Finally, we questioned if the above changes in epitope-specific CTL would also be associated with changes in immunity to LCMV infections. We performed virus titration from the spleens at different days p.i. after initializing first cross-priming in the same protocol as shown in Fig. 7A. The data in Fig. 7B demonstrate that immunization with ADC encoding LCMV proteins caused significant reduction in virus titers 4 days p.i. with LCMV compared control mice. By day 7, this effect was unambiguous with no detectable virus in the cross-priming condition. These results suggest that initial cross-priming of LCMV proteins

prior to viral infection can enhance anti-viral immunity due to increased frequencies of CTL as shown in Fig. 7A. Induction of protective immune responses against viruses or tumors can be achieved via cross-priming 4–6. In this study, we employed the LCMV infection model OSI-906 ic50 to study how cross-presentation GSI-IX of multiple epitopes translates when studying cross-priming. We used LyUV treatment of the ADC because it fully inactivated the virus and allowed for efficient NP396 cross-presentation similar to HEK-NP cells 7, 8. We initially expected that GP33 would fail to cross-present because it is located in the signal peptide

of LCMV-GP 12, but it was cross-presented with low efficiency, probably due to its exceptional long half-life of 6 h 15. Although NP396, located in the long-lived nucleoprotein 21, was efficient at cross-presentation, NP205 was poor, possibly due to inefficient processing by the phagosomal/proteasomal machineries. GP276 is another epitope that cross-presented with low efficiency, probably due to its low binding affinity to MHC 22, which could be critical when the antigen supply is limited. Immunoproteasomes, which have been implicated in cross-presentation 23, could Interleukin-3 receptor also account for GP276 poor cross-presentation since it can downregulate GP276 presentation 24. We have

detected LMP7 expression in DC2.4 (unpublished data), which concurs with the previous data showing that immunoproteasomes are expressed in DC irrespective of their maturation states 25. Considering the direct presentation of the NP and GP-derived epitopes, it has been shown that NP epitopes can be detected as early as 2 h p.i., whereas GP epitopes were detected 4–6 h p.i. 20. Our results show that infected ADC can provide antigens for cross-presentation in a comparable time frame. Such kinetics would probably be dependent on the stress status of the infected cell 26 and would be sufficient to supply antigens for cross-presentation before the completion of the replication cycle. Thus far, one would expect that during cross-priming, NP396 will dominate the CTL response in vivo.

Without CD8 expression, only the two highest affinity TCRs (19LF6 and 16LD6) showed significant tetramer staining (Fig. 2B and Supporting Information Fig.1C and D). The co-expression of CD8 significantly enhanced the mean fluorescence intensity (MFI) of tetramer staining for all T cells (Fig. 2B and Supporting Information NVP-BGJ398 cell line Fig. 1C). The tetramer MFI increased

with the TCR affinity by SPR (Fig. 2C); the increase was most significant from the lowest to the second lowest affinity TCRs (W2C8 with a KD ∼100 μM and L2G2 with a KD ∼60 μM). This observation is similar to our previous study performed using primary mouse CD8+ cells [36] and to other studies [8]. Similar to 3D TCR affinity, tetramer staining had no statistically significant correlation with TCR function (R2 = 0.46, p = 0.14, Fig. 2D). Furthermore, the off-rates of tetramer dissociation from hybridoma cells measured by the tetramer decay assay [5, 24] (Supporting Information

Fig. 1D and E) did not correlate with TCR functional activity (R2 = 0.046, p = 0.68, Supporting Information Fig. 1F). A possible reason for the lack of correlation between 3D kinetic parameters measured by SPR and T-cell functional activities could be that the soluble αβ TCR in SPR measurement no longer connects with the cellular environment and hence misses its regulation or constraints [30]. Indeed, recent studies on several mouse TCR systems [26-28, 33] suggest that 2D TCR–pMHC kinetic measurements, which are performed in the native membrane environment, show better Dimethyl sulfoxide correlation with T-cell responsiveness. However, human Romidepsin self-antigen-specific TCR systems have not been investigated. Furthermore, the previous 2D TCR–pMHC kinetic measurements varied the pMHC as opposed to the TCR. Therefore, we asked whether 2D measurements would better correlate the kinetics with responsiveness in our

system. Using the micropipette adhesion frequency assay [37], we first measured the 2D TCR–pMHC interaction using CD8− hybridoma cells. Despite the slow 3D off-rates for some of the TCRs [36], the adhesion frequency (Pa) versus contact time (tc) curves had already reached plateaus at the shortest tc (0.1 s) for all six TCRs (Fig. 3A and Supporting Information Fig. 2A–E). The lack of a gradual transient phase in the binding curves indicates that the 2D off-rates are too fast to be measured by the micropipette system due to its limited temporal resolution (∼0.2 s). Using Eq. (1) (see Materials and methods), we calculated the effective affinities for the panel of TCRs from the plateau Pa levels (Fig. 3C). These 2D affinities showed a positive correlation (R2 = 0.75; p = 0.025) with, but a two-log broader range than their 3D counterparts (Supporting Information Fig. 3A). Because of the fast TCR–pMHC dissociation, we used the thermal fluctuation assay [38] to determine the off-rates (Supporting Information Fig. 4).

In subsequent experiments, after treatment with plain medium, empty vector, gC1qR vector, negative control siRNA vector or gC1qR siRNA vector for the indicated time periods, ROS generation was determined

using H2DCFDA fluorescence and quantified by flow cytometric analysis. The data showed that ROS generation reached a maximal level at 60 hr after the initial manipulation, and ROS levels in the gC1qR vector group were increased by approximately 3.18-fold compared with empty HM781-36B clinical trial vector-treated HTR-8/SVneo and HPT-8 cells (Fig. 3C). Cytosolic Ca2+ levels were determined using a fluorescent ELISA reader, and the results revealed a notable increase at 84 hr after the initial manipulation (Fig. 3D). At this time, the [Ca2+]i concentration

in the gC1qR vector group was 3.6-fold higher than that in the empty vector-treated HTR-8/SVneo and HPT-8 cells. The gC1qR siRNA vector group showed no changes compared with the negative siRNA vector-treated HTR-8/SVneo and HPT-8 cells. Time-dependent changes in relative Δψm values in gC1qR-overexpressing Selleckchem KU-60019 HTR-8/SVneo and HPT-8 cells were also explored. We used the JC-1 dye to monitor the estimated Δψm using the 590:527 nm emission ratio at specific time points from 0 hr to 84 hr following transfection. The value of Δψm in the gC1qR vector group decreased approximately 61.8% compared with the empty vector group at 84 hr. There was no difference in Δψm in HTR-8/SVneo and HPT-8 cells between the negative control siRNA and gC1qR siRNA groups after the initial manipulation (Fig. 3E). Additionally, we evaluated the production of ATP. As shown in Fig. 3F, the ATP concentration was notably decreased in cells transfected with the gC1qR vector compared with the empty vector group. In contrast, no significant change in the ATP concentration was observed in cells treated with the negative control siRNA and gC1qR siRNA

vectors (P > 0.05). To investigate whether the effects of the gC1qR gene on ROS generation and intracellular Ca2+ influx were interlinked, gC1qR vector-mediated gC1qR-overexpressing cells were treated either with the antioxidant Oxymatrine PDTC (25 μm) or with EGTA a Ca2+ ion chelator (30 μm). As shown in Fig. 4A, there was a twofold decrease in ROS generation in the presence of the Ca2+ ion chelator EGTA. Furthermore, the intracellular Ca2+ level was diminished by 68% after treatment with PDTC (Fig. 4B). The data indicated that inhibition of Ca2+ accumulation by EGTA diminished ROS generation. Similar results were also demonstrated in that blocking excess ROS generation with PDTC decreased the Ca2+ levels. Metformin can promote mitochondrial biosynthesis.

“
“Objectives: The current study was undertaken to characterize the binding of propiverine to muscarinic receptors in mouse tissues by measuring plasma concentrations of the drug

and its metabolite. Methods: At 0.5–24 h after the oral administration of propiverine at pharmacologically relevant doses, muscarinic receptors in tissue homogenates were measured by a radioligand www.selleckchem.com/products/fg-4592.html binding assay using [N-methyl- 3H]scopolamine (NMS), along with the drug’s concentration in plasma by the liquid chromatography-tandem mass spectrometric method. Results:In the in vitro experiments, propiverine and its metabolite 1-methy-4-piperidyl benzilate N-oxide competed with [3H]NMS for binding sites in

the bladder, submaxillary gland and heart of mice in a concentration-dependent manner. After the oral administration of propiverine, dose- and time-dependent increases in the dissociation constant for specific [3H]NMS binding were observed in the bladder and other tissues Hydroxychloroquine of mice, indicating that orally administered propiverine and/or its metabolite undergo significant binding to muscarinic receptors in mouse tissues. A longer-lasting binding of muscarinic receptor was seen in the bladder than in the submaxillary gland at relatively low doses of propiverine. Furthermore, the decrease in maximal number of binding sites values for [3H]NMS binding was more remarkable in the bladder than submaxillary gland of propiverine treated mice. There was a dose-dependent rise in the plasma concentrations of propiverine and 1-methy-4-piperidyl benzilate N-oxide in mice after the oral administration of propiverine. Conclusion: The oral

administration of propiverine exerts a more prominent and longer-lasting effect in the bladder than in the submaxillary gland Immune system of mice. The N-oxide metabolite may contribute significantly to the blockade of muscarinic receptors caused by oral propiverine. “
“Patients with lower urinary tract diseases often have a constellation of symptoms, and the degree of distress due to individual symptoms varies. In particular, some symptoms are more bothersome to patients and lead to treatment. However, traditional outcomes, such as urodynamic data, voiding diaries, and standardized patient-reported outcomes, may fail to address the individual factors. In contrast, patient-centered outcomes rely on patients to assess treatment outcomes in terms of their concerns or goals. Goal achievement is a patient-centered outcome that was pioneered in prolapse surgery. Recently, this most individualized outcome measure has been evaluated in the context of lower urinary tract symptoms (LUTS). According to the studies, most patients with LUTS have symptom-related goals.

baumannii expression properties that augment the organism’s ability to transition from exponential to stationary phase, as opposed to strain-dependent characteristics. Moreover, characterizing conserved biological processes may, in turn, provide rationale for developing strategies PI3K inhibitor for the therapeutic intervention of A. baumannii infections. Accordingly, each strain was cultured in LB medium, aliquots were removed during each growth phase, and RNA

was isolated and subjected to microarray analysis. The results presented here are refined to only those changes in gene expression that are conserved across both strains; individual strain expression properties are provided in Supporting Information, Table S1. Results revealed that the gene expression profiles of exponential- and stationary-phase A. baumannii differ dramatically and these differences are relatively well conserved

across the two strains studied. A total of 502 ORFs were determined to exhibit at least a twofold increase (t-test; P ≤ 0.05) in expression during exponential as opposed to stationary phase of growth regardless of the strain studied. Most of these genes belonged to distinct clusters of orthologous functional groups that are related to aspects of cell growth (Fig. 1). For instance, genes associated with amino acid metabolism (n = 43), translation (n = 93), cell wall/envelope selleck screening library biogenesis (n = 43), nucleotide transport (n = 28), transcription (n = 22), and replication (n = 21) were upregulated during exponential as opposed to stationary phase growth. Conversely, the mRNA levels of 175 genes were upregulated during stationary as opposed to exponential phase

in both strains. Of these, the majority were associated with energy production and conversion (n = 23), lipid transport all and metabolism (n = 15), and post-translational modification (n = 11). As described below, a more elaborate analysis of the data indicated that several genes that are likely to contribute to the organism’s ability to cause disease were found to be differentially expressed in a growth phase-dependent manner. Acinetobacter baumannii possesses the ability to survive on common hospital surfaces for weeks at a time, due in part to its ability to tolerate desiccation and form biofilms, subsequently providing a means for the organism to persist in the environment and act as a source for bacterial transmission to susceptible patients (Wendt et al., 1997; Jawad et al., 1998; Espinal et al., 2012). Our microarray data provided potential insight with regard to the biological systems that may contribute to the organism’s ability to form biofilms. More specifically, two members of the trehalose metabolic pathway, trehalose-6-phosphate synthase (A1S_0803), and trehalose-6-phosphate phosphatase (A1S_0804) were among the most highly upregulated stationary phase genes.

Intriguingly, the closest insect ortholog of the intracellular sensors RIG-I and MDA5 is Dicer-2 and virus infection in Drosophila initiates a specific transcriptional response, including the induction of the antiviral effector Vago, whose expression is dependent MK-2206 supplier upon Dicer-2 [32]. This suggests that Dicer-2-driven signaling contributes to the induction of a specific set of antiviral effectors during infection. The spectrum of Dicer-2-dependent downstream signaling events, and whether this function of Dicer-2 is conserved in shrimp and

other invertebrates, has yet to be elucidated. One potential mechanism to explain the nonspecific immunity triggered by dsRNA in shrimp is that the detection

of dsRNA, either by Dicer-2 or an additional sensor (Fig. 1B), triggers a feed-forward loop, whereby the RNAi machinery itself is transcriptionally upregulated, thus setting up a cellular environment that is poised to attack and degrade additional foreign nucleic acids. A recent study found that injection of dsRNA leads to the specific upregulation of Ago2 and Sid-1 mRNA in the shrimp Litopenaeus vannamei [28]. Moreover, WSSV infection induced Dicer-2 mRNA in Litopenaeus vannamei [33]. Recent work in our laboratory has shown that virus infection of Drosophila induces the upregulation of the RNAi pathway components Dicer-2 and Ars2 [34]. However, the viral PAMPs involved in inducing this response are not likely dsRNA, since the transcriptional upregulation Daporinad order Bumetanide of antiviral effectors occurs prior to viral replication. The shrimp Ars2 ortholog was recently identified and cloned [35]; it will therefore be important to investigate whether Ars2 and additional members of the RNA-silencing pathways in shrimp are regulated by infection. Although vsiRNAs are produced in an infected cell, whether these small RNAs or other viral RNA species, such as dsRNA, are released from infected cells

remains unknown. It is possible that the release of nucleic acids from infected cells alerts neighboring cells or even distant cells to the presence of infection. Accordingly, a local infection could lead to systemic antiviral defenses. This would also present opportunities for synergies between sequence-specific responses, which act cell-autonomously, and sequence-independent responses, which generate a nonautonomous anti-viral state. Studies in Drosophila have demonstrated that systemic RNAi can suppress viral replication [36]. Further exploration of these possibilities will likely reveal additional aspects of immunity to viral pathogens, but altogether will reinforce the fact that the initiation of antiviral immunity in response to the detection of viral PAMPs, including dsRNA, is a defense strategy conserved through evolution.

05). Conclusion: EPA improves the urinary protein in association with an increase in the EPA/AA ratio in CKD patients with dyslipidemia. EPA may have renoprotective role by reduction of proteinuria in CKD patients. The mechanisms of reduction of proteinuria by EPA would be clarified in the ongoing study. GULATI SANJEEV, KUMAR KAPIL, GUPTA UMESH, selleck chemicals KALRA VIKRAM, TIWARI S C Fortis Institute of Renal Sciences Introduction: Interstitial fibrosis &

tubular atrophy is the leading cause of graft loss in kidney transplant patient. Proliferation signal inhibitors may help in reducing calcineurin inhibitor exposure without increasing acute rejection episodes. Current study evaluated efficacy of conversion from mycophenolate to everolimus with CNI minimization in patients with biopsy proven

IFTA and deteriorating renal function. Methods: Prospective single center trial, study cohort selected from 200 live related renal transplant recipients in followup. All had received basiliximab induction and triple drug immunosupression (tacrolimus, MMF/EC-MFS, steroids). Inclusion criteria: biopsy proven IFTA, absence of significance proteinuria (<400 mg/24 hour), progressive graft dysfunction (decline of GFR > 15% DZNeP solubility dmso over 1 month), eGFR > 40 ml/min/1.73 m2. All underwent conversion from mycophenolate to everolimus with CNI minimization. Results: The study group composed of 22 patients (M : F = 19:3), mean age 37 years (range 24–58). Conversion done at 24 months Galeterone (IQR: 8.5–24.5) post-transplantation and median follow-up is 22 (IQR: 5–9) months. The tacrolimus trough levels decreased from 5.1 ± 1.6 ng/ml to 3.6 ± 1.1 ng/ml (p = 0.03). The everolimus levels achieved were 6.68 ± 2.4 ng/ml and 5.7 ± 1.4 ng/ml at 1 and 3 months. The eGFR that had declined from best stable values of 59.3 ± 11.9 ml/min to 48.2 ± 9.5 ml/min at conversion stabilized and improved to 50.7 ± 11, 53.3 ± 13.1, 54.9 ± 13.9 and 57.1 ± 10.1 ml/min at 1, 3, 6 and 12 months post conversion respectively (p = 0.028 at 3 months). There were no episodes of rejection, 2 patients was withdrawn at 3 months & 24 months due to proteinuria. Conclusion: Conversion from mycophenolate to everolimus

with CNI minimization resulted in stabilization of renal function. OJIMA SAKI, IO HIROAKI, WAKABAYASHI KEIICHI, KANDA REO, YANAGAWA HIROYUKI, AOKI TATSUYA, NAKATA JUNICHIRO, YAMADA KAORI, NOHARA NAO, SHIMIZU YOSHIO, HAMADA CHIEKO, HORIKOSHI SATOSHI, TOMINO YASUHIKO Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine Introduction: Previous study reported that dialysis patients are easy to occur carnitine deficiency. Thus, they have shown the weakness of the skeletal muscle, cardiomyopathy, heart failure and renal anemia. In the randomized controlled trial of L-carnitine in dialysis patients who had dilated cardiomyopathy, the survival rate of the carnitine administrated group was significantly better than the controled group for 3 years (Rizos I.

The rat AG-014699 purchase myeloid cell line RMW [5] and BWN3G [27] were generated in our laboratory. 293T cells were obtained from ATCC. Resident peritoneal macrophages were collected by peritoneal lavage, left to adhere to plastic dishes for 2 h, and washed. Remaining adherent cells were cultured overnight in either M-CSF (20 ng/mL), IL-4 (20 ng/mL), or IFN-γ (20 ng/mL) plus LPS (10 ng/mL) (cytokines from PeproTech and LPS from InvivoGen). Cells were analyzed by flow cytometry the next day. Expression constructs consisting of the full-length

open reading frames of rat Mincle, DCIR-1, or KLRH1 followed by a C-terminal FLAG epitope tag; rat FcεRI-γ with an N-terminal HA tag; or rat MCL without tag were generated. 293T cells were transiently transfected using polyethylenimine “Max” (m.w. 25 000, Polysciences) [28]. A rat Mincle-Fcγ2b Fc fusion protein was used to immunize female BALB/c mice by i.p. injections. Hybridomas were generated using standard techniques. One clone of IgG1 isotype, WEN43,

was selected and shown by flow cytometry to react specifically with cells transfected with rat Mincle, and not other APLEC-encoded receptors (Fig. 1). WEN42 (anti-MCL), WEN43 (anti-Mincle), and OX-42 (anti-CD11b/c) were produced in-house; STOK9 (anti-KLRH1) was a gift from Bent Rolstad; commercial antibodies were OX-41 (anti-CD172a/SIRP-α, Accurate Chemical & Scientific Co.), OX-22 (anti-CD45RC, BioLegend), and OX6 (anti-MHC class II, AbD Serotec). Data were acquired using a FACSCanto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). Transfected 293T cells were lysed in 1% digitonin (Calbiochem) lysis selleck compound buffer. Lysates were immunoprecipitated with Protein G Dynabeads (Invitrogen), separated by SDS-PAGE, and detected by ECL as detailed previously [5]. For immunoprecipitation, mAbs used were: anti-MCL (WEN42), see more isotype control (W6/32, IgG2a), or anti-FLAG (M2, Sigma-Aldrich). Immunoblotting: anti-FLAG-biotin (M2, Sigma-Aldrich), anti-MCL-biotin (WEN42), or rabbit anti-FcεRI-γ (Upstate Biotechnology) were used as primary antibodies, followed by streptavidin-HRP or HRP-conjugated anti-rabbit IgG

(Jackson ImmunoReseach Laboratories). 293T cells were incubated with Ab-coated yellow-green fluorescent 1-μm microspheres, counterstained with streptavidin-DyLight594, and analyzed by imaging flow cytometry on an ImageStream X (Amnis) as described previously [5]. For blocking, a cocktail of three mAbs specific for rat MCL was used. To compare efficiency of phagocytosis, data are expressed as phagocytic ratio: (fraction of bead-binding cells with internalized beads)/(fraction of bead-binding cells with no internalized beads). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison posttest (GraphPad InStat). We would like to thank Wendi Jensen for her technical assistance. This work was supported by grants from the University of Oslo (to M.R.D.) and the Norwegian Research Council, Norway (to M.R.D., S.F.

Two patients were not plasma exchanged and died. Four patients received plasma exchange and are alive. One patient had a clinical relapse 6 years after their initial presentation and is on renal replacement Ferroptosis inhibitor cancer therapy. Conclusion:  Concurrent ANCA and anti-GBM disease is rare. The mortality rate is high. Aggressive immunosuppression with steroids, cyclophosphamide and plasma exchange can

induce remission and preserve renal function. Long-term monitoring for relapses should occur. “
“Both enalapril and losartan are effective and widely used in patients with chronic kidney disease (CKD). This review aimed to evaluate the benefits of enalapril and losartan in adults with CKD. PubMed, EMBASE, the Cochrane Library and ClinicalTrials.gov were searched, without language limitations, for randomized controlled trials (RCT), in which enalapril and losartan were compared in adults with CKD. Standard methods, consistent with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, were used. Reviewer Manager software, ver. 5.2, was used for meta-analysis. Of 318 citations retrieved, 17 RCT (14 parallel-group and three cross-over) met our inclusion criteria. The pooled analysis for parallel RCT showed that the effects of enalapril and losartan on blood pressure, renal function and serum uric acid (UA) were similar. Meta-analysis Cytoskeletal Signaling inhibitor indicated that patients taking

enalapril had a higher risk of dry cough (risk ratio, 2.88; 95% CI, 1.11–7.48; P = 0.03). Sensitivity analysis showed good robustness of

these findings. Enalapril has similar effects to losartan on systemic blood pressure, renal function and serum UA in patients with CKD, but carries a higher risk of dry cough. Larger trials are required to evaluate the effects of these medications on clinical outcomes. “
“Aim:  To determine: (i) the proportion of stable asymptomatic haemodialysis patients with elevated troponin; (ii) stability of troponin values after dialysis and over a 2-week interval; and (iii) whether high-sensitivity troponin T (hsTnT) was associated with higher prevalence of cardiovascular risk factors or cardiovascular disease in these patients. Methods:  We measured hsTnT and the fourth generation troponin I before and after dialysis in Molecular motor 103 stable in-centre haemodialysis patients without ischaemic symptoms. Patients were divided into quartiles to test for associations with established cardiovascular risk factors or disease. Results:  hsTnT was above the 99th percentile for the general population in 99% of haemodialysis patients compared with only 13% elevation for the troponin I assay (P < 0.001). Median pre-dialysis hsTnT concentrations were unchanged after a 2-week interval (69 vs 69 ng/L, P = 0.55) but fell slightly immediately following dialysis (69 vs 61 ng/L, P < 0.001). Established coronary artery disease (59% vs 22%), peripheral vascular disease (38% vs 4%) and diabetes (18% vs 7%) were more prevalent (P < 0.

CD137−/− mice were provided by Professor Dr Robert Mittler from Emory University (Atlanta, GA, USA). C57BL/6J control mice

were purchased from Charles River (Sulzfeld, Germany). The animal protocols were constructed according to institutional and state guidelines and approved by the local animal welfare committee. Eight to 10-week-old age- and sex-matched mice were immunized with ovalbumin [OVA, lipopolysaccharide (LPS) content-reduced <10 EU/mg protein, as described previously [28]] using two protocols (Fig. 1), as follows. Allergy protocol.  Mice were sensitized twice intraperitoneally (i.p.) with OVA (20 µg in 200 µl 0·9% NaCl) in adjuvant (Imject Pexidartinib order Alum®, PerbioScience, Bonn, Germany) followed by six challenges in which 20 µg OVA in 40 µl of 0·9% NaCl was given intranasally (i.n.) (allergy protocol). Control mice (Alum) received injections and challenges of 0·9% NaCl. Mice were killed 24 h after the last local allergen provocation. Tolerance protocol.  To induce respiratory tolerance, MI-503 cell line WT and CD137−/− mice were pretreated twice i.n. with 500 µg OVA in 40 µl of 0·9% NaCl. Thereafter, mice underwent allergy protocol as described above. BALF from each individual mouse was obtained by flushing the lungs with PBS/2 mM ethylenediamine tetraacetic acid (EDTA); the total number and

differentiation of BALF cells were then determined as described previously [28]. Lung histological sections and computer-based quantification of the degree of pulmonary inflammation [haematoxylin and eosin (H&E)] and mucus production [periodic acid-Schiff (PAS)] were performed as described previously

[28]. Serum levels of OVA-specific IgE, IgG1 and IgG2a were measured by enzyme-linked immunosorbent assay (ELISA), according to standard protocol. IgE concentrations PD184352 (CI-1040) (pg/ml) were calculated using a standard curve for mouse anti-OVA IgE (AbD Serotec, Oxford, UK). Data for IgG1 and IgG2a are presented as titre values derived from analysis of optical density (OD) values versus factors of serum dilution series using a logarithmic curve-fitting model. Spleen and bronchial lymph node (bLN) isolated cells were restimulated in vitro with OVA (200 µg/ml) in RPMI-1640 containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cytokines (IL-4, IL-5, IL-13, IFN-γ) were measured in supernatants after 3 days using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Cell cultures were pulsed with 3[H]-thymidine and incorporated activity was measured in a Betaplate scintillation counter. Single-cell suspensions from spleen, lung and bLN were incubated with fluorescently labelled antibodies for 20 min at 4°C in phosphate-buffered saline (PBS)/0·5% bovine serum albumin (BSA). Intracellular staining of forkhead box protein 3 (FoxP3) was performed using the eBioscience kit, according to the manufacturer’s instructions. Briefly, cells were surface-stained, fixed and incubated with antibody to FoxP3 for 30 min at 4°C.