The virus was prevalent in more than 213 countries and caused IWR-1 in vivo at least 16,931 deaths (3). In Japan, the virus was first detected on 8 May 2009 at Narita airport in returnees from Canada; more than 20,000 confirmed cases and at least 99 deaths had been reported by 16 May 2010 (4). Although the A(H1N1)pdm09 has moderate virulence, it has mostly infected the young, especially those of school-age (5) and disproportionately impacted them (6). Pandemic (H1N1) 2009 virus is a novel reassortant, containing the PB1, PB2, PA, HA, NP and NS gene segments from North American triple-reassortant

swine viruses, and the NA and M gene segments from the Eurasian swine lineage (7). The latest whole-genome phylogenetic analysis of A(H1N1)pdm09 has shown

that at least seven different clades of viruses have been circulating globally (8). In this report, we analyzed the HA genes of 70 strains isolated from a single university population in order to describe the phylogenetic relationships of the strains circulating in the university. Nasal swab specimens from 71 patients, most of whom were current students and a few of Ivacaftor mouse whom were trainee doctors, were collected in the Dental and Medical Clinic attached to Health Sciences University of Hokkaido at Tobetsu in neighboring Sapporo. The Tobetsu campus, which is attended by approximately 2,500 students, consists of three faculties; Pharmaceutical Sciences, Dentistry, and Nursing and Social Services. Most of the students live in Tobetsu or commute from Sapporo on the same train line. The collected specimens were kept in a sample medium of DMEM supplemented with 1% BSA and 50 μg/mL gentamicin. The specimens were collected between 3 September and 15 December 2009, during which time the school was closed twice (from 21 to 23 October and 10 to 13 November) due to influenza epidemics (Fig. 1). All study patients tested positive for type A influenza by Meloxicam a rapid

test for influenza A and B viruses. The study was approved by the ethics committee of the Health Sciences University of Hokkaido and all patients gave informed consent. Madin-Darby canine kidney cells were cultured in DMEM supplemented with 10% FBS and 50 μg/mL gentamicin. The sample medium containing the specimen was clarified by centrifugation and inoculated into a monolayer of MDCK cells. After 1hr incubation at 35°C, the medium was removed and the cells were incubated in DMEM supplemented with 1% BSA, 50 μg/mL gentamicin and 5 μg/mL trypsin at 35°C for 2–3 days. When extensive cytopathic effects had been observed, the supernatants were collected, clarified and passaged once in MDCK cells. The HA genes of influenza A virus were amplified and analyzed as previously described (9). Briefly, 10 mL of the culture fluid of virus-infected cells was ultracentrifuged and the pellet was used for RNA extraction.

© 2011

Wiley-Liss, Inc. Microsurgery, 2011. “
“Perforator flaps as an innovative method for soft tissue transfer that maximizes Fludarabine function preservation, were originally introduced primarily as free flaps. Their reliability and versatility has been found to not differ from other sources of free flaps where total failure is an uncommon event. Partial failure should also be recognized as a possible dilemma that is perhaps more of a unique untoward sequela of perforator flaps. A retrospective review of our flap experience over the past decade included 310 perforator free flaps. Partial perforator flap failure that required a second free flap for salvage was selected in 6 patients. All perforator free flaps in our experience that had some form of partial failure were anterolateral thigh [ALT] free flaps. Clinically initially unrecognizable but ultimately distal flap ischemia could be attributed to poor flap design, and was the cause of immediate partial flap necrosis in 2 cases. Delayed difficulties were complications not specific to perforator flaps. In all cases, a free flap was considered the best option, and a second perforator free flap proved to resolve all reconstructive

objectives. The root cause of partial failure of a perforator free flap was found to be either iatrogenic or de novo in origin. The proper design requires an awareness of the correct topographic axis and an understanding of the perforasome concept to better insure adequate flap perfusion. If a free flap is still find more considered the best solution after a partial failure, the advantages and benefit of a second perforator free flap should not be overlooked. © 2013 Wiley Periodicals, Inc. Microsurgery 34:177–182, Sodium butyrate 2014. “
“Ultrasound (US) has been used in the management of carpal tunnel syndrome since the 1980s. The first report of US-guided carpal tunnel release (CTR) was published in 1997, with cadaver and clinical reports confirming the safe navigation of surgical tools

with US for division of the transverse carpal ligament. The MANOS CTR device was recently reported as a minimally invasive tool for CTR, and may be well suited for use with US guidance. The authors report three cases of US-guided CTR using the MANOS CTR device. The MANOS device was inserted in a blunt configuration into the safe zone, and the cutting surface was deployed with a thumb-activated trigger that simultaneously ejected a sharp through the palm. The transverse carpal ligament was divided safely and confirmed with US. US allowed for clear identification of the median nerve, safe zones, transverse carpal ligament, and the MANOS CTR device in relation to all pertinent structures of the carpal tunnel. Complete division of the transverse carpal ligament was confirmed in all three cases.

Nuclear extracts from Jurkat cells were used as negative control, and nuclear extracts from

Raji cells included in the kit and from MoT cells served as positive controls in the assay. In order to address the potential cytotoxic effects of signaling pathway pyrrolidine dithiocarbamate (PDTC) on mononuclear cells, experiments were performed treating PBMCs with PDTC for 1 h at three different concentrations (1 μM, 10 μM, 30 μM) or left them untreated, then washed three times with RPMIc. After 3 h in culture, cell viability was measured by the trypan blue exclusion method. PDTC-treated cells were also subjected to apoptosis determination by fluorescence activated cell sorter (FACS) using the annexin-V/7-aminoactinomycin D (7AAD) kit (BD Bioscience Pharmingen). More than 95% viable cells were determined in trypan blue exclusion assay for PBMCs treated with PDTC under these concentrations. In addition, the PDTC agent did not affect the viability of the cells as assessed by annexin-V and 7AAD staining (data not shown), and therefore pretreatment of PBMCs was performed with 30 μM of PDTC. To examine the role of NF-κB in Tax-mediated CC-chemokine secretion, PBMCs were pretreated with 30 μM of PDTC, a potent inhibitor of NF-κB, for 1 h then washed three times with RPMIc, followed by

treatment with Tax proteins (100 pM) for 3 h, shown to be the optimal time-point to assess levels of CC-chemokines in Tax-treated PBMCs (Fig. 1). In other experiments, see more PBMCs were transduced with the NF-κB super-repressor (NF-κB/SR) at an MOI of 25 using lipofectamine plus reagent (Invitrogen) for 20 h prior to Tax protein treatment (3 h). PBMCs were also co-transduced with NF-κB/SR and Ad-Tax2 or Ad-GFP. Cell-free supernatants were harvested after 24 h of incubation and assayed for MIP-1α, MIP-1β and RANTES expression, as described above. All statistical analyses were performed using GraphPad Prism version 6·00 for Windows (GraphPad

GNA12 Software http://www.graphpad.com) and the data expressed as mean ± standard error of the mean. One-way analysis of variance (anova) with Bonferroni’s multiple post-test comparison were used to evaluate three or more groups. Statistical comparisons for two groups were assessed by two samples assuming equal variances Student’s t-test. P-values <0·05 were considered statistically significant. We have reported recently that extracellular Tax2 and Tax1 proteins induced high levels of CC-chemokines in mononuclear cells [24, 25]. The optimal dose of protein required to detect CC-chemokine secretion was determined previously by exposing PBMCs to increased concentrations of Tax proteins [24]; the concentration of 100 pM was optimal, and therefore used in all subsequent experiments. In order to determine the time of MIP-1α, MIP-1β and RANTES release, PBMCs were treated once with Tax2A (subtype A), Tax1 or mock-treated control and then cell-free supernatants were harvested after 1, 2, 3, 6, 12 or 24 h of incubation.

In MS, the precise distribution of different laminin isoforms is reported to be important for integrin-mediated leucocyte extravasation to the active lesion, where ‘perivascular cuffs’ of inflammatory infiltrates

specifically associate with patches of laminin α4 but not laminin α5 expression [347,348]. In the chronic lesion, increased perivascular expression of fibrillar collagens (types I, III and V) and the SLRPs decorin and biglycan was suggested to reduce monocytic expression of the leucocyte attractant chemokine CCL2 (MCP1) [349]. Similarly to the approaches discussed earlier with regards to traumatic CNS injury, manipulating the https://www.selleckchem.com/autophagy.html ECM therefore a represents a potential therapeutic strategy to overcome www.selleckchem.com/products/abc294640.html demyelination (recently reviewed in [350]). Indeed, reduction of CSPG synthesis using xyloside, in vivo, was shown to increase OPC and oligodendrocyte numbers in lesions and improve remyelination in a lysolecithin murine model [351]. Thus, there is promise for future studies to apply ECM modification strategies to models

of MS and it will be of great interest to determine whether these strategies can improve disease pathology and lead to functional repair. The ECM plays a critical role during development and following disease or injury to the CNS. Rather than mere provision of a supportive

environment, the ECM is actively involved in many fundamental processes such as cell signalling, axon guidance and synaptic plasticity. Following disease or damage to the CNS, the composition Enzalutamide chemical structure of the ECM can prove detrimental to axonal regeneration, plasticity and repair. Manipulating the ECM represents a powerful therapeutic approach, with the aim of recapitulating beneficial processes that occur during development and/or reducing negative remodelling after injury, either by targeting specific ECM components or by global targeting of families of ECM molecules. There is now much pre-clinical evidence to suggest that beneficial outcomes can be achieved following traumatic brain and spinal cord injury with therapies involving matrix manipulation and encouragingly, some of these strategies are progressing closer to clinical application. We may only be beginning to understand the complexities of ECM interactions in neurodegenerative disorders but it appears that manipulations of the ECM may well have wide applications in future strategies to promote repair following CNS injury or disease. “
“F. Mori, K. Tanji, Y. Miki, A. Kakita, H. Takahashi and K.

In this present study, we characterise the global transcriptional signatures at this time point in ovine afferent lymph cells as they migrate from the injection site into the lymphatics following vaccination with a liposome antigen formulation incorporating CpG. We show that at 72h post vaccination,

liposomes alone find more induce no changes in gene expression and inflammatory profiles within afferent lymph; however the incorporation of CpG drives interferon, antiviral and cytotoxic gene programs. This study also measures the expression of key genes within individual cell types in afferent lymph. Antiviral gene signatures are most prominent in lymphocytes, which may play a significant and unexpected role in sustaining the immune response to vaccination at the site of injection. These findings provide a comprehensive analysis of the in vivo immunological pathways that connect the injection site with the local draining lymph node following vaccination.

This article is protected by copyright. All rights reserved. “
“IFN-α/β link innate and adaptive immune responses by directly acting on naïve CD8+ T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8+CD45RO− cells were stimulated with beads coated with anti-CD3 and anti-CD28 mAb, mimicking Ag (type-1) and selleck screening library co-stimulatory (type-2) signals, in the presence or absence of IFN-α and their transcriptional profiles were defined by cDNA-microarrays. We show that IFN-α provides a strong third signal directly to human CD8+ T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated

at the protein level and verified by functional assays. Interestingly, the biological effects derived from next this stimulation vary depending on the CD8+ T-cell population. Thus, whereas IFN-α increases the proliferative capacity of naïve CD8+ T cells, it inhibits or does not affect the proliferation of Ag-experienced cells, such as memory and effector CTL, including CMV-specific lymphocytes. Cytolysis and IFN-γ-secretion of all these populations are enhanced by IFN-α-derived signals, which are critical in naïve CD8+ T cells for acquisition of effector functions. Our findings in human CD8+ T cells are informative to understand and improve IFN-α-based therapies for viral and malignant diseases. Type I IFN (IFN-I) comprises a cytokine family that in humans includes 13 IFN-α subtypes and single proteins for IFN-β, IFN-ε, IFN-κ and IFN-ω 1. IFN-α/β are produced in response to viruses and are critical for viral defense. IFN-I signals through a common receptor (IFNAR) composed of two subunits, IFNAR1 and IFNAR2 2. The JAK-STAT pathway is critical for IFNAR signaling 3.

Depletion of HIV-specific CD8+ IL-10+ cells from PBMCs led to upregulation of CD38 on CD14+ monocytes together

with increased IL-6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL-10+ population and was also induced by exposure of monocytes to HIV-1 in vitro. Production https://www.selleckchem.com/products/ink128.html of IL-10 by HIV-specific CD8+ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection. Interleukin-10 (IL-10) plays a critical role in limiting proinflammatory immune responses that might otherwise cause damage to the host. During infection, the timing and cellular source of IL-10 production PCI-32765 ic50 are essential to the balance between successful pathogen clearance by innate and adaptive responses and the prevention of immune pathology. Mistimed or excessive IL-10 production can interfere with elimination or control of various bacteria, viruses, and protozoa [1]. For example, in the murine lymphocytic choriomeningitis virus model, blockade of IL-10 signalling resulted in clearance of a chronic viral infection by host and vaccine-induced cell-mediated immune responses [2, 3]. It was noted nearly two decades

ago that IL-10 is upregulated from an early stage of HIV-1 infection and this was proposed to underlie Th cell dysfunction [4, 5]. More recent studies reporting enhancement of HIV-specific effector T-cell responses following in vitro depletion of virus-specific IL-10-producing ‘suppressor’ cells or antibody-mediated blockade of IL-10

support this notion [6, 7]. However, IL-10 gene transcription is upregulated in multiple cell types in the peripheral blood during chronic HIV-1 infection [7]. Whether the reported immune suppressive effects are limited to a specific cell subset is unresolved [8]. This is of critical importance for the development of new therapeutic interventions aiming to ameliorate CD8+ and CD4+ T-cell dysfunction in chronic viral infections including HIV-1. An additional consideration learn more is that IL-10 induction in HIV-1 infection may protect the host from excessive immune activation, since diverse pathogens that cause chronic infections drive the expansion of IL-10-producing adaptive or induced T regulatory (Treg) cells in the periphery [9-11]. In support of this notion, rapid induction of strong Treg-cell responses, together with TGF-β and IL-10, was observed in primary SIV infection of African green monkeys, which is typically nonpathogenic, while these responses were delayed in pathogenic SIV infection in macaques [12]. Furthermore, the presence of an IL-10 promoter polymorphism conferring increased cytokine expression was associated with delayed CD4+ T-cell decline in HIV-1 infection [13].

On day 6, fresh medium containing GM-CSF, IL-4, IL-1β, IL-6,

PGE2, and TNF was added to the culture. After additional 48 h of culture, nonadherent cells were harvested and used as APCs. find more Purified CD4+, CD8+ and DN T cells (1×105/well) from donor A were cocultured with allogeneic mature DC (2.5×104/well) from donor B or with anti-CD3/CD28-coated beads (2.5×104/well; Dynabeads CD3/CD28, Invitrogen) in 96-well U-bottom plates in complete medium supplemented with 3% TCGF. T cells were restimulated weekly with fresh allogeneic DC. Viability and purity of the T cells were monitored by flow cytometry. Further purification via magnetic separation was performed if purity decreased to lower than 95%. T cells were used for functional assays 6 days after last stimulation. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ, anti-CD4, anti-CD8, anti-TCR-γδ, phycoerythrin (PE)-conjugated anti-CD25, anti-CD45RO, anti-TCR-, and allophycocyanin-conjugated anti-CD38, anti-CD45RA, anti-CTLA4 monoclonal antibodies (mAb) (all from BD Biosciences, Heidelberg, Germany). Isotype control mAb, FITC-labeled annexin V, and 7AAD were purchased from BD. Foxp3 stains were performed

with allophycocyanin-conjugated anti-Foxp3 mAb and the respective control from eBioscience (San Diego, USA). For intracellular IFN-γ staining, activated CD4+ T cells were cocultured with DC and DN T cells in the presence BGB324 of monensin (GolgiStop, BD) for 5 h. After washing, cells were stained for surface markers, fixed and permeabilized (Cytofix/Cytoperm kit, BD), and then stained for intracellular cytokines. Flow cytometry was performed on a FACSCanto II (BD); cell sorting was accomplished on a MoFlo (Beckman Coulter). Acyl CoA dehydrogenase Data were analyzed with FlowJo software (Treestar, Ashland, OR, USA). CFSE (Sigma, Munich, Germany) labeled CD4+ and CD8+ T cells (5×104/well) from donor A were stimulated in 96-well U-bottom plates with allogeneic DC (2.5×104/well) from donor B, anti-CD3/CD28

beads (2.5×104/well, Invitrogen/Dynal, Oslo, Norway), or plate-bound anti-CD3 (0.25 μg/well, Orthoclone OKT3, Janssen-Cilag) in complete medium in the presence or absence of DN T cells or CD4+CD25+ Tregs (5×104/well). Anti-CD2/CD3/CD28 loaded particles (Treg Suppression Inspector, Miltenyi Biotec) were used according to the manufactures instructions. After 5–6 days of culture, cells were harvested and stained with anti-CD4, anti-CD8, anti-TCR-αβ, and anti-CD25 mAb. Proliferation of cells was determined by flow cytometry. For blocking experiments, mAb to IL-10 (10 μg/mL JES3-19F1; BD), TGF-β (10 μg/mL 1D11; R&D Systems), Fas (10 μg/mL ZB4; Biomol), or isotype-matched controls were added to the MLR. To block TCR-signaling and protein translocation, DN T cells were incubated with Lck-inhibitor II (100 μM; Calbiochem, Darmstadt, Germany) or with monensin (GolgiStop, according to the manufacture’s protocol; BD) for 3 h, and then used as suppressor cells in the MLR.

003, Wilcoxon-test). Male-target cells pulsed with the control-peptide I540S did not influence T cell reactivity compared with naïve cells (I540S: 12–29/100,000; median: 23; P < 0.106 to P < 0.066). In vivo-primed female T cells recognized peptide-loaded T2-cells (W248: 85 ± 28/100,000 T cells; T368: 35 ± 12/100,000; K1234: 50 ± 17/100,000) being UTY-specific as indicated by Anti-MHC-I-antibody-blockage

(W248: 30 ± 10/100,000 T cells; T368: 26 ± 9/100,000; K1234: 10 ± 3/100,000; P < 0.026 to P < 0.018, Wilcoxon-test). In contrast, T2-cells alone or loaded with the I540S-control-peptide demonstrated only low unspecific-reactions (20–1/100,000 T cells, median: 9; P < 0.113 to P < 0.018, Wilcoxon-test). According to Cobimetinib the in vitro experiments (Table 2, Fig. 3) in vivo primed female T cells mostly reacted with male-BM (<45 specific-spots/100,000 CD3+T cells)

followed by monocytes (<29 spots) and PBMCs (<15 spots) and in vivo immunogenicity of the hUTY-peptides was comparable with those in vitro: W248 exhibited the most immunogenic potential on T2-cells (85 spots/100,000 T cells > K1234 (50 spots) >T368 (35 spots)). We provide evidence that hUTY-derived male-peptides specifically expand T lymphocytes derived from female-DLA-identical-dogs either using autologous-peptide-pulsed-female DCs as APC in vitro or male-DLA-identical PBMCs in vivo. The expanded female T cells recognized HLA-A2-binding hUTY-derived endogenously presented peptides W248, K1234 and T368 only on BGB324 in vivo DLA-identical

male-cells (mostly BM) representing a male-specific restriction. Thereby, W248 appeared to be the most immunogenic-peptide. Importantly, no response against autologous- and female-DLA-identical cells, not expressing the male-specific-UTY antigen, was detected. Therefore, we conclude that the mHA UTY is very homologous Cell press in male-humans and dogs, and the canine-system could serve as a large-animal model to study T cell applications in terms of immunotherapeutic approaches after alloSCT in male patients with female donors. Consequently hUTY-(especially W248)-pulsed female DCs might be used in male hematopoietic-SCT recipients with female stem-cell donors [3, 6, 7]. CD8+T cell-proliferation was induced up to 3-fold within 3–4 weeks (Fig. 1). After in vitro stimulation expanded CD8+T cells specifically reacted against the hUTY-derived peptides presented on autologous-female DCs in up to 3.1% of all T cells (IFN-γ-ELISPOT assay, Fig. 2), but not against autologous-naïve DCs and monocytes. This proves that HLA-A2-restricted peptides selected from human-UTY protein bind to canine-DLA-identical molecule(s), and these peptides are immunogenic in dogs and can induce UTY-specific T cell reactivity. Detected amounts of reactive-UTY-specific CD8+T cells after in vitro culture with IFN-γ-ELISPOT and [51Cr]-release-assays were comparable. This is in accordance with findings by others, although both the assays address different CTL-mechanisms [41].

g. reactive oxygen species) and non-oxidative (e.g. various proteases) mechanisms.[17] The importance of neutrophil function is evident in individuals who have defects in neutrophil chemotaxis,

phagocytic functions or who have neutropenia.[18, 19] These individuals are more prone to bacterial infections. On the other hand, microbicidal molecules released from activated and dying neutrophils can cause bystander damage selleck screening library to healthy tissue. The consequent cell injury and death can itself cause or aggravate disease. Accordingly, it is important to elucidate the factors controlling neutrophilic inflammation. In this study we describe the surprising finding that the gut flora influences the ability of animals to mount a systemic acute neutrophilic inflammatory response

in the peritoneum and characterize the underlying basis for this observation. Specific pathogen free (SPF) C57BL/6 mice and IL-1R−/− mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Germ-free C57BL/6 selleck products mice were obtained from The National Gnotobiotic Rodent Resource Center, North Carolina State University Gnotobiotics Unit and Gnotobiotic Research Resource, Medical University of South Carolina. MyD88−/− mice were provided by Dr Shizuo Akira, Osaka University, Osaka, Japan or purchased from The Jackson Laboratories. RIP2−/− mice were provided by Dr Michelle Kelliher and RIG-I−/− and MDA5−/− mice were provided by Dr Kate Fitzgerald (University of Massachusetts Medical School, Worcester, MA). NOD1−/− mice were Acesulfame Potassium provided by Dr Grace Chen, University of Michigan, Ann Arbor, MI. For generating the tamoxifen-inducible deletion mutant mice of MyD88, we used a strategy similar to the one described

previously.[20] MyD88−/− mice were crossed to the whole tissue, tamoxifen-inducible Cre transgenic mice (Rosa26-Cre/ESR+/+) (provided by Dr Roger Davis, University of Massachusetts Medical School, Worcester, MA). The resultant offspring, MyD88+/− Rosa26-Cre/ESR+/− mice were crossed to the MyD88flox/flox mice (provided by Dr Robert Finberg, University of Massachusetts Medical School, Worcester, MA) to generate the MyD88−/flox Rosa26-Cre/ESR+/− (conditional knockout; cKO). Animals were housed and handled according to protocols approved by the University of Massachusetts animal care and use committee. Mice were injected intraperitoneally with 0·2 mg zymosan (Sigma-Aldrich, St Louis, MO), 0·5 mg silica crystals (Sigma-Aldrich), 0·5 mg monosodium urate crystals or 5 ng recombinant murine MIP-2 (R&D Systems, Minneapolis, MN) in 0·2 ml PBS. For the thioglycollate injections, 1 ml of 3% thioglycollate (Thermoscientific, Lenexa, KS) was used. The monosodium urate crystals were prepared as described before.[21] Mice were killed by exposure to isoflourane 4–16 hr after the injection. The peritoneum was lavaged with 2 ml Dulbecco’s modified Eagle’s medium with 2% fetal calf serum, 3 mm EDTA and 10 U/ml heparin.

We believe that our present experimental observations further support a possible benefit of MZR in the treatment of lupus nephritis. Poly IC was from Sigma (St. Louis, MO, USA). Primer oligo(dT)12–18, dNTP mix, and Moloney murine leukemia virus (MMLV) reverse transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). SsoFast EvaGreen

Supermix was from Bio-Rad (Hercules, CA, USA). Oligonucleotide primers for polymerase chain reaction (PCR) were custom synthesized by Greiner Japan (Atsugi, Japan). Enzyme-linked immunosorbent assay (ELISA) kits for MCP-1, CCL5, fractalkine and IL-8 were from R&D Systems (Minneapolis, MN, USA). Dexamethasone (DEX) was from Roche Diagnostics buy PI3K Inhibitor Library (Basel, Switzerland). MZR was from Asahi Kasei Pharma Corporation (Tokyo, Japan). Tacrolimus (Tac) was from Astellas Pharma Corporation (Tokyo, Japan).

Normal human mesangial cells (MCs) were purchased from Lonza (Walkersville, MD, USA), and the cells were cultured according to the manufacturer’s protocol.[13-17] Poly IC was dissolved in phosphate-buffered saline (PBS) and the cells were treated with 2–50 μg/mL poly IC for up to 48 h.[13-17] In the experiments using immunosuppressive reagents, the see more cells were pretreated, with 1–100 μg/mL MZR, 10 μM DEX, or 5 μg/mL Tac, 1 h before the treatment with 30 μg/mL poly IC. We have already confirmed that viability of cells was not affected by the treatment of these reagents (not shown). To examine the effect of MZR in

more detail in this setting, the cells at the time of 16 h after the stimulation with 30 μg/mL poly IC were post-treated with 100 μg/mL of MZR for 24 h. Total RNA was extracted from cells using RNeasy RNA extraction kit. Single-strand cDNA was synthesized from 1 μg of total RNA using oligo(dT)12–18 primer and MMLV reverse transcriptase. The cDNA for MCP-1, CCL5, fractalkine, IL-8, or glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was amplified using SsoFast EvaGreen Supermix, as reported previously.[13-17] The primers were custom-synthesized by Greiner Japan (Atsugi, Japan), and the sequences of the primers were as follows: MCP-1: -forward, 5′-AAACTGAAGCTCGCACTCTCGC−3′, reverse, RG7420 in vitro 5′-ATTCTTGGGTTGTTGAGTGAGT−3′; CCL5: -forward, 5′-CTACTCGGGAGGCTAAGGCAGGAA−3′, reverse, 5′-GAGGGGTTGAGACGGCGGAAGC−3′; fractalkine: -forward, 5′-GACCCCTAAGGCTGAGGAAC-3′, reverse, 5′-CTCTCCTGCCATCTTTCGAG-3′; IL-8: -forward, 5′-AGGAGTGCTAAAGAACTTCGA−3′, reverse, 5′-TGAATTCTCAGCCCTCTTCAA-3′, and GAPDH: -forward, 5′-GCACCGTCAAGGCTGAGAAC−3′, reverse, 5′-ATGGTGGTGAAGACGCCAGT−3′. Each sample was run in triplicate. The concentration of MCP-1, CCL5, fractalkine and IL-8 in cell-conditioned medium was measured in triplicate in each, using an ELISA kit according to the manufacturer’s protocol. Statistical significance was evaluated using the paired t-test.