e. exchange of oxygen and carbon dioxide.[25] Bellanger et al. [25] studied the interaction of alveolar epithelial cell lines with various antigenic sources including L. corymbifera by measuring the amount of IL-8

and IL-13, inflammatory and allergic cytokines, respectively. In their study, L. corymbifera was the only microorganism with increased up-regulation of IL-8 and IL-13 after 8 h of exposure in epithelial cells. This strongly indicates the possibility of L. corymbifera playing a crucial role in development of FLD. Generally fungi are considered as the most common microbes encountered by mammalian Tyrosine Kinase Inhibitor Library hosts. Fungi accounts for up to 4–11% of fine particle mass in urban and rural air.[26, 27] Fröhlich-Nowoisky et al. [27] stated that in their investigated air, nearly all detected fungal species were Basidiomycota (64%) or Ascomycota (34%), and with 2% from the Zygomycota. Mucorales are airborne and common inhabitant of soil. Therefore, it is agreeable that the route of entry to the host of the fungi is mainly via the respiratory tract. However, the infection does not occur as frequently despite of ubiquity of the fungal nature. Thanks to our efficient immunity, we have many different barriers

against the fungal invasion. Our immunity Dinaciclib supplier is comprised of two types; innate and adaptive. First one gives more rapid response compared to the later one. In this review, innate immune system will be scrutinised along with the cases of zygomycetes. The innate immune system allows immediate defence against foreign molecules such as pathogens. This system consists of various cellular components such as granulocytes, macrophages, mast cells, dendritic cells (DCs) and natural killer (NK) cells and soluble factors like complement proteins leading to clearance of pathogen, in this case, zygomycetes by phagocytic cells. The key players of the innate immune system participating in fungal invasion are illustrated in Fig. 3. According to many studies, innate immunity plays

a crucial role in mucormycosis by suppressing spore germination and/or hyphal growth. This statement is well met by high susceptibility 4-Aminobutyrate aminotransferase to mucormycosis among diabetic patients as they found to have altered or dysfunctional innate immunity.[7] A few studies were engaged in the comparison between zygomycetes with Aspergillus fumigatus, which is the most causative agent of mycoses. One of the reasons why cases of mucormycosis were less reported than those caused by A. fumigatus might be due to the size of the spores. Clearly, A. fumigatus spores are less in size than Mucorales and this by itself is likely to aid A. fumigatus spores to more easily deposited in the alveolar space when compared to the spores of the Mucorales, which are up to 6 times larger than A. fumigatus (average spore size 2–3 μm). Neutrophils are most abundant type of leucocytes in blood.

Furthermore, testing of sera with individual peptides of each protein showed that rabbit antibodies recognized several linear epitopes that were scattered throughout the sequence of each protein. Interestingly, in previous studies using pools of synthetic peptides, all of these proteins have been shown as major T cell antigens in humans, and the linear T cell epitopes of Rv3874 and Rv3875 were found scattered throughout the sequence https://www.selleckchem.com/products/AP24534.html of these proteins [10, 11]. A further

analysis of the sequence of each protein for B cell epitope prediction using ABCPred Prediction server, which is based on artificial neural network [37], also showed that B cell epitopes are scattered throughout the sequence of each protein (Fig. 5A, B and C for Rv3874, Rv3875 and Rv3619c, respectively).

Thus, both prediction and experimental results for B cell epitopes confirm the strong immunogenicity of Rv3874, Rv3875 and Rv3619c proteins for inducing polyclonal and antigen-specific antibody AZD5363 in vivo reactivity in rabbits. In conclusion, the present study shows that pGES-TH-1 vector is useful in obtaining highly purified recombinant preparations of Rv3874, Rv3875 and Rv3619c proteins of M. tuberculosis. All of these recombinant proteins were immunogenic in rabbits, and antibody epitopes were scattered throughout the sequence of each protein. These results suggest that pGES-TH-1 vector could be useful in obtaining pure recombinant proteins, predicted to be encoded by hypothetical genes present in M. tuberculosis-specific genomic regions, for their immunological characterization. This work was supported by the Research Administration Grant YM 01/03 and the College of Graduate Studies, Kuwait University, Kuwait. We are thankful to Prof. Suhail Ahmed for providing pGES-TH-1 vector. Rabbits were immunized and handled according to established IACUC-approved protocols

at Kuwait University, Kuwait. “
“Sarcoidosis is an inflammatory disease. Epidemiological and treatment studies suggest that fungi play a part in the pathogenesis. The aim of this work was to study Terminal deoxynucleotidyl transferase the effect of fungal cell wall agents (FCWA) on the in vitro secretion of cytokines from peripheral blood monocytes from subjects with sarcoidosis and relate the results to fungal exposure at home and clinical findings. Subjects with sarcoidosis (n = 22) and controls (n = 20) participated. Peripheral blood mononuclear cells were stimulated with soluble or particulate β-glucan (S-glucan, P-glucan), chitin or lipopolysaccharide (LPS), whereafter tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 were measured. The severity of sarcoidosis was determined using a chest X-ray-based score. Serum cytokines (IL-2R, IL-6, IL-10 and IL-12) were determined. To measure domestic fungal exposure, air in the bedrooms was sampled on filters.

2 mm) was significantly higher in non-responder group (p = 0.038). Among 70 patients in 2nd study population, 45 patients were responder (64.2%), and the proportion of patients who had larger parathyroid glands than cutoff value was significantly higher in nonresponder group (responsder vs nonresponder 60.5 vs 87.0%, p = 0.028). Conclusions: Measurement of parathyroid gland diameters with CT scan was useful to predict the response of cinacalcet therapy. KURASHIGE MAHIRO1,2, HANAOKA KAZUSHIGE1, IMAMURA MINAKO2, UDAGAWA TAKASHI1, KAWAGUCHI YOSHINDO1,3, HASEGAWA TOSHIO1,3, HOSOYA TATSUO1, YOKOO TAKASHI1, MAEDA

SHIRO2 1Division of Kidney and Hypertension, Department of Internal Medicine, The Jikei University, School of Medicine, Minato, Tokyo, Japan; 2Laboratory for Endocrinology, Metabolism and MAPK Inhibitor Library Kidney Diseases, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan; 3Department of Medicine, learn more Kanagawa Prefectural Shiomidai Hospital, Yokohama, Kanagawa, Japan Introduction: Autosomal

Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary kidney disorder, and most of its heritability could be explained by mutations in two genes, PKD1 and PKD2 in populations of European descent. However little is known about Asian ADPKD including Japanese. To elucidate the genotypic and phenotypic characteristics of ADPKD in Japanese populations, we performed a comprehensive search for mutations in PKD1 and PKD2 in 180 Japanese ADPKD patients from 161 unrelated Cepharanthine families. Methods: We screened the entire coding regions and their flanking regions of the PKD1/PKD2 by direct sequencing, and evaluated candidates for causal variants by subsequent in-silico and/or bio-analyses. We also searched for large genomic rearrangements within PKD1/PKD2 loci by using quantitative PCR. Results: We identified 111 mutations within 134 families (detection rate = 83.2%), including 88 PKD1 mutations (48 truncating, 6 atypical splice, 29 missense and 5 in-frame mutations) in 96 families, and 23 PKD2 mutations (18 truncating, 1

atypical splice and 3 missense mutations and 1 large deletion) in 38 families. Patients with PKD2 mutations account for 23.6% of all Japanese ADPKD families in this study. Seventy-four out of the 111 mutations have not been reported previously. The estimated glomerular filtration rate (eGFR) decline was significantly faster in patients with PKD1 mutations than in those with PKD2 mutations (−3.25 and −2.08 ml·min−1·year−1 for PKD1 and PKD2, respectively, p < 0.01). Conclusion: Mutations within PKD1 and PKD2 can be linked to most of the cases of Japanese ADPKD, and the renal function decline was faster in patients with PKD1 mutations than in those with PKD2 mutations also in the Japanese ADPKD. We also found that PKD2 mutations were more frequent in Japanese ADPKD than that in European or American ADPKD.

The plateau seems to depend on the local, non-neurally mediated release of nitric oxide (NO), because it is suppressed by inhibitors of NO synthase [11,12,16] and insensitive to local anesthesia [16]. In contrast, the early peak shows little dependence on NO, and is largely mediated by the stimulation of nociceptive C-fibers that trigger vasodilation through an axon reflex [13]. Accordingly, it is diminished by local anesthesia [7,16,21]. In short, the prevailing view [15] is that the early part of thermal hyperemia is due to the transient

activation of an axon reflex, which progressively gives way, as heating is pursued, to a non-neural, NO-dependent mechanism. Thermal hyperemia can easily be DAPT price recorded in the skin in a non-invasive fashion, using laser-Doppler flowmetry to evaluate SkBF. Indeed, thermal hyperemia has been proposed as a test of microvascular function. This test has been used to document microvascular Selleckchem Erastin dysfunction in diabetes [1,22,23] and other conditions [14,19]. In a previous study, we found that the repeat application of a local thermal stimulus on the same skin patch was associated with a reduction in the elicited vasodilatory response,

a phenomenon hereafter termed desensitization [3]. This result is of some practical importance, for example, if thermal hyperemia is to be used as an end point in acute interventional trials. However, other groups [4,20] found no evidence for desensitization, when recording two thermal hyperemia either one or two hours apart on the same skin site, as we had done. The aim of this study was to understand the reasons for

this apparent discrepancy and, more specifically, to test whether it was related to differences in instrumentation. We had measured SkBF with laser-Doppler imaging (LDI) at a wavelength of 633 nm [3], whereas the cited studies used single-point laser-Doppler flowmetry (LDF) at 780 nm [4,20]. In comparison with 633 nm, the latter wavelength has greater skin penetration, and thus the potential to explore different vessels. In addition, the heating chambers used in our study were custom-made, as opposed to the commercial equipment employed by these other authors. We therefore set out to establish Protein Tyrosine Kinase inhibitor whether desensitization to thermal hyperemia occurred under four sets of conditions, i.e., measuring SkBF with LDI or LDF, and heating the skin with our custom-made or with commercially available chambers. Twenty-eight healthy male subjects, aged from 18 to 32 years, were included. They were all non-smokers, had no personal history of hypertension, diabetes, or hypercholesterolemia, and no dermographism. None took any drugs or reported being sick in the last 15 days before the start of the study. The volunteers were fully informed about the protocol, and gave their written informed consent.

[9, 10] It should, however, be noted that microglial activation is a continuum that depends on the stimulus encountered in their microenvironment.[11] It has been suggested that

under different pathological conditions, different stimuli act on different microglial receptors to orchestrate microglial 3-MA price response with a shift towards a more deleterious or a more neuroprotective phenotype.[12] The dynamic microglia interacts with different types of cells in the inflammatory environment, both of neural and immune origin. In particular, T cells, a component of the neuroinflammatory reaction in CNS diseases, can modulate microglial activation through secretion of pro-inflammatory and anti-inflammatory cytokines.[13] In this context, interferon-γ (IFN-γ) secreted by T helper type 1 T cells induces a classically activated phenotype in microglia upon binding to the IFN-γ receptors 1/2,[14] with up-regulation of MHC class II and selleck chemicals co-stimulatory molecules and enhancement of their function as antigen-presenting

cells,[13] possibly through microglia–T-cell cross-talk via the CD40–CD40 ligand interaction.[11] In contrast, low doses of IFN-γ or the anti-inflammatory cytokine IL-4, which is released by T helper type 2 cells, promote an alternatively activated profile with a release of neurotrophic factors.[15] In addition to Toll-like receptors (TLR) and other pattern recognition receptors through which they perceive, and react to, the presence of pathogens, microglia express a number of other receptors, whose up- or down-regulation depends on microglial activation status under pathological conditions. In vitro stimulation of mouse microglia with TLR agonists, including lipopolysaccharide (LPS) for TLR4 and CpG DNA for TLR9, leads to increased secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-12, as well as nitric oxide, that in turn Farnesyltransferase cause neuronal injury.[16] Recently, microRNA let-7 was shown to activate microglia, acting as a signalling activator of TLR7.[17] Activation of microglial TLR-signalling

pathway(s) plays a role also in non-infectious CNS diseases, as a response to endogenous danger signals.[16] For example, heat-shock protein 60 released from injured CNS cells binds microglia through TLR4 and triggers neuronal injury in a TLR4-dependent and myeloid differentiation factor 88-dependent manner, inducing release of neurotoxic nitric oxide from microglia.[18] Maintenance of the interaction between CD200 expressed on neurons and its receptor CD200R expressed on microglia is an off signal that is essential for preventing the expression of a classically activated microglial profile with over-activation of microglia and subsequent neurotoxicity.[19] Similarly, disruption of the CX3CL1–CX3CR1 interaction results in highly activated microglia with increased IL-1β production that may induce neurotoxicity.

6b(1)). The selected peptide–H-2Kb interface as the template from crystal structures is presented in Fig. 6b(2).50 NS2:114–121, GQ and FG

peptides are simulated with the same H-2Kb and TCR from the template crystal structure (Fig. 6b(3,4,5)). As the backbones of several H-2Kb-bound peptides adopt the same conformation, we have speculated on many features of the critical contact residues to be the main factors to affect specific recognition by TCR (Figs 6a(2),b). At the fifth anchor motif, substitution of phenylalanine (F) with glycine (G) could undermine the binding forces of GQ to H-2Kb because of the lack of an inward benzyl group without compromising the recognition of the outward side chain via TCR (Fig. 6b(3,4)). The substitution of glutamine (Q) with glycine (G) at the sixth TCR contact site has removed the outward amide side chain selleck chemicals from recognition by specific TCR (Fig. 6b (3,5)). Simulation results are compatible with those obtained

from laboratory experiments (Tables 2 and 3; Figs 2 and 5). The simulation approach with TCR contact information has more accurate prediction results on epitope identification than all previous computing programmes. Respiratory syncytial virus causes bronchiolitis and pneumonia in infants and young children.51 Influenza A virus still represents one of the major respiratory viruses causing significant morbidity and mortality in severe respiratory tract infections.52 Cobimetinib ic50 In the 1960s, the trials of formalin-inactivated vaccines not only failed to protect those people who were vaccinated from RSV infection but induced deviant pathological consequences.53 The lack of CD8 T-lymphocyte responses has been associated with pulmonary eosinophilia that was observed in vaccinated people or experimental animals.7,53,54 Antigenic drifts and heterotypic influenza A viruses continue to

cause annual epidemics and pandemic outbreaks.4,6 It is critical to identify the important elements constituting the epitope to enable CD8 T-lymphocyte recognition as well as to map mutant epitopes from mutable pathogens, either for experimental research or for immunoinformatical programmes. The role of anchor motifs Tau-protein kinase of peptides in the binding to MHC class I molecules is known and well-studied.19–22 Immunologists and microbiologists have long relied on these anchor motifs to predict MHC class I-restricted epitopes from the protein sequences of viral pathogens. Several peptide–MHC class I binding methods have been developed to map CD8 T-lymphocyte epitopes. Consistent with the previous publication of competitive binding experiments, M2:82–90 had the highest binding affinity to H-2Kd molecules to be detected by RMA-S-Kd cells22 (Figs 1a,c and Supplementary material, Fig. S2).

Several authors [2, 37, 38] described protective effect

of antibodies against experimental disseminated candidiasis in vivo. Prepared monoclonal antibodies showed enhanced ingestion and killing of yeast cells by PMN (MAb B6.1) or macrophages (MAb C7) in the presence of serum complement [37, 38]. They proposed that complement activation might contribute to the protection by antibodies in vivo and that during initiation of candidiasis protective antibodies induce prompt complement opsonization, which results into an association of Candida cells with host phagocytes. Non-protective antibodies may lead to reduced complement activation kinetics. According these results, we could assume enhanced candidacidal Sirolimus nmr activity induced by serum opsonization in vitro as a possible precondition for protection in vivo. Differences concerning the antibody quantity, specificity and isotype composition of polyclonal sera could explain why antibody protection against Candida infection has been observed in some studies but not in the others. Presented study indicates limited effectiveness of branched α-mannooligosides to induce production of highly protective antibodies. Additional and more detailed immunomodulatory properties

investigation of α-mannooligosides of different structure should bring significant information to successful protective anti-Candida subcellular vaccine development. This project was supported by grants from Grant Agency of Slovak Academy of Sciences VEGA No.

2/0026/13, by the Slovak Research MK-8669 cell line and Development Agency under the contract No. APVV- 0032-06. This contribution is the result of the project implementation: Centre of excellence for Glycomics, ITMS 26240120031, supported by the Research & Development Operational Programme funded by the ERDF. “
“Biological Research Department, Drug Discovery and Biomedical Technology Unit, Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan Germinal centers (GCs) are generally considered to be the sole site of memory B-cell generation. However, recent studies demonstrate that Montelukast Sodium memory B cells can also develop in response to a T-cell dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B-cell types requires classical T-cell help, the generation of GC-dependent memory B cells requires TFH-cell help, while the generation of GC-independent memory cells does not. These findings led to the conclusion that B-cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC-independent pathway of memory B-cell development, and discuss how the unique features of memory B cells are maintained in the GC-independent pathway.

[10] The discovery that the mechanism of action of FTY720 occurs via S1PR modulation[11] spurred interest in immunological functions of S1P signalling. Later studies demonstrated amelioration of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, with low-dose FTY720,[12] which has since been approved as a first-line oral agent for treatment of relapsing–remitting multiple sclerosis.[13-15] The pharmacology and biology of FTY720 are covered in great depth by other reviews.[16, 17] Studies to characterize the mechanisms underlying the induction of lymphopenia by FTY720 paved the

way to better this website understanding of the basic biological principles of lymphocyte circulation and revealed the importance of S1P1 in this process[4] (Fig. 1a). Using fetal liver from S1pr1−/− embryos to create bone marrow chimeric mice, Matloubian, et al. demonstrated that egress of lymphocytes from thymus and secondary lymphoid organs did not occur in the absence of S1P signalling, establishing

a requirement for S1P–S1P1 interaction in regulating lymphocyte egress. Additional MK-1775 molecular weight studies established that S1P1 expression was temporally regulated during T-cell development, culminating in high expression by mature single-positive CD4 or CD8 thymocytes and that conditional deletion of S1pr1 in T cells alone was sufficient to block their egress from the thymus. As S1P1 provides a critical chemotactic cue, and levels of S1P are high in the blood and lymph and low in most tissues,[7] it was postulated that this

‘S1P gradient’ would play a role in lymphocyte egress. Indeed, disruption of the S1P gradient by 2-acetyl-4-tetrahydroxyimidazole, an inhibitor of the S1P degradative enzyme S1P lyase, led to lymphopenia and blocked T-cell egress from the thymus.[18] This effect was mediated by increases in tissue concentrations of S1P and S1P-mediated down-regulation of surface S1P1, so impairing chemotactic responses.[18] Studies using conditional deletion of the S1P biosynthetic enzymes, sphingosine kinases 1 and 2 (Sphk1/2) demonstrated that an almost complete loss of S1P in the blood and lymph correlated with high cell surface expression Oxalosuccinic acid of S1P1 on naive T cells in the circulation. Lymphopenia was also evident, but infusion of S1P (in the form of S1P-producing erythrocytes) into sphingosine kinase-deficient mice, led to the release of lymphocytes into the blood concomitant with decreased cell surface expression of S1P1.[19] Mutant mice that express an internalization-defective S1P1 that is signalling competent have delayed lymphopenia kinetics in response to FTY720 or 2-acetyl-4-tetrahydroxyimidazole treatment, further supporting the premise that cell surface residency of S1P1 is a primary determinant of lymphocyte egress.[20] These observations combine to create a model whereby high concentrations of ligand lead to S1P1 surface down-regulation and so to non-responsiveness to S1P chemotactic cues.

Before performance of DGGE, the PCR products were analyzed by electrophoresis on a 1.7% agarose gel containing 0.5 μg/ml ethidium bromide to confirm

equal loading of the samples (data not shown). The conditions of DGGE and the visualization of the gels were the same as above. Phoretix 1D software package (Nonlinear Dynamics, Newcastle, United Kingdom) elimination followed by manual correction was performed to create a synthetic reference lane for each gel. Each lane on the gel was then compared to the reference lane, allowing generation of a matching profile for each lane (Fig. 2). UPGMA dendrograms were then used to generate the clustering patterns shown click here in Figure 2 (14). For a single sample, the number of bands on DGGE gel ranged from 23–47 for the V3-V5 region and 20–49 for the V6-V8 region without significant differences (P > 0.05), although there was a trend towards the average numbers in the V6-V8 region being higher than for the V3-V5 region (Fig. 3a). In samples from the same periodontal pockets, there were no significant differences in the number of bands at the baseline and 6 weeks after mechanical debridement in either www.selleckchem.com/products/PD-0332991.html the V3-V5 or V6-V8 regions (P > 0.05, Fig. 3a), suggesting that re-colonization of bacteria may indeed occur, as reported by Zijnge et

al. (7). These authors analyzed the Cediranib (AZD2171) DGGE fingerprints of the microbial population from four patients at baseline, one day after treatment and 3 months after treatment (7). They observed that two patients showed a pronounced decrease in the DGGE bands one day after treatment, but that by 3 months after treatment the number of the bands had increased back to the baseline level. In addition, in that report the Cs of the DGGE profiles of the four patients was 33–47% between baseline and 3 month after treatment. The Cs

of the DGGE profiles of the six patients in the present research was also calculated by the same method using the following equation: (7) DGGE analysis has been thought to be a good alternative in periodontal microbial diagnostics (7, 8, 14). However, the comparability of plaque bacterial DGGE patterns generated by different primer pairs remains unclear. To elucidate which region can best be used to characterize subgingival communities by DGGE, type strains of periodontal pathogens of P. gingivalis, F. nucleatium and P. nigrascens were used in the present study to generate 16S rDNA fragments of V3, V3-V5, and V6-V8 regions. From the present results, the authors speculate that the primer pairs of V3-s and V3-a, which target the DNA fragment in Escherichia coli 16S RNA between positions 341 to 534, may make it difficult to estimate the bacterial population, since multiple bands for single pathogenic bacteria appeared in the lanes.

coli serotype 055:B5, Sigma-Aldrich), lipoteichoic acid (LTA, Invivogen), flagellin (FLA-ST Ultrapure, Invivogen), CpG (ODN 2336, Invivogen), Polyinosinic-polycytidylic acid (Poly(I:C), Sigma-Aldrich), IFN-β (Invitrogen), R848 (Invivogen),

ssRNA40-LyoVec (Invivogen), Poly(I:C) high molecular weight (HMW, Invivogen), Poly(I:C)-LyoVec LMW (Invivogen), Poly(I:C)-LyoVec HMW (Invivogen), or combinations of ligands. Combinations of ligands were, unless described otherwise, added simultaneous. For LPS, an extra purification step was performed as described previously [[48]]. For determination of the viral titer, A549 cells (ATCC, CCL-185) were infected with RSV A2 for 24 h, trypsinized and fixed with 80% acetone. Cells were immunostained with FITC-conjugated mouse monoclonal antibody to RSV nucleoprotein (Abcam), followed by FACS analysis. Determination of the percentage of infection Selleckchem CAL 101 was repeated three times and the viral titer was calculated from the dilution at which 50% infection was seen. After 4 and 24 h, the supernatants were collected and stored at −20°C for cytokine measurement. The cells were resuspended in 150 μL RLT buffer with 1% β-mercaptoethanol and stored at −80°C for quantitative PCR. TNF-α, IL-1β, and IL-10 concentrations were measured in the cell supernatants by commercial ELISA kits (Pelikine

Compact, Selleckchem Y27632 Sanquin, Amsterdam, The Netherlands) according to the instructions of the manufacturer. TNF-α and IL-1β had a detection limit of 20 pg/mL, for IL-10 the detection limit was 7 pg/mL. Synergy was expressed as the ratio of cytokine response to

the combination of two ligands divided by the sum of cytokine responses obtained with both ligands alone; (virus + ligand)/((virus) + (ligand)). When cytokine response was as low as detection threshold for all individual ligands as well as the combination of ligands, we set the Transmembrane Transproters inhibitor ratio to 1 in order to prevent a false positive down regulation. Total RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany), genomic DNA was removed using TurboDNase (Ambion, Foster City, CA, USA) and cDNA was synthesized using SuperScripttm Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Quantitative PCR measurements for IFN-β (NM_002176.2), TNF-α (NM_000594.2), IL-1β (NM_000576.2), NOD2 (NM_022162.1), RIG-I (NM_014314.3), TLR3 (NM_003265.2), and GAPDH (NM_002046.3) were performed using commercially available Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, USA). The PCR conditions were as follows: initial denaturation for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1min at 60°C. Mean relative mRNA expression from two replicate measurements was normalized to GAPDH expression in each sample.