Serological studies conducted in countries where malaria is endemic suggest that high titres of cytophilic IgG3 and IgG1 or weakly cytophilic IgG2 antibody subclasses are associated with protection against severe malaria [81]. Malaria parasites were shown to be killed in vitro by monocytes, and this was enhanced in the presence of various antibody subclasses [82], ICG-001 in vivo which facilitated phagocytosis of parasites by binding to Fcγ receptors on the phagocytes through their Fc domain; the parasites were then killed by the respiratory burst generated by Fc receptor cross-linking. This antibody-dependent

cellular inhibition (ADCI) of parasites was positively associated with protection against malaria [83-85]. Antibody responses against three P. falciparum blood-stage antigens–MSP-1 [86], MSP2 [87] and AMA-1 [88]–were skewed towards the cytophilic isotypes

IgG1 and IgG3, responses associated with protection against malaria. How long protective antibody responses are retained after recovery from malaria is of great interest. The absence of a memory B-cell response (MBC), or the presence of a dysregulated B-cell response, has been attributed to the highly polymorphic Gefitinib solubility dmso and clonally variant nature of P. falciparum blood-stage antigens. However memory B cells apparently existed in vaccinated mice that acquired sterilizing immunity after rechallenge [89]. During self-resolving P. chabaudi infections, the expression of a memory B-cell phenotype was detectable for at least 60 days after primary Metformin datasheet infection, and after rechallenge, they rapidly formed germinal centres in the spleen and differentiated into plasma cells giving a more efficient and rapid antibody response than in the primary infection [90]. Studies with transgenic mice carrying a TCR specific for an epitope of MSP-1 of P. chabaudi showed that some MSP-1-specific B cells were found in the spleen up to 8 months after a primary infection, although not in high numbers [91]. While present in the spleens of immune mice, similar B cells

have been found more recently among peripheral blood mononuclear cells in human malaria infections [92-95]. The cellular basis of humoral immunity has been clarified by the introduction of the B-cell ELISPOT assay that has enabled the prevalence, specificity and life-span of malaria-specific memory B cells to be determined, both after natural infections with P. falciparum [93-95] and in studies in mice [91]. The number of individuals with malaria-specific memory B cells has been found to increase with age [93, 94, 96], indicating that protective immunity depends on the range of these cells as well as the antibody response [97]. Over 75 years ago, Taliaferro and Mulligan demonstrated that blood-stage malaria in mice was associated with activation and expansion of the mononuclear phagocyte system.

Blots were scanned and densitometry was performed with ImageJ (v1.44p). Total RNA was isolated

from tissue GSK126 mouse with Trizol© according to the manufacturer’s instructions. Tissue was washed in PBS and homogenized using the power homogenizer in 1 ml Trizol© per 100 mg of tissue. 1 µg RNA was incubated with 1 μl DNase and 1 μl DNase buffer made up to 10 μl volume with diethylpyrocarbonate-treated water for 15 min at room temperature for removal of contaminating DNA. Eight microlitres of the DNAse-treated mix was incubated with 1 μl 10 mm dNTP and 1 μl oligo-dT(12–18) (0·5 µg/ml) for 5 min at 65°. To this mix, 2 μl 10X RT buffer, 4 μl 25 mm MgCl2, 2 μl 0·1 mm dithiothreitol, 1 μl RNAse Out and 1 μl Superscript III was added. (In the reverse transcriptase controls no Superscript

III was added.) The mix was incubated at 42° for 10 min and the reaction was terminated at 70° for 15 min. Then 0·5 μl RNAse H was added and the mix was incubated at 37° for 20 min. Samples were stored at −20° until further use. PCR was used to selleck inhibitor amplify the cDNA. Paired oligonucleotide primers for amplification of the genes of interest were designed to produce amplicons where the intron/exon boundary was crossed wherever possible. Non-template reverse transcriptase controls were used. Table 1 provides the primers for CRTH2, L-19, COX-2 and the cytokines IL-4, IL-10, interferon-γ (IFN-γ) and TNF-α. The mesoscale discovery multi-spot ultrasensitive mouse Th1/Th2 9-plex assay click here was used as per the manufacturer’s protocol for the detection of the following cytokines: IL-12, IFN-γ, TNF-α, IL-1β, KC/GRO, IL-4, IL-5, IL-10 and IL-2. Cytokines were quantified against an eight-point calibration curve from 0 to 2500 pg/ml, constructed from serially diluted standards provided by the kit. The 96-well multi-spot plate was blocked in 1% BSA in PBS for 1 hr before the addition of 40 μg of murine myometrium or 100 μg of pup brain protein lysate and incubated

for 2 hr at room temperature. The multi-spot ELISA plate was read using a Sanger 2400 imager. The quantities of cytokines were determined against the standard curve and transferred into an excel spreadsheet for further analysis. Mice were killed by cervical dislocation at E15–16 of gestation; the uterus was harvested, kept in PBS on ice and was used within 5 hr of harvesting. The uterus was dissected either in the longitudinal or horizontal direction to expel the fetuses and the placentas. Vasculature and decidua were removed macroscopically, and 5 × 10 mm strips were mounted on the DMT myograph (DMT, Aarhus, Denmark) in the orientation dependent on the muscle type being examined; longitudinal direction for longitudinal muscle and horizontally for the circular muscle orientation.

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 BVD-523 mw 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 selleck chemical 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 PFKL 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].

Further cholinergic mechanisms affecting Aβ metabolism were previously reviewed [41-44]. While some authors suggested that any potential deficits within the cholinergic system do not significantly contribute to the pathophysiology of AD [45, 46], Mesulam et al. [20] as well as Mufson and co-workers [47] clearly stated that the cytopathology in cholinergic pathways involving CPN is a very early event in the course of the continuum that leads from advanced age to mild cognitive impairment and AD. Remarkably, altered cholinergic processes find more but no loss of CPN were observed in single and double transgenic

animal models harbouring mutated APPs and/or presenilins as transgenes [48-52]. In TauPS2APP mice with human mutations of APP, presenilin 2 and tau, the cholinergic medial septum remained unaffected, but in parallel Loreth et al. [46] found a degeneration of parvalbumin-containing septo-hippocampal projection neurones targeting GABAergic hippocampal interneurones [53]. In contrast, very old 3xTg mice displayed a slight reduction of cholinergic MS/DB neurones and age-dependent cholinotrophic alterations in the hippocampus [21-23]. Here we show that 4 months following cholinolesion of 12-month-old NVP-BEZ235 3xTg mice, the elimination of CPN induced

a drastic increase of Aβ and the C99 fragment from APP. This is in line with a report by Gil-Bea et al. [54], who used Tg2576 mice with a similarly induced cholinergic hypofunction and found a drastically enhanced soluble Aβ1–42 and a lowered expression of α-secretase ADAM17, which apparently favours the amyloidogenic route of APP processing.

Furthermore, treatment of Tg2576 mice with scopolamine, an antagonist of muscarinic acetylcholine receptors, caused increased levels of fibrillar Aβ and diminished Ribose-5-phosphate isomerase α-secretase activity [55]. The impact of experimentally altered Aβ deposits in numerous studies and drastically enhanced levels of APP, its C99 fragment and total Aβ after cholinolesion in the present work remain at least partially controversial. Whereas it is now widely accepted that a correlation between age-dependent total plaque load and dementia is lacking [56], there are interrelations between cognitive impairment and fibrillar Aβ, known to be toxic [57] and causing synaptic abnormalities as well as neurite breakage [58, 59]. Furthermore, Aβ oligomers are known to be highly toxic Aβ species [60-63] and have been shown to cause Ca2+ elevation, missorting of endogenous tau into dendrites, tau phosphorylation, and destruction of microtubules and spines [64]. The increased levels of monomeric Aβ extracted from the hippocampus of immunolesioned 16-month-old 3xTg mice using a buffer devoid of detergents, points to a detrimental role of soluble Aβ species in the current model.

Setting a conservative haematocrit target of 30% for CKD patients by the NHI of Taiwan in 1996 was not evidence-based but might be purely due to economic concerns. Unexpectedly, the Normal Hematocrit Trial published in 1998 demonstrated that there was a strong trend toward increased mortality or nonfatal myocardial infarction DMXAA mw in HD patients assigned to a higher haematocrit target of 42%, compared with a lower haematocrit target of 30%.[4] Later

on, the results from CHOIR, CREATE, and TREAT studies all demonstrated an increased risk of adverse outcomes at higher haemoglobin targets and higher ESA dosage.[5-7] In 2012, the KDIGO Anaemia Guideline recommended that for patients with anaemia of CKD on dialysis, ESA treatment should be initiated when the haemoglobin concentration is between 9–10 g/dL to avoid having the fall of haemoglobin below 9.0 g/dL.[15] It is

worthy of note that this recommendation had been complied within Taiwan since 1996. Under bundling, it is of paramount importance to determine a cost-effective ESA and iron protocols. In 1996, nephrology experts from nine medical centres in Taiwan reached consensus on the diagnostic criteria for iron deficiency. We recommended that iron supplementation should be considered when a ferritin <300 ng/mL and/or transferrin saturation (TSAT) < 30% in dialysis patients and to maintain a ferritin level of 300−500 ng/mL and TSAT of 30%−50%. The consensus was based on several previous studies performed in Taiwan and provided guidance on the use of intravenous iron to correct CKD anaemia.[16-19] This recommendation on GDC 0068 the management of anaemia and iron deficiency in patients with CKD was years ahead of the current major CKD guidelines (Table 1).[15, 20, 21] According to the results of our study, a serum ferritin of 300 ng/mL has a 100% ability to separate patients with or without initial response to ESAs.[16] TSAT is a good indicator for the balance

of supply and demand of plasma iron. Abiraterone mouse Since there is a great need for iron during increased erythropoiesis mediated by ESAs, a TSAT of 30% is a cut-off for the diagnosis of functional iron deficiency.[18, 19] The studies by Fishbane, Frei, and Maesaka[22] and Besarab et al.[23] demonstrated more reductions in ESA requirements by the use of intravenous iron supplementation to increase the ferritin to higher than 300 ng/mL and TSAT to 30–50%. As shown in the yearly distributions of serum ferritin and TSAT levels from 1995 to 2012 (Fig. 2), 51% of HD patients and 47% of PD patients had ferritin levels <300 ng/mL, and nearly 30% of HD and PD patients had TSAT levels <20% in 1995. Notably, the proportion of HD patients with ferritin levels <300 ng/mL fell to 23% until 2012. The proportion of HD and PD patients with TSAT <20% had also halved from 1995 to 2012.

Our understanding of the basic immunobiological properties of DC has been significantly advanced over the years. This has not only provided good explanations for the problems encountered, but also stimulated many new

ideas regarding the potential ways forward aimed to improve DC therapy in a more fundamental way. The important issues lie within DC heterogeneity and functional plasticity, and hence their immunogenic versus tolerogenic properties or potentials. GSK3235025 clinical trial It has gradually become clear that DC are not a homogeneous population, and questions have also been raised about the origin and nature of the monocyte-derived, DC-like cells generated in vitro 27. The ability of these cells to provide activation signals, of both antigen-specific and non-specific triggers, can vary vastly among DC subsets or lineages, and depends on their functional status 28–31. Among them, a unique human DC subset (CD11c+CD141+), with superior antigen cross-presentation capacity and expressing the XC chemokine

receptor 1 (XCR1+), has recently been identified by several groups as the homologue of mouse CD8α+ DC 32–35. As with their murine counterparts, this type of DC was found to be effective activators of CD8+ cytotoxic T cells, which IWR-1 manufacturer may have important implications in the design of new human DC vaccines. Moreover, in addition to subset-dependence, the functional properties of DC are also associated with the maturation status of the cell. Immature DC are in a so-called “antigen-uptake mode”, with low cell surface expression of MHC class I and class II molecules, which

can be rapidly enhanced upon exposure to maturation or activation signals, acquiring subsequently the “antigen-presenting mode”. The low MHC expression may therefore affect the ability of immature DC to present antigen to T cells. Under certain conditions, DC can even exert tolerogenic effects by producing immunosuppressive molecules, oxyclozanide or by inducing regulatory T cells, to inhibit the immune system 1, 8, 24, 36. The concept of tolerogenic DC has become far more appreciated. It is now recognised that while immunogenic DC play an important role in host defence, their tolerogenic counterparts are crucial for the maintenance of self-tolerance, being part of a built-in mechanism to avoid autoimmunity 37. It has been demonstrated that, under the tumourigenic microenvironment, the host DC possessed a typical tolerogenic, or regulatory, phenotype 38. DC, as a double-edged sword, can therefore induce either active immunity or tolerance depending on their functional conditions. The types and functional status of DC, hence the immunogenic “quality” or nature of the cell vectors employed for tumour vaccine delivery, are therefore of critical importance. Various attempts have subsequently been made in order to generate DC with a highly immunogenic phenotype.

The day before adoptive transfer, recipient mice were treated where indicated with 25 mg/kg CTLA-4-Ig. Five hours after adoptive transfer the recipient groups were challenged with DNFB by the standard procedure and ear swelling measured 24, 48 and 72 h post-challenge. A second adoptive transfer experiment was conducted where biopsies were taken from the inflamed ear 48 h post-challenge. These were analysed for their content of different cytokines

and chemokines, as described previously, in order to investigate whether the changed cytokine and chemokine expression after CTLA-4-Ig treatment is due to a direct suppressive effect on the keratinocytes or if it can be explained by a decreased infiltration AZD2014 of effector cells after CTLA-4-Ig treatment. To investigate binding of CTLA-4-Ig on lymph node cells in the inguinal lymph node after sensitization, groups of mice (n = 5) were treated with CTLA-4-Ig or isotype control (25 mg/kg). The next day all mice were sensitized with 0·5% DNFB, as described above. Subsequently, mice were killed 3, LY2835219 supplier 4 and 5 days after sensitization and single cells

from the inguinal lymph node were prepared for flow cytometric analysis as described above and the cell suspensions were blocked with anti-CD32/CD16 (Fc block; BDBiosciences) for 10 min and stained with the following anti-mouse monoclonal antibodies (mAb): anti-human IgG1-APC (Jackson Immunoresearch, West Grove, PA, USA), CD45-Efluor605 (eBiosciences), TCR-β-Qdot655 (Invitrogen), CD19-V450 (BDBiosciences), CD11c-PECy7 (BDBiosciences), I-A/E-FITC (eBiosciences) and CD86-PE (eBiosciences) for 30 min. Flow cytometric analysis of samples was analysed on a BD LSRII flow cytometer equipped with a blue, red and violet laser and data were analysed in BD fluorescence activated cell

sorter (FACS) Diva software, version 6·1.3. DCs were gated as CD45+TCR-β–CD19−, MHCII+ and CD11c+, while B cells were gated as CD45+CD19+ cells, and the level of human IgG1+ DCs and B cells together with CD86+ DCs and B cells were investigated. To investigate whether CTLA-4-Ig is able to suppress hapten-induced inflammation in vivo, two mouse models of contact hypersensitivity very were analysed: the DNFB- and oxazolone-induced CHS models, respectively. BALB/c mice were treated with CTLA-4-Ig or control proteins (hIgG1Fc) and subsequently sensitized on day 0. Five (DNFB) or 6 (oxazolone) days later, mice were challenged with hapten, and ear thickness measured 24, 48 and 72 h later. Control groups included mice which were sensitized with acetone/olive oil but challenged with DNFB or oxazolone, and mice which were treated with only acetone/olive oil in both the sensitization and challenge phases. Figure 1 shows the ear-swelling response after 24 h (Fig. 1a,c) and summarized as area under the curve (AUC) from 0–72 h (Fig. 1b,d); the data confirm that CTLA-4-Ig mediates a dose-dependent suppression of the ear-swelling response in both models.

Initial encounter with a pathogen and, hence, initial Th-cell

polarization will most likely occur solely by the tissue-resident DCs or, in case of tse-tse fly-mediated blood infection with trypanosomes, steady-state DCs. Tip-DCs develop later during infection from recruited monocytes and by GM-CSF secreted from T cells at the site of inflammation. Others reported that the steady-state occurring splenic DC subsets (CD8α−, CD8α+ or plasmacytoid DCs) show intrinsic differences to mount preferentially a Th1- or Th2-cell biased response 8, 55, 56. Thus, our BM-DC equivalents to Tip-DCs might play a selleck products decisive role in dampening or modulating the initially mounted Th-cell response to effectively eliminate the invading pathogen, a process also referred to as “success-driven”

Th-cell modulation 57. The functional difference of inflammatory vs steady-state occurring DCs might explain the reason why DCs indirectly activated by inflammatory mediators in vivo failed to mount Th2-cell responses, but inflammation drives Th2-cell differentiation at the Tip-DC level 27, 52. The analyses of our microarray data indicated that (i) TNF, the AnTat1.1 mfVSG and the MiTat1.5 sVSG regulated only Erlotinib manufacturer a limited set of genes in DCs as compared with LPS, (ii) the regulation patterns of TNF, AnTat1.1 mfVSG, and the MiTat1.5 sVSG are widely overlapping, and (iii) the differences between TNF (only proinflammatory) and AnTat1.1 mfVSG or the MiTat1.5 sVSG (presumed antiparasitic Th2-cell immunity) are remarkably

few. Our findings that TNF induces less gene regulation as compared with LPS is in agreement with the findings using a DC line 58 and also the general inflammatory pattern of 24 genes we found, shared remarkable overlap with the 44 genes that have been found by others 40, sharing key factors such as CD40, IL-1β, and IL-6. While LPS induced the same 24 genes, it regulated many more others, suggesting that inflammatory semi-maturation may represent more a quantitatively different state of maturation, rather than a completely L-gulonolactone oxidase different quality. One marked difference is the absence of IL-12p40 in our general inflammatory profile of 24 genes, which appeared only after LPS stimulation. This may be due to the fact that in the studies with the D1 line only pathogens but not inflammatory mediators were included and IL-12p40 thereby reflects pathogen stimulation. In addition, the lack of genes specifically regulated by mfVSG and MiTat1.5 sVSG would indicate an immune response against T. brucei is missing. The Th2-cell response generated by mfVSG and MiTat1.5 sVSG-matured DCs was expected to result in an enhanced isotype switches and IgG1 and IgE production in the asthma model. However, here the two VSG antigens behaved like TNF, i.e. “only inflammatory.

We have proposed a template-based scoring

function to determine the reliability of protein–protein interactions36 and to identify template-based homologous protein complexes42 derived from a structural complex. To measure the protein–peptide Buparlisib concentration interaction score, the scoring function is defined as: in which Evdw is the interacting van der Waals force; and ESF are special bonds, for instance the hydrogen bond, electrostatic forces and the disulphide bond. Esim is the similarity score of template interfaces, whereas Econs is the couple-conserved amino acid score. W constant has been set to 3, based on our previous research on protein–protein interactions. To some extent, anchor motifs have been successful in the prediction of CD8 T-lymphocyte epitopes.19,43,44 The substitution drug discovery of anchor motifs at P2

tyrosine (Y) or at P9 isoleucine (I) with glycine (G) abolished the binding of variant peptides, such as SG, to H-2Kd molecules (Table 1, Fig. 1a and Supplementary material, Fig. S2). The replacement of the anchor motif P5 phenylalanine (F) with glycine (G) blocked the binding of the variant peptide GQ to H-2Kb molecules (Table 1; Fig. 1b). These results have demonstrated the decisive role of anchor motifs in the binding of epitopes to MHC class I molecules. In contrast to this observation, very previous studies have shown that many immunogenic and protective epitopes do not contain known anchor motifs.22,45,46

In our experimental systems, exclusive of glycine (G), any substitution of known anchor motifs that reduced the binding of peptides to MHC class I molecules was still recognised by virus-specific CD8 T lymphocytes for fewer IFN-γ responses, for instance histidine (H) or cysteine (C) (Table 1; Figs 1c and 2a). These observations have indicated the limitation of anchor motifs to sort all potential epitopes with less binding affinity to MHC class I molecules.22 The substitution of the anchor motif P2 (Y) with phenylalanine (F) did not affect the binding affinity of SF to H-2Kd molecules, which was comparable to M2:82–90 (Table 1; Fig. 1c). The placement of cysteine (C), histidine (H) or tryptophan (W) at the P2 anchor motif reduced the binding affinity of variant peptides to H-2Kd molecules, resembling SC, SH and SW (Table 1; Fig. 1c and Supplementary material, Fig. S3). Side chains of anchor motifs have a significant impact on the binding affinity of epitopes to MHC class I molecules. In contrast to the positive correlation between MHC class I binding affinity and epitope predictability, in recent years many epitopes with lower binding affinity to MHC class I molecules and subdominant epitopes have been identified as protective.

Due to Akt inhibitor the high non-specific background of polyclonal anti-HAX1 Ab generated in mouse, immunoprecipitation of HAX1 with anti-HAX1 mAb (clone 52/HAX1, BD Biosciences, Heidelberg, Germany) was performed prior to detection.

Immunoprecipitates and cell lysates were separated by SDS-PAGE and transferred to an Immobilon™-P polyvinylidene difluoride (0.45 μm) membrane. The membrane was probed with primary (anti-HAX1 (clone E-20), anti-β-actin (clone C-4, both Santa Cruz Biotechnology, Santa Cruz, CA, USA)) and secondary Ab conjugated with HRP. The signal was detected with Pierce SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL, USA). Serial dilutions (in PBS/0.1% BSA) of mouse sera were incubated on NUNC Maxi-Sorp™ High Protein-Binding Capacity ELISA 96-well plates coated with goat anti-mouse IgM, goat anti-mouse IgG1, goat anti-mouse IgG2a or rat anti-mouse IgE (all from EGFR inhibitor SouthernBiotech, Birmingham, AL, USA). Plates were developed using alkaline phosphatase-conjugated goat anti-mouse IgG1, goat anti-mouse IgG2a and goat anti-mouse IgM (all from SouthernBiotech) or rat anti-mouse IgE (BD Biosciences). Signals were visualized by addition of p-nitrophenyl phosphate substrate (Sigma-Aldrich, St. Louis, MO, USA) and optical densities were measured at 405 nm (with 492 nm as the reference wavelength). Ten-week-old mice were sacrificed and resting splenic B and CD4+ T cells were isolated

by negative selection (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany). For in vitro proliferation assays, lymphocytes (5×106–1×107/mL) were incubated in the dark with 5 μM CFSE (Invitrogen, Carlsbad, CA, USA) in PBS for 10 min at RT and washed with complete RPMI 1640 medium (PAA Laboratories, Pasching, Austria). Before flow cytrometric analysis, B lymphocytes were seeded in triplicates dipyridamole at a density of 105/well in 96-well

flat-bottom plates and cultured in RPMI 1640 complete medium (RPMI 1640 supplemented with 10% FBS (Gibco®, Invitrogen), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 mg/mL streptomycin and 50 μM 2-ME (all PAA Laboratories) with the following additives for 3 days: LPS (10 μg/mL; Sigma-Aldrich), or anti-IgM F(ab’)2 (5 μg/mL, 61–5900; Zymed, San Francisco, CA, USA) plus anti-CD40 (5 μg/mL, 3/23; BD Biosciences), or anti-CD40 (5 μg/mL, BD Biosciences) plus IL-4 (10 ng/mL, R&D Systems, Minneapolis, MN, USA). T lymphocytes were seeded in triplicates at a density of 2–5×105/well in 96-well flat-bottom plates and cultured in MEM complete medium (αMEM supplemented with 1% mouse serum, 1× non-essential aa (Gibco®, Invitrogen), 2 mM L-glutamine, 1 mM sodium pyruvate, 20 mM HEPES, 50 U/mL penicillin, 50 mg/mL streptomycin and 50 μM 2-ME (all PAA Laboratories) with ConA (2 μg/mL; (Sigma-Aldrich) alone, or anti-CD3e (0.025–0.25 μg/well, 17A2; eBioscience, San Diego, CA, USA) plus anti-CD28 (0.3 μg/mL, 37.