The impact of this antiseptic following such exposure on CSH of C. dubliniensis isolates has not been investigated. Hence, the main objective of this study was to investigate the effect of brief exposure to sub-therapeutic concentrations of chlorhexidine gluconate on the CSH of RO4929097 nmr C. dubliniensis isolates. Twelve oral isolates of C. dubliniensis were briefly exposed to three sub-therapeutic concentrations

of 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate for 30 min. Following subsequent removal of the drug, the CSH of the isolates was determined by a biphasic aqueous-hydrocarbon assay. Compared with the controls, exposure to 0.005% and 0.0025% chlorhexidine gluconate suppressed the relative CSH of the total sample tested by 44.49% (P < 0.001) and 21.82% (P < 0.018), respectively, with all isolates being significantly affected. Although exposure JQ1 to 0.00125% of chlorhexidine gluconate did not elicit a significant suppression on the total sample tested (7.01%; P > 0.05), four isolates of the

group were significantly affected. These findings imply that exposure to sub-therapeutic concentrations of chlorhexidine gluconate may suppress CSH of C. dublinienis isolates, thereby reducing its pathogenicity and highlights further the pharmacodynamics of chlorhexidine gluconate. “
“Photodynamic therapy is a treatment that combines the use of three non-toxic components, viz. photosensitiser, light and oxygen to cause localised oxidative photodamage. In the present study, the antifungal effect of the photosensitiser, BAM-SiPc, an unsymmetrical bisamino phthalocyanine, was investigated. BAM-SiPc was effective in photo-inactivating Candida albicans in a dose-dependent manner. The cell viability as determined by the clonogenic assay was reduced to c. 10% at 0.02 μmol l−1 BAM-SiPc with a total fluence of 12 J cm−2 at a cell density of 107 cells ml−1. A short incubation time of 5–15 min was sufficient to allow the photosensitiser

to exert its optimal antifungal PRKACG activity. Microscopical analysis showed that BAM-SiPc was effectively internalised by the fungal cells. Photodynamic treatment led to an increase in the intracellular reactive oxygen species level and disturbed the membrane integrity of the fungal cells. “
“Candidiosis is a mycosis that is currently increasingly affecting the population in consequence of its frequency and the severity of its complications, especially among immunocompromised hosts. In this work, the in vitro anticandidal activities of two phenothiazines (PTZs), chlorpromazine (CPZ) and trifluoperazine (TFP), and their combinations with amphotericin B (AMB) were tested against 12 different Candida strains representing 12 species (Candida albicans, Candida glabrata, Candida guillermondii, Candida inconspicua, Candida krusei, Candida lusitaniae, Candida lypolitica, Candida norvegica, Candida parapsilosis, Candida pulcherrima, Candida tropicalis and Candida zeylanoides).

1). We found that 104 was the optimal number of pmel-1 spleen cells that could be mixed with 107 WT spleen cells. Compared with WT spleen cells, donor spleen cells from IL-15 KO mice has a significantly less suppressive effect on the primary response of pmel-1 T cells to peptide-pulsed Torin 1 DC than spleen cells from WT mice (Supporting Information Fig. 2).

The suppression mediated by co-transfer of WT spleen cells was even more dramatic when the secondary response of pmel-1 T cells to DC vaccination was measured. Surprisingly, the co-transfer of spleen cells from IL-15 KO mice did not suppress but increased the secondary response of pmel-1 T cells. IL-15 KO mice are known to have deficient numbers of CD122+CD8+ memory-like https://www.selleckchem.com/products/nivolumab.html (sometimes referred to as “memory-phenotype” or “innate”) T cells, NK, and NKT cells, but have sufficient numbers of CD25+CD4+ Treg (see review 11, and Supporting Information Fig. 2), suggesting that lymphocytes other than CD25+CD4+ Treg played the key suppressive role in our model. Consistent with this notion, CD122+CD8+ memory-like cells constituted the major population of lymphocytes that underwent lymphopenia-driven proliferation when adoptively transferred into sub-lethally irradiated mice (Supporting Information

Fig. 3). To substantiate our initial observations and determine the effect of CD122 depletion on the therapeutic efficacy of adoptive T-cell therapy in lymphopenic

mice, we treated mice bearing BCKDHB 6-day subcutaneous F10 tumors with irradiation, followed by adoptive transfer of 104 pmel-1 spleen cells and 107 congenic spleen cells with or without prior depletion of CD122+ cells, and vaccination with peptide-pulsed DC. The absolute numbers of pmel-1, congenic, and host T cells in the blood were enumerated at different intervals after vaccination. We found that depletion of CD122+ cells doubled the number of pmel-1 T cells found in the blood of vaccinated mice 2 wk after vaccination (Fig. 1A), and there was no recovery of congenic T cells when CD122+ cells were depleted (Fig. 1B). CD122+ lymphocytes rather than CD122− cells were the primary lymphocyte subpopulation that underwent lymphopenia-driven proliferation. In contrast, host T-cell recovery, which is reflected by the thymic output of naïve T cells, did not differ in recipients of CD122-depleted and non-depleted T cells. Most importantly, depletion of CD122+ lymphocytes resulted in a greater antitumor efficacy (Fig. 1C and D). Depletion of CD122+ cells from congenic donor spleen cells led to a significantly longer delay of tumor growth and an increase in median survival of tumor-bearing mice (from 38 days to 58 days).

Statistical analysis.  Genotype frequencies Napabucasin cell line were determined by direct counting of the individual positive for a particular KIR phenotype specificity. Chi square was used to test for statistical significance of the genotypes or haplotypes between the patients and controls. P values < 0.05 were regarded as statistically significant. The strength of association was estimated by calculating the odds ratio (OR) and 95% confidence interval (95% CI). Statistical analysis was carried out using the spss 13.0 software package (IBM Corporation, West Harrison, NY, USA). All the tested KIR genes were present in different frequencies in control

and patient groups in this study. Framework genes KIR2DL4, KIR3DL2, KIR3DL3 and KIR3DP1 were present in all individuals. All KIR genotypes and haplotypes were determined in this study according to the model described by Hsu et al. [4]. In this study, we found 25 genotypes, including 11 new genotypes of NF1∼NF11, which had not been observed in Caucasians so far [4]. Among these genotypes, 21 were determined in healthy controls, and 22 in patients with syphilis (Table 2). In healthy controls, three genotypes with higher frequency in

rank order were AJ (34.90%), P (14.06%) and AH (10.42%). In patients AZD4547 mw with syphilis, the genotypes AJ (28.95%), AH (14.2%) and AF (10.00%) were three higher genotypes. Of interesting, the frequencies of genotype AE and AG were higher in patients with syphilis than those in healthy controls (P = 0.020 and P = 0.041, respectively), while the frequency of genotype P was lower in patients with syphilis than that in healthy controls (P = 0.002) and its OR was 0.304. The other KIR genotypes did not show significantly different distribution in the two groups. According to previous description [4], the genotypes P and AE contain combinations of haplotype 2 and 17 and haplotype 1 and 6, respectively, while genotype AG contains combination of homozygous haplotype 1. Next, we reanalysed the distribution of KIR haplotype in both patients

with syphilis and controls. In this study, all the 25 genotypes could be resolved into corresponding pairs of haplotypes as shown in Table 3. Both healthy controls and patients with syphilis had 17 different haplotypes. Haplotype 2 was the most frequent, followed by haplotype 1 and 5 in the two groups. Interestingly, the frequencies of haplotype PAK6 1 and 6 were lower in healthy controls than those in patients with syphilis, while the frequency of haplotype 17 was higher in healthy controls compared with that in patients with syphilis, and its OR was 0.321. The other KIR haplotypes did not show significantly different distribution in the two groups. All haplotypes mentioned earlier belong to either haplotype A or haplotype B. The frequencies of haplotype A and B were shown in Table 4. The frequency of haplotype A was higher than that of haplotype B in both healthy controls and patients with syphilis.

However, recent reports have described a protective role of IL-17A in IBD 21–23. In this regard, it is of interest that the lck-DPP2

kd mice showed no signs of IBD (results not shown). In summary, the data presented here on the activation phenotype of T cells from lck-DPP2 kd mice point to a model in which DPP2 lifts the threshold of T-cell activation, preventing spontaneous cell division. Upon knock down Angiogenesis chemical of DPP2, cells may drift into early G1 of the cell cycle and may proliferate faster upon stimulation, because they have an advantage by being poised to enter S phase sooner. This would provide an explanation for the hyper-proliferative behavior of DPP2 kd T cells upon stimulation. Activated DPP2 kd CD4+ cells differentiate into Th17 cells through a default pathway bypassing the required cytokines, IL-6, IL-1 and/or

TGF-β, for Th17-cell differentiation. Interestingly, DPP2 kd CD8+ T cells also generate increased amounts of IL-17A, PD98059 datasheet suggesting that IL-17 production is the default pathway for all T cells. In the presence of DPP2, exogenous factors are required to overcome this threshold of activation, allowing differentiation into effector cells. Collectively, these results imply that DPP2 is an essential protease that is intricately involved in the G0/G1 transition in T cells, preventing their differentiation into IL-17-producing effector cells. The shRNAs against mouse DPP2 were generated using the pSicoOligomaker1.5, which can be found at http://web.mit.edu/jacks-lab/protocols/pSico.html, and were verified on the Dharmacon Web site http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx. The selected oligos were cloned in pSicoR and pSico vectors 24, according to the protocol described on the Tyler Jacks Web site. Double-stranded RNA was synthesized by Dharmacon (Lafayette, CO). All DNA sequencing was done at the Tufts University Core Facility. shRNA sequences that had the most significant kd of mouse Lck DPP2 measured by qRT-PCR was selected to

infect 129/SVEV ES cells (♯CMT1-1, Chemicon). The empty lentiviral vector was used as a control. Sense strand against mouse DPP2 (shDPP2): 5′-TGG TTC CTA GTG TCA GAT AA-3. Lentiviruses were generated essentially as described in 41. Briefly, 10 μg of lentiviral vector and 4 μg of each packaging vector were cotransfected in 293T cells by using the calcium phosphate method (Current Protocols in Molecular Biology). Supernatants were collected 36–40 h after transfection, filtered through a 0.45-μm filter, followed by centrifugation of the viral supernatant at 25 000 rpm in a Bechman SW28 rotor for 1.5 h to concentrate the virus. The viral pellet was resuspended in 200 μL ES cell media and added to 10 000–20 000 ES cells that were plated on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) and incubated for 6 h at 37°C.

pylori detected in the stomach.

The challenge procedure is relatively well established in the literature, but its efficiency varies at different institutions and is mainly dependent on the infecting strain utilized. In our laboratory, the H. pylori challenge has been effective in inducing infection in ∼80% of mice. Infected mice tended to have either a high number (∼1 × 104) of H. pylori copies μg−1 DNA – which likely indicates no protection as that was the level shown by unvaccinated mice – or a low number (∼1 × 102.5× 104) GSK1120212 of H. pylori copies μg−1 DNA – which likely indicates partial protection. The challenge method utilizes a high dose (1 × 109) of H. pylori organisms over a brief period, which is unlike natural human infection that occurs through exposure to low levels of H. pylori over a prolonged period. This artificial way of find more infection may partially explain why some properly immunized mice missed protection. We could not find a good serological correlate of protection. Even though as a group, those with the highest serum IgG and IgA had the lowest geometric mean H. pylori copies μg−1 DNA, the correlation

was very poor at the individual level (r2=0.3037 and 0.0577 for IgG and IgA, respectively). This finding suggests that serum antibodies are markers of immune response but, by themselves, play a limited role in protection, and that other arms of the immune system (innate, cellular, mucosal) are more important. Unfortunately, in this set of experiments, we could not detect any stool antibodies. We expressed the level of infection as the number of H. pylori copies μg−1 purified DNA. This is unconventional as most studies express the level of infection as H. pylori copies mg−1 Uroporphyrinogen III synthase of stomach. We decided to use DNA as the denominator because our detection method was based on PCR

of purified DNA and the purification efficiency may have varied for each specimen. Indeed, even though there was a good correlation between the weight of the stomach and the amount of DNA purified, it was less than perfect (r2=0.59). So that our results can be compared with the ones reported in other studies, in our experiments, on average, 3.4 H. pylori copies μg−1 DNA corresponded to 1 copy mg−1 stomach. In conclusion, our study adds to the evidence that rUreB is a promising H. pylori vaccine candidate, that aluminum hydroxide has a significant but modest adjuvant effect and that better adjuvants must be pursued. This work was partially funded by NIH grant R03CA128048. The authors have no competing interests. “
“Mammalian TLRs in adult animals serve indispensable functions in establishing innate and adaptive immunity and contributing to the homeostasis of surrounding tissues. However, the expression and function of TLRs during mammalian embryonic development has not been studied so far. Here, we show that CD45+ CD11b+ F4/80+ macrophages from 10.5-day embryo (E10.5) co-express TLRs and CD14.

The increased expression of suppressors of cytokine signaling (SOCS) proteins in periodontitis was recently reported [[45]]. Both SOCS-1 and SOCS-3 are able to inhibit MxA expression [[46]]. In conclusion, this study demonstrates that α-defensins, antimicrobial peptides constitutively expressed in healthy periodontal tissue, induce expression of a classical antiviral protein, MxA, in gingival epithelium. Strong MxA activity at the strategic gingival sulcus, in close proximity to microbial

plaque, may serve as one of the important innate tools in maintaining periodontal homeostasis. We believe that our findings warrant further research into the physiological role of α-defensin-induced MxA in the antiviral response of the periodontal tissue. Antimicrobial peptides: human α-defensin-1, -2, and -3, human β-defensin-1, -2, and -3, and LL-37 Afatinib manufacturer were obtained from Innovagen (Lund, Sweden). IFN-α and neutralizing antibodies against IFN-α and IFN-β were

purchased from PBL Biomedical Laboratory (Piscataway, NJ, USA). Neutralizing antibody against α-defensins was obtained from Hycult biotech (Uden, The Netherlands). Polymorphprep was purchased from Axis-Shield PoC AS (Oslo, Norway). Tissue specimens were collected from patients (one biopsy SCH727965 research buy per one patient) at Periodontal Clinic and Department of Oral Maxillofacial Surgery, Faculty of Dentistry, Chulalongkorn University. The ethical approval by the ethics committee of Faculty of Dentistry, Chulalongkorn University and informed consent of all participating

subjects were obtained before operation. Healthy periodontal tissue samples were collected from sites with clinically healthy gingiva (no gingival inflammation, probing depth < 4 mm, and no radiographic bone loss) during crown-lengthening procedure for prosthetic reasons. Severe periodontitis tissue samples were collected from sites of extracted teeth with hopeless prognosis (inflamed gingiva, probing depth > 6 mm, and bone loss Racecadotril > 60% of the root). Periodontal tissue specimens used for immunostaining, real-time quantitative RT-PCR, and in vitro cultures were derived from different donors. The primary HGECs, derived from healthy periodontal tissue, were obtained following established procedure [[9]]. In brief, the excised tissues were immediately washed with Dulbecco’s phosphate buffered saline and digested in 0.2% dispase for 24 h at 4°C. The separated epithelial layer was washed, minced, and cultured in a serum-free keratinocyte growth medium (Clonetics, Walkersville, MD, USA) supplemented with human recombinant epidermal growth factor, hydrocortisone, bovine insulin, bovine pituitary extract, gentamicin sulfate, amphotericin B, and 0.15 mM CaCl2. The HGEC cultures at passage two to four were used throughout the study. Total RNA from periodontal tissue samples and HGECs were isolated by using an RNeasy Mini kit from Qiagen (Hilden, Germany).

The purpose of this study is to

examine the fine specificity of autoantibodies targeting MPO. This continuing effort could define epitopes that have pathogenic implications, provide insight into the initiation of this autoimmune response and identify potential therapeutic targets. The Oklahoma Clinical Immunology Serum Repository (Oklahoma City, OK, USA) contains more than 120 000 coded samples from 70 000 individuals. Sixty-eight samples from patients that tested positive for p-ANCA, and had adequate sera stored within the repository, were obtained for further analysis. Frequency matched healthy controls were selected to run in parallel experiments. This work was conducted with appropriate Institutional Review Board approval from the Oklahoma Medical Research Foundation and the University of Oklahoma Health Sciences Center (OUHSC). Patient Selumetinib sera were tested for ANCA using indirect immunofluorescence (IIF) following the protocol provided by the manufacturer (Inova Diagnostics, Inc., San Diego, CA, USA). Patient samples with a positive p-ANCA buy NVP-BGJ398 titre by IIF were also tested for MPO antibodies by enzyme-linked immunosorbent assay (ELISA) from the same manufacturer to verify the presence of antibodies to myeloperoxidase. The published sequence of MPO (Accession number: PO5164) was used to construct 369 decapeptides of the 745 amino acid protein overlapping by eight amino acids. The peptides were synthesized on the ends of

polyethylene pins using f-moc side-chain protection chemistry and arranged in the format of 96-well microtitre plates (Chiron Mimotopes Pty Ltd, Dimethyl sulfoxide Clayton, Victoria, Australia), as described previously [8,9]. Positive control peptides were synthesized on each plate using a peptide with known positive reactivity by a patient serum

sample. Solid-phase peptides were then tested for antibody reactivity using a modified enzyme-linked immunosorbent assay (ELISA) procedure described previously in detail [8,9]. Assay steps were executed by lowering the pins into microtitre plate wells and incubations were carried out in sealed plastic containers. The peptides were blocked in a 3% low-fat milk phosphate-buffered saline (PBS) solution and then incubated with sera containing primary antibodies. The solid-phase supports were washed with PBS with 0·05% Tween and then incubated with anti-human immunoglobulin (Ig)G as a secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Following another wash period, the peptides were incubated in a para-nitrophenyl phosphate solution in order to induce a colour change if an antibody–peptide interaction was present. The colour change was measured using a micro-ELISA plate reader (Dynex Technologies, Chantilly, VA, USA) at 410 nm and the absorbance values were recorded. Positive controls were developed and normalized to an optical density (OD) of 1·0 to standardize results across plates and assays.

Data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 as compared to control. Figure S3. (A) Fleshly isolated CD4+CD25- and CD4+CD25+ T cells were stimulated with anti-CD3 mAb (0.5 ìg / mL) and IL-12 receptor (IL-12R) ® chain expression was analyzed with flowcytometry. Bold line : CD4+CD25- T cells, Thin line: CD4+CD25+ T cells, filled grey : isotype control. (B) Expression level of IL-12R®2

after siRNA treatment was confirmed. 1 × 106 siRNA transfected or untreated CD4+CD25- T cells were cultured with 1 × 105 irradiated autologous CD4-depleted PBMCs and anti-CD3 mAb. Three days later, cells were harvested and RNA was extracted to confirm knockdown of IL-12R®2 expression by real-time RT-PCR. The relative differences in gene expression were calculated using threshold

cycle (Ct) values that were normalized to those of Trichostatin A TATA-box-binding protein gene, and compared with the relative Ct value of untreated CD4+CD25- T cells by the 2-ddCt. (C)) 1 × 104 CD4+CD25- T cells with/without siRNA treatment were cultured with 1 × 105 irradiated autologous CD4-depleted PBMCs and anti-CD3 mAb in the presence or absence of 5 × 103 CD4+CD25high Tregs with OK-432 (1 ìg / mL). Proliferation was evaluated as described in Materials and Methods. These experiments were performed independently at least twice with similar results. Data are expressed as mean ± SD. “
“Citation Noronha LE, Antczak DF. Maternal immune responses to trophoblast: www.selleckchem.com/products/VX-809.html the contribution of the horse to pregnancy immunology. Am J Reprod Immunol 2010 The horse has proven to be a distinctively informative species in the study of pregnancy immunology for several reasons. First, unique aspects of the anatomy and physiology of the equine conceptus facilitate approaches that are not possible in other model organisms, such as non-surgical Sorafenib chemical structure recovery of early stage embryos and conceptuses and isolation of pure trophoblast cell populations. Second, pregnant mares make strong cytotoxic antibody responses to paternal major histocompatibility complex class I antigens expressed by the chorionic girdle cells, permitting detailed evaluation of the antigenicity of

these invasive trophoblasts and how they affect the maternal immune system. Third, there is abundant evidence for local maternal cellular immune responses to the invading trophoblasts in the pregnant mare. The survival of the equine fetus in the face of strong maternal immune responses highlights the complex immunoregulatory mechanisms that result in materno–fetal tolerance. Finally, the parallels between human and horse trophoblast cell types, their gene expression, and function make the study of equine pregnancy highly relevant to human health. Here, we review the most pertinent aspects of equine reproductive immunology and how studies of the pregnant mare have contributed to our understanding of maternal acceptance of the allogeneic fetus.

[21-23] To date the endogenous and microbial antigens are weaker activators of iNKT cells, and it is possible learn more that lipids as potent as synthetic

αGalCer do not occur in a physiological setting. In addition to recognition of lipids on CD1d through their TCR (Signal 1), iNKT cells can be activated by co-stimulatory signals. However, the co-stimulatory signals for iNKT cells are most often cytokines like IL-12 and IL-18, and these cytokines co-stimulate iNKT cells in many important physiological examples of iNKT cell activation.[24, 25] Unlike naive adaptive MHC class I and class II restricted T cells, iNKT cells display an effector/memory phenotype and are poised for rapid effector function at steady state.[26] Their rapid response, lack of memory and expression of NK receptors have led to them being considered “innate” T cells. Invariant NKT Veliparib cells characteristically express high levels of the BTB–POZ-ZF family [broad complex, tramtrack, bric-a‘-brac (BTB) or poxvirus and zinc finger (POZ)-zinc finger] transcription factor promyelocytic leukaemia zinc finger (PLZF) encoded by Zbtb16.[27, 28] PLZF is also expressed by human mucosal-associated invariant T cells, which are another population of invariant T cells, as well a subset of γδ T cells. PLZF is thought to control the innate phenotype and rapid cytokine response of these

and forced expression of PLZF on CD4 T cells induced an innate-like iNKT cell phenotype.[28] Known functions of iNKT cells are diverse because of their striking ability to kill targets and also produce both T helper type 1 (Th1) and Th2 cytokines upon

activation.[29, 30] A major function of iNKT cells is in transactivating other immune cells through their rapid cytokine production. Therefore they can both kick-start an immune response, and skew the type of response, Orotic acid as well as regulate homeostasis of other cell types. As well as cytokine production, iNKT cells, or at least a subset of iNKT cells, have cytotoxic activity. Indeed, one of the first functions reported for iNKT cells was cytotoxicity again tumour cells. In a B16 model of melanoma with liver metastasis, αGalCer administration completely protected wild-type mice from tumour development, but mice lacking iNKT cells had no protection,[31] suggesting that activation of iNKT cells led to their potent cytotoxicity against tumour cells. However, as their role in transactivating other immune cells, like natural killer (NK) cells, through IL-2 or interferon-γ (IFN-γ) production became accepted, it is thought that tumour protection induced by αGalCer could be due to subsequent NK cell activation and cytotoxicity. This scenario seems likely to occur, but in addition, iNKT cells themselves have cytotoxic activity and can also kill tumour cells that express CD1d, but not CD1d-negative tumour cells.

Co-transfection experiments designed to validate the miR-155 binding site present in the 3′UTR of SOCS-1 were also performed using FA-associated liposomes. Two hundred microlitres of FA-lipoplexes, containing pmiR-155 and a plasmid encoding the reporter gene luciferase and the 3′UTR of SOCS-1 (pSOCS-1 3′UTR) were delivered to N9 cells to obtain a final plasmid concentration Palbociclib of 1 μg/well for each plasmid. In parallel experiments, plasmid (p) GFP was used in addition to pSOCS-1

3′UTR to serve as a control. In all transfection protocols, after 4 hr of incubation, the medium was replaced by new RPMI-1640 medium and N9 microglia cells were incubated for different periods of time, before further analysis. Luciferase expression following

co-transfection of pSOCS-1 3′UTR and pmiR-155 or pGFP was evaluated by assessing luciferase activity. Briefly, 48 hr after transfection, cells were washed twice with PBS and 100 μl lysis buffer (1 mm dithiothreitol, 1 mm EDTA, 25 mm Tris–phosphate, 8 mm MgCl2, 15% glycerol, 1% [volume/volume (v/v)] Triton X-100, pH 7·8) were added to each well. After cell lysis at Selleckchem AZD6244 −80°, 50 μl of each lysate were incubated with luciferin and ATP and light production was determined in a luminometer (Lmax II384; Molecular Devices, San Jose, CA). The protein content of the lysates was evaluated through the DC Protein Assay reagent (Bio-Rad, Hercules, CA), using BSA as the standard.

Data were expressed as relative light units of luciferase per mg of total cell protein and presented as fold change with respect to control (untransfected cells). Total RNA, including small RNA species, was extracted from N9 microglia cells or primary microglia cultures using the miRCURY™ Isolation Kit – Cells (Exiqon), according to the manufacturer’s recommendations for cultured cells. Briefly, after cell lysis, the total RNA was adsorbed to a silica matrix, washed with the recommended buffers and eluted with 35 μl RNase-free water by centrifugation. After RNA quantification, cDNA conversion for miRNA quantification was performed Thiamet G using the Universal cDNA Synthesis Kit (Exiqon). For each sample, cDNA for miRNA detection was produced from 20 ng total RNA according to the following protocol: 60 min at 42° followed by heat-inactivation of the reverse transcriptase for 5 min at 95°. The cDNA was diluted 80 × with RNase-free water before quantification by qRT-PCR. Synthesis of cDNA for mRNA quantification was performed using the iScript cDNA Synthesis Kit (Bio-Rad) and employing 1 μg total RNA for each reaction, by applying the following protocol: 5 min at 25°, 30 min at 42° and 5 min at 85°. Finally, the cDNA was diluted 1 : 4 with RNase free water. Quantitative PCR was performed in an iQ5 thermocycler (Bio-Rad) using 96-well microtitre plates.