In addition to higher basal proliferation, draining LN cells from B10.S mice immunized with 3B3/PLP139–151/CFA showed much higher proliferation upon antigen restimulation (Fig. 5A). The treatment dramatically enhanced both IFN-γ- and IL-17-producing CD4+ T cells, while the treatment did not increase IL-4/IL10-producing T cells (Fig. 5B). Consistently, the 3B3-treated mice became susceptible to the development of EAE, with over 70% of B10.S mice developing PD98059 EAE (Fig. 5C and Table 2). To further examine the effect of high-avidity anti-Tim-1 as a co-adjuvant on DCs and effector and regulatory T cells, we generated B10.S Foxp3/GFP ‘knock-in’ mice. The ‘knock-in’ mice were immunized with

3B3 or control rIgG in immunogenic emulsion. DCs, Foxp3−CD4+ effector T cells (Teffs), and Foxp3+CD4+ Tregs were Y27632 isolated from spleen and lymph nodes of the mice and analyzed in criss-cross proliferation assays (Fig. 6A). Teffs from 3B3-treated

mice showed stronger proliferation and produced higher levels of IFN-γ and IL-17 upon antigen restimulation than Teffs from rIgG-treated mice. More interestingly, DCs from 3B3-treated mice induced higher Teffs proliferation and IFN-γ and IL-17 production than DCs from rIgG-treated mice (Fig. 6A). The frequency of Foxp3+ Tregs in spleens, lymph nodes, or the CNS was not significantly affected by 3B3 treatment (Fig. 6D and data not shown). However, Foxp3+ Tregs from 3B3-treated mice was less efficient in suppressing Teff proliferation in the cultures where Foxp3− Teffs and DCs were obtained from

rIgG-treated B10.S mice (Fig. 6B). Phenotypically, 3B3 in PLP139–151/CFA emulsion promoted DC activation as the treatment significantly upregulated the intensity of costimulatory molecules CD80, CD86, and MHC class II (Fig. 6C). In the CNS, treatment with the high-avidity anti-Tim-1 resulted in more mononuclear cell infiltration, containing high frequencies/numbers of CD11c+ DCs and CD4+ T cells (Fig. 6D and data not shown). Although the frequency of CD4+Foxp3+ Tregs in 3B3-treated mice was not dramatically decreased, significantly more Foxp3+ Tregs in the CNS of 3B3-treated Ceramide glucosyltransferase mice produced proinflammatory cytokine IL-17 (7.85±2.36% from 3B3-treated mice versus 1.85±0.96% from rIgG-treated mice, n=3; p<0.05). In addition, the frequency of CNS-infiltrating CD4+Foxp3− Teffs producing IFN-γ and/or IL-17 was also increased in 3B3-treated mice (Fig. 6D). Moreover, similar to the observation in Fig. 5B, control rIgG-treated B10.S mice showed a very low percentage of IL-17-producing Teffs in the CNS, which was dramatically increased by the high-avidity anti-Tim-1 treatment (Fig. 6D). DCs are professional APCs with a remarkable capacity to activate naïve T cells and prime T-cell responses, therefore providing a link between innate and adaptive immunity.

In histological sections, the occurrence of numerous alcian blue–positive mucous cells was observed among the intestinal epithelial cells of infected fish notably within the epithelia in close proximity to the nodule (Figure 2a). RCs in variable numbers (Figure 3a) were seen among the epithelia of both M. wageneri-infected Selleckchem CX-5461 tench (i.e. in close proximity to the point of cestode attachment and at a distance) and in uninfected specimens. Interestingly, within the parasitized intestines, RCs were found to co-occur with granulocytes within the submucosa of the nodule (Figure 3b) and in close proximity to blood vessels and/or within the capillaries. The inflammatory swellings surrounding the M. wageneri

primarily consisted of fibroblasts but also included a large number of neutrophils and MCs. Neutrophils (Figure 3c) and MCs were seen within the connective tissue surrounding capillaries and within the blood vessels within the submucosa and muscularis layer.

In some intestinal sections taken from infected tench, neutrophils were also observed within the epithelia (not shown). Neutrophils appeared round to oval in shape although their outline was commonly irregular (Figure 3c). These cells also contained a round nucleus and a cytoplasm selleck chemicals that contained dark, elongated granules that were fibrous in appearance (Figure 3c). Very few mitochondria and fragments of rough endoplasmic reticulum were observed in the cytoplasm of the neutrophils. The MCs, which were frequently observed within the epithelia of

infected hosts (Figure 3a), were irregular in shape with an eccentric, polar nucleus, and a cytoplasm characterized by numerous large, electron-dense, membrane-bounded granules (Figure 3d). The cytoplasm typically contained two to three mitochondria and an inconspicuous Golgi apparatus. Accurate counts of MCs and neutrophils were obtained from two intestinal grids from each infected fish. Neutrophils were found to be numerous within the nodule, in close proximity to the tegument of the cestode, but their number was seen to decrease towards the periphery of the nodule. Neutrophils were significantly more abundant than MCs (Table 1; anova, P < 0·01) SPTLC1 in host tissue close to the point of cestode attachment. At a distance of 200 μm from the site of parasite attachment, however, the number of neutrophils was significantly lower than the MCs (Table 1; anova, P < 0·01). There were significant differences in the number of neutrophils in close proximity to and at a distance of 200 μm from the point of cestode attachment (Table 1; anova, P < 0·01). Likewise, there were significant differences in the number of MCs at the site of infection and 200 μm away (Table 1; anova, P < 0·01). Commonly, the neutrophils and MCs adjacent to the M. wageneri scolex tegument had a cytoplasm that appeared vacuolized (Figure 4a) and contained very few organelles.

In recent years T cell biology has been enriched and enlivened

by the description of two further subsets. Interleukin (IL)-17-producing T cells were identified as important drivers of autoimmune pathology, forcing the re-evaluation of the role of Th1 cells in models of autoimmunity [2–4]. Elucidation of the factors promoting development of these Th17 cells [transforming growth factor (TGF)-β, IL-6 and IL-21][5–8] and the regulators of their transcriptional profile (RORγt and RORα[9,10]) established Th17 cells as a third effector T cell subset (reviewed in [11]). The Crizotinib three effector subsets appear to have evolved to cope with the threat posed by distinct classes of pathogen. Th1 cells are associated classically with intracellular bacteria and viral infections, Th2 responses are elicited by parasitic helminths, Pexidartinib molecular weight while Th17 responses are protective against certain extracellular bacterial and fungal infections [11]. Dysregulated Th2 responses promote the development of allergy and asthma, while uncontrolled Th1 and Th17 responses can result in autoimmune inflammation; therefore, the actions of these effector CD4+ cells need to be controlled strictly. The

identification of a minor subpopulation of CD4+ cells capable of preventing the development of autoimmunity [12,13] revolutionized our concept of T cell regulation. Identification of forkhead box P3 (FoxP3) as the lineage-specific transcriptional Tyrosine-protein kinase BLK regulator determining this suppressive

phenotype [14,15] confirmed the status of FoxP3+ regulatory T cells (Tregs), as distinct from previously described effector subsets [16]. In the scurfy mouse, a frameshift mutation in FoxP3 results in production of non-functional product and a lethal lymphoproliferative disorder [17,18] caused by over-activation of CD4+ T cells [19]. Similarly, the human condition immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FoxP3 [20]. ‘Natural’ Treg (nTreg) provide the thymically derived FoxP3+ cells that prevent spontaneous inflammatory disease and provide the Treg population that are assessed in vitro when using naive mice [21]. In addition, T cell receptor (TCR) stimulation of naive T cells in the presence of TGF-β can drive de novo expression of FoxP3 in uncommitted naive T cells, providing a population of ‘induced’ Tregs (iTregs). Antigenic stimulation, therefore, can drive the polarization of naive T cells to become Th1, Th2, Th17 and/or iTreg cells, in addition to the activation of antigen-responsive nTregs. The balance of (and timing in the appearance of) these different populations is dependent upon the nature of the antigen presentation and the cytokine milieu.

Sciatic nerve was transected, and end-to-end neurorrhaphy was performed on 32 male Sprague-Dawley rats, which were randomly divided into four groups (n = 8 per group): nerve coaptation without treatment (group I); nerve coaptation covered with HA film sheath (group II); nerve coaptation with intramuscular VEGF gene in plasmid injection (group III); and nerve coaptation combined with HA film selleck screening library sheath and intramuscular VEGF gene in plasmid injection (group IV). Contralateral sciatic nerves were used as control. VEGF

expression was verified from gluteal muscle biopsies surrounding the sciatic nerve by reverse transcriptase-PCR. Electrophysiological, histopathological, and electron microscopic evaluations were performed after 4 weeks. Mean peak amplitude of groups I–IV and nonoperated sciatic nerve were 4.5 ± 0.6 mV, 6.4 ± 0.4 mV, 6.7 ± 0.5 mV, 8.5 ± 0.4 mV, and 9.8 ± 0.5 mV, respectively. Mean myelinated axonal counts of groups I–IV and nonoperated sciatic nerve were 105 ± 24, 165 ± 19, 181 ± 22, 271 ± 23, and 344 ± 17, respectively. Treatment with HA film sheath coverage combined with intramuscular VEGF gene in plasmid injection yielded statistically significant

higher peak amplitudes and myelinated axonal counts this website (P < 0.001). In addition, significantly less scar formation with HA administration (groups II and IV; P < 0.001) was found. Thus, it was found that VEGF might crucially regulate nerve regeneration processes and that HA can reduce the scar

formation. This study showed that the combination of HA film sheath and VEGF gene may synergistically promote peripheral nerve regeneration. © 2013 Wiley Periodicals, Inc. Microsurgery 34:209–216, 2014. “
“Venous flow-through flaps are well-described options for Beta adrenergic receptor kinase small defects where donor site morbidity is undesirable or in areas where useful local veins are in close proximity to the defect, particularly in the extremities. However, higher rates of flap loss have limited their utility. The saphenous venous flap in particular has been widely sought as a useful flap, and while arterialization of this flap improved survival rates, congestion has remained a limiting feature. We describe report a modification in the design of saphenous venous flaps, whereby an arterialized flap is provided with a separate source of venous drainage, and demonstrate survival of substantially larger venous flaps than previously reported. In five consecutive patients, we describe three main modifications to the saphenous venous flap as previously described: (a) Using arterialized flaps only; (b) Reversing the flap to allow unimpeded flow during arterialization; and (c) Anastomosing additional vein(s) that are not connected to the central vein—especially at the periphery of the flap for true venous drainage.

Anti-CD3 mAb-induced redirected cytotoxicity against B7-H3/P815

cells was higher than that against P815 in both CD4+ and CD8+ T cells (Fig. 1c). We examined OVA-specific cytotoxicity against E.G7 cells that express peptide antigens derived from OVA protein, using OT-I-derived CD8+ T cells to Vemurafenib investigate whether B7-H3 on target cells up-regulated antigen-specific cytotoxicity of CD8+ T cells. B7-H3 expression on parental E.G7 and B7-H3/E.G7 cells is shown in Fig. S1. Cytotoxicity against B7-H3/E.G7 cells by freshly isolated OT-I CD8+ T cells was consistently higher than that against parental E.G7 cells (Fig. 2a). When the in vitro-sensitized OT-I CD8+ T cells were used as effectors, cytotoxicity against B7-H3/E.G7 was seen

even at lower effector : target (E : T) ratios (E : T = 1 and E : T = 5) and consistently showed higher cytotoxicity than that against parental E.G7 cells (Fig. 2b). These results indicate that tumour-associated B7-H3 enhanced antigen-specific cytotoxicity of CD8+ T cells. To investigate whether CD8+ T cells selectively lyse tumour cells that express B7-H3, different fluorochrome-labelled parental E.G7 and/or B7-H3/E.G7 cell combinations were injected into the peritoneal cavity of OT-I mice, and PEC were analysed after 24 hr by flow cytometry. In the mix of CMTMR-labelled E.G7 and CFSE-labelled E.G7 (1 : 2) (A-mix; i) cells, the ratio of recovered CFSE-labelled cells : CMTMR-labelled cells was approximately 2 (Fig. 2c). MRIP This was similar to the injected cell ratio, suggesting that the respective fluorochrome-labelled E.G7 cells were lysed equally. In contrast, for the mix of CMTMR-labelled CH5424802 E.G7 and CFSE-labelled B7-H3/E.G7 (1 : 2) (B-mix; ii), the ratio of CFSE-labelled B7-H3/E.G7 to CMTMR-labelled WT E.G7 was dramatically reduced (Fig. 2c; centre and right panels), suggesting a selective deletion of B7-H3/E.G7 cells. Similar experiments with different fluorescent protein-expressing J588L and B7-H3/J558L cells injected into syngeneic mice also showed the selective elimination of B7-H3/J558L at 14 days (data not shown). The

selective elimination of the B7-H3-expressing target cells suggests preferential activation of CD8+ T cells in the interactions with CD8+ T cells and B7-H3-expressing tumour cells. We next examined whether B7-H3 on tumour cells enhances CD8+ T-cell activation at either the induction or effector phases using two different models. B6 and OT-I mice were sensitized in vivo with P815 or B7-H3/P815 cells as alloantigen-expressing cells and E.G7 or B7-H3/E.G7 cells as OVA-peptide-expressing cells, respectively, and then cytotoxicity against parental tumour cells was analysed. The in vivo sensitization with either alloantigen or OVA antigen by B7-H3-expressing tumour cells did not affect the induced cytotoxicity (Fig. 3a). These results suggest that B7-H3 expressed on tumour cells did not enhance antigen-specific priming of CD8+ T cells in the induction phase.

In addition, Rorγt+ ILC numbers were also reduced upon specific deletion of AhR in Rorγt-expressing cells (including ILCs) [[56]]. Together these data indicate that the effects of AhR-deficiency on

Rorγt+ ILCs are cell intrinsic. Interestingly, the reduction of Rorγt+ ILC numbers, induced by ablation of AhR, was observed only after birth. buy Temsirolimus During fetal development, and early after birth, the ILC22 numbers in AhR-deficient mice are comparable to those in wild type mice, indicating that AhR is not required for development of these cells [[54]]. However, after weaning, the numbers of Rorγt+ ILCs in AhR-deficient mice steadily decrease [[54]]. Maintenance of ILC numbers is not a consequence of AhR activation by products of colonizing microbiota, because the difference in ILC22 numbers between wt and AhR-deficient animals is not affected by treatment with a mix of antibiotics [[54]]. Also, the observation that germ-free animals do not show reductions in gut residing Rorγt+ ILC numbers [[55, 57]] is consistent with the notion

that products from commensals are not required for the maintenance of these cells. It is controversial whether dietary products are the AhR ligands responsible for the maintenance of gut-residing Rorγt+ ILCs, as observed for IELs [[53]]. In one study, it was found that mice fed with a diet free of AhR-binding phytochemicals showed decreased numbers of Rorγt+ ILCs, causing a lack of CPs and PIK3C2G ILFs [[55]]. Addition of indole-3-carbinol, a dietary product, restored the Rorγt+ ILC numbers [[55]]. Another study, however, suggested that endogenous INCB024360 clinical trial AhR ligands, including the tryptophane catabolite kynurenine, were potent regulators of Rorγt+ ILC maintenance as removal of dietary AhR ligands in that study did not disturb Rorγt+ ILC homeostasis and function [[56]]. The differences may be due to different types of controlled diets used by the different groups.

Further experiments should aim to resolve these discrepancies. The mechanisms by which AhR controls Rorγt+ cell numbers are not fully understood. Microarray analysis of Rorγt+ cells from wt and AhR-deficient mice suggested that Notch 1 is a downstream target of AhR [[56]]. Consistent with this, administration by gavage of the toxin TCCD (2,3,7,8-tetrachlorodibenzo-p-dioxin) resulted in the upregulation of Notch1 and Notch2 in gut Rorγt+ ILCs. Evidence for a role of Notch in AhR-mediated maintenance of Rorγt+ ILCs was provided by the observation that mice deficient for RBP-Jk, an essential partner of Notch, showed substantially reduced numbers of NKp46-expressing Rorγt+ ILCs and, although less prominently, of CD4+ Rorγt+ ILCs (LTi cells) also [[56]]. However, there were differences between the AhR- and RBP-Jk-deficient mice, in that in the latter, cryptopatches and ILFs were largely intact, whereas they were greatly reduced in AhR-deficient mice [[56]].

To determine the molecular parameters that determine this major functional effect in the NOD mouse we measured the affinity of hCD47 for SIRPα from various mouse strains. PD0325901 ic50 Human CD47 bound SIRPα from the NOD mouse with an affinity 65 times greater than SIRPα from other mouse strains. This is due mainly to the NOD SIRPα lacking two amino acids

in domain 1 compared with other mouse strains. Remarkably the SIRPα(NOD) binds hCD47 with 10 times the affinity of the syngeneic hCD47/hSIRPα interaction. This affinity is outside the normal range for affinities for leucocyte surface protein interactions and raises questions as to what is the optimal affinity of this interaction for engraftment and what other xenogeneic interactions involved in homeostasis may also not be optimal. “
“This represents an overview of the use of animal models to study the adverse

pregnancy outcomes seen in humans. The purpose is to entice clinicians to utilize some of this information to seek out the literature and have more meaningful and profitable discussions with their academic colleagues and enhance transdisciplinary research in reproductive health. This represents an overview and not an exhaustive (or systematic literature) review of the use of animal models to study the adverse pregnancy outcomes seen in humans. For several of the outcomes mentioned herein, there exist more in-depth reviews and there likely will be more to follow. Nor is this a review almost of all the data and mechanisms relating to normal and abnormal pregnancy and see more parturition. I have decided to include a balance between older reports and observations and reviews by revered scientists, as well as newer observations

and reviews by seasoned and perhaps less-seasoned investigators. My hope is that clinicians will be able to utilize some of this information to seek out the literature and have more meaningful and profitable discussions with their academic colleagues. I further hope that they will be enticed to engage in regular interactions that will enhance transdisciplinary research in reproductive health. My ultimate agenda is to eliminate the tendency to dismiss work in animal models out of hand because they do not exactly capture human physiology. In addition, I want to prevent the thinking that little can be learned from observations in humans because of inability to modulate and study-specific mechanisms. I would like to see more support for conversations starting from both sides with ‘This is how I understand how the model behaves and how it might (or not) be reflected in humans. What is your understanding?’ I would also like to see the literature, including titles of manuscripts and keywords increase visibility of the animal models (e.g. including the words ‘animal model’ and species name) involved in the observations conveyed.

The all too slow evolution of eukaryotes to encode a new recognition became no match for the evolutionary potential of the prokaryotes to rapidly encode escape from that recognition. The only solution was to somatically generate a random recognitive repertoire that divided the antigenic universe into combinatorials of determinants referred BVD-523 ic50 to as epitopes. This somatically generated repertoire characterizes what is referred to as the adaptive immune system. While this made it very difficult for an infectious agent to escape recognition, a random somatically generated repertoire posed two new problems that demanded

concurrent solutions. First, the repertoire had to be sorted into those specificities which if expressed would debilitate the host [i.e. anti-self (S)] and those specificities which if not expressed would result in the debilitation of the host by infection [i.e. anti-nonself (NS)]. The anti-S had to be purged leaving as a residue the anti-NS to protect the host. This process is metaphorically referred to as ‘the S-NS discrimination’. Second, the sorted anti-NS repertoire had to be selectively coupled to largely

the same panoply of effector functions that were used by the recognitive repertoire RXDX-106 supplier of the innate system. These two problems need comment. It is the fact that the output is just as biodestructive Thalidomide and ridding for the host as it is for the pathogen that mandates a mechanism to sort the repertoire. The innate repertoire is sorted by germline selection over evolutionary time with the result

that it distinguishes the self-of-the-species from the pathogenic universe. On the one hand, any mutation in the innate repertoire that resulted in recognition of a self-component of the species would be lethal in the offspring of a mating between that mutant and an individual expressing that self-component. On the other hand, any mutation that resulted in the recognition of an antigenic determinant common to many pathogens would be distinctly advantageous. As a consequence, the innate repertoire is blind to the self-of-the-species and recognizes a limited number of epitopes shared by many pathogens. This can be easily seen as hosts without adaptive immune systems permit grafting without rejection between individuals of a species and in many cases between species. In the presence of the adaptive system, grafts between individuals of a randomly mating species are rejected. The adaptive system is individual-specific; the innate system is species-specific. Specificity of the epitope-recognitive receptors (paratopes) is evolutionarily driven by the necessity to make a S-NS discrimination. For the innate system, its specificity must be sufficient to distinguish the pathogen from the self-of-the-species.

p.m. versus 3000 c.p.m.; P < 0·03). From these data, along with

those shown in Figs 2 and 3, we speculate that eosinophils not only present antigens to CD4+ T cells in an MHC class II pathway, but also present antigens to CD8+ T cells by using their MHC class I molecules. To test this hypothesis, experiments were performed to determine whether the induction of C. neoformans-primed T-cell proliferation was caused by the presentation of CX-5461 mouse antigens by eosinophils in conjunction with MHC class I and MHC class II molecules. C. neoformans-pulsed eosinophils were treated with anti-MHC class I or anti-MHC class II mAbs before incubation with C. neoformans-primed CD4+ and CD8+ T cells. The blocking of MHC molecules on the eosinophil surface was found to suppress the ability of C. neoformans-pulsed eosinophils to stimulate C. neoformans-primed T-cell proliferation (Fig. 6d). Moreover, the suppression seen www.selleckchem.com/products/Everolimus(RAD001).html in the lymphocyte proliferation was more pronounced with anti-MHC class II, which coincided

with the higher proliferation of CD4+ T cells shown in Fig. 6c. In conclusion, C. neoformans-pulsed eosinophils stimulated C. neoformans-primed MSCs and T cells (CD4+ as well as CD8+) in an MHC class II- or class I-dependent manner. This stimulation of proliferation, however, was not observed for naive T cells or when C. neoformans-pulsed Mφ were used as APCs. To characterize and differentiate the T-cell profile seen after co-culture with C. neoformans-pulsed eosinophils, C. neoformans-primed purified T cells (CD4+ and CD8+) were analyzed SPTLC1 by flow cytometry to determine the intracellular expression levels of IFN-γ and IL-4 after 4 days of culture with C. neoformans-pulsed eosinophils or medium alone. Figure 7 shows a significant increase in the percentage of IFN-γ-producing cells when T cells were incubated with C. neoformans-pulsed eosinophils compared with T cells cultured in medium alone (6·56% versus 1·61%; P < 0·02). With regard to the IL-4-producing T-cell population, the percentage

with C. neoformans-pulsed eosinophils (2·42%) was similar to that for medium alone (2·35%). These results allowed us to conclude that C. neoformans-pulsed eosinophils were able to induce the expansion of IFN-γ-producing Th1 cells, but not of IL-4-producing Th2 cells. To analyze the production of cytokines by CD4+ and CD8+ T cells in supernatants, the concentrations of IFN-γ, TNF-α, IL-4, IL-10 and IL-13 were measured after 4 days of culture. The results presented in Fig. 8(a,b) show that there was a significant increase in the production of IFN-γ and TNF-α generated by C. neoformans-primed T cells cultured with C. neoformans-pulsed eosinophils compared to the cytokine production by T cells cultured in medium alone, with fixed yeasts of C. neoformans or with unpulsed eosinophils. In contrast, no differences in the levels of IL-4, IL-13 or IL-10 were detected in supernatants of C. neoformans-primed T cells cultured with C.

Mice were treated i.p. with anti-CCR3 in three different doses (30–300 μg/animal in 500 μl PBS) or isotype control (100 μg/animal in 500 μl PBS, rat IgG2b, clone R35-38; BD-Bioscience Europe, Erembodegem, Belgium) 1 hr before allergen exposure on the first day of exposure. Cytospin preparations from BM and BAL were stained for CD34 using a biotinylated rat anti-mouse CD34 mAb (clone

RAM34; BD Biosciences). Bound antibodies were visualized with a Vector Red Alkaline Phosphatase Substrate kit (Vector Laboratories Inc., Burlingame, CA). The slides Kinase Inhibitor Library concentration were also stained with Luxol Fast Blue and counterstained with Mayer’s haematoxylin (DAKO) to identify these cells as eosinophil-lineage precursors. Five hundred cells were evaluated in random fields of view. Cytospins from BAL were stained with a rat anti-mouse CD34 mAb (clone RAM34; BD Biosciences). A rabbit anti-rat immunoglobulin

(DAKO) was used as a link antibody before incubation with alkaline phosphatase–anti-alkaline phosphatase (DAKO). Bound antibodies were visualized with the Vector Red Alkaline Phosphatase Substrate kit. Slides were then treated with a biotin blocking system (DAKO) and incubated overnight at 4° with a biotinylated rat anti-mouse Sca-1/Ly6 mAb (Clone 177228; R&D Systems). Next day, the slides were washed and incubated with streptavidin-β-galactosidase and X-Gal substrate (β-Gal Sorafenib staining set; Roche) and counterstained with Mayer’s haematoxylin. Four hundred cells were counted in random fields of view. All data are expressed as mean ± SEM. Statistical analysis was carried out using a non-parametric analysis of variance (Kruskal–Wallis test) to determine the

variance among more than two groups. If significant variance was found, an unpaired two-group test (Mann–Whitney U-test) was used to determine significant differences between individual groups. Wilcoxon signed rank test was used to analyse changes within the same group. P < 0·05 was considered statistically significant. Flow cytometric analysis for CD34+ CCR3+ cells in BM, blood, lung and BAL showed a significant increase of this Tryptophan synthase cell population in all three compartments of OVA-sensitized/exposed animals when compared with OVA-sensitized but saline-exposed control animals (Fig. 1a). Triple staining for CD34+ CCR3+ Sca-1+ on lung cells was performed to determine if a part of the CD34+ CCR3+ cells also expressed Sca-1. Allergen exposure induced a significant increase in the number of CD34+ CCR3+ Sca-1+ lung cells both in the SSChigh gated population (i.e. eosinophils) and in the SSClow gated cell population (i.e. eosinophil-lineage-committed progenitors) when compared with saline-exposed animals (Fig. 1b). CCR3+, Sca-1+ CCR3+ and CD34+ CCR3+ cells were also increased in the SSChigh and SSClow gated cell populations in allergen-exposed mice when compared with saline-exposed mice (Fig. 1c,d).