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FcgammaR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA)
Blom, Arjen B
van Lent, Peter L
van Vuuren, Hanneke
Holthuysen, Astrid E
Jacobs, Cor
van de Putte, Leo B
van de Winkel, Jan G
van den Berg, Wim B
Arthritis Research
We investigated the role of Fcγ receptors (FcγRs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) γ-chain-/- C57BL/6 mice, which lack functional FcγRI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of naïve DBA/1 mice expressed a significantly higher level of FcγRs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that FcγR expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.
Fcγ receptors (FcγRs) present on cells of the haematopoietic lineage communicate with IgG-containing immune complexes that are abundant in the synovial tissue of patients with rheumatoid arthritis (RA). In mice, three classes of FcγR (RI, RII, and RIII) have been described. Binding of these receptors leads to either activation (FcγRI and RIII) or deactivation (FcγRII) of intracellular transduction pathways. Together, the expression of activating and inhibitory receptors is thought to drive immune-complex-mediated diseases.
Earlier studies in our laboratory showed that macrophages of the synovial lining are of utmost importance in the onset and propagation of immune-complex-driven arthritic diseases. Selective depletion of macrophages in the joint downregulated both inflammation and cartilage destruction. As all three classes of FcγR are expressed on synovial macrophages, these cells are among the first that come in contact with immune complexes deposited in the joint. Recently, we observed that when immune complexes were injected into the knee joints of mice, strains susceptible to collagen-type-II arthritis (DBA/1, B10.RIII) developed more severe arthritis than nonsusceptible strains did, or even developed chronic arthritis. One reason why these strains are more susceptible might be their higher levels of FcγRs on macrophage membranes. To test this hypothesis, we investigated the role of FcγRs in inflammation and cartilage damage during immune-complex-mediated arthritis (ICA). First, we studied arthritis and subsequent cartilage damage in mice lacking functional FcγRI and RIII (FcR γ-chain-/- mice). Next, DBA/1 mice, which are prone to develop collagen-type-II arthritis (`collagen-induced arthritis'; CIA) and are hypersensitive to immune complexes, were compared with control C57BL/6 mice as regards cartilage damage and the expression and function of FcγRs on their macrophages.
To examine whether FcγR expression on macrophages is related to severity of synovial inflammation and cartilage destruction during immune-complex-mediated joint inflammation.
ICA was induced in three strains of mice (FcR γ-chain-/-, C57BL/6, and DBA/1, which have, respectively, no functional FcγRI and RIII, intermediate basal expression of FcγRs, and high basal expression of FcγRs) by passive immunisation using rabbit anti-lysozyme antibodies, followed by poly-L-lysine lysozyme injection into the right knee joint 1 day later. In other experiments, streptococcal-cell-wall (SCW)- or zymosan-induced arthritis was induced by injecting SCW (25 μg) or zymosan (180 μg) directly into the knee joint. At several time points after arthritis induction, knee joints were dissected and studied either histologically (using haematoxylin/eosin or safranin O staining) or immuno-histochemically. The arthritis severity and the cartilage damage were scored separately on an arbitrary scale of 0-3.
FcγRs were immunohistochemically detected using the monoclonal antibody 2.4G2, which detects both FcγRII and RIII. Deposition of IgG and C3c in the arthritic joint tissue was also detected immunohistochemically. Expression of FcγRs by murine peritoneal macrophages was measured using a fluorescence-activated cell sorter (FACS).
Peritoneal macrophages were stimulated using heat-aggregated gamma globulins (HAGGs), and production of IL-1 was measured using a bioassay. To assess the levels of IL-1 and its receptor antagonist (IL-1Ra) during arthritis, tissue was dissected and washed in RPMI medium. Washouts were tested for levels of IL-1 and IL-1Ra using radioimmunoassay and enzyme-linked immunosorbent assay. mRNA was isolated from the tissue, and levels of macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, IL-1, and IL-1Ra were determined using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR).
ICA induced in knee joints of C57BL/6 mice caused a florid inflammation at day 3 after induction. To investigate whether this arthritis was FcγR-mediated, ICA was induced in FcR γ-chain-/- mice, which lack functional FcγRI and RIII. At day3, virtually no inflammatory cells were found in their knee joints. Levels of mRNA of IL-1, IL-1Ra, MCP-1, and MIP-2, which are involved in the onset of this arthritis, were significantly lower in FcR γ-chain-/- mice than in control C57BL/6 mice. Levels of IL-1 protein were also measured. At 6 h after ICA induction, FcR γ-chain-/- mice and control C57BL/6 mice showed similar IL-1 production as measured by protein level. By 24 h after induction, however, IL-1 production in the FcR γ-chain-/- mice was below the detection limit, whereas the controls were still producing a significant amount. To investigate whether the difference in reaction to immune complexes between the DBA/1 and C57BL/6 mice might be due to variable expression of FcγRs in the knee joint, expression in situ of FcγRs in naïve knee joints of these mice was determined. The monoclonal antibody 2.4G2, which detects both FcγRII and RIII, stained macrophages from the synovial lining of DBA/1 mice more intensely than those from C57BL/6 mice. This finding suggests a higher constitutive expression of FcγRs by macrophages of the autoimmune-prone DBA/1 mice. To quantify the difference in FcγR expression on macrophages of the two strains, we determined the occurrence of FcγRs on peritoneal macrophages by FACS analysis. The levels of FcγR expressed by macrophages were twice as high in the DBA/1 mice as in the C57BL/6 mice (mean fluorescence, respectively, 440 ± 50 and 240 ± 30 intensity per cell). When peritoneal macrophages of both strains were stimulated with immune complexes (HAGGs), we found that the difference in basal FcγR expression was functional. The stimulated macrophages from DBA/1 mice had significantly higher IL-1α levels (120 and 135 pg/ml at 24 and 48 h, respectively) than cells from C57BL/6 mice (45 and 50 pg/ml, respectively).
When arthritis was induced using other arthritogenic triggers than immune complexes (zymosan, SCW), all the mouse strains tested (DBA/1, FcR γ-chain-/-, and C57BL/6) showed similar inflammation, indicating that the differences described above are found only when immune complexes are used to elicit arthritis.
We next compared articular cartilage damage in arthritic joints of the three mouse strains FcR γ-chain-/-, C57BL/6 (intermediate basal expression of FcγRs), and DBA/1 (high basal expression of FcγRs). Three indicators of cartilage damage were investigated: depletion of PGs, chondrocyte death, and erosion of the cartilage matrix. At day 3 after induction of ICA, there was no PG depletion in FcR γ-chain-/- mice, whereas PG depletion in the matrix of the C57BL/6 mice was marked and that in the arthritic DBA/1 mice was even greater. PG depletion was still massive at days 7 and 14 in the DBA/1 mice, whereas by day 14 the PG content was almost completely restored in knee joints of the C57BL/6 mice. Chondrocyte death and erosion of cartilage matrix, two indicators of more severe cartilage destruction, were significantly higher in the DBA/1 than in the C57BL/6 mice, while both indicators were completely absent in the FcR γ-chain-/- mice. Again, when arthritis was induced using other triggers (SCW, zymosan), all strains showed similar PG depletion and no chondrocyte death or matrix erosion. These findings underline the important role of immune complexes and FcγRs in irreversible cartilage damage.
Our findings indicate that inflammation and subsequent cartilage damage caused by immune complexes may be related to the occurrence of FcγRs on macrophages. The absence of functional FcγRI and RIII prevented inflammation and cartilage destruction after induction of ICA, whereas high basal expression of FcγRs on resident joint macrophages of similarly treated mice susceptible to autoimmune arthritis was correlated with markedly more synovial inflammation and cartilage destruction. The difference in joint inflammation between the three strains was not due to different susceptibilities to inflammation per se, since intra-articular injection of zymosan or SCW caused comparable inflammation. Although extensive inflammatory cell mass was found in the synovium of all strains after intra-articular injection of zymosan, no irreversible cartilage damage (chondrocyte death or matrix erosion) was found. ICA induced in C57BL/6 and DBA/1 mice did cause irreversible cartilage damage at later time points, indicating that immune complexes and FcγRs play an important role in inducing irreversible cartilage damage. Macrophages communicate with immune complexes via Fcγ receptors. Absence of functional activating receptors completely abrogates the synovial inflammation, as was shown after ICA induction in FcR γ-chain-/- mice. However, the γ-chain is essential not only in FcγRI and RIII but also for FcεRI (found on mast cells) and the T cell receptor (TcR)-CD3 (Tcells) complex of γδT cells. However, T, B, or mast cells do not play a role in this arthritis that is induced by passive immunisation. Furthermore, this effect was not caused by a difference in clearance of IgG or complement deposition in the tissue. In this study, DBA/1 mice, which are susceptible to collagen-induced autoimmune arthritis and in a recent study have been shown to react hypersensitively to immune complexes, are shown to express higher levels of FcγRs on both synovial and peritoneal macrophages. Because antibodies directed against the different subclasses of FcγR are not available, no distinction could be made between FcγRII and RIII. Genetic differences in DBA/1 mice in genes coding for or regulating FcγRs may be responsible for altered FcγR expression. If so, these mouse strains would have a heightened risk for immune-complex-mediated diseases.
To provide conclusive evidence for the roles of the various classes of FcγR during ICA, experiments are needed in which FcγRs are blocked with specific antibodies, or in which knockout mice lacking one specific class of FcγR are used. The only available specific antibody to FcγR (2.4G2) has a stimulatory effect on cells once bound to the receptor, and therefore cannot be used in blocking experiments. Experiments using specific knockout mice are now being done in our laboratory.
Macrophages are the dominant type of cell present in chronic inflammation during RA and their number has been shown to correlate well with severe cartilage destruction. Apart from that, in humans, these synovial tissue macrophages express activating FcRs, mainly FcγIIIa, which may lead to activation of these macrophages by IgG-containing immune complexes. The expression of FcRs on the surface of these cells may have important implications for joint inflammation and severe cartilage destruction and therefore FCRs may constitute a new target for therapeutic intervention.
autoimmunity; cytokines; Fc receptors; inflammation; macrophages
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Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response
Serafino, Annalucia
Vallebona, Paola Sinibaldi
Andreola, Federica
Zonfrillo, Manuela
Mercuri, Luana
Federici, Memmo
Rasi, Guido
Garaci, Enrico
Pierimarchi, Pasquale
BMC Immunology
Besides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence of Eucalyptus oil (EO) extract on the monocytic/macrophagic system, one of the primary cellular effectors of the immune response against pathogen attacks. The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether EO extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU).
Morphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i) in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit.
EO is able to induce activation of MDMs, dramatically stimulating their phagocytic response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that EO may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after EO administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly decreased by the chemotherapic.
Our data, demonstrating that Eucalyptus oil extract is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate further investigations also using single components of this essential oil. This might drive development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-suppressive pathologies, in infectious disease and after tumour chemotherapy.
Related Articles
Pandey, Shivanand
Journal of Advanced Pharmaceutical Technology & Research
Arthritis is an auto immune disorder characterized by pain, swelling and stiffness. Its prevalence depends upon age. It occurs more frequently in women than in men. It is an inflammation of synovial joint due to immuno mediated response. All anti inflammatory drugs are not anti arthritic because it does not suppress T-cell and B-cell mediated response. Epidemiological studies overall show a female to male ratio of about 3:1. There are many class of anti-arthritic drugs are available like NSAIDS, Monoclonal anti-bodies, uricosuric agents, gold compounds, anti-cytokinine immunosuppressant like glucocorticoids, etc. But this all class of drugs is responsible for symptomatic relief. To evaluate the drug which actually prevent cause of arthritic or act during various step of arthritis there is requirement of evaluative model which produce arthritis in (vial same that produce in humans. Animal models of arthritis are used to study pathogenesis of disease and to evaluate potential anti-arthritic drugs for clinical use. Therefore morphological similarities to human disease and capacity of the model to predict efficacy in humans are important criteria in model selection.
Monoclonal anti-bodies; uricosuric agents; gold compounds; anti-cytokinine immunosuppressant
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CD134 as target for specific drug delivery to auto-aggressive CD4+ T cells in adjuvant arthritis
Boot, Elmieke PJ
Koning, Gerben A
Storm, Gert
Wagenaar-Hilbers, Josée PA
van Eden, Willem
Everse, Linda A
Wauben, Marca HM
Arthritis Research & Therapy
T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4+ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134+ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4+CD134+ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134+ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4+ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.
Related Articles
Mactinin: a modulator of the monocyte response to inflammation
Luikart, Sharon D
Krug, Hollis E
Nelson, Robert D
Hinkel, Timothy
Majeski, Peter
Gupta, Pankaj
Mahowald, Maren L
Oegema, Theodore
Arthritis Research & Therapy
During inflammatory processes, monocytes leave the blood stream at increased rates and enter inflammation tissue, where they undergo phenotypic transformation to mature macrophages with enhanced phagocytic activity. α-Actinin, a cytoskeletal protein, is present in focal adhesion complexes and left in the microenvironment as a result of cell movement. Mactinin, a 31 kDa amino-terminal fragment of α-actinin, is generated by the degradation of extracellular α-actinin by monocyte-secreted urokinase. We have previously demonstrated that mactinin promotes monocyte/macrophage maturation. We now report that 0.5–10 nM mactinin has significant chemotactic activity for monocytes. Mactinin seems to be present in inflammatory arthritis synovial fluid, because affinity-purified antisera reacted with a protein of the expected molecular mass in various types of arthritis fluids that were immunoaffinity-purified and subjected to Western analysis. Thus, six of seven samples from patients with psoriatic arthritis, reactive arthritis, gout, or ankylosing spondylitis contained mactinin at levels that are active in vitro. Initially, mactinin was not found in affinity-purified rheumatoid arthritis samples. However, it was detectable after the dissociation of immune complexes, suggesting that it was complexed to anti-microfilament auto-antibodies. In addition, mactinin was found in the lavage fluid from the arthritic knee joints of rabbits with antigen-induced arthritis and was absent from the contralateral control knee fluids. We conclude that mactinin is present in several types of inflammatory arthritis and might modulate mononuclear phagocyte response to inflammation.
arthritis; chemotaxis; inflammation; monocytes
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Protection against TNFα-Dependent Liver Toxicity by Intraperitoneal Liposome Delivered DsiRNA Targeting TNFα In Vivo
Lundberg, Patric
Yang, Hui-Jung
Jung, Seung-Jae
Behlke, Mark A.
Rose, Scott D.
Cantin, Edouard M.
Journal of Controlled Release
Tumor necrosis factor alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathogenesis of several autoimmune and inflammatory diseases including viral encephalitis. Macrophages being major producers of TNFα are thus attractive targets for in vivo RNA interference (RNAi) mediated down regulation of TNFα. The application of RNAi technology to in vivo models however presents obstacles, including rapid degradation of RNA duplexes in plasma, insufficient delivery to the target cell population and toxicity associated with intravenous administration of synthetic RNAs and carrier compounds.
We exploited the phagocytic ability of macrophages for delivery of Dicer-substrate small interfering RNAs (DsiRNAs) targeting TNFα (DsiTNFα) by intraperitoneal administration of lipid-DsiRNA complexes that were efficiently taken up by peritoneal macrophages and other phagocytic cells. We report that DsiTNFα-lipid complexes delivered intraperitoneally altered the disease outcome in an acute sepsis model. Down-regulation of TNFα in peritoneal CD11b+ monocytes reduced liver damage in C57BL/6 mice and significantly delayed acute mortality in mice treated with low dose LPS plus D-galactosamine (D-GalN).
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THE ROLE OF DISEASE STAGE, PLASMA VIRAL LOAD AND Tregs ON AUTO-ANTIBODY PRODUCTION IN THE SIV INFECTED NON-HUMAN PRIMATES1
Journal of autoimmunity
Auto-antibodies appear in the sera of rhesus macaques following SIV infection. The present study was conducted to examine the role of viral load, anti-viral chemotherapy and stage of disease on the titers of such auto-antibodies and the spectrum of auto-antigens that become the target of such auto-immune responses. In addition, the role of regulatory T cells (Tregs) was also examined. Results of these studies showed that the highest auto-antibody titers were noted in animals with lower relative plasma viral loads with a wider spectrum of auto-antigens that are the target of such responses as compared with lower auto-antibody titers in animals with relatively higher plasma viral loads and a narrower spectrum of auto-antigens. Short term anti-viral chemotherapy did not influence the titers of auto-antibodies. While there was a gradual decrease in the frequency and absolute number of Tregs, the levels of Tregs was inversely correlated with viral load and lower autoantibody titers. The mechanisms for these differences remain unknown and suggest complex relationships exist between levels of immuno-suppression, auto-immune response, homeostatic proliferation and the spectrum of auto-antigens that become the target of such auto-immune responses.
Auto-immunity; SIV infection; Non-human primates; Regulatory T-cells; immunodeficiency disease; AIDS
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CCR2 Deficiency Promotes Exacerbated Chronic Erosive Neutrophil-Dominated Chikungunya Virus Arthritis
Poo, Yee Suan
Nakaya, Helder
Gardner, Joy
Larcher, Thibaut
Schroder, Wayne A.
Le, Thuy T.
Major, Lee D.
Suhrbier, Andreas
Journal of Virology
Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2−/− mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2−/− mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2−/− mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2−/− mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2+ monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides.
IMPORTANCE Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.
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The anti-inflammatory mechanisms of Hsp70
Borges, Thiago J.
Wieten, Lotte
van Herwijnen, Martijn J. C.
Broere, Femke
van der Zee, Ruurd
Bonorino, Cristina
van Eden, Willem
Frontiers in Immunology
Immune responses to heat shock proteins (Hsp) develop in virtually all inflammatory diseases; however, the significance of such responses is only now becoming clear. In experimental disease models, Hsp administration can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory diseases, Hsp peptides have been shown to promote the production of anti-inflammatory cytokines, indicating immunoregulatory potential of Hsp. Therefore, the presence of immune responses to Hsp in inflammatory diseases can be seen as an attempt of the immune system to correct the inflammatory condition. Hsp70 can modulate inflammatory responses in models of arthritis, colitis and graft rejection, and the mechanisms underlying this effect are now being elucidated. Incubation with microbial Hsp70 was seen to induce tolerogenic dendritic cells (DCs) and to promote a suppressive phenotype in myeloid-derived suppressor cells and monocytes. These DC could induce regulatory T cells (Tregs), independently of the antigens they presented. Some Hsp70 family members are associated with autophagy, leading to a preferential uploading of Hsp70 peptides in MHC class II molecules of stressed cells. Henceforth, conserved Hsp70 peptides may be presented in these situations and constitute targets of Tregs, contributing to downregulation of inflammation. Finally, an interfering effect in multiple intracellular inflammatory signaling pathways is also known for Hsp70. Altogether it seems attractive to use Hsp70, or its derivative peptides, for modulation of inflammation. This is a physiological immunotherapy approach, without the immediate necessity of defining disease-specific auto-antigens. In this article, we present the evidence on anti-inflammatory effects of Hsp70 and discuss the need for experiments that will be crucial for the further exploration of the immunosuppressive potential of this protein.
Hsp70; stress proteins; immunomodulation; adaptive immunity; innate immunity
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Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4
Kim, Seon Hee
Evans, Christopher H
Kim, Sunyoung
Oligino, Thomas
Ghivizzani, Steven C
Robbins, Paul D
Arthritis Research
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
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Treg Inducing Adjuvants for Therapeutic Vaccination Against Chronic Inflammatory Diseases
Keijzer, Chantal
van der Zee, Ruurd
van Eden, Willem
Broere, Femke
Frontiers in Immunology
Many existing therapies in autoimmune diseases are based on systemic suppression of inflammation and the observed side effects of these therapies illustrate the pressing need for more specific interventions. Regulatory T-cells (Treg) are pivotal controllers of (auto-aggressive) immune responses and inflammation, and decreased Treg numbers and/or functioning have been associated with autoimmune disease. Therefore, Treg became frequently studied targets for more specific immunotherapy. Especially antigen-specific targeting of Treg would enable local and tailor made interventions, while obviating the negative side effect of general immuno-suppression. Self-antigens that participate in inflammation, irrespective of the etiology of the different autoimmune diseases, are held to be candidate antigens for antigen-specific interventions. Rather than tolerance induction to disease inciting self-antigens, which are frequently unknown, general self-antigens expressed at sites of inflammation would allow targeting of disease independent, but inflammatory-site specific, regulatory mechanisms. Preferably, such self-antigens should be abundantly expressed and up-regulated at the inflammatory-site. In this perspective heat shock proteins (Hsp) have several characteristics that make them highly attractive targets for antigen-specific Treg inducing therapy. The development of an antigen-specific Treg inducing vaccine is a major novel goal in the field of immunotherapy in autoimmune diseases. However, progress is hampered not only by the lack of effective antigens, but also by the fact that other factors such as dose, route, and the presence or absence of an adjuvant, turned out to be critical unknowns, with respect to the effective induction of Treg. In addition, the use of a Treg inducing adjuvant might be required to achieve an effective regulatory response, in the case of ongoing inflammation. Future goals in clinical trials will be the optimization of natural Treg expansion (or the induction of adaptive Treg) without loss of their suppressive function or the concomitant induction of non-regulatory T-cells. Here, we will discuss the potential use of protein/peptide-based vaccines combined with Treg inducing adjuvants for the development of therapeutic vaccines against chronic inflammatory conditions.
regulatory T-cells; immunologic adjuvants; therapeutic vaccines; Treg inducing adjuvants; autoimmunity
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Enhancement of Immune Responses by Co-delivery of CCL19/MIP-3beta Chemokine Plasmid With HCV Core DNA/Protein Immunization
Hartoonian, Christine
Sepehrizadeh, Zargham
Tabatabai Yazdi, Mojtaba
Jang, Yong Suk
Langroudi, Lida
Amir Kalvanagh, Parisa
Negahdari, Babak
Karami, Ali
Ebtekar, Massoumeh
Azadmanesh, Kayhan
Hepatitis Monthly
Using molecular adjuvants offers an attractive strategy to augment DNA vaccine-mediated immune responses. Several studies have revealed that an efficient HCV vaccine model should be able to induce both humoral and cell mediated immune responses targeting the conserved regions of the virus to circumvent the immune escape mutants. The beta chemokine Macrophage Inflammatory Protein 3-beta (MIP-3beta) is a key modulator of dendritic cells (DCs) and T-cells interaction, functions during immune response induction and is secreted specifically by cells in the lymphoid tissues.
In the present study, we questioned whether co-administration of MIP-3beta gene could enhance the immune responses to HCV core in DNA vaccination.
Materials and Methods:
Expression and biological activity of MIP-3beta expressing plasmid were evaluated by ELISA and transwell migration assays, respectively. HCV core DNA vaccine ± plasmid expressing MIP-3beta were electroporated subcutaneously to the front foot pads of BALB/c mice on days 0 and 14, and HCV core protein booster was applied to all core-DNA-vaccine received mice on the day 28. Both cell mediated immunity (proliferation, IFN-γ and IL-4 cytokine release, IFN-γ ELISpot and cytotoxic Granzyme B release assays) and humoral immune responses (total IgG and IgG2a/IgG1 subtyping) were evaluated ten days after final immunization.
Mice covaccinated with MIP-3beta elicited an enhanced Th1 biased systemic immune response as evidenced by higher IFN-γ/IL-4 and anti-core IgG2a/IgG1 ratio, lymphoproliferation, strong cytolytic GrzB release and enhanced population of IFN-γ producing immunocytes. Likewise, the humoral immune response assumed as the total anti-core IgG level was augmented by MIP-3beta co-delivery.
These results exhibited the immuno potentiator effects of MIP-3beta plasmid when coadministrated with the HCV core DNA vaccine. Complimentary studies integrating MIP-3beta as a genetic adjuvant in HCV-core-DNA vaccination models are warranted.
Chemokine CCL19; Hepatitis C; Hepatitis C Antigens; Adjuvants
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Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells
Novobrantseva, Tatiana I
Borodovsky, Anna
Wong, Jamie
Klebanov, Boris
Zafari, Mohammad
Yucius, Kristina
Querbes, William
Ge, Pei
Ruda, Vera M
Milstein, Stuart
Speciner, Lauren
Duncan, Rick
Barros, Scott
Basha, Genc
Cullis, Pieter
Akinc, Akin
Donahoe, Jessica S
Narayanannair Jayaprakash, K
Jayaraman, Muthusamy
Bogorad, Roman L
Love, Kevin
Whitehead, Katie
Levins, Chris
Manoharan, Muthiah
Swirski, Filip K
Weissleder, Ralph
Langer, Robert
Anderson, Daniel G
de Fougerolles, Antonin
Nahrendorf, Matthias
Koteliansky, Victor
Molecular therapy. Nucleic acids
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
delivery; immune cell; siRNA
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A New Arthritis Therapy with Oxidative Burst Inducers
Hultqvist, Malin
Olofsson, Peter
Gelderman, Kyra A
Holmberg, Jens
Holmdahl, Rikard
PLoS Medicine
Despite recent successes with biological agents as therapy for autoimmune inflammatory diseases such as rheumatoid arthritis (RA), many patients fail to respond adequately to these treatments, making a continued search for new therapies extremely important. Recently, the prevailing hypothesis that reactive oxygen species (ROS) promote inflammation was challenged when polymorphisms in Ncf1, that decrease oxidative burst, were shown to increase disease severity in mouse and rat arthritis models. Based on these findings we developed a new therapy for arthritis using oxidative burst-inducing substances.
Methods and Findings
Treatment of rats with phytol (3,7,11,15-tetramethyl-2-hexadecene-1-ol) increased oxidative burst in vivo and thereby corrected the effect of the genetic polymorphism in arthritis-prone Ncf1DA rats. Importantly, phytol treatment also decreased the autoimmune response and ameliorated both the acute and chronic phases of arthritis. When compared to standard therapies for RA, anti-tumour necrosis factor-α and methotrexate, phytol showed equally good or better therapeutic properties. Finally, phytol mediated its effect within hours of administration and involved modulation of T cell activation, as injection prevented adoptive transfer of disease with arthritogenic T cells.
Treatment of arthritis with ROS-promoting substances such as phytol targets a newly discovered pathway leading to autoimmune inflammatory disease and introduces a novel class of therapeutics for treatment of RA and possibly other chronic inflammatory diseases.
Treatment of arthritis in rats with phytol, a reactive oxygen species promoting substance, suggests a novel pathway of autoimmune inflammatory disease and possibly a novel therapeutic strategy.
Editors' Summary
Rheumatoid arthritis (RA) is a chronic illness that affects between 0.3% and 1% of people worldwide, causing pain and swelling in joints, tendons, and other tissues, and frequently leading to permanent deformity and disability. RA involves an abnormal attack by cells of the immune system against the body's own connective tissues (so-called autoimmunity). Current drugs for RA work by counteracting the molecules that cause the pain and swelling (inflammation). By reducing the severity of autoimmune inflammation, these drugs may also reduce the disease's long-term damage to joints.
Inflammation is not always abnormal, but in fact plays an important part in the body's defense against infection. As part of their activity against disease-causing bacteria, the white blood cells known as granulocytes generate reactive oxygen species (ROS), sometimes known as "free radicals." After engulfing invading bacteria, neutrophils release an "oxidative burst" of ROS—essentially the subcellular equivalent of pouring hydrogen peroxide on a wound to disinfect it. A complex of molecules known collectively as the NADPH oxidase complex has the specific function of generating ROS to fuel the oxidative burst. Interestingly, recent experiments in arthritis-prone rats found that animals with an altered form of one of the subunits of this complex, Ncf1, that decreased the production of ROS also had greater susceptibility to arthritis. This finding was surprising because free radicals have generally been associated with inflammation and long-term damage to cells, so that a reduction in ROS might have been expected to decrease susceptibility to an inflammatory disease like RA.
Why Was This Study Done?
Because many patients with autoimmune inflammatory illnesses like RA do not respond to currently available therapies, new approaches to treatment merit investigation. Based on the observed association between reduced ROS and increased susceptibility to arthritis, the researchers wanted to find out whether treatment with a compound that increases ROS production by the NADPH oxidase complex would cause an improvement in arthritis.
What Did the Researchers Do and Find?
The researchers tested a compound called phytol in arthritis-prone rats to see how it affected inflammation. It is known that arthritis can be induced in these rats by injecting them with an oil called pristane. The researchers found that phytol caused a strong oxidative burst in human granulocyte cells grown in the laboratory, but did not cause arthritis in rats; whereas pristane, which does cause arthritis, caused a lower oxidative burst in the granulocytes.
They then studied whether phytol prevented arthritis in rats. They found that rats injected with phytol were protected from arthritis following a later injection of pristane. Given this result, they wanted to know if phytol increased ROS in the rats as it did in laboratory cell cultures. Studying granulocytes taken from rats that had been treated with phytol, they found that the oxidative burst of these cells was indeed increased, and remained increased for several weeks after treatment. They went on to test phytol as a treatment for active arthritis, and found that it dramatically reduced swollen joints and destruction of cartilage when given to rats with acute pristane-induced arthritis.
The beneficial effects of phytol were seen not only in rats bred with a form of Ncf1 that produces abnormally low amounts of ROS, but also in rats whose granulocytes produce normal oxidative bursts. When compared (in rats) to drugs licensed for RA (etanercept and methotrexate), phytol appeared to be at least as effective. The activity of phytol against arthritis was shown to involve T lymphocytes, as injection of phytol inhibited transfer of pristane-induced arthritis with these cells.
What Do These Findings Mean?
These experiments raise the intriguing possibility of an entirely new modality for treating autoimmune diseases; namely, through drugs designed to increase the production of ROS. This study raises a number of practical and scientific issues. For example, it is not known whether reduced capacity to produce ROS is a significant factor in human RA. Also, the connection between ROS production (by granulocytes) and autoimmune arthritis (which involves activity by T lymphocytes) remains to be clarified. Finally, the destructive effects typically associated with free radicals (such as damage to DNA and blockage of blood vessels) could complicate the use of this approach in humans, and like any new drugs, those that increase ROS production might have other, unanticipated side effects. Whatever the outcome of drug development efforts, however, this study is an excellent reminder that there are no "good" or "evil" biochemicals—in the intricacies of cellular metabolism, it's all a matter of balance.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030348.
The Arthritis Foundation: Rheumatoid Arthritis pages
Medical Inflammation Research pages (R. Holmdahl research group)
Wikipedia chapter on Rheumatoid Arthritis (note: Wikipedia is a free Internet encyclopedia that anyone can edit)
Wikipedia chapter on Reactive Oxygen Species (note: Wikipedia is a free Internet encyclopedia that anyone can edit)
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A panoramic spectrum of complex interplay between the immune system and IL-32 during pathogenesis of various systemic infections and inflammation
Khawar, Babar
Abbasi, Muddasir Hassan
Sheikh, Nadeem
European Journal of Medical Research
Cytokines have always been of great interest due to their vast potential and participation in the progression and pathogenesis of various ailments. Interleukin-32 (IL-32) is a recently identified cytokine, whose gene is located on human chromosome 16 p13.3, with eight exons and six splice variants (IL-32α to IL-32ζ). IL-32α, the most abundant form, is secreted by different types of cells including T cells, natural killer (NK) cells, monocytes, endothelial cells and epithelial cells. It acts as a preferential mediator and effector of abnormal immune responses to multiple inflammatory and auto immune diseases including rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), etc. It was found to stimulate the induction of various chemokines, pro-inflammatory cytokines including IL-1β, IL-6, IL-8, TNF-α and macrophage inflammatory protein-2 (MIP-2). Hence, IL-32 mediates the crucial interplay among immune system and body cells during pathogenesis of various insults. The aim of the present effort is to summarize the role, mechanism of pathogenesis and potential therapeutic applications of IL-32 in different systemic infections and diseased conditions.
Auto immune disease; Inflammatory bowel disease; Interleukin-32; Rheumatoid arthritis
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Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease
Thurlings, Rogier M.
Wijbrandts, Carla A.
Bennink, Roelof J.
Dohmen, Serge E.
Voermans, Carlijn
Wouters, Diana
Izmailova, Elena S.
Gerlag, Danielle M.
van Eck-Smit, Berthe L. F.
Tak, Paul P.
PLoS ONE
Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man.
Methods/Principal Findings
We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99mTc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4×10−3 (0.95−5.1×10−3) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion.
The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.
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A systematic analysis of the effect of target RNA structure on RNA interference
Westerhout, Ellen M.
Berkhout, Ben
Nucleic Acids Research
RNAi efficiency is influenced by local RNA structure of the target sequence. We studied this structure-based resistance in detail by targeting a perfect RNA hairpin and subsequently destabilized its tight structure by mutation, thereby gradually exposing the target sequence. Although the tightest RNA hairpins were completely resistant to RNAi, we observed an inverse correlation between the overall target hairpin stability and RNAi efficiency within a specific thermodynamic stability (ΔG) range. Increased RNAi efficiency was shown to be caused by improved binding of the siRNA to the destabilized target RNA hairpins. The mutational effects vary for different target regions. We find an accessible target 3′ end to be most important for RNAi-mediated inhibition. However, these 3′ end effects cannot be reproduced in siRNA-target RNA-binding studies in vitro, indicating the important role of RISC components in the in vivo RNAi reaction. The results provide a more detailed insight into the impact of target RNA structure on RNAi and we discuss several possible implications. With respect to lentiviral-mediated delivery of shRNA expression cassettes, we present a ΔG window to destabilize the shRNA insert for vector improvement, while avoiding RNAi-mediated self-targeting during lentiviral vector production.
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An Inflammation Loop Orchestrated by S100A9 and Calprotectin Is Critical for Development of Arthritis
Cesaro, Annabelle
Anceriz, Nadia
Plante, Audrey
Pagé, Nathalie
Tardif, Mélanie R.
Tessier, Philippe A.
PLoS ONE
The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.
In this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.
Treatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα, IL-1β and IL-6, and of chemokines like MIP-1α and MCP-1.
The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed.
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Systemic JIA: New Developments in the Understanding of the Pathophysiology and Therapy
Vastert, Sebastiaan J.
Kuis, Wietse
Grom, Alexei A.
Best practice & research. Clinical rheumatology
Systemic juvenile idiopathic arthritis (sJIA) is a rare, systemic inflammatory disease classified as a subtype of JIA. Besides arthritis, it is characterised by systemic features like spiking fever, skin rash, hepatosplenomegaly or serositis. It is becoming clear now that abnormalities in the innate immunity (cytokines like IL-1, IL-6 and IL-18, and neutrophils and monocytes/macrophages rather than lymphocytes) play a major role in the pathogenesis of sJIA, distinguishing sJIA from other JIA-subtypes. Another distinctive feature of sJIA is its strong association with macrophage activation syndrome (MAS). Based on this, consensus is emerging that sJIA should be viewed as an auto-inflammatory syndrome rather than a 'classic' autoimmune disease.
As a consequence of the progression in understanding the underlying mechanisms of sJIA, major changes in the management are evolving. Sofar, treatment has been based on glucocorticosteroids in combination with disease modifying drugs like methotrexate. Recently, remarkable improvement has been observed with IL-1 and IL-6 targeted therapies. These therapies might also change the long term outcome of this disease. However, controlled trials set up in international collaboration are needed to determine the optimal treatment strategies for all sJIA patients.
systemic juvenile idiopathic arthritis; innate immunity; myeloid related proteins (MRP´s); IL-1; IL-6; Macrophage Activation Syndrome (MAS)
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Proteasome inhibitors as experimental therapeutics of autoimmune diseases
Verbrugge, Sue Ellen
Scheper, Rik J
Lems, Willem F
de Gruijl, Tanja D
Jansen, Gerrit
Arthritis Research & Therapy
Current treatment strategies for rheumatoid arthritis (RA) consisting of disease-modifying anti-rheumatic drugs or biological agents are not always effective, hence driving the demand for new experimental therapeutics. The antiproliferative capacity of proteasome inhibitors (PIs) has received considerable attention given the success of their first prototypical representative, bortezomib (BTZ), in the treatment of B cell and plasma cell-related hematological malignancies. Therapeutic application of PIs in an autoimmune disease setting is much less explored, despite a clear rationale of (immuno) proteasome involvement in (auto)antigen presentation, and PIs harboring the capacity to inhibit the activation of nuclear factor-κB and suppress the release of pro-inflammatory cytokines such as tumor necrosis factor alpha and interleukin-6. Here, we review the clinical positioning of (immuno) proteasomes in autoimmune diseases, in particular RA, systemic lupus erythematosus, Sjögren's syndrome and sclerodema, and elaborate on (pre)clinical data related to the impact of BTZ and next generation PIs on immune effector cells (T cells, B cells, dendritic cells, macrophages, osteoclasts) implicated in their pathophysiology. Finally, factors influencing long-term efficacy of PIs, their current (pre)clinical status and future perspectives as anti-inflammatory and anti-arthritic agents are discussed.
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Suppression of HLA Expression by Lentivirus-mediated Gene Transfer of siRNA Cassettes and In Vivo Chemoselection to Enhance Hematopoietic Stem Cell Transplantation
Hacke, Katrin
Falahati, Rustom
Flebbe-Rehwaldt, Linda
Kasahara, Noriyuki
Gaensler, Karin M. L.
Immunologic research
Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation.
The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing.
Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps "universally" compatible cellular grafts.
HLA – Human Leukocyte Antigen; MHC – Major Histocompability Complex; shRNA - short hairpin-type RNAi; RNAi – RNA interference; MGMT - O6-methylguanine-DNA-methyltransferase; MAG - MGMTP140K-2A-GFP lentivirus vector; BU - Busulfan; BG - Benzylguanine; BCNU - 1,3-bis(2-chloroethyl1-nitrosourea); Lentivirus
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Permanent, Lowered HLA Class I Expression Using Lentivirus Vectors With shRNA Constructs: Averting Cytotoxicity by Alloreactive T Lymphocytes
Haga, K.
Lemp, N.A.
Logg, C.R.
Nagashima, J.
Faure-Kumar, E.
Gomez, G.G.
Kruse, C.A.
Mendez, R.
Stripecke, R.
Kasahara, N.A.
Cicciarelli, J.C.
Transplantation proceedings
Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.
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How can we cure a heart "in flame"? A translational view on inflammation in heart failure
Hofmann, Ulrich
Frantz, Stefan
Basic Research in Cardiology
The prevalence of chronic heart failure is still increasing making it a major health issue in the 21st century. Tremendous evidence has emerged over the past decades that heart failure is associated with a wide array of mechanisms subsumed under the term "inflammation". Based on the great success of immuno-suppressive treatments in auto-immunity and transplantation, clinical trials were launched targeting inflammatory mediators in patients with chronic heart failure. However, they widely lacked positive outcomes. The failure of the initial study program directed against tumor necrosis factor-α led to the search for alternative therapeutic targets involving a broader spectrum of mechanisms besides cytokines. We here provide an overview of the current knowledge on immune activation in chronic heart failure of different etiologies, summarize clinical studies in the field, address unresolved key questions, and highlight some promising novel therapeutic targets for clinical trials from a translational basic science and clinical perspective.
Cytokines; Heart failure; Immuno-modulation
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Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica
Rinaldi, Gabriel
Morales, Maria E.
Cancela, Martín
Castillo, Estela
Brindley, Paul J.
Tort, José F.
PLoS Neglected Tropical Diseases
The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.
Author Summary
Reverse genetics tools allow assessing the function of unknown genes. Their application for the study of neglected infectious diseases could lead eventually to the identification of relevant gene products to be used in diagnosis, or as drug targets or immunization candidates. Being technically more simple and less demanding than other reverse genetics tools such as transgenesis or knockouts, the suppression of gene activity mediated by double-stranded RNA has emerged as a powerful tool for the analysis of gene function. RNAi appeared as an obvious alternative to apply in complex biological systems where information is still scarce, a situation common to several infectious and parasitic diseases. However, several technical or practical difficulties have hampered the development of this technique in parasites to the expectations originally generated. We developed a simple method to test the presence of a viable RNAi pathway by silencing an exogenous reporter gene. The method was tested in F. hepatica, describing the conditions for transfection and confirming the existence of a viable RNAi pathway in this parasite. The experimental design created can be useful as a first approach in organisms where genetic analysis is still unavailable, providing a tool to unravel gene function and probably advancing new candidates relevant in pathobiology, prevention or treatment.
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Molecular Blocking of CD23 Supports Its Role in the Pathogenesis of Arthritis
Rambert, Jérôme
Mamani-Matsuda, Maria
Moynet, Daniel
Dubus, Pierre
Desplat, Vanessa
Kauss, Tina
Dehais, Joël
Schaeverbeke, Thierry
Ezzedine, Khaled
Malvy, Denis
Vincendeau, Philippe
Mossalayi, M. Djavad
PLoS ONE
CD23 is a differentiation/activation antigen expressed by a variety of hematopoietic and epithelial cells. It can also be detected in soluble forms in biological fluids. Initially known as the low-affinity receptor for immunoglobulin E (FcεRII), CD23 displays various other physiologic ligands such as CD21, CD11b/c, CD47-vitronectin, and mannose-containing proteins. CD23 mediates numerous immune responses by enhancing IgE-specific antigen presentation, regulating IgE synthesis, influencing cell differentiation and growth of both B- and T-cells. CD23-crosslinking promotes the secretion of pro-inflammatory mediators from human monocytes/macrophages, eosinophils and epithelial cells. Increased CD23 expression is found in patients during allergic reactions and rheumatoid arthritis while its physiopathologic role in these diseases remains to be clarified.
Methodology/Principal Findings
We previously generated heptapeptidic countrestructures of human CD23. Based on in vitro studies on healthy and arthritic patients' cells, we showed that CD23-specific peptide addition to human macrophages greatly diminished the transcription of genes encoding inflammatory cytokines. This was also confirmed by significant reduction of mediator levels in cell supernatants. We also show that CD23 peptide decreased IgE-mediated activation of both human and rat CD23+ macrophages. In vivo studies in rat model of arthritis showed that CD23-blocking peptide ameliorates clinical scores and prevent bone destruction in a dose dependent manner. Ex-vivo analysis of rat macrophages further confirmed the inhibitory effect of peptides on their activation. Taken together our results support the role of CD23 activation and subsequent inflammatory response in arthritis.
CD23-blocking peptide (p30A) prevents the activation of monocytes/macrophages without cell toxicity. Thus, targeting CD23 by antagonistic peptide decreases inflammatory markers and may have clinical value in the treatment of human arthritis and allergic reactions involving CD23.
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Sync & Viz Of aRbitrary Streams (Savors)
Savors is a visualization framework that supports the ingestion of data streams created by arbitrary command pipelines. Any number of data streams can be shown synchronized by time in the same or different views, which can be arranged in any layout. These capabilities combined with a powerful parallelization mechanism and interaction models already familiar to administrators allows Savors to display complex visualizations of data streamed from many different systems with minimal effort.3 weekly downloads
The NodeMon distributed monitoring tool.
Nodemon is a visualization tool for monitoring system resource utilization. It was developed for monitoring the Columbia supercomputer, a 10,240-processor Linux system at NASA Ames Research Center. It can monitor resources on any Linux system or cluster3 weekly downloads
Biocentrica Voyager Antivirus
It moves by itself inside networks like virus infection & plagues, it is being written to solve computer virus problem drastically and responsibly. It is legal, free and open for public domain to improve W3 ICT Security.
Dynamic Perimeter Enforcement (Dyper)
Dyper is a framework for adding connection tracking and dynamic pinhole capabilities to stock routers/switches that allows multiport protocol communication to be maintained with other sites under least privilege while achieving maximum performance.
Packet Mangler
System to delay, drop, and corrupt IP packets to test network capabilities in a closed environment. The packets can be selected in a variety of ways.

subset: commoncrawl


text:
WASHINGTON – On Wednesday, November 20, President Barack Obama will award the Presidential Medal of Freedom.
The Medal of Freedom is our Nation's highest civilian honor, presented to individuals who have made especially meritorious contributions to the security or national interests of the United States, to world peace, or to cultural or other significant public or private endeavors.
Additional details about the event will be released at a later date.


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Skip to Main Content
Public key cryptography (PKC) has been the enabling technology underlying many security services and protocols in traditional networks such as the Internet. In the context of wireless sensor networks, elliptic curve cryptography (ECC), one of the most efficient types of PKC, is being investigated to provide PKC support in sensor network applications so that the existing PKC-based solutions can be exploited. This paper presents the design, implementation, and evaluation of TinyECC, a configurable library for ECC operations in wireless sensor networks. The primary objective of TinyECC is to provide a ready-to-use, publicly available software package for ECC-based PKC operations that can be flexibly configured and integrated into sensor network applications. TinyECC provides a number of optimization switches, which can turn specific optimizations on or off based on developers' needs. Different combinations of the optimizations have different execution time and resource consumptions, giving developers great flexibility in integrating TinyECC into sensor network applications. This paper also reports the experimental evaluation of TinyECC on several common sensor platforms, including MICAz, Tmote Sky, and Imotel. The evaluation results show the impacts of individual optimizations on the execution time and resource consumptions, and give the most computationally efficient and the most storage efficient configuration of TinyECC.


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Hotels near Adventure Science Center
Search hotels near Adventure Science Center in Nashville
Trending now in Nashville
Hotels within 0.5 miles of Adventure Science Center
134 people looked at this hotel in the last hour
Most recent review
"An overnight stop...but we wanted easy (walking) access to downtown Nashville...this was a good location and a reasonable price."
A Traveller, May 2016, US
Hotels within 1 mile of Adventure Science Center
234 people looked at this hotel in the last hour
Most recent review
"Motel was great except they were doing work in the pool area so machinery started up at 8am every day. Complimentary breakfast was good with range of choices. 5-10 min Uber to honky tonk strip. Free parking if you have a car. Room was clean with fridge / wifi - if possible get room away from the ..."
H, May 2016, AU
214 people looked at this hotel in the last hour
Most recent review
"The hotel is great. Great location. Good breakfast. Can walk everywhere.New and clean. Staff were very nice. I would have liked to have a microwave in the room ."
A Traveller, Jun 2016, CA
144 people looked at this hotel in the last hour
Most recent review
"The Omni is centrally located and within walking distance to most of Nashville's great entertainment venues and the lower Broadway bar and music scene. Was an awesome time, I'm coming back"
A Traveller, Jun 2016, US
118 people looked at this hotel in the last hour
Most recent review
"Went to Nashville to experience the local music, country music hall of fame.& bbq "
A Traveller, Jun 2016, US
114 people looked at this hotel in the last hour
Most recent review
"Very pleasant stay; the hotel is older but very clean and well-maintained. The staff was all very professional, courteous, and friendly. We walked to everything we wanted to do in Nashville, takes about 20 minutes to get right to the center of town. Also very convenient to Vanderbilt University."
A Traveller, Jun 2016, US
92 people looked at this hotel in the last hour
Most recent review
"Stay there every year. Just gets better every time"
W, Jun 2016, US
- Exclusive by Nashville VacationsNo Guest Reviews yet
30 people looked at this hotel in the last hour
Most recent review
"Very comfortable accommodations. It was a home away from home. Although not a deal breaker, the cable/sat selections could have been better. Very nice. I would definitely stay here again!"
A Traveller, Jan 2016, US
Most recent review
"I thought I was renting a room in a hotel; this is a condo. The listing should clearly state this. There is only parking for 1 vehicle. The ladies that met us were very pleasant and even helped up carry our luggage up. Unfortunately, there were not enough linens or towels for our party of 4. They..."
A Traveller, Dec 2015, US
Map of hotels near Adventure Science Center
- Nissan Stadium
- Grand Ole Opry
- Bridgestone Arena
- Music City Center
- Vanderbilt University
- Opry Mills
- CoolSprings Galleria
- Ryman Auditorium
- Country Music Hall of Fame and Museum
- Nashville Shores Waterpark
- Nashville Zoo
- Tennessee State University
- Opryland Hotel Gardens
- Belmont University
- Riverfront Park
- Nashville Convention Center
- Mall at Green Hills
- Tennessee State Fairgrounds
- Nashville Municipal Auditorium
- Marathon Music Works
Hotels in Nashville near Adventure Science Center
Are you looking for a cheap Adventure Science Center hotel, a 5 star Adventure Science Center hotel or a family friendly Adventure Science Center hotel? You just landed in the best site to find the best deals and offers on the most amazing accommodations for your stay.
When you search for hotels near Adventure Science Center with Hotels.com, you need to first check our online map and see the distance you will be from Adventure Science Center, Tennessee.
Our maps are based on hotel search and display areas and neighborhoods of each hotel so you can see how close you are from Adventure Science Center and refine your search within Nashville or Tennessee based on closest public transportation, restaurants and entertainment so you can easily get around the city. All the hotels details page show an option for free or paid onsite parking.
If you wish to see the hotels with the highest featuring discounts and deals near Adventure Science Center, simply filter by price/ average nightly rate. We recommend you filter by star rating and read our genuine guest reviews so you can get the best quality hotel with the best discount.
One of the new features on Hotels.com guest reviews is that also show reviews from Expedia for Adventure Science Center hotels and the TripAdvisor Adventure Science Center hotels reviews so you can make sure that you checking with a reliable source. See the review scores on our Nashville hotel information pages.
Make the most out of your family vacation when you book your accommodation with Hotels.com – book your hotel near Adventure Science Center, Nashville after reviewing the facilities and amenities listed for each hotel.
After booking your hotel near Adventure Science Center, expect to receive your reservation confirmation in the mail in less than 10 minutes. The confirmation email contains more information on all nearby attractions, local directions and weather forecast, so you can better plan the days during your trip.
After getting the best hotel rates you can still save more by winning 1 free night! That's right, book 10 nights in any hotel near Adventure Science Center, Nashville and after you sign up for the Welcome Rewards program, you are eligible hotel you receive 1 night free*
The best hotel deals are here: We have Adventure Science Center hotel deals, Adventure Science Center last minute deals and offers to get you the cheapest Adventure Science Center hotel with our lowest price guarantee.
Nashville travel guides
Where to Stay in Nashville – a neighborhood guide
Nashville's all about spending nights drifting from one buzzing country music venue to the next, and...
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Nashville's retail spots range from vast shopping malls to tiny family-run boutiques. Whether you're...
Where to Eat in Nashville – a food and dining guide
Nashville's booming culinary scene creates the perfect counterpart to its well-established, music sc...


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ODT Online
ODT /directory
Police hunting a suspected kidnapper who escaped custody are
door-knocking his known associates.
Brett Makiri, 23 was arrested yesterday afternoon after he
allegedly kidnapped and assaulted a woman on Monday.
Makiri was being held in an interview room in the Auckland
Central police station when he escaped about 6pm.
Police, who already warned the public against helping Makiri,
this afternoon said they had already visited 22 addresses of
Makiri's known associates in Auckland and Waikato.
"Our focus remains on Mr Makiri's recapture and we will
continue to visit his associates until we locate him,"
Detective Senior Sergeant John Sutton said.
"It would be wise of Mr Makiri to turn himself as soon as
"We remind members of the public and associates of Mr Makiri
once again that assisting an escaped prisoner is an offence."
The 29-year-old woman involved in the kidnapping on Monday
night was picked up in Auckland and driven to Coromandel.
She was assaulted several times before escaping when the car
she was in ran out of petrol near the Coromandel township.
Makiri was known to the woman and her partner.
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publication, Property
Times South Canterbury


text:
I'm using the email to post function for a client. I haven't had issues with getting it to work before so I'm not sure where to go from here. I've actually set up too addresses for the purpose of this exercise and neither work, but give different errors.
The first is an address that is not the same domain as the wordpress installation, the second is the same domain as the wordpress installation.
The first gives me this error:
POP3 connect: Error [110] [Connection timeout]
The second gives me the error:
POP3 connect: Error [111] [Connection refused]
I know the post to email works. I've never actually had a problem with it so I assume that the issue is with the connection to the POP server or on the mail side, but any ideas are helpful. I actually haven't seen any of the POP3 connect: Error [111] [Connection refused] errors in the forums, a few of the 110, so I wonder if this is working but the authentication isn't working out.
Any ideas?


text:
Excerpts From Opinion and Dissents in Supreme Court Case on Cross Burning
Published: April 8, 2003
WASHINGTON, April 7— Following are excerpts from the Supreme Court's 6-to-3 ruling in Virginia v. Black that states can outlaw cross burning that is meant to intimidate. Justice Sandra Day O'Connor wrote the majority opinion; Justice David H. Souter and Justice Clarence Thomas wrote dissents. A full transcript is online at nytimes.com/national.
By Justice O'Connor
The First Amendment, applicable to the states through the Fourteenth Amendment, provides that ''Congress shall make no law . . . abridging the freedom of speech.'' The hallmark of the protection of free speech is to allow ''free trade in ideas,'' even ideas that the overwhelming majority of people might find distasteful or discomforting. Thus, the First Amendment ''ordinarily'' denies a state ''the power to prohibit dissemination of social, economic and political doctrine which a vast majority of its citizens believes to be false and fraught with evil consequence.'' The First Amendment affords protection to symbolic or expressive conduct as well as to actual speech.
The protections afforded by the First Amendment, however, are not absolute, and we have long recognized that the government may regulate certain categories of expression consistent with the Constitution. The First Amendment permits ''restrictions upon the content of speech in a few limited areas, which are of such slight social value as a step to truth that any benefit that may be derived from them is clearly outweighed by the social interest in order and morality.''
Thus, for example, a state may punish those words ''which by their very utterance inflict injury or tend to incite an immediate breach of the peace.'' We have consequently held that fighting words -- those personally abusive epithets which, when addressed to the ordinary citizen, are, as a matter of common knowledge, inherently likely to provoke violent reaction'' -- are generally proscribable under the First Amendment. Furthermore, ''the constitutional guarantees of free speech and free press do not permit a state to forbid or proscribe advocacy of the use of force or of law violation except where such advocacy is directed to inciting or producing imminent lawless action and is likely to incite or produce such action.'' And the First Amendment also permits a state to ban a ''true threat.''
''True threats'' encompass those statements where the speaker means to communicate a serious expression of an intent to commit an act of unlawful violence to a particular individual or group of individuals. The speaker need not actually intend to carry out the threat. Rather, a prohibition on true threats ''protects individuals from the fear of violence'' and ''from the disruption that fear engenders,'' in addition to protecting people ''from the possibility that the threatened violence will occur.'' Intimidation in the constitutionally proscribable sense of the word is a type of true threat, where a speaker directs a threat to a person or group of persons with the intent of placing the victim in fear of bodily harm or death. Respondents do not contest that some cross burnings fit within this meaning of intimidating speech, and rightly so. The history of cross burning in this country shows that cross burning is often intimidating, intended to create a pervasive fear in victims that they are a target of violence.
The Supreme Court of Virginia ruled that in light of R.A.V. v. City of St. Paul, even if it is constitutional to ban cross burning in a content-neutral manner, the Virginia cross-burning statute is unconstitutional because it discriminates on the basis of content and viewpoint.
It is true, as the Supreme Court of Virginia held, that the burning of a cross is symbolic expression. The reason why the Klan burns a cross at its rallies, or individuals place a burning cross on someone else's lawn, is that the burning cross represents the message that the speaker wishes to communicate. Individuals burn crosses as opposed to other means of communication because cross burning carries a message in an effective and dramatic manner.
The fact that cross burning is symbolic expression, however, does not resolve the constitutional question. The Supreme Court of Virginia relied upon R.A.V. v. City of St. Paul to conclude that once a statute discriminates on the basis of this type of content, the law is unconstitutional. We disagree.
In R.A.V., we held that a local ordinance that banned certain symbolic conduct, including cross burning, when done with the knowledge that such conduct would ''arouse anger, alarm or resentment in others on the basis of race, color, creed, religion or gender'' was unconstitutional. We held that the ordinance did not pass constitutional muster because it discriminated on the basis of content by targeting only those individuals who ''provoke violence'' on a basis specified in the law. The ordinance did not cover ''those who wish to use 'fighting words' in connection with other ideas -- to express hostility, for example, on the basis of political affiliation, union membership, or homosexuality.'' This content-based discrimination was unconstitutional because it allowed the city ''to impose special prohibitions on those speakers who express views on disfavored subjects.''


text:
While finance minster displayed good endeavors to maintain country's journey on path of fiscal consolidation, he disappointed big businesses who expected to see FM snip corporate tax rate.
01 Mar 2016, 3.46 pm | Source: Moneycontrol.com
Abhijit Roy, MD and CEO of Berger Paints, says the company's high-end products have done better than economic segment in Q3.
19 Feb 2016, 1.06 pm | Source: CNBC-TV18
Strengthening NPL recovery teams/processes, rebalancing its loan mix by increasing share of better rated corporate loans as well as retail loans, and scaling-up its digital platform are few initiatives it is taking to spearhead it...
15 Feb 2016, 12.46 pm | Source: Moneycontrol.com
Shantanu Dasgupta, Vice President-Corporate Affairs and Strategy-Asia South, Whirlpool says the company's profit after tax (PAT) was aided by low material costs.
08 Feb 2016, 4.43 pm | Source: CNBC-TV18


text:
Boy's High School basketball post-season
PHS, and Saranac get first round winsUPDATED 12:50 AM EST Feb 21, 2013
Saranac hosts Saranac Lake, and Plattsburgh, NCCS add another chapter to their rivalry, in boy's basketball.
More Video
CommentsThe views expressed are not those of this site, this station or its affiliated companies. By posting your comments you agree to accept our terms of use.blog comments powered by Disqus


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The deleterious effects of inactivity on the functional capacities of several organ systems in the human are effectively documented and described by Kottke elsewhere in this issue (p 825). For many years physiologists have studied the effects of exercise on the cardiovascular and neuromuscular systems, so that we are now well aware of many specific physiologic effects of mild and strenuous physical activity on cardiac function, circulation, and muscular strength, and thus have the basic knowledge useful in the management of many patients. However, only relatively recently have physiologists become actively concerned in evaluating physiologic effects of bed rest, immobilization, and varying states of inactivity. The knowledge obtained from these and future studies should be of vital interest to physicians and others concerned with the guidance and regulation of the activities of patients, aging citizens, astronauts, and space travelers, as well as average persons.
Our contemporary civilization tends to foster
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2 Recommendations
1002's recommendation
a fitting tribute to those in love. birthstones nestled against one another will remind her who her true love is.
My Collections:
whosdiary's recommendation
Couple's birthstone necklace:- * sterling-silver diamond-cut snake chain extends from 16"-18" long * lobster clasp * pendant measures 3/4" x 5/8" high * made in USA. * $155.00 - $790.00
My Collections:


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These adorable saint statues will bring a smile to young and old alike. Each is presented in a full color gift box with easy to understand life stories, a perfect way to introduce the most beloved saints. Build your collection and share their inspiring lives. Great for birthdays, feast days, and sacramental celebrations.
|h koczur (May 8, 2015)
|Fun collection for anyone...so cute!
|Cecelia Mangan (Pittsburgh, March 26, 2015)
|I gave this to my granddaughter (who is 5). When she went to bed the first night she asked her mother if St. Jude came alive at night and her mother said she didn't know, but he would protect her all the time. She said I hope he doesn't come alive because Magic (who is her service dog) would bite him.lol Her mother said he would not, that he would protect Magic too.


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Information and cosmetic advice for women battling cancer. Hands on makeup and skin care application. Cosmetics are provided free of charge. Register by calling the American Cancer Society at 800.227.2345.
24/7 Physician Referral.
Contact Methodist Hospitals
© 2009 - 2016 Methodist Hospitals. All rights reserved. Web Development by alligatortek.
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Get Driving Directions


text:
Definition of enchorial in English:
Denoting or relating to the kind of language used by ordinary people; colloquial or demotic.
Example sentences
- The enchorial language was not placed on sacred edifices.
- Difficulties of no ordinary kind presented themselves to all who had applied to make out even the second, or enchorial inscription.
- The writing is enchorial, but of a form so closely allied to hieroglyphics that it shows how the one passes into the other.
Early 19th century: from Greek egkhōrios 'in or of the country' (from en- 'within' + khōra 'country') + -al.
For editors and proofreaders
Line breaks: en|chor|ial
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= trending


text:
Published on
You're Solr powered, and needing to customize its capabilities. Apache Solr is flexibly architected, with practically everything pluggable. Under the hood, Solr is driven by the well-known Apache Lucene. Lucene for Solr Developers will guide you through the various ways in which Solr can be extended, customized, and enhanced with a bit of Lucene API know-how. We'll delve into improving analysis with custom character mapping, tokenizing, and token filtering extensions; show why and how to implement specialized query parsing, and how to add your own search and update request handling.