Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and monocytes isolated from MS-affected individuals as compared with those from healthy donors, which was rescued by IFN-β therapy. Collectively, our data unveil a novel TLR7-regulated mechanism in in vivo IFN-β-stimulated whole leukocytes that could be exploited to define new TLR7-based strategies for the treatment of MS. Growing evidence is accumulating

on the central role that B lymphocytes play in the immunopathology of multiple sclerosis (MS) [1, 2]. For example, oligoclonal IgG bands, found in the cerebrospinal fluid of more than 90% of patients with MS, indicate an intrathecal Ab production [3]. The presence of clonally expanded B cells, plasma cells, complement and myelin-specific Abs in chronic MS lesions also suggested an intrathecal, Epigenetics inhibitor Ag-driven humoral immune response in the central nervous system of MS

sufferers [4-6]. In addition, B-cell follicle-like structures are detectable in the meninges of MS patients [7, 8]. More recently, B-cell depleting therapies, such as Rituximab (that targets the B lymphocyte surface antigen CD20 [9-11]), together with Ocrelizumab and Ofatumumab (two other humanized anti-CD20 monoclonal Abs), are proving their efficacy at various stages of clinical development [12]. All together these findings contribute to the compelling evidence that B cells and the humoral

immune response are implicated in the pathogenesis of MS and suggest the therapeutic implications that all this may have for the treatment of this disease. B BTK inhibition lymphocytes play an essential role in bridging innate and adaptive immunity. To differentiate into specialized cells capable of communicating with helper T cells and undergo Ab diversification, clonal expansion, and Ig secretion, B lymphocytes need the support of a coordinated network of cytokines, growth factors, adhesion, and ligand-receptor signals [13]. Among B-cell receptors, the TLRs and their natural agonists have raised interest since Branched chain aminotransferase they elicit direct effects on human B cells [14]. TLRs are germ-line encoded pattern recognition receptors that can detect conserved molecular patterns either expressed on microorganisms or of self-origin. Targeting them or modulating their functions may have therapeutic potential in autoimmune diseases, including MS [15]. B lymphocytes selectively express TLR7 and TLR9 and are activated by their specific ligands [16, 17]. At variance with other TLRs, TLR7 and TLR9 share relevant properties. Indeed, they both recognize microbial and endogenous nucleic acids; in particular, TLR7 specifically binds guanosine- and uridine-rich ssRNAs while TLR9 senses hypomethylated CpG-rich dsDNAs. Furthermore, they both reside in the endosomal compartments, unlike the other TLRs present on the cell surface.

[31] To make things more complicated, not all inflammatory milieux produce the same outcome. Some studies have indicated an important role

for toll-like receptors (TLR), membrane-spanning, non-catalytic receptors that recognize structurally conserved molecules derived from microbes and mediate the activation of immune responses of both innate and adaptive types. Mesenchymal stromal cells express a large number of TLR, the stimulation of which has been shown to profoundly affect MSC immunomodulatory properties as well as their migratory phenotype. It is well established that MSC express a number of TLR at both RNA and protein Fluorouracil levels. High mRNA expression of TLR1, TLR2, TLR3, TLR4, TLR5 and TLR6 has been consistently detected, whereas TLR2, TLR3, TLR4, TLR7 and TLR9 expression has been reported by flow cytometry. Unfortunately, there is little consensus about the pattern of their expression in MSC with the major confounding factors being the heterogeneity of MSC preparations and the modality of TLR detection. The expression of TLR on MSC has also been functionally assessed. Although

TLR3 and TLR4 binding antagonises MSC immunosuppressive activity,[60] the stimulation of the same receptor on isolated MSC before their use in culture boosts MSC immunosuppressive activity.[61] It has been shown that TLR3 and TLR4 activation induces the production of pro-inflammatory mediators, such as IL-1, IL-6, IL-8 and CCL5 together with the expression of iNOS, and TNF-related C59 wnt apoptosis-inducing ligand (TRAIL).[62] It should be noted however that the time of exposure to TLR ligands and the concomitant presence of other cytokines are likely to

add layers of complexity. Following low-level, short-term TLR-priming, Waterman[63] observed opposing effects of TLR3 or TLR4 stimulation. Last, targeting TLR2 results in the up-regulation of galectin-3, known to modulate T-cell proliferation.[64] The possibility of alternating the immunomodulatory properties of MSC depending on the inflammatory environment to which they are exposed has profound implications on how to harness Non-specific serine/threonine protein kinase their therapeutic potentials. The studies conducted to investigate MSC therapeutics in graft-versus-host disease (GvHD) are fully consistent with the biological features so far identified. Irrespective of the animal models used, MSC are effective at treating GvHD only when administered at fairly specific intervals.[65-67] At these time-points, the levels of inflammatory cytokines like IFN-γ are particularly high and therefore more likely to promote MSC immunosuppressive activity. The clinical studies are fully in accord with these data.

Jin et al. [32] demonstrated that besides strain differences in mice, the context in which B cells were activated influenced their fate. IL-21-driven apoptosis and inhibition of proliferation were dominant when B cells were activated through TLR-4

and TLR-9. Co-stimulation and low apoptosis were observed in B cells stimulated with anti-IgM or anti-IgM plus anti-CD40, whereas both apoptosis and co-stimulation were detected when IL-21 acted on anti-CD40 previously activated B cells. This raised the possibility that different subsets of B cells responded differentially to IL-21. In our hands, although IL-21 rescues SCH772984 in vitro unstimulated CD27– B cells from spontaneous apoptosis, it reduces the protective effect of most of the stimuli both in CD27– and CD27+ B cells. On the contrary, IL-21 increases the protective effect of anti-CD40 in CD27+ B cells. This suggests that IL-21

per se increases survival of CD27– (mostly RXDX-106 solubility dmso naive and transitional) B cells, but this effect is lost after these cells are activated. However, CD27+ B cells become sensitive to rescue from apoptosis if they are prestimulated with a surrogate T-dependent stimulus (anti-CD40). Stimulation through the BCR or with a T-independent stimulus (CpG-ODN) renders CD27+ B cells insensitive to the protective effect of IL-21. IL-21 acts as a checkpoint for a productive B cell response. Only memory and marginal zone B cells (contained in the CD27+ population) that receive cognate T cell help in the presence of IL-21 would be protected from apoptosis and directed to proliferation and eventually differentiation to antibody secreting cells. We also report that rescue from apoptosis is independent of proliferation. This is particularly evident with anti-CD40 that, although it does not induce proliferation, it rescues most CD27– B cells from apoptosis.

Our present results support that the inability of CVID B cells to produce normal levels of immunoglobulins in vitro (and in vivo) can be the consequence of an increased susceptibility to apoptosis upon stimulation. That would result in a reduced number of cells during an immune response. Thiamet G CD27–, but particularly CD27+ B cells, from our CVID MB0 patients are less sensitive to rescue from apoptosis than MB1 patients and controls. Moreover, CD27+ B cells from CVID MB0 patients showed significantly higher expression of TRAIL than controls or CVID MB1 patients. TRAIL is a member of the TNF superfamily of cytokines able to induce programmed cell death in tumour cells. Different subpopulations of B cells show distinct sensitivity to TRAIL-mediated apoptosis. BCR triggering sensitizes peripheral blood memory, but not naive human B cells, to TRAIL-mediated apoptosis [33] and TRAIL promotes death of normal plasma cells [34]. In agreement with our results, van Grevenynghe et al. [13] demonstrated that memory B cell survival was decreased significantly in aviraemic successfully treated (ST) HIV subjects compared with uninfected controls.

Use of anti-infectives that do not kill bacteria, https://www.selleckchem.com/products/apo866-fk866.html rather than traditional antibiotics, theoretically lifts the strong selective pressure for the evolution of resistance. Our laboratory initially developed a manual liquid-based assay for identifying compounds that cure Enterococcus faecalis infection and used the assay to screen ∼6000 compounds in a proof-of-principle experiment [61]. We identified 18 compounds that cured the infection, having in vivo efficacious

doses substantially lower than their in vitro minimal inhibitory concentrations (MIC). In contrast, the in vivo effective doses of traditional antibiotics such as tetracycline were much higher than their in vitro MICs. These data showed that, in contrast to traditional antibiotic screens, the C. elegans–E. faecalis curing assay identifies compounds that affect the virulence of the pathogen, that suppress pathogen survival in vivo or that enhance the immune response of the host. Because these latter compounds have activity in vivo only in the whole-animal assay, it www.selleckchem.com/products/epz-6438.html provides proof-of-principle for a proposed drug discovery approach that exploits (and

blocks) pathogen adaptation to host physiology. Figure 2 illustrates a newly developed automated scoring assay that discriminates between live and dead worms [62]. The assay uses the fluorescent dye SYTOX that is excluded from living cells and tissues, but stains dead organisms, including C. elegans. To test the assay, a pilot screen of 33 931 small molecules and 3283

natural product extracts has been carried out using the C. elegans–E. faecalis infection model. Of these 37 214 compounds and extracts, 136 and 108 tested positive in primary and secondary screens, respectively. Of the 108 compounds, 28 were not previously known anti-microbials. Nine of these 21 compounds were able to promote nematode survival at concentrations lower than their MIC values in vitro, a hallmark of anti-infective compounds that could be targeting bacterial virulence or host immunity [62]. These nine compounds are now undergoing in-depth chemical and biological characterization. The next couple of years will probably see fast progress in a number of areas related to host–pathogen interactions in C. elegans and beyond. In C. elegans, important areas that mafosfamide require further study include extensive characterization of the signalling networks that influence the outcome of infection and host response, and the cell types in which they function. At the whole organism level, different tissues and organ functions are co-ordinated during infection through systemic endocrine signals that remain to be delineated precisely. Further insight will be gained by precise examination of the actual mechanisms involved in pathogenesis for each pathogen type and infection process. Because the study of C. elegans immunity highlights the role of epithelial innate immunity, it is important to explore further the generality of such findings. How many features of C.

Repetition of ATCMR promotes chronic change of allograft tissue, which results

in the poor allograft outcome. Therefore, our results suggest that the IL-17-dominant state may involve in the development of chronic change by repeat ATCMR. We investigated C4d positivity to evaluate whether the FOXP3/IL-17 ratio is associated with humoral immunity. Our results showed that C4d positivity and the coexistence of acute antibody-mediated rejection did not differ significantly between Tanespimycin in vivo the two groups. In addition, glomerulopathy or vasculopathy, which is associated with humoral immunity, was not different between the two groups.31–33 These findings suggest that the impact of the Th17–Treg axis on humoral immunity is not as strong as its effect on T-cell-mediated immunity. The results of our study demonstrated that the ratio between Treg and IL-17-secreting

cell infiltration in the renal allograft represents the severity of ATCMR. But it is uncertain whether a similar ratio between these two cells is observed in peripheral blood mononuclear cells (PBMCs). In a previous report, significantly higher Treg infiltration in allograft tissue was observed even though its proportion in PBMCs was not elevated.34 It may be because the allograft is a more active site of immune stimulation than PBMCs. Therefore, it is possible that the ratio between Treg and IL-17-secreting cells in PBMCs click here is different from that in allograft. Our study has some limitations. First, this study is retrospective and non-randomized. For example, the proportion of basiliximab induction therapy was significantly

higher in the FOXP3 high group. However, basiliximab induction was not a significant prognostic factor for allograft outcome in this study. In addition, the FOXP3/IL-17 ratio did not differ significantly between the patients who took basiliximab induction and the patients who did not (data not shown). The above findings suggest that basiliximab induction did not have a significant effect on the development of an IL-17-secreting cell or FOXP3+ Treg dominant condition, and allograft outcome Resveratrol after ATCMR. Second, the microenvironment, which is associated with the IL-17-driven or the FOXP3+ Treg-driven condition, was not assessed. Therefore, randomized controlled trials investigating the inflammatory cytokines associated with IL-17-producing cell development, such as IL-6, IL-21 and tumour necrosis factor-α, may help to understand clearly the underlying mechanisms that drive the IL-17 high or FOXP3 high condition.35 In summary, it is helpful to assess IL-17-secreting cell infiltration combined with FOXP3+ Treg in predicting the clinical outcome after ATCMR. The ratio between FOXP3 and IL-17 was closely associated with allograft function and the severity of tissue injury. Their ratio was also associated with the clinical outcome of ATCMR and long-term allograft survival.

1a–c). Maximal levels of expression were detected at 24 h for MIP-1α and at 6 h for MIP-1β and RANTES following Tax1 treatment. Interestingly, higher levels of MIP-1α were observed at 6 and 12 h when PBMCs were treated with Tax2A compared to Tax1 (Fig. 1a), while higher levels of MIP-1β and RANTES were detected after 3 and 6 h for Tax1 treatment compared to Tax2A (Fig. 1b,c).

These results indicated that HTLV-2 Tax protein induced a rapid and sustained production of MIP-1α, MIP-1β and RANTES. Tax1 and Tax2A recombinant proteins were assessed for their potential to activate the p65/RelA subunit, which is a well-established indicator of the canonical NF-κB pathway [34], a rapid-acting primary transcription factor. We also employed Tax2A/1–198 and Tax2A/135–331 recombinant Tax2A fragments containing NF-κB domains [28, 29] to evaluate their Cilomilast ic50 potential to activate the NF-κB pathway compared to the entire Tax2A protein. Treated cells were immunolabelled

for the detection of phosphorylated p65/RelA by immunofluorescence. After 1 h, both the entire compound screening assay Tax2A and the Tax2A/1–198 fragment induced p65/RelA activation significantly over controls (14- and 10-fold, respectively, P < 0·05) (Fig. 2a). Significantly higher levels of activation were also observed when the entire Tax2A and the Tax2A/135–331 fragment were used to treat PBMCs for 2 h (27- and ninefold, respectively, P < 0·05). The complete Tax2A protein also induced significantly higher levels of p65/RelA activation compared to Tax1 and both Tax2A fragments after 2 h of treatment (Fig. 2b). Tax1 protein induced significant levels of p65/RelA activation at 1 (12-fold) and 2 h (eightfold) (P < 0·05). The Jurkat cell BCKDHA line served as a negative control and the HTLV-2-infected MoT cell line, displaying constitutive activation of NF-κB [27], served as positive control in the assay (Fig. 2c). It was observed that the activation of p65/RelA (Fig. 2a,b) by Tax2A preceded the secretion of MIP-1α, MIP-1β and RANTES in all conditions tested (Fig. 1). Next, the

binding activity of p65/RelA and p50 NF-κB subunits was assessed quantitatively in nuclear extracts from PBMCs treated with Tax2A or Tax1 proteins using the TransAM assay. Tax2A significantly enhanced the activation of both p65/RelA and p50 after 1 and 2 h compared to untreated and mock-treated controls (P < 0·001). Although Tax1 also induced high levels of both p65 and p50 activation by 1 (P < 0·05) and 2 h (P < 0·001) after treatment compared to controls (Fig. 3a,c), Tax2A induced significantly higher levels of p65/RelA activation than Tax1 following 1 h of treatment (P < 0·05) (Fig. 3a). Nuclear extracts from MoT and Raji nuclear extracts, used as positive controls, induced high levels of both p65/RelA and p50 activation (Fig. 3b,d).

Double- and triple-colour fluorescence images were acquired using a Leica microscope. CXCR3 expression was detected on acetone-fixed tissue sections using a polyclonal rabbit

anti-mouse antibody to CXCR3 (0·5 µg/ml final concentration; Zytomed) followed by the tyramide signal amplification (TSA) system with peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) (5 µg/ml; Jackson Immunoresearch) and FITC-tyramide (PerkinElmer Life Sciences, Boston, MA, USA). CD117+ lin- precursor-enriched lamina propria mononuclear cells (lamina propria MCs) were finally isolated subsequently using lineage-marker [negative depletion with antibodies to CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), 7-4 and Ter-119] and c-kit microbeads (positive selection) and MACS techniques (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s click here www.selleckchem.com/products/AZD6244.html instructions. Total RNA of isolated precursor cells and bone marrow-derived dendritic cells (bmDCs) was isolated

using TRIzol (Sigma-Aldrich, Hamburg, Germany) according to the manufacturer’s recommendations. Reverse transcription into complementary DNA was performed using the Moloney murine leukaemia virus (MMLV) reverse transcriptase (Life Technologies Inc., Carlsbad, CA, USA) method. Chemokine receptor expression was analysed using two multiplex PCR kits (Maxim Biotech, San Francisco, CA, USA) including CCR1-9 and CX3CR1, according to the manufacturer’s instructions. Notch 1–4 expression by Sodium butyrate IEL precursors and mature IEL was analysed by RT–PCR as described elsewhere [11]. Notch-ligand expression on bmDC was analysed 24 h after incubation with various concentrations of rmMip3a (R&D Systems) by real-time PCR as described elsewhere [11]. For isolation of bmDC, bone marrow was isolated from femur and tibia and erythrocytes were lysed. The remaining cells were plated at a density of 106 per ml in six-well plates in RPMI-1640 (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) and containing 10 ng/ml of murine granulocyte–macrophage colony-stimulating factor (GM-CSF) and 1 ng/ml of murine IL-4 (Peprotech, Rocky Hill, NJ, USA). The cells were incubated

at 37°C with 5% CO2. After 2 days of culture the cells were washed gently and replaced with RPMI-10 containing the same concentration of GM-CSF and IL-4 for an additional 5 days and semi-adherent cells were harvested for further experiments. For maturation, bmDC were stimulated further with 1 µg/ml LPS for 24 h and incubated with variable concentrations of rmMip3a (R&D Systems). Colitis was induced by addition of 3% DSS (molecular weight 40 000; ICN Biomedicals, Aurora, OH, USA) to drinking water for 7 days. Citrobacter rodentium was grown overnight in Luria–Bertani broth at a concentration of 2·5 × 109/ml. Adult (10-week-old) CCR6 heterozygous mice were infected with 200 µl of the bacterial suspension (5 × 108 bacteria) by oral gavage.

Genetic information for receptor chains is carried by a germline pool of variable (V), joining (J), and diversity (D) genes that undergo somatic DNA rearrangements

to generate receptors with diverse-binding specificity see more [2]. The “innate-like” γδ T cells have unique features when compared with the more abundant αβ T cells, e.g. a preferential distribution in both epithelial and mucosal sites, an immunoglobulin (IG)-like antigen recognition mechanism in addition to the MHC-restricted one. Moreover, their percentage in peripheral blood cells, depending on age and species, differs strikingly from that of αβ T cells [3]. Artiodactyls are referred to as “γδ-high species” since they exhibit a higher frequency and a wider physiological distribution of γδ T cells with respect to other mammalian species, including humans and

mice which are referred to as “γδ-low species” [4]. The locus organization and expression of TCRG and TCRD genes have been characterized in ruminants; these species have been shown to possess a large TCRG [5, 6] and TCRD [7] germline repertoire. Camelus dromedarius (often referred to as the Arabian or one-humped camel) Romidepsin ic50 is arguably the most famous member of the Camelidae family for its historical and economic importance. Despite this, the dromedary literature is far less extensive than that on other domestic animals. Even the relative phylogenetic placement of Camelidae within Cetartiodactyla remains uncertain [8]. Indeed, it should be noted that the immune system of the camelids has so far been considered unique: in addition to the conventional tetrameric IgGs, camelids have special smaller heavy chain-only antibodies [9]. Here, we report an extensive analysis of the locus organization and expression of the TCRG genes in dromedary. The germline locus is composed

of only a few genes: two TCRGVs, four TCRGJs, and two TCRGCs. Indeed our gene expression data suggest that in this organism, γ chain diversity is likely to be generated not only by V-J rearrangement but also by somatic hypermutation (SHM) in the variable domain. It is generally accepted that SHM occurs primarily in germinal center B cells and is Protirelin the driving force for antibody affinity maturation. It introduces mainly point mutations into the variable domains of IG genes, at a rate of 10−5 to 10−3 per base per generation [10]. G-C and A-T base pairs are mutated at roughly equal frequencies with certain “”hotspot”" DNA motifs ((A/G/T)G(C/T)(A/T) motif (or DGYW) and (A/T)A (or WA), as well as their reverse complements) being preferentially targeted by the enzyme activation-induced cytidine deaminase (AID) [10-12]. Recently, it has been reported that SHM occurs also in the TCRGV region of the sandbar shark [13]. In our opinion, our findings support the important conclusion that, as for TCRDV genes [14], the C. dromedarius TCRG gene repertoire is also likely to have been shaped by SHM.

These studies were supported by the Crohn’s and Colitis Foundation of Canada. The authors have no conflict of interest to report with regard to this manuscript. “
“Memory cross-reactive CD8+ T-cell responses may induce protection or immunopathology upon secondary viral challenge. To elucidate the potential role of T cells in sequential flavivirus infection,

we characterized cross-reactive CD4+ and CD8+ T-cell responses between attenuated and pathogenic Japanese encephalitis virus (JEV) and pathogenic West Nile virus (WNV). A previously reported WNV NS4b CD8+ T-cell epitope and its JEV variant elicited CD8+ T-cell responses in both JEV- and WNV-infected mice. The peptide variant homologous to the immunizing virus induced greater cytokine secretion and activated higher frequencies of epitope-specific Epigenetics inhibitor Rucaparib chemical structure CD8+ T cells. However, there was a virus-dependent, peptide variant-independent pattern of

cytokine secretion; the IFNγ+-to-IFNγ+TNFα+ CD8+ T-cell ratio was greater in JEV- than in WNV-infected mice. Despite similarities in viral burden for pathogenic WNV and JEV viruses, CD8+ T cells from pathogenic JEV-immunized mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. Patterns of killer cell lectin-like receptor G1 (KLRG1) and CD127 expression differed by virus type, with a rapid expansion and contraction of short-lived effector cells in JEV infection and persistence of high levels of short-lived effector cells in WNV infection. Such cross-reactive T-cell responses to primary infection may affect the outcomes of sequential flavivirus infections. The arthropod-borne Flaviviruses co-circulate in different geographic regions worldwide and include important human pathogens. The Japanese encephalitis serogroup includes Japanese encephalitis virus (JEV), the leading cause of viral encephalitis among children in Southeast Asia, and West Nile virus (WNV), which causes neuroinvasive disease in adults in temperate regions 1. A live-attenuated JEV vaccine, SA14-14-2,

has been licensed in China, but currently, there is no licensed WNV vaccine selleck for humans 2. The flavivirus genome encodes three structural (C, prM, envelope (E)) and seven nonstructural genes (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Both the humoral and cellular arms of the immune system are vital to protect mice from JEV and WNV encephalitis 3–6. Protective CD8+ and CD4+ T-cell epitopes residing in the WNV NS4b and NS3 proteins, respectively, play an important antiviral role through cytokine production and cytotoxic activity 7–9. Heterologous immunity to related or unrelated viral pathogens induces protection or immunopathology upon a secondary viral challenge due to cross-reactive memory CD8+ T-cell responses 10, 11.

3a). Because SOCS-1 is expressed in microglia, acting as a negative regulator of several inflammatory pathways triggered by cytokines and LPS, we investigated the contribution of miR-155 to the regulation of SOCS-1 expression in these cells. A recent study, using a luciferase reporter assay, has provided functional evidence that miR-155 is able to bind to the 3′UTR of SOCS-1 mRNA in HEK293T cells.27

Using a similar assay, which comprises the co-transfection of pmiR-155 and a plasmid encoding both the luciferase gene and the 3′UTR sequence of SOCS-1 (pSOCS-1 3′UTR), followed by the evaluation of luciferase activity 48 hr after transfection, we were also able to validate miR-155 binding to the untranslated repeat of this protein in N9 cells (Fig. 3b). With this experiment, it was possible to observe the expected Fluorouracil manufacturer increase in luciferase activity following the delivery of both pSOCS-1 3′UTR and the pGFP plasmids. However, delivery of pmiR-155 in addition to pSOCS-1 3′UTR resulted

in reduced luciferase activity levels, which were significantly lower than those obtained following transfection https://www.selleckchem.com/products/wnt-c59-c59.html with the control plasmid (pGFP) and pSOCS-1 3′UTR. These results indicate that, similar to what was reported in HEK293T cells, miR-155 expression in N9 cells is able to block luciferase expression through binding to the 3′UTR sequence of SOCS-1, which precedes the luciferase gene. The miR-155–mRNA pairing leads to post-transcription repression

or mRNA degradation, decreasing luciferase expression and hence luciferase activity, so validating SOCS-1 as a target of miR-155. Aiming at ascertaining a possible temporal relation between miR-155 and SOCS-1 expression levels, we performed a qRT-PCR time–course study to identify changes in SOCS-1 levels following microglia incubation with LPS (0·1 μg/ml). The results displayed in Fig. 3(c) show that following 2 hr of incubation with LPS, SOCS-1 mRNA levels present a sharp increase of fivefold, but decrease afterwards, approaching only a twofold increase after 4 hr of incubation and reaching basal levels at 18 hr. These results correlate temporally with those shown in Fig. 1(c) and support the hypothesis that miR-155 may contribute directly to the observed decrease in SOCS-1 levels by targeting SOCS-1 mRNA. To confirm this possibility Non-specific serine/threonine protein kinase we determined whether over-expression or inhibition of miR-155 would lead to significant changes in SOCS-1 mRNA and protein levels. For this purpose, N9 microglia cells were transfected with a plasmid encoding miR-155 (p155) or with anti-miR-155 oligonucleotides, which bind with high affinity to miR-155 and avoid miRNA–target mRNA interactions. N9 cells were exposed 24 hr later to LPS (0·1 μg/ml). A non-inhibitory oligonucleotide (control oligonucleotide) and a plasmid encoding GFP (pGFP) were used as negative controls, to detect possible transfection-related unspecific changes in SOCS-1.