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“Cockroaches have been identified as one of the major indoor allergens inducing perennial rhinitis and asthma. Per a 1s are a group of the major allergens from American cockroach. Although Per a 1s are major allergens from American cockroach, factors

contributing to the allergenicity of Per a 1s are still poorly defined. To investigate the effects of Per a 1s on the expression Selleckchem IWR1 of PARs and the release of proinflammatory cytokines from mast cells. Per a 1.0101 and Per a 1.0104 were cloned from American cockroach and then expressed in Ivacaftor supplier Eschericia coli. The purified allergens were used to stimulate P815 mast cells, and the expression of protease-activated receptors (PARs) was determined by real-time RT-PCR and flow cytometry. The levels of IL-4 and IL-13 in culture

media were detected with ELISA. Sera from 80 and 77.3% of cockroach allergy patients reacted to recombinant Per a (rPer a) 1.0101 and rPer a 1.0104, confirming they are major allergens. Both rPer a 1.0101 and rPer a 1.0104 had no enzymatic activity, but rPer a 1.0101 upregulated the expression of PAR-1 and PAR-2, and rPer a 1.0104 enhanced Meloxicam the expression of PAR-1 and PAR-4 proteins. Both recombinant allergens were able to increase the release of IL-4 and IL-13 from P815 mast cells. This is the first study aiming to investigate functions of group 1 allergens of American cockroach. rPer a 1.0101 and rPer a 1.0104 have the capacity to upregulate

the expression of PARs and to enhance Th2 cytokine production in mast cells. Cockroach allergens have been identified as one of the major indoor allergens, which induce IgE-mediated allergic respiratory illness such as perennial rhinitis and asthma. Sensitization to cockroaches is well recognized in human beings throughout the world. The two most common domiciliary species associated with allergic diseases are the American cockroach (Periplaneta americana) and German cockroach (Blattella germanica) [1]. Three different types of major allergens have been identified from American cockroach, named Per a 1, Per a 3 and Per a 7 [2]. Per a 1 is a group of major allergens consisting of five members, Per a 1.0101, Per a 1.0102, Per a 1.0103, Per a 1.0104, Per a 1.0105 and Per a 1.02, known as isoallergens [3]. Among them, Per a 1.0101 showed 79.2% and 94% amino acid sequence identity with Per a 1.0104 and Per a 1.0102, respectively [4]. There is no cysteine and potential N-glycosylation site in Per a 1 molecules [3].

Thus, in the absence of SAP or CD84, CD4+ T cells are unable to form stable conjugates with B cells and cannot deliver help to B cells.47,51 In addition, this prevents the CD4+ T cells from receiving signals from B cells that regulate the formation or maintenance of Tfh cells. While

it is thought that Tfh cell development is a multi-step process with initial activation on DCs, followed by secondary signals provided by B cells, several recent findings have challenged this view. Many reports have demonstrated that Tfh cell numbers are decreased in the absence of B cells or when T–B cell interactions are disrupted.5,9,16,35,36 However, we recently showed that in the absence of antigen presentation by B cells, Tfh cell development (as indicated by surface phenotype and GC localization) could at least partially be CHIR-99021 ic50 rescued in the presence of abundant antigen, which prolonged presentation by DCs.9 Consistent with this, a recent study found that mTOR inhibitor Tfh cells also developed in B cell-deficient mice in response

to chronic viral infection.52 This suggests that the requirement for B cells results not from a unique signal that B cells provide, but because Tfh cells need prolonged antigen stimulation and B cells often quickly become the only cells capable of presenting antigen to the T cells.9 A requirement for prolonged antigen presentation is consistent with data indicating a crucial role of TCR signalling in Tfh cell development. For example, many of the features of Tfh cells, such as up-regulation of CXCR5 and PD-1 and down-regulation of CD127, are observed in T cells following TCR stimulation.3,6,53,54 Moreover, it has been shown that high-affinity Cytidine deaminase T cells are preferentially selected to become Tfh cells.55 The restriction of antigen presentation to the B cells presumably occurs ordinarily because, first, the B cell receptor allows for efficient uptake of antigen and secondly, as the T cells move

into the B cell follicle and then the GC, these are the antigen-presenting cells (APCs) which the T cells encounter. Furthermore, several new papers support the idea that early activation on DCs is able to drive differentiation of Tfh cells. They demonstrated that CD4+ T cells with a Tfh cell phenotype – high CXCR5, PD1, IL-21 and Bcl-6 expression – could be identified early on in the response (e.g. day 3)21–23 in the interfollicular zone or outer follicle.21,22 This early appearance of Tfh-like cells was independent of B cells;21,23 however, the continued maintenance of these cells was disrupted in the absence of B cells.21–23 This suggests that a role of the second round of signalling, usually provided by B cells, may be to maintain a Tfh cell phenotype or the survival of Tfh cells rather than to drive unique differentiation events. Generation of the different Th lineages is associated with the action of particular cytokines.

Results presented in Supporting Information Fig. 3 show that the polyclonal anti-EBI3 Ab is specific for EBI3. The monoclonal Ab against p35 (clone27537), IL-12p40 (mAb609), IL-12p70 (mAb611), IL-27 (mAb25261), and TGF-β (mAb240) were purchased from R&D Systems. The neutralizing polyclonal anti–IL-10 Ab (PAL-hIL10) was obtained from Strathmann Biotech (Hannover, Germany). MAb EB-I against IFN-α

was kindly provided by G. Adolf (Boehringer Ingelheim). PBMC were isolated from buffy coats obtained from the Red Cross in Austria. Heparinized whole blood of healthy donors was separated by standard density gradient centrifugation with Ficoll-PaqueTM Plus (GE Healthcare Chalfont St. Giles, UK). Subsequently, T cells (total-CD3+ T cells used unless stated otherwise), CD4+, CD8+ find more and CD25– T cells, and monocytes were separated by magnetic sorting using the MACS technique (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously Navitoclax solubility dmso 34. Naïve T cells were isolated from CB. CB samples from healthy

donors were collected during healthy full-term deliveries. Approval was obtained from the Medical University of Vienna institutional review board for these studies. CB-T cells used in this study were CD45RA+ (92±3%) and CD45RO−. DC were generated by culturing purified blood monocytes for 7 days with a combination of GM-CSF (50 ng/mL) and IL-4 (100 U/mL). Preparation and purification of rhinoviruses were performed as described 34. DC were treated with HRV14 for 1 day (R-DC) at a titer of 1 TCID50 (50% tissue culture infectious

dose) per cell. To examine the suppressor activity of the SN of R-DC-induced Treg, T cells were added to R-DC or DC in a 10:1 or 5:1/T-cell:DC ratio. These SN were harvested after 1–3 days of coculture and 100 μL/well were added to different MLR. Centricon YM-50 filters (Millipore, Bedford, MA, USA) were used for size fractionation of the SN. The fraction containing molecules >50 kDa was compared to the fraction containing molecules <50 kDa in an allogeneic MLR. The T cells of the coculture were also investigated by intracellular staining or analyzed via real-time PCR. For the MLR, allogeneic, purified T cells (1×105) were incubated with graded numbers of DC. Experiments were performed in Alectinib nmr 96-well round bottom cell culture plates in RPMI 1640 medium supplemented with 10% FBS. Proliferation of T cells was monitored by measuring (methyl-3H)TdR (ICN Pharmaceuticals, Irvine, CA, USA) incorporation on day 5 of culture. Cells were harvested 18 h later, and radioactivity was determined on a microplate scintillation counter (Packard Instruments, Meriden, CT, USA). Assays were performed in triplicates. For Fig. 1 preactivated T cells were harvested, irradiated (30 Gy, 137Cs source) and tested for their suppressive function, for Supporting Information Fig. 1 and 4A preactivated T cells were not irradiated.

Maximum concentrations of these cytokines were observed in animals treated with the combination of cisplatin + 78 kDa along with MPL-A. As compared to this group, the mice immunized with cisplatin + 78 kDa showed significantly (P < 0·05) lesser concentration of these cytokines. Least concentration of these cytokines was observed in the animals treated with the immunotherapy alone (Figure 4a,b). The levels of Th2-regulated cytokine, IL-10 and IL-4, were significantly lesser in treated animals as compared to the infected controls. Maximum levels

of this cytokine were observed in the infected controls. Animals treated with cisplatin + 78 kDa + MPL-A showed least concentration SCH 900776 clinical trial selleck kinase inhibitor of IL-10 and IL-4 (Figure 5a,b). As compared to this group, the concentration of the cytokine was significantly (P < 0·05) higher in the animals treated with cisplatin + 78kD followed by cisplatin. It has been well established that the success of any chemotherapy is often dependent on the type of immune response generated by the infected host, and in leishmaniasis, a drug is considered successful if it results in generation of antigen-specific T cells and delayed hypersensitivity. Due to the existence of close association

between chemotherapy and cell-mediated immunity, immunochemotherapy is thought to be more agreeable for treatment for VL. Therefore, in the present study, the therapeutic potential of immunochemotherapy was tested by treating the BALB/c mice with a novel antileishmanial drug cisplatin along with a 78 kDa antigen formulated with an adjuvant (MPL-A). Earlier studies have shown that significant inhibition was observed in golden hamsters infected with L. donovani when treated with a combination of low doses

of both Stibanate and poly ICLC plus l-arginine [16]. Similarly, we tested a low dose of cisplatin (0·5 mg/kg body wt.) and combined it with 78 kDa antigen along with the adjuvant as parasite antigens that preferentially stimulate the induction of significant protection through Th1 response represents a rational approach for vaccines Dimethyl sulfoxide against leishmaniasis. This has been demonstrated in our earlier study carried out by Nagill and Kaur, [6] where experimental infection of mice immunized with 78 kDa antigen along with MPL-A induced significant protection against L. donovani infection. Maximum reduction in parasite load in the present study was observed in animals treated with cisplatin +78 kDa + MPL-A followed by cisplatin + 78 kDa more than any individual therapy. This is in consistence to an earlier study carried out by Tan et al., [24] which showed that both low-dose cisplatin (0·6 mg/kg) and xenogeneic endoglin (10 μg/mouse) resulted in significant tumour growth inhibition.

*Remarks: The Thailand peritonitis study group included (by alphabet list) SOHARA EISEI, SUSA KOICHIRO, RAI TATEMITSU, ZENIYA MOKO, MORI YUTARO, SASAKI SEI, learn more UCHIDA

SHINICHI Department of Nephrology, Tokyo Medical and Dental University Introduction: Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding the WNK1 and WNK4 kinases were known to be responsible. Recently, two genes (KLHL3 and Cullin3) were newly identified as responsible for PHAII. KLHL was identified as substrate adaptors in the Cullin3-based ubiquitin E3 ligase. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and NCC were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is true substrate(s) of KLHL3-Cullin3 E3 ligase, in other words, what is the true pathogenesis of PHAII caused buy IWR-1 by KLHL3 mutation. Methods: To investigate the pathogenesis of PHAII by KLHL3 mutation, we generated and analyzed KLHL3R528H/+ knock-in mice. Results: Under high-salt diet, the systolic blood pressure Sclareol of KLHL3R528H/+ mice was higher

than that of wild-type mice. Metabolic acidosis and hyperkalemia were also observed in KLHL3R528H/+ mice. Moreover, the phosphorylation of OSR1, SPAK and NCC were also increased in KLHL3R528H/+ mice kidney. These data clearly indicated that the KLHL3R528H/+ knock-in mice are ideal mouse model of PHAII. Interestingly, both of WNK1 and WNK4 protein expression was significantly increased in KLHL3R528H/+ mouse kidney, indicating that these

increased WNK kinases caused the activation of WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3R528H/+ knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptide to the whole KLHL3, using fluorescence correlation spectroscopy. The diffusion time of TAMRA-labeled WNK1 and WNK4 peptide was not affected by the addition of mutant KLHL3 R528H protein, indicating that neither WNK1 nor WNK4 bind to mutant KLHL3 R528H. Conclusion: Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases, due to impaired KLHL3-Cullin3 mediated ubiquitination, cause PHAII by KLHL3 R528H mutant. Our findings also implicated that both WNK1 and WNK4 are physiologically regulated by KLHL3-Cullin3 mediated ubiquitination.

Additional features were detected in this tumor that are known to be associated with an unfavorable this website prognosis, including loss of

p16 expression and gains of chromosomes 1q and 12. The patient experienced the most rapid downhill course reported to date for intracranial Ewing sarcoma, developing multiple extracranial metastases at 2 months and dying 6 months after the initial operation. “
“V. Leinonen, A. M. Koivisto, S. Savolainen, J. Rummukainen, A. Sutela, R. Vanninen, J. E. Jääskeläinen, H. Soininen and I. Alafuzoff (2012) Neuropathology and Applied Neurobiology38, 72–86 Post-mortem findings in 10 patients with presumed normal-pressure hydrocephalus and review of the literature Aims: Neuropathological features of idiopathic normal-pressure hydrocephalus (iNPH) are poorly characterized. Brain biopsy during life may help in the differential diagnosis of dementia, but post-mortem validation of biopsy findings is scarce. Here we review and

report brain biopsy and post-mortem neuropathological findings in patients with presumed NPH. Methods: We evaluated 10 patients initially investigated by intraventricular pressure monitoring and a frontal cortical biopsy for histological and immunohistochemical assessment ABT-263 ic50 as a diagnostic procedure for presumed NPH. Results: Out of the 10 patients, eight were shunted and seven benefited. Until death, six had developed severe and two mild cognitive impairment. One was cognitively unimpaired, and one was mentally retarded. Three subjects displayed amyloid-β (Aβ) aggregates in their frontal cortical biopsy obtained at the initial procedure. One of these patients developed Alzheimer’s disease during a follow-up time of nearly 10 years. One patient with cognitive impairment and NPH suffered from corticobasal degeneration. In six patients

Fludarabine clinical trial various vascular lesions were seen at the final neuropathological investigation. Five of them were cognitively impaired, and in four vascular lesions were seen sufficient in extent to be considered as causative regarding their symptoms. Conclusions: The frequent finding of vascular pathology in NPH is intriguing, suggesting that vascular alterations might be causative of cognitive impairment in a notable number of patients with NPH and dementia. Brain biopsy can be used to detect Aβ aggregates, but neuropathological characteristics of iNPH as a distinct disease still need to be discovered. “
“Wnt activation in medulloblastomas is associated with good outcome. Upfront testing and risk-adapted stratification of patients will be done in future clinical studies. In a cohort of 186 pediatric medulloblastomas our aim was to identify the optimal methods in standard clinical practice to detect this subgroup. Nuclear accumulation of ß-catenin was analyzed by immunohistochemistry (IHC). DNA of FFPE tissue was amplified by PCR for single-strand conformation polymorphism analysis and direct sequencing of CTNNB1 exon 3.

The prevalence of low serum bicarbonate at baseline was 17.3%. Lower estimated glomerular filtration rate had the strongest relationship Deforolimus in vivo with low serum bicarbonate. Factors associated with higher odds of low serum bicarbonate, independent of estimated glomerular filtration rate, were urinary albumin/creatinine ≥10 mg/g, smoking, anaemia, hyperkalaemia, non-diuretic use and higher serum albumin. These and younger age, higher waist circumference,

and use of angiotensin converting enzyme inhibitors or angiotensin receptor blockers associated with negative Δ serum bicarbonate in linear regression models. Several factors not typically considered to associate with reduced serum bicarbonate in chronic kidney disease were identified including albuminuria ≥10 mg/g, anaemia, smoking, higher serum albumin, higher waist circumference, and use of angiotensin converting enzyme inhibitors or angiotensin receptor blockers. Future studies should explore the longitudinal effect of these factors on serum bicarbonate concentration. “
“Nephrotic syndrome is one of the most this website commonly diagnosed

primary kidney diseases and its progressive forms can lead to chronic kidney disease and or end-stage renal disease. Steroid-resistant nephrotic syndrome is defined by resistance to standard steroid therapy and it remains one of the most intractable causes of kidney failure. Mutations in NPHS2, which encodes for podocin, an integral membrane protein of the glomerular epithelial cells (podocytes), represent a frequent cause of steroid-resistant nephrotic syndrome worldwide. This study was aimed at screening for known NPHS2 mutations in Indians with nephrotic syndrome. We screened a cohort of 484 subjects from the southern Indian population for the presence of four missense mutations G92C, P118L, R138Q and D160G within the NPHS2 gene using

tetra primer ARMS PCR. Our results revealed that these mutations were seen only among the patients (14.02%) and were absent in the controls, suggesting their disease-causing nature. Further categorization revealed that these mutations were together responsible for 18.5% of steroid-resistant cases buy Gemcitabine in our study group. Conversely, the studied mutations were not found in the controls as well as in the patients with steroid-sensitive nephrotic syndrome. This is the first such report from India. More studies are warranted to establish the frequency of NPHS2 mutations in the Asian–Indian population and such analysis may help in developing mutation(s)-specific therapeutic interventions in the future. “
“Patients with end-stage kidney disease have significantly increased morbidity and mortality. While greater attention has been focused on advanced care planning, end-of-life decisions, conservative therapy and withdrawal from dialysis these must be supported by adequate palliative care incorporating symptom control.

In the common form, there was no difference

between the two, while in the pure form, Japanese cases were usually of young onset with parkinsonism as the chief symptom and Euro-American cases were of older onset with progressive dementia as the chief symptom, similar to the common form. Around that time, the term “senile dementia of Lewy body type” was proposed by Perry et al.,13 and the term “Lewy body variant of Alzheimer’s disease by Hansen et al.14 in 1990. In 1995, the first International Hydroxychloroquine cell line Workshop7 was held in Newcastle-upon-Tyne, UK. Then, the term “dementia with Lewy bodies” (DLB) was proposed, and the clinical and pathological diagnostic criteria (Consortium on Dementia with Lewey Bodies guidelines)8 were published in Neurology in 1996. In 1996, we proposed the cerebral type of Lewy body disease,15 in which progressive dementia without parkinsonism was the main symptom, and cortical Lewy bodies were marked in the cerebral cortex, but only rare Lewy bodies were present in the brain stem. The presence of the cerebral type means that Lewy bodies could occur first in the cerebral cortex and later develop in the brain stem. As above-mentioned,

we proposed the term Lewy body disease in 1980,11 and since then, we have insisted that DLB(D), PD, and Parkinson’s disease with dementia Copanlisib cost (PDD) should be understood within the spectrum of Lewy body disease.16 This insistence has been recently accepted by the International Workshop and the International Working Group on DLB and PDD in 200517 and in 2006,18 respectively. In 1997, two very important findings

were reported. Polymeropoulas et al.19 reported the mutation of the alpha-synuclein gene in familial PD, and Spillantini et al.20 reported alpha-synuclein in Lewy bodies. Since then, alpha-synuclein has received attention in neuropathological and molecular biological studies. Our alpha-synuclein immunohistochemical examination of materials from the first DLBD case disclosed much more marked Lewy pathology in the cerebral cortex than we had expected. Only this Lewy pathology could explain the profound dementia in this case. In addition, only this case also had both PD and AD. Therefore, the case is now diagnosed as having a common form9 (especially AD form10) of DLB(D). The authors thank Mrs Chie Haga, and Dr Haruhiko Akiyama, Tokyo Institute of Pschiatry for technical assistance. “
“Heterozygous LIS1 mutations are the most common cause of human lissencephaly, a human neuronal migration defect, and DCX mutations are the most common cause of X-linked lissencephaly. Lissencephaly is characterized by a smooth cerebral surface, thick cortex and dilated lateral ventricles associated with mental retardation and seizures due to defective neuronal migration. Lissencephaly due to the heterozygous loss of the gene LIS1 is a good example of a haploinsufficiency disorder.

1 and 6.3 μm. The relative standard deviation in the diameters was in the range of 4% for resting and swollen spores but increased to about 10% for opsonised spores. The comparison of phagocytosis assays is done by computing characteristic quantities referred to as phagocytosis ratio, ratio for phagocyte-adhesion and the fungal aggregation ratio. These quantities, which are introduced and computed in the subsequent sections, can be retrieved only from an image-based analysis Doxorubicin in vitro of phagocytosis assays. The phagocytosis ratio is defined by In Fig. 4, the ratios for adhesion, phagocytosis and aggregation are shown. Adhesion is lower in the virulent than in the attenuated strain if resting

and opsonised spores were applied, but higher if swollen spores were utilised. The phagocytosis ratio was higher in the virulent than in the attenuated strain for all three spore conditions, but regressive

if opsonised spores of the attenuated strain were used in the phagocytosis assay. Generally, phagocytosis ratios pr differ significantly between the virulent and the attenuated strain (Fig. 4). The spores from the virulent strain are phagocytosed to a higher extent as the attenuated strain for all three spore conditions, resting, swollen selleck compound and opsonised spores (Fig. 4a). Interestingly, for the virulent strain we observed pr(resting) ≈ pr(swollen) < over pr(opsonised), whereas for the attenuated strain pr(resting) < pr(swollen) ≈ pr(opsonised). It can be concluded that opsonisation has a negligible effect for phagocytosis of spores from the attenuated strain, however, has a pronounced

effect in the virulent strain. We performed statistical tests for the significance of the phagocytosis ratio. Since the data were not normally distributed, we applied the Wilcoxon rank-sum test for any two pairs of the two strains and the three conditions. In Fig. 4b, we show a significance map, where the P-value was colour-coded as follows: black for P ≥ 0.05, red for P < 0.05, green for P < 0.01 and blue for P < 0.001. It turns out that all differences in the phagocytosis ratios are significant (P < 0.001), except for the virulent strain between resting and swollen spores as well as for the attenuated strain between swollen and opsonised spores with P-values P ≥ 0.05. The ratio for phagocyte-adhesion is defined as In analogy to the procedure for the phagocytosis ratio, we performed the Wilcoxon rank-sum test to determine the significance of the results for the adhesion ratio. We found that there is no significant difference between the resting and the opsonised spores in the virulent strain, except for the virulent strain between resting and opsonised spores as well as between the swollen spores of the virulent and resting spores of the attenuated strain (Fig. 5b).

Cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin,

100 mg/ml streptomycin, 50 μg/ml gentamicin and 2 mm l-glutamine (all from Invitrogen, Eugene, OR) at 37° in a humidified 5% CO2 incubator. Purified CD4+ subsets were activated in the presence of anti-CD3 antibody (purified OKT3 0·5 μg/ml) and autologous PBMCs irradiated with 40 Gy gamma-radiation, as a source of multiple co-stimulatory ligands provided AZD2014 purchase by B cells, dendritic cells and macrophages found in these populations.28 In other experiments, cells were cultured in the presence of recombinant human (rh) IL-2 (5 ng/ml), click here IL-7 (10 ng/ml) or IL-15 (5 ng/ml) (all from R&D Systems, Minneapolis, MN). Cytokines were added at the beginning of the cell culture and not replenished. These cells were harvested at different times for phenotypic

and functional analyses. The PBMCs were stimulated with 10 μg/ml of purified protein derivative (PPD; Statens Serum Institut, Copenhagen, Denmark), 1/50 dilution of varicella zoster virus (VZV) -infected cell lysate, 1/200 dilution of Epstein–Barr virus (EBV) -infected cell lysate or 1/50 dilution of herpes simplex virus (HSV) -infected cell lysate (all from Virusys, Taneytown, MD). A CMV-infected cell lysate (used at 1/10 dilution) was prepared by infecting human embryonic lung fibroblasts with the Towne strain of CMV (European Collection of Animal Cell Cultures) at a multiplicity of infection

of 2. After 5 days, the infected cells were lysed by repeated freeze–thaw cycles. The PBMCs were left unstimulated or stimulated with antigenic lysates for 15 hr at 37° in a humidified CO2 atmosphere, with 5 μg/ml brefeldin A (Sigma-Aldrich) added after 2 hr. The cells were surface stained with peridinin chlorophyll protein-conjugated (-PerCP) CD4, phycoerythrin-conjugated Acetophenone (-PE) CD27 and phycoerythrin-Cy7-conjugated CD45RA (BD Biosciences) on ice. After being fixed and permeabilized (Fix & Perm Cell Permeabilization kit; Caltag Laboratories, Buckingham, UK), cells were stained with allophycocyanin-conjugated (-APC) interferon-γ (IFN-γ). Samples were acquired on an LSR I flow cytometer (BD Biosciences). For bone marrow experiments, paired peripheral blood and bone marrow samples were stimulated and analysed in parallel.