vice-Chancellor is exercised through the University's Senior Executive Group (SEG). Operating within a context set by the Senate, the Senate's rules and in particular its delegations of decision-making authority to officers of the University, SEG's terms of reference are to:
- oversee the implementation of the University's strategic plan
- account to Senate for the financial and academic viability and sustainability of the University
- protect and enhance the position and reputation of the University
- oversee performance monitoring and improvement, regulatory compliance and quality assurance processes
- maintain the University's risk register and ensure appropriate risk mitigation within the context of the overall Risk Framework
- oversee the University's curriculum, teaching, research and research training, in conjunction with the Academic Board
- oversee the University's human resources management and capability planning
- review the University's budget prior to its presentation for Senate approval
- prioritise digital and physical infrastructure projects
- oversee, in conjunction with the Academic Board, the programs of faculty reviews conducted jointly by the Curriculum Committee and the Academic Board
- review and approve SEG committee and divisional board recommendations regarding policies, procedures, programs and initiatives
- negotiate compacts with divisions regarding their research and teaching excellence strategies.
SEG's membership comprises:
- the Vice-Chancellor (chair)
- all deputy vice-chancellors
- the Chair of the Academic Board
- proportional representation of each of the seven academic divisions (including the dean of each of the University's 16 faculties)
- the Director of Human Resources and
- the Chief Financial Officer.
A number of senior members of staff routinely attend SEG meetings to assist with the conduct of its business and with the communication and implementation of its decisions.
SEG's decision-making is underpinned by a number of committees and divisional boards that undertake detailed analysis and planning in particular areas of expertise and interest. The work of these committees and divisional boards intersects at many points.
SEG committees
The SEG committees focus on key University-wide themes, such as education and research training, finance and infrastructure, and curriculum and course planning. They provide expert, high-level strategic advice to SEG, and engage SEG members, along with other experts, in the detailed preparation of matters for SEG consideration.
There are 14 SEG committees:
- Aboriginal and Torres Strait Islander Strategy and Services Committee
- Alumni, Development and Marketing Committee
- Charles Perkins Centre Governing Committee
- Colleges Consultative Committee
- Cultural Resources Committee
- Curriculum and Course Planning Committee
- Disability Action Plan Committee
- Education Committee
- Finance and Infrastructure Committee
- Human Resources and Equity Committee
- International Committee
- Research Committee
- Research Training Committee
- Work Health and Safety Committee
Divisional boards
The SEG divisional boards are charged with detailed strategic planning, portfolio analysis, and educational and research reform for groups of cognate faculties. Their focus is not on University-wide strategies, but on the particularities of diverse educational and research communities.
The divisional boards are composed as follows:
- Division of Architecture and Creative Arts (Faculty of Architecture, Design and Planning; Sydney College of the Arts; Sydney Conservatorium of Music)
- Division of Business (The University of Sydney Business School)
- Division of Engineering and Information Technologies (Faculty of Engineering and Information Technologies)
- Division of Health Sciences (Faculty of Health Sciences)
- Division of Humanities and Social Sciences (Faculty of Arts and Social Sciences; Faculty of Education and Social Work; Faculty of Law)
- Division of Medicine, Dentistry, Nursing and Pharmacy (Faculty of Dentistry; Faculty of Medicine; Faculty of Nursing and Midwifery; Faculty of Pharmacy)
- Division of Natural Sciences (Faculty of Agriculture and Environment; Faculty of Science; Faculty of Veterinary Science).
More information about the membership and terms of reference of SEG and its committees is available at sydney.edu.au/senior_executive_group


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Towns & Parishes
|Nearby places
"SOUTH CADBURY, a parish in the hundred of Catsash, in the county of Somerset, 1 mile to the S. of North Cadbury. Wincanton is its post town. The living is a rectory* in the diocese of Bath and Wells, in the patronage of J. Bennett, Esq. The church is dedicated to St. Thomas-a-Becket. Near this village, on a ridge of hills, is the extensive ancient camp called Camalet, or Cadbury Castle, which was partly hewn out of the solid rock (blue lias), and defended by four trenches. Within the area is another entrenchment, called King Arthur's Palace, which has but one entrance, and is surrounded by six or seven trenches. The centre camp, the form of which is irregular and determined by the shape of the hill, covers an area of 30 acres. Its origin is unknown, but from the Roman relics discovered here it appears certain that it was at one time occupied by the Romans, if not first formed by them." From The National Gazetteer of Great Britain and Ireland (1868) Transcribed by Colin Hinson © 2003
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I have owned horses my whole life with the exception of the past couple of years. I am now boarding in South FL for the first time. It seems like a whole new horse-owning world down here! Time to learn how to ride English it seems.
I have been lurking for some time and figured since I finally have a horse again, it was about time I finally joined. I really enjoy all of the good information and have learned a lot.