To answer the question of whether affinity or stability is the better correlate of immunogenicity, we extracted 12 affinity-balanced pairs each consisting of an “immunogenic binder” and a “nonimmunogenic binder” according to Sette and colleagues [6]. These peptides were synthesized and affinity and stability of their interactions with HLA-A*02:01 was measured. This representative analysis showed that “immunogenic binders” were significantly more stably bound to HLA-A*02:01 than “nonimmunogenic binders” (p = 0.0007, paired two-tailed

Student’s t-test) (Table 1, Fig. 4B), whereas no significant difference in affinity was observed between the two groups (Table 1, Fig. 4A). Note that one of the reported immunogenic peptides, RTLLGLILFV, in our hands was a low-affinity, low-stability-binding peptide. Upon closer inspection, the N-terminally truncated peptide, TLLGLILFV, appeared to be a likely HLA-A*02:01-binding peptide. This peptide FG-4592 cost was synthesized and found to be a high-stability (half-life 33 h) peptide. We would like to suggest that TLLGLILFV is the real HLA-A*02:01-restricted CTL epitope. Depicting this data in a log(stability) versus log(affinity) plot showed that the increased

stability of peptide-HLA-A*02:01 complexes involving “immunogenic binders” (y = 0.65x − 5.1, R2 = 0.65) versus Doxorubicin research buy “nonimmunogenic binders” (y = 0.75x − 4.5, R2 = 0.53) was seen throughout the binding range KD < 100 nM (Fig. 4C). When we inspected the 2 × 12 affinity-paired peptides (24 ever in total), we noted that 10 of 12 peptides with optimal amino acids residues in both anchor position 2 (LM) and C-terminal (VLI) had a half-life of more than 5 h, whereas nine of 12 peptides with a suboptimal amino acid residue (typically T or Q in position 2 or C-terminally) had a half-life of less than 5 h. At face value, this highly significant distribution (p = 0.014, Chi-square test with Yates correction) suggests that peptide-HLA-A*02:01 complexes are destabilized by just

one of the anchor positions being occupied with a suboptimal amino acid. For the seven peptides with suboptimal anchor residues, we substituted the suboptimal anchor residue with an optimal residue (leucine or methionine in position 2 and valine in C-terminal), and repeated the stability experiment. In all seven cases, the stability was improved (in six of the seven peptides, stability was increased by seven to tenfold), and four of the seven previously unstable peptides achieved a half-life better than 5 h, see Table 2. Thus, there appear to be a subtle difference in the specificity of high-affinity peptides, which may tolerate a suboptimal amino acid residue in an anchor position, and the specificity of high-stability peptides, which seems to be less inclined to tolerate suboptimal amino acid residue in anchor positions (in particular not in position 2).

IL-10 KO mice naturally develop inflammation in the colon from 10 to 12 weeks of age [43]; however, in the present study, the NKG2D ligand expression on small IECs was investigated in the IL-10 KO mice before any development of clinical sign of colitis. Nonetheless, we cannot exclude that NKG2D

ligand upregulation is induced by an inflammatory molecule produced in these mice, especially as we in the present study found no alterations in the intestinal IL-10 levels of the antibiotic-treated mice. In addition, decreased level of IFN-γ and IL-15 in the small intestine was observed in the vancomycin-treated mice similar to the NKG2D ligand expression and IL-15 was furthermore increased in the ampicillin-treated mice as was the NKG2D ligand expression. www.selleckchem.com/products/birinapant-tl32711.html This is interesting, as IL-15 is known to be directly involved in NKG2D ligand upregulation on IELs during celiac disease [5], and it is thus tempting to speculate that a less proinflammatory state, kept in check by the commensal microbes, actively keeps the NKG2D ligand expression low, although such a scenario needs experimental verification. IL-17 was however downregulated in both ampicillin-

and vancomycin-treated mice which suggests that this cytokine is not involved in the regulation of NKG2D ligands on IECs. Instead, both antibiotic treatments most likely eradicated important bacteria, for example segmented filamentous bacteria which can induce IL-17 [31, BMN 673 molecular weight 44]. The commensal microbiota may also directly express or secrete molecules that affect NKG2D ligand surface expression. We have previously shown that propionate from propionic bacteria is involved in the opposite scenario, as it increases

NKG2D ligand expression [17]. Further studies are however needed to establish the mechanisms behind these interesting 5-Fluoracil mw observations. It is noteworthy that the level of NKG2D ligand expression was substantially lower in the B6 mice housed in the Novo Nordisk animal facility compared with that in B6 mice housed at the University of Copenhagen. Differences in gut microbiota compositions in the groups of untreated control mice because of the different facility environments, sex, and animal vendors from which the mice were purchased, may explain the observed differences in NKG2D ligand expression. In general, we believe that it is important to take differences in microbiota composition into account, when comparing levels of NKG2D ligands measured by different laboratories. This could, at least partly, explain differences observed in the past. NKG2D ligand regulation by microbial interaction is supported by a growing body of data. Tieng et al. [7] have shown increased expression of NKG2D ligands on IECs after infection with certain pathogenic strains of E. coli and IECs have also been shown to express NKG2D ligands upon TLR3-dependent poly I:C treatment [45].

7B). In addition, the proliferation of LPL knock-down T cells was attenuated (Fig. 7C). Since the shorter calcium signal of knock-down Volasertib purchase T cells is the consequence of a reduced contact time with APC (Fig. 7A and B), it was tempting to speculate that antibody stimulation of T cells neutralizes this effect as this kind of stimulation is independent on T-cell/APC contact time. This was indeed the case (Supporting Information Fig. 8). In marked contrast to stimulation via APC, stimulation via antibodies induced an equal calcium influx in both control and LPL knock-down T cells. Furthermore, there was no inhibition

of proliferation if LPL knock-down T cells were stimulated via crosslinked antibodies (Supporting Information Fig. 8). Thus, the attenuated proliferation of LPL knock-down T cells upon stimulation with APC may at least rely in part on the reduced contact time and the short calcium signals. The activation of antigen-specific T cells is initiated by interaction of T cells with APC bearing the cognate antigen. Thereby, an ordered contact zone, called IS,

is established. The actin cytoskeleton is indispensable for spatial arrangements of a multitude of receptors and proteins that finally define a mature IS at the T-cell/APC contact zone 9, 30. Intracellular proteins regulating the selective spatio-temporal receptor accumulation to the IS were, however, so far not known. Employing RNAi-guided experiments here, we demonstrate that the actin-bundling AP24534 protein LPL is crucial for the stabilization of LFA-1 in the IS, the duration of T-cell/APC contact formation and sustained calcium signaling. Thus, LPL is an important regulator for the temporal organization of IS formation and T-cell

activation. There are a couple of evidences that the initial relocalization of LPL in the IS is dependent on actin polymerization. Thus, LPL completely colocalized with F-actin in the IS and deletion of the actin-binding domains of LPL interfered with its relocalization. Moreover, it was described that T-plastin binds only to newly formed actin Thymidine kinase filaments, a process which can be considered as actin–plastin copolymerization 14, 15. Therefore, it is likely that the actin/LPL copolymerization in the IS is the major force that brings LPL in the IS. Vice versa, LPL seemed to stabilize F-actin since LPL knock-down T cells had a reduced amount of total F-actin. Given that LPL might protect F-actin from being depolymerized by cofilin 16, F-actin depolymerization may be enhanced in LPL knock-down cells, whereas actin polymerization may be comparable with control cells. In this scenario, F-actin fibers were smaller, which could contribute to the reduced synapse size. Our data show that actin polymerization and accumulation in the T-cell/APC contact zone are required but not sufficient to establish a mature IS.

With the next set of experiments we addressed the question whether surface IgE-positive B cells can be detected in IgE knock-in mice. First, we stimulated total spleen cells for 5 days with LPS and IL-4. We used IgE knock-in mice on the CD23−/− background in order to avoid passive binding of soluble learn more IgE to the low

affinity IgE receptor (CD23) on B cells [23]. Surface IgE and IgG1 were detected by flow cytometry. LPS alone neither induced significant IgE nor IgG1 expression (0.4–1.5%) (Fig. 2A and Supporting Information Fig. 1). In B cells from WT mice LPS+IL-4 induces IgG1 (23%), but only very little IgE (1.5%). In contrast, both cells isolated from either heterozygous or homozygous IgE knock-in mice express comparably high amounts of IgE (ca. 15%) on the cell surface. However, the selleck screening library small fraction of positively stained cells might be due to a cross-reactivity or background staining of

the detection antibodies (see also Fig. 2E). WT mice express 23% and heterozygous IgE knock-in mice 10% IgG1 and, as predicted, no IgG1 was found in IgEki/ki mice. These results suggest that in vitro the chimeric membrane IgE molecule can be transported to the surface with a slightly lower efficiency than natural IgG1. To confirm these results, we performed a RT-PCR analysis of the membrane forms of IgE, IgG1, and the chimeric membrane IgG1-IgE form (Fig. 2B). The results of LPS+IL-4 stimulated cultures are in line with the protein expression data (Fig. 2A); however, LPS alone induces mRNA transcripts with little IgG1 or chimeric IgE being expressed on the surface of the cells (Fig. 2B). Second, we analyzed B cells from bone marrow, lymph nodes (data not shown), and spleens of heterozygous IgE knock-in mice and their WT littermates. We could find a normal B-cell subset distribution in vivo (data not shown). However, we could not detect membrane IgE-positive B cells (Fig. 2C) in the

spleen. The absence of CD23 demonstrates that the increase in IgE expression is not a result of an increase in membrane IgE expressing B cells in unchallenged, naïve mice (Fig. 2C) [23]. Additionally, immunization and boost with the T-dependent antigen Cetuximab research buy trinitro-phenyl-chicken ovalbumin (TNP-OVA) and the subsequent immunohistochemical analysis of splenic B-cell follicles shows only very rare IgE-positive cells located at the edge of the B-cell follicle in IgE knock-in mice of the CD23−/− background (Fig. 2D). Surface IgE and IgG1 expression in vivo were then analyzed after infection with the helminth Nippostrongyus brasiliensis (Nb), which leads to pronounced Th-2 responses [29]. Mesenteric lymph nodes of IgEki/ki, IgEki/wt, and WT mice were taken at day 14 after infection, at the peak of the germinal center response. IgEki/ki mice, as expected, showed no staining for IgG1, whereas IgEki/wt had intermediate expression of surface IgG1 when compared to WT.

4C, D). However, not only γδCD8αα+ iIEL but also αβCD8α+ iIEL cells showed a basal [Ca2+]i decrease. This was unlikely to be a direct effect of the GL3 mAb on αβ iIEL but may be due to changes in the composition of αβCD8α+ iIEL, e.g. through attraction of systemic αβ+CD8+ cells with lower basal [Ca2+]i levels into the gut epithelium 40. In contrast, basal [Ca2+]i levels of neither systemic CD8− p-γδ nor CD8− i-γδ were altered by GL3-treatment (Fig. 4C and D). These data suggest that the observed high basal [Ca2+]i levels of γδCD8αα+ Navitoclax nmr iIEL reflect a constant TCR-specific activation in vivo,

which could be partially blocked by anti-γδ TCR mAb treatment. Next, we investigated how γδ T cells from GL3-treated γδ reporter mice responded to TCR stimulation. As shown in Fig. 4A, the TCR complex was down-regulated

but still present at residual levels on the cell surface of these γδ T cells. We found that anti-CD3 and anti-γδ PD-0332991 chemical structure TCR mAb clustering still elicited Ca2+-fluxes in CD8− p-γδ and CD8− i-γδ from mice injected with GL3, albeit with lower or almost flat amplitudes compared with those from mock-treated animals. The iIEL populations CD8+ i-γδ and CD8+ i-αβ only showed a decrease of basal [Ca2+]i, without evident mAb-induced Ca2+-flux neither in PBS nor in GL3 treated mice (Fig. 5A). The quantification of these changes, displayed as fold of basal [Ca2+]i Cyclin-dependent kinase 3 levels after anti-CD3 and anti-γδ TCR mAb clustering, showed that CD8− p-γδ and CD8− i-γδ were affected by the GL3 treatment (Fig. 5B). In addition, iIEL from PBS- and GL3-treated γδ reporter mice were analyzed

for responsiveness to ex vivo stimulation with GL3 and GL4, a different anti-γδ TCR mAb. In vivo treatment with GL3 reduced the TCR-dependent CCL4 and IFN-γ production of γδ iIEL (Fig. 5C). Surprisingly, the CCL4 and IFN-γ production capability of γβ iIEL from GL3-treated γδ reporter mice stimulated ex vivo with the anti-αβ TCR (H57) was increased (Fig. 5D). In conclusion, γδ iIEL suffered a loss of function in response to TCR stimuli when their TCR was modulated by GL3 treatment for 6 days. Together, this suggests that the iIEL do not become exhausted and do not change their activated phenotype with repeated high-dose anti-γδ TCR treatment. However, the down-modulation of their surface TCR in combination with the decoration of residual surface γδ TCR is likely to be the reason for the diminished TCR responsiveness and cytokine production. This further implies a role for the TCR in the physiology of γδ T cells. However, it is at present not clear to what extent the responsiveness of γδ T cells to other stimuli, e.g. engagement of other receptors such as NKG2D or TLR, may be also altered by TCR modulation. The question whether, after thymic selection, the TCR on γδ T cells had a physiological role at all was not unanticipated 19, 23.

infantum[14], have been reported previously to down-regulate CD1a expression. L. donovani was also shown to prevent activation of CD1-restricted T cells by DCs, which may represent a survival strategy by avoiding parasite glycolipid recognition [12]. L. amazonensis https://www.selleckchem.com/products/mitomycin-c.html was able to alter DC differentiation by inducing a significant decrease in CD1a and CD80 expression and a significant increase in CD86 expression, causing down-regulation of the Th1 adaptive immune response [16]. We did not observe significant down-regulation of CD80 or increase of CD86. This could

be attributed to differences in the biology of Lm and L. amazonensis. In the last part of our work we showed that, despite their intracellular location, Lm clones did not stimulate IL-12p70, TNF-α or IL-10

production by DCs. In agreement with our results, others have reported that the uptake of the parasites alone by immature DCs provided an insufficient stimulus for cytokine production [6,11–13,25]. However, in the presence of an appropriate co-stimulation, and depending on the life stage and species involved, Leishmania parasites were shown to be able to modulate cytokine production by human DCs. We showed that, independently of their virulence, Lm clones were able to induce a decrease of IL-12p70 secretion during LPS-induced maturation of DC. Interestingly, although the LV Lm clone www.selleckchem.com/products/poziotinib-hm781-36b.html was not internalized by DCs, it was able to down-regulate IL-12p70 production during DC maturation similarly to the high virulent clone. It has been suggested that Leishmania-induced maturation does not require infection of DC and that direct recognition of parasites by DCs could be sufficient [28]. In agreement with our data, altered DC responsiveness to exogenous stimuli in the presence of Leishmania parasites and antigens has been reported by others [12,16,25]. L. donovani parasites crotamiton and

excreted–secreted antigens from L. donovani and Lm inhibited strongly IL-12p70 secretion by mature DCs [25]. Leishmania phosphoglycans family of virulence-associated antigens were able to inhibit DC maturation [29]. Conversely, it was reported that Lm was able to prime DC for CD40L-dependent IL-12p70 production [6,11,30] in a life stage and species- and strain-dependent manner [11]. This variability of Leishmania parasites ability to modulate a human DCs cytokine response could be explained not only by intrinsic differences between Leishmania species or strains or infective stage, but also by differences in the specific culture conditions such as the nature of priming and triggering signals used to induce maturation.

First, pDC express the immunoregulatory enzyme IDO 75, 76, which promotes tryptophan catabolism, depleting

the tryptophan pool that T cells need to generate effective responses. IDO-expressing cells in TDLN of patients living with breast cancer correlate with worse clinical outcome 56. Similarly, studies performed in a mouse model of malignant melanoma have demonstrated that cells resembling pDC expressed IDO in TDLN 56 and activated Treg 57. Second, activated human pDC express ICOS ligand, which promotes the generation of IL-10-producing Treg from naïve T cells 77. In addition to infiltrating TDLN, pDC can be directly recruited to tumors by factors such as stromal-derived factor-1 30, 35 and induce IL-10-producing Treg. Moreover, human pDC can directly suppress T-cell responses through Olaparib this website the expression of granzyme B 78. The ability of pDC to induce Treg can also impact responses to HIV infection. Human pDC exposed to HIV in vitro express IDO and promote the differentiation of naïve CD4+ T cells into Treg that suppress proliferation of effector T cells 79 and impair DC maturation 80. Therefore, pDC accumulation during HIV infection may be detrimental. Although damaging in some cases, pDC-mediated recruitment of CTL and IFN-I secretion might be essential in the control of several infections, such as murine hepatitis virus, RSV, HSV-1 and HSV-2, where pDC depletion dramatically impairs host antiviral responses 44, 45, 48, 49, 81, 82. pDC

induction of Treg is also beneficial in many situations. Despite inducing tolerance to tumor cells, Phosphoprotein phosphatase pDC mediate tolerance to harmless Ag and alloAg through the induction of Treg 83–86. In homeostatic conditions,

self-reactive T cells are kept in check by Treg. Genetic defect of the Treg-specific transcription factor Foxp3 results in Treg deficiency and development of fatal autoimmune pathology 87. pDC also reside in the thymus 88, 89 and may directly participate in the generation of Treg in this organ 90, 91. Despite the negative impact pDC may have during HIV infection, evidence suggests that pDC may serve a protective role, at least early on during infection. Initially, it was observed that pDC numbers were dramatically reduced in the blood of patients chronically infected with HIV. Loss of pDC correlates with high viral loads, decreased numbers of CD4+ T cells and the onset of opportunistic infections 92–100. pDC stimulated in vitro with HIV secrete IFN-I and other immune mediators 101, 102 and can cross-present HIV-derived Ag to CD8+ T cells 103. HIV-activated pDC may also contribute to host responses by inducing DC maturation through the secretion of IFN-I and TNF-α 101. Furthermore, pDC-derived IFN-I induces an antiviral state and limits replication of HIV in CD4+ T cells 104, 105. pDC secretion of IFN-I also limits HCV replication in hepatocyte cell lines 106. Therefore, pDC may be capable of eliciting protective responses to HIV and HCV in vivo.

1A, B). H & E stain from a biopsy of one nodule showed normal

tissue being replaced by anaplastic cells suggestive of a malignancy, and ICH for placental alkaline phosphatase was positive indicating a primary germ cell tumour (probably a metastasis) of unknown location. (Fig. 1C, D, respectively). Despite this, ERT was continued along with palliative therapy for pain management until the patient eventually died at the age of 67 months due to septic shock. To investigate the molecular basis SB525334 cell line of immune deficiency in the patient, we obtained genomic DNA from whole blood and buccal epithelial cells at the age of 30 months, and sequenced all the exons of the ADA gene. As shown in Fig. 2 (upper panel, A and B), a homozygous missense Vemurafenib chemical structure mutation in

exon 4 was found (g.29009 T > C) that leads to a replacement of a leucine for a proline in the position 107 of the protein (L107P). This mutation has been reported previously and results in ≤0.05% of ADA activity in vitro, correlating with the clinical phenotype of severe early-onset ADA deficiency in our patient [5]; in addition, both parents were heterozygous for this mutation (Fig. 2 upper panel, C and D). We also measured ADA activity in the blood spots obtained from the patient and found no activity on his RBC (0 vs. 25.5 nmol/h per mg protein in the control) (Table 2, 30 months old); moreover, both parents showed approximately MRIP half of the ADA activity observed in the healthy control. However, dAXP were modestly elevated (14.1% vs. 0% for

the healthy control and 50.3 ± 18% for patients with ADA-SCID), and this finding is more consistent with a delayed-onset phenotype. An unexpected increase in the numbers of T lymphocytes in patients with SCID could be explained either by spontaneous engraftment of maternal lymphocytes or alternatively, by transfusion of HLA-mismatched non-irradiated blood products [3]. As no records of previous blood transfusions were found, we karyotyped the PBL and performed HLA typing on the patient and his parents and found that he was both 46 (X, Y) and HLA haploidentical to his parents, excluding maternal and transfusion-related engraftment of T cells (data not shown). The possibility of somatic mosaicism caused by a de novo mutation was excluded because both parents were carriers of the same mutation (Fig. 2). A small number of ADA-deficient patients reported to date exhibit variable counts of T lymphocytes that result from an in vivo reversion of inherited mutations in the ADA gene [9–13].

Calcineurin upregulation was also found in the podocytes of FSGS patients, in which miR-30s were downregulated. Finally, luciferase reporter

assays confirmed that TRPC6, PPP3ca, PPP3cb, PPP3r1 and NFATc3 are the direct targets of miR-30s inpodocytes. Conclusion: miR-30s inhibit calcineurin signaling in podocytes by directly targeting calcineurin signaling components. Downregulation of miR-30s and the consequent upregulation of calcineurin signaling may be an alternative mechanism by which TGF-beta, LPS or PAN damages podocytes. FUKUDA AKIHIRO1, SATO YUJI1, IWAKIRI TAKASHI1, KOMATSU HIROYUKI1, KIKUCHI MASAO1, KITAMURA KAZUO1, FUJIMOTO SHOUICHI1,2 1First Department of Internal Medicine, University of Miyazaki; 2Department of Hemovascular Medicine and Artificial Organs, University of Miyazaki Introduction: Proteinuria and/or albuminuria are widely used selleck compound for noninvasive

assessment of kidney diseases. Proteinuria is a nonspecific marker of diverse forms of kidney injury, therefore, more glomerular disease specific biomarkers are required. Podocyte depletion is a major mechanism driving glomerulosclerosis, and that persistent podocyte loss check details is the likely driver for most forms of glomerular disease progression. Podocyte cell lineage specific mRNAs can be recovered from urine pellets of model systems and man. We have previously reported that urine podocyte mRNAs could serve as useful glomerular disease biomarker in model system (Sato Y et al. J Am Soc Nephrol 2009, Fukuda A et al. Kidney Int 2012). The purpose of this study was to test whether the urine podocyte mRNAs could Nutlin-3 solubility dmso be useful biomarker in human glomerular diseases. Methods: From January 2008 to October 2013, early morning voided urine samples were obtained from 121 patients with histology-proven glomerular diseases (minimal change nephrotic syndrome (MCNS, n = 16), crescentic glomerulonephritis (Crescentic GN, n = 14), membranous nephropathy (MN, n = 21), IgA nephropathy (IgAN, n = 60) and lupus nephritis (LN, n = 10)). A total of 29 urine samples were collected

from healthy volunteer who had no known kidney disease or hypertension. We examined urine podocyte mRNAs (urine podocin/AQP2 mRNA ratio, urine podocin/nephrin mRNA ratio and urine podocin/creatinine ratio) and urine protein/creatinine ratio (U-PCR)), also examined the relationships between urine podocyte mRNAs and U-PCR, or renal histological findings. Results: Compared with controls, urine podocyte mRNAs significantly increased in patients with glomerular diseases except MCNS. Compared with no proteinuria (U-PCR < 0.3), urine podocyte mRNAs were significantly increased in with proteinuria (U-PCR ≥ 0.3), however, urine podocyte mRNAs did not reveal linear correlation with U-PCR in any glomerular diseases.

In this study, in an attempt to determine IL-17 could mediate the asthma allergic reaction associated with A. simplex infection and to characterize the mechanism of innate immune response, we analyzed the immune responses in an experimental airway

inflammation mouse model treated with A. simplex larva excretory–secretory (ES) selleck chemicals llc proteins. The Anisakis type I larvae were collected manually from the viscera, flesh and body cavities of naturally infected blue whiting (Micromesistius poutassou), and were thoroughly washed in sterile phosphate-buffered saline (PBS). After collection, to prevent any contamination with the host material, the worms were thoroughly and carefully washed several times over a 3 h period in PBS. Anisakis simplex larvae were classified on Quiazon’s criteria (21). They were then introduced into sterile flasks with serum-free RPMI 1640 medium supplemented with antibiotics (100 μg/mL penicillin/streptomycin; Gibco, Grand Island, NY, USA). The culture Gamma-secretase inhibitor was then maintained for seven

consecutive days at 37°C in 5% CO2. It was confirmed that, during this time, all of the larvae remained alive and evidenced good mobility. Following centrifugation (12 000 g for 30 min), the supernatants were concentrated by pressure applied in a concentrator (Amicon, Millipore Corporations, Billerica, MA, USA) with 3000 Da pore size membranes. Various proteins (3 kDa to above 100 kDa) were detected in SDS page gel electrophoresis. The find more unnecessary excessive salts were eliminated from collected medium using HiTrap Desalting™ (Amersham Bio-Sciences AB, Uppsala, Sweden) and dialyzed against PBS for 24 h with continuous agitation in a cold room to eliminate any antibiotic remnants. Lipopolysaccharide (LPS) was depleted (endotoxin levels <0·01 μg/mL) from ES proteins using Detoxi-Gel Affinity Pak prepacked columns (Pierce Biotechnology, Rockford, IL, USA), in accordance

with the manufacturer’s instructions. RNase I (6 mg) from bovine pancreas (EC 3·1·4·22; 50 Kunitz units/rag; BDH Chemicals Ltd, Poole, England), and RNase A type III (10 mg) (RNase C; Sigma-Aldrich, Saint Louis, MO, USA) were dissolved in 1 mL of PBS (pH 7·4). Then, 2 mL (10 μg/mL) of ES proteins and 0·1 mL of RNase A and C solution were mixed and incubated for 1 h at room temperature. Chicken egg OVA (Sigma-Aldrich) were reconstituted in sterile PBS at 1 mg/mL and stored at −20°C. For intranasal challenge, 10 μL (10 μg) of ES proteins was added to 40 μL (40 μg) of OVA immediately prior to intranasal administration. C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were induced with airway inflammation by ES proteins for six total challenges, as described previously (22,23). One day after the final challenge, the mice were killed for analysis of bronchoalveolar lavage fluid (BALF). At the time of lavage, the mice were killed with 200 μL of ketamine : lumpun : PBS (2 : 3 : 5).