These organs were disrupted and filtered through

a nylon mesh, and the cells were adjusted to 2·5 × 106 and then surface-labelled with fluorescein isothiocyanate (FITC) anti-rat CD4 (0·5 μg) and allophycocyanin (APC) anti-rat CD25 (0·25 μg). After this step, a staining for Foxp3 by using the phycoerythrin (PE) anti-mouse/rat Foxp3 Staining Set (eBioscience, San Diego, CA, USA) was performed according to the manufacturer instructions. After incubation with these antibodies, the cells were fixed in paraformaldehyde 1% and analysed with a FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometer and Flow Jo software (TreeStar, Ashland, OR, USA). EAE was induced by inoculation of 25 μg of myelin basic protein (MBP; Sigma, St Louis, MO, USA) emulsified with complete Freund’s adjuvant (CFA) containing 5 mg/mL of Mycobacterium butyricum, in the hind left footpad. Animals were daily Cell Cycle inhibitor evaluated for weight loss and clinical score. Signs of disease were graded as 0 (zero): no disease; 1: loss of tonicity in the distal portion of the tail; 2: total check details loss of tail tonicity; 3: hind limb weakness (partial paralysis); 4: complete hind limb paralysis and urinary incontinence and 5: moribund. The presence and amount of brain and spinal cord inflammatory infiltrates were assessed during EAE recovery phase (20 days after immunization) as previously described

(12). IFN-γ and IL-10 production G protein-coupled receptor kinase were also determined at this phase. For this, lymph node (popliteal + inguinal) cells were collected and adjusted to 2·5 × 106 cells/mL in RPMI supplemented with 10% fetal calf serum, 2 mm l-glutamine and 40 mg/L of gentamicin, in the presence of 10 μg/mL of myelin or 5 μg/mL of concanavalin A (ConA; Sigma). Cytokine levels were evaluated by ELISA in culture supernatants collected 72 h later, according to manufacturer’s instructions (R & D Systems, Minneapolis, MN, USA). ELISA sensitivity for IFN-γ and IL-10 was 19 and 31 pg/mL, respectively. Data were expressed as mean ± SD. Comparisons between groups were made by Student’s t-test or one-way anova with post-hoc Holm–Sidak test for

parameters with normal distribution and by Mann–Whitney U-test or Kruskal–Wallis test for parameters with non-normal distribution. Significance level was P < 0·05. Statistical analysis was accomplished with SigmaStat for Windows v 3.5 (Systat Software Inc., Witzenhausen, Hesse, Germany). A high number of EPG was detected 8 days after the first worm inoculation. The amount of eggs decreased by day 13 and was very low at days 20 and 27. No more eggs were detected 34 days after initial infection (Figure 1a). Evaluation of specific antibody levels by ELISA indicated significant production of IgG1 but not IgG2b (Figure 1b). The frequency of cells expressing the regulatory foxp3 marker was determined in spleen and lymph node cells.

e. characteristics TSA HDAC of the different agonistic mAb) deserves attention. In this regard, CD300e associates with DAP-12 in transfected cells. Yet, identification of the adaptor molecule(s) responsible for CD300e signaling in monocytes remains thus far elusive. Remarkably, CD300e ligation triggered functional effects in mDC resembling those induced by LPS, but different from the response previously reported in moDC

upon engagement of TREM-2 35 or hOSCAR 29. Although all these stimuli upregulated surface expression of co-stimulatory molecules (i.e. CD40 or CD86), CD300e cross-linking triggered a strong production of different pro-inflammatory cytokines (TNF-α, IL-6 and IL-8/CXCL8), whereas TREM-2 35 did not induce any detectable cytokine secretion and hOSCAR triggered only IL-8/CXCL8 release in moDC 29. These results suggested that mDC activation via CD300e might effectively contribute to the generation of an adaptive immune response. This hypothesis was further supported by the ability of CD300e-stimulated mDC to enhance the alloreactivity of naive T cells. Upon serum starvation and in the absence of growth factors, myeloid cells have been shown to undergo apoptosis. In monocytes, programmed cell death may involve CD95 (Fas) and the mitochondrial-mediated

pathway 36. Signaling through CD95 upon engagement by CD95L (FasL) results in the sequential activation of caspase 8 and caspase 3, ultimately leading to apoptotic cell death 37. It has Selleck ABT-263 been shown that this pathway can be inhibited in monocytes by LPS

or TNF-α 36. Accordingly, it was conceivable that the ability of CD300e engagement to prevent monocyte and mDC apoptosis might depend on an autocrine TNF-α-dependent inhibition of caspase 3. Yet, the lack of effect of neutralizing TNF-α ruled out this possibility. It is of note that hOSCAR is also able to prevent apoptosis of moDC despite not inducing secretion of TNF-α 29 consistent with the involvement of other mechanisms. Although Phloretin the function of activating receptors associated with ITAM-bearing adaptors expressed by myeloid cells has been extensively studied, their ligand specificity remains often ill defined. Some of these molecules may function as pathogen-associated molecular pattern receptors or, alternatively, could contribute to sensing self stress-inducible molecules 30, 38. Thus, the identification of the CD300e ligand is warranted to precisely understand its physiological role. Human peripheral blood samples were obtained from healthy donors according to guidelines approved by the Clinical Research Ethical Committee (CEIC-IMAS). PBMC were separated from fresh blood by Ficoll-Paque PLUS centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and extensively washed with PBS for platelet removal.

A link between low-grade inflammation and the presence of LVDD has been suggested by this study. Cytokine gene polymorphism plays important role in the risk of many diseases, including cardiovascular diseases (CVDs). Yilmaz et al. [124] have evaluated the role of cytokine gene polymorphism in carotid intima-media thickness (CIMT) and left ventricular mass index (LVMI) progression in non-diabetic haemodialysis (HD) patients. TNF-α and IL-10 polymorphisms were determined in the study. Risk factors for cardiovascular diseases have no difference between TNF-alpha rs1800629 high-/low-producer genotype Rapamycin groups. CIMT and LVMI progressions were detected

at higher levels in patients with high-producer genotypes (AA + AG) than in patients with the low-producer genotype (GG). The

rs1800629 polymorphism was strongly associated with C-reactive protein (CRP). Analysis also showed that the combination of high production of TNF-α and learn more low production of IL-10 was associated with higher average IMT, LVMI progression and elevated average CRP levels compared with a combination of low production of TNF-α and high production of IL-10. Association of TNF-α gene with spontaneous deep intracerebral haemorrhage was investigated by Chen et al. [125] in the Taiwan population. Deep parenchymal structure including the basal ganglia, thalamus, brainstem and cerebellum is the most frequently affected site of spontaneous intracerebral haemorrhage (SICH). Rost et al. [126] comprehensively reviewed the candidate genes of SICH reported during 1996–2007. Reported candidate genes that CYTH4 show association with SICH were involved in the

pathways of the vessel wall integrity (ACE, APOE, neprilysin, endoglin, TGF-β1), endothelial dysfunction (ACE), inflammation markers (IL-6, TNF) and haemostasis (APOE, CD-14, Factor VII and XIII, VKORC1). Spontaneous deep intracerebral hemorrhage (SDICH) risks were positively associated with TNF (rs1799964 C and rs1800629 A) in men but inversely associated with (rs1800630 A) in females [126]. There were significant interaction effects between gender and SNPs (rs1799964, rs1800630 and rs1800629) on SDICH risks. Kim et al. [127] carried out case–control studies including patients with ischaemic stroke, patients with silent brain infarctions SBIs and controls. Significant differences in the frequency of the TNF-α rs1800629 polymorphism were found between the patients with ischaemic stroke and the control group. The frequency of the TNF-α (rs1800629 GA + AA) genotype was higher in the group having highest homocysteine (tHcy) levels than in the group having lowest tHcy levels. The tHcy levels were significantly and inversely correlated with folate levels in the TNF-α (rs1800629 GG) and TNF-α (rs361525 GG) genotypes in the ischaemic stroke, SBI and control groups.

Emerging clinical and experimental data suggest that the injury to the conduction system may happen through a two-stage process, which is detailed in a review by Wahren-Herlenius and Sonesson [21]. In the first step, maternal anti-Ro autoantibodies bind to foetal cardiomyocytes,

which leads to calcium dysregulation, calcium overload and subsequent apoptosis. Anti-La antibodies then subsequently bind to apoptotic cardiomyocytes, which escalate the inflammatory cascade, activating infiltrating macrophages that secrete proinflammatory and profibrotic cytokines. The subsequent evolution of more severe tissue damage, including fibrosis and calcification of conduction tissue and surrounding myocardium, the second step of the process, probably requires a genetic predisposition or susceptibility in the foetus particularly given the discordant influence of the maternal autoantibodies in twins and siblings of affected foetuses and lack of consistent findings find more in the offspring of sera-positive women. Approximately 1–3% of foetuses and infants whose mothers are autoantibody positive develop AVB, and the risk of recurrence in subsequent offspring

is 17–18% [22–24]. Although 20–30% of the mothers have well-defined autoimmune disease, most are clinically asymptomatic and are only recognized to have the autoantibodies after the diagnosis Selleck Seliciclib of AVB is made in the foetus [14, 22]. In a prospective study of 15,000 pregnant women in the metropolitan Toronto area, we found 2.8% to have anti-Ro and/or anti-La autoantibodies (unpublished data, Maternal Autoantibodies in Pregnancy prospective study in Metropolitan Toronto). Although the subgroup

of sera-positive women at greatest risk of having an affected foetus is still not fully known, clinical observations have identified risk factors. In addition to those with a previously affected foetus [22–24], women with anti-52 kD-Ro antibodies appear to be at increased risk of having an affected foetus, and the nearly universal presence of anti-52 kD-Ro in affected mothers has suggested an important role in Cyclin-dependent kinase 3 the pathogenesis of AVB [23, 25]. Although absolute antibody titres have not been previously consistently linked to risk, a recent single centre investigation by Jaeggi et al. in 186 autoantibody-positive women, including 59 asymptomatic mothers, suggested that cardiac manifestations of NLE in general are associated with moderate (≥50 U/ml, 15% incidence) or high (≥100 U/ml, 85% incidence) maternal anti-Ro antibody titres [26]. This study further found foetal and neonatal cardiac manifestations to be independent of anti-La titres. This finding is in contrast with an earlier multicentre retrospective study of Gordon et al. which examined antibody titres in 125 mostly clinically symptomatic mothers of children with NLE [25]. In their cohort, they found the child of an anti-Ro (52 kD)-positive mother to have a risk of 2% of having AVB, which increased to 3.1% if the mother was anti-La positive as well [25].

Methods: The spinal cord and brain tissues of 13 sporadic ALS

(SALS) patients were investigated using immunohistochemical analysis. Results: TDP-43-positive inclusions in lower motor neurones of SALS patients were immunopositive for Smurf2 and pSmad2/3. find more Multiple immunofluorescence staining for Smurf2, pSmad2/3, TDP-43 and ubiquitin revealed co-localization of these four proteins within the inclusions in lower motor neurones of SALS patients. Furthermore, the loss of nuclear pSmad2/3 immunoreactivity was observed in cells bearing TDP-43 inclusions. In contrast, TDP-43-positive inclusions in the extramotor neurones in the brain of SALS patients were noticeably negative for Smurf2 and pSmad2/3. In addition, pSmad2/3 immunoreactivity was preserved in the nuclei of inclusion-bearing cells. Conclusions: This regional difference in the expression of Smurf2 and pSmad2/3 within TDP-43-positive inclusions might be one of the pathomechanisms underlying the loss of lower motor neurones and comparatively spared cortical neurones seen in ALS. “
“A case of unusual fibro-osseous lesion resembling osteoblastoma of the pineal region is reported, in a 50-year-old selleck kinase inhibitor man. The patient presented with a history

of headache, vomiting and generalized tonic-clonic seizures. CT scan showed a hyperdense lesion in the posterior third ventricle with obstructive hydrocephalus. On histopathology the lesion showed cellular areas with oval to polygonal cells showing clear to eosinophilic cytoplasm along with focal anastomosing network of osetoid-like extracellular material lined by similar cells. The extracellular material was seen densely calcified at places with cement lines and Haversian canal formation. The cells were strongly immunoreactive for epithelial membrane antigen and focally for S-100 protein and negative for glial fibrillary acidic protein. “
“We have reported an autopsy case of primary granulomatous angiitis of the CNS preferentially involving the small veins with a granulomatous Thymidine kinase leukoencepalitis-like lesion

in the cerebral white matter of a 48-year-old man. The latter lesion was ischemic necrosis due to circumferential multiple perivenous granulomas in the adjacent Virchow-Robin space. Multifocal progressive involvement of venular adventitia by granulomas, leaving behind mural fibrosis and luminal stenosis, was related clinically to the prolonged stepwise deterioration observed in the patient, and pathologically to diffuse loosening with dilated veins in the deep cerebral white matter and subcortical hemorrhagic infarction in the left parietal lobe through chronic venous stagnation. PCR demonstrated negativity for Mycobacterium tuberculosis and Propionibacterium acnes, and in situ hybridization with EBV-encoded small nuclear RNA probe was also negative.

Moreover, T cell responses to nucleosomes were increased in SLE patents [14]. If Fas-mediated apoptosis of T cells is defective, activated T cells reactive to self-antigens may escape apoptosis and proliferate abnormally, resulting in the destruction of target tissues. Given that oestrogen triggers SLE activity, www.selleckchem.com/products/Rapamycin.html which correlates with

an apoptotic defect of T cells [15], it can be postulated that oestrogen may affect the survival of activated T cells and their associated molecules, although the direct effects of oestrogen on SLE T cells have not yet been tested. The aim of this study was to determine whether oestrogen acts as a regulator of AICD and FasL expression in SLE T cells. This work was approved by the institutional review committees of the Catholic Medical Center

(Seoul, Republic of Korea). Heparinized peripheral blood (100 ml) was collected aseptically from SLE patients. Informed consent for usage of cells was obtained from all the SLE patients included in this study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation on a Ficoll-Hypaque. Sorting of CD3+, CD4+ and CD8+ T cells (1 × 105 cells) was performed using anti-CD3, anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec, Auburn, CA, USA), respectively. T cells were then cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. Each culture was performed Belnacasan purchase in triplicate in 96-well plates. Cells were incubated for the predetermined times at 37°C in a 5% CO2 atmosphere and then stimulated

with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) plus ionomycin (5 µg/ml) in the absence or presence of 17β-oestradiol (Sigma, PDK4 St Louis, MO, USA), ranging from 10−8 M to 10−6 M. Assessment of T cells undergoing apoptosis was accomplished using a cellular DNA fragmentation enzyme-linked immunosorbent assay (ELISA), as described previously [16]. Briefly, an anti-DNA antibody was fixed in the wells of a microtitre plate. The bromodeoxyuridine (BrdU)-labelled DNA fragments contained in the sample were then bound to the immobilized anti-DNA Ab. Following this, the immune-complexed BrdU-labelled DNA fragments were denatured and fixed on the surface of the plate through microwave irradiation. In the final step, the anti-BrdU peroxidase conjugate was reacted with the BrdU incorporated into the DNA. After removing the unbound peroxidase conjugates, the quantity of peroxidase bound in the immune complex was determined photometrically with 3,3,5′,5′-tetramethylbenzidine dihydrochloride (TMB) as a substrate.

Rosiglitazone had

no effect on these responses. Further investigations on compounds that nullify the downstream effects of these AGE are warranted. “
“Aim:  To better understand the health-care needs of adolescents and young adults (AYA) with end-stage kidney disease (ESKD), we sought to describe the demographic characteristics of a national cohort. Methods:  Data were retrieved from the Australia and New Zealand Dialysis and Transplant Registry. We included all patients aged 15–25 years, living in Australia and receiving renal replacement therapy (RRT) on 31 December 2009. Data included race, aetiology of kidney disease, postal code, transition and migration history. Results:  A total of 495 AYA were receiving RRT in Australia giving a prevalence of 143 per million age-related population. Sixty-three per cent had a functioning transplant, 24% were receiving CHIR-99021 haemodialysis and 13% peritoneal dialysis. Median current age was 22 years (interquartile range (IQR) 19–24). The most prevalent cause of ESKD was glomerulonephritis (33%). The majority

of patients lived in capital cities. Indigenous patients were more likely to live in more remote areas. Eighty-five per cent of patients were currently receiving care at an adult unit and 35% of these patients had transitioned from a paediatric unit since starting RRT. The median number of patients per adult unit was 5 (IQR 3–10). Conclusions:  The majority of Australian AYA with ESKD are managed in adult check details units; however, the number at any one unit is low. As most live in the capital cities there may be an opportunity to establish centralized services designed to cater for the needs of AYA patients. However, the needs of patients

living in more remote areas, including a significant proportion of Indigenous patients, may not be met by such a model. “
“Aim:  The goal of the present study was to investigate the changes in sulfur metabolism in erythrocytes of end-stage renal failure patients. Methods:  The following substances were determined in erythrocytes of chronic kidney disease patients before dialysis, patients treated with continuous ambulatory peritoneal ID-8 dialysis, and in a group of healthy volunteers: (i) sulfane sulfur level and activity of the enzymes involved in its metabolism and in cyanide detoxification; (ii) concentration of total and non-protein sulfhydryl groups -SH; and (iii) protein carbonylation rate. Results:  Erythrocytes of chronic kidney disease patients in predialysis period contained lower levels of sulfane sulfur, non-protein thiols, total thiols and 3-mercaptopyruvate sulfotransferase. On the other hand, in erythrocytes of end-stage renal failure patients treated with continuous ambulatory peritoneal dialysis, sulfane sulfur, non-protein thiols, total thiols and 3-mercaptopyruvate sulfotransferase activity remained at the level observed in healthy controls.

The authors would like to thank the people of SP600125 cost Um-Zukra village for their continuous cooperation. This study was supported by the Institute of Nuclear Medicine, Molecular Biology and Oncology, University of Gezira, Sudan. Our thanks are also due to the Ministry of Higher Education and Scientific Research for their partial financial support. “
“Thyroid disease is one of the most common endocrine conditions affecting women during reproductive age. A link between thyroid and assisted reproduction outcome is debated. Serum TSH levels, number and scoring of oocytes and embryos, and number of clinical pregnancies were retrospectively recorded

in 164 women undergoing assisted reproduction technologies (ART) at an University–based fertility center, to evaluate the outcome of the first steps of assisted reproduction (ovarian stimulation, oocyte pickup and fertilization, embryo transfer and implantation) in relation to thyroid function and autoimmunity. No significant relationship was found between TSH and all parameters, except clinical pregnancy rate (22.3% in TSH ≤ 2.5 group versus 8.9% in TSH > 2.5 mUI/L group; P = 0.045).

Fludarabine No pregnancy occurred in women with anti-thyroperoxidase autoantibodies, while pregnancy occurred in 23.9% of cycles without autoimmunity (P = 0.02). Further studies must be conducted in order to shed light on the link between infertility and thyroid dysfunction. “
“The

mammalian target of rapamycin (mTOR) is a key regulator of cell growth and metabolism. It associates with multiple proteins and forms two distinct signaling complexes, mTORC1 and mTORC2. Accumulating evidence has revealed critical roles for intact mTOR signaling during T-cell activation and responses to microbial infection. However, the importance of mTOR regulation Staurosporine solubility dmso in T cells has yet to be explored. The TSC1/TSC2 complex has been shown to inhibit mTORC1 signaling in cell line models. We show here that deletion of TSC1 in the murine T-cell lineage results in a dramatic reduction of the peripheral T-cell pool, correlating with increased cell death. While mTORC1 is constitutively activated, mTORC2 signaling, reflected by Akt phosphorylation and activity, is decreased in TSC1-deficient T cells. Furthermore, TSC1-deficient T cells contain elevated reactive oxygen species (ROS) and exhibit decreased mitochondrial content and membrane potential, which is correlated with the activation of the intrinsic death pathway. Overall, our results demonstrate that TSC1 differentially regulates mTORC1 and mTORC2 activity, promotes T-cell survival, and is critical for normal mitochondrial homeostasis in T cells. The induction of the adaptive immune response is, in part, characterized by the aggressive expansion of an antigen-specific T-cell pool, coincident with the production of cytokines by said population.

If the CCM has a histologically aggressive appearance as in our case, we suggest that postoperative adjuvant radiotherapy should be performed despite total resection of the tumor. “
“We present an extremely rare case of pinealoblastoma with retinoblastic differentiation in a 32-year-old woman who presented with a history of intermittent headache of 2 years duration and diminution of vision for 2 months which eventually lead to total loss of vision. The fundus examination showed bilateral secondary optic atrophy. She did not have any previous history of retinoblastoma. The family history was non-contributory. Paraffin

section of the tumor showed a primitive neuroectodermal tumor with numerous Flexner-Wintersteiner

rosettes and the tumor cells were strongly positive for synaptophysin and negative for GFAP, S-100 protein and FDA approved Drug Library manufacturer epithelial membrane antigen. This is the first case in the literature of a sporadic case of pinealoblastoma with prominent retinoblastic differentiation as evidenced histomorphologically by the presence of numerous Flexner-Wintersteiner rosettes in an adult female. “
“We treated a 56-year-old woman who had a right temporal lobe tumor found by chance after a traffic accident. MRI confirmed a heterogeneously enhanced tumor in the temporal lobe with large peritumoral edema extending to the superior parietal lobe. The patient underwent tumor resection. The JQ1 nmr tumor consisted largely of distinct cells with discrete borders and granular cytoplasm. In granular cells, the accumulation of PAS-positive granules was observed. Immunohistochemical analysis demonstrated positive staining for GFAP, S-100, and oligodendrocyte Palmatine transcription factor 2 and negative staining for synaptophysin. CD68 was negative in granular cells, but positive in stromal cells. Ki-67 labeling index was quite

low. The tumor was diagnosed as a granular cell astrocytoma (GCA). Postoperative radiotherapy combined with temozolomide was administered. One month after chemoradiotherapy, the tumor occurred in the parietal lobe, and a tumorectomy was performed. The tumor was composed of poorly differentiated astrocytic tumor cells with prominent microvascular proliferation and necrosis. A small number of granular cells were locally observed and the tumor was diagnosed as a glioblastoma. O6-methylguanine–DNA methyltransferase promoter methylation was detected in the GCA but not in the glioblastoma. Isocitrate dehydrogenase mutations were not detected in either tumor. Comparative genomic hybridization analysis demonstrated that no chromosomal abnormality was found in the GCA; however, a gain of chromosomes 7 and 19 and a loss of chromosomes 10 and 9p21 (CDKN2A) were found in the glioblastoma. p53 was strongly expressed in both the GCA and glioblastoma. The tumor progressed despite extensive chemotherapy, and the patient died 1 year after the initial treatment.

OVA257–264 (SIINFEKL), tyrosine-related protein-2 tyrosinase-related protein (TRP)-2180–188 (SVDYDFFDWL), OVA323–339 (ISQAVHAAHAEINEAGR) and lymphocytic choriomeningitis virus–glycoprotein (LCMV GP)61–80 (GLKGPDIYKGVYQFKSVEFD) were obtained from A&A Laboratories (San Diego, CA, USA). DC were isolated from

spleens of naive mice or mice treated for 9 days with 10 µg human recombinant (hr)FLT3L as described previously [34]. hrFLT3L was a kind gift check details from Amgen (Thousand Oaks, CA, USA). DC were analysed for the expression of CD4, CD8α, CD11b, CD11c, CD40, CD54, CD80, CD86, Kb, Db and I-A/E by flow cytometric analysis (antibodies/isotype controls; eBioscience/Biolegend, San Diego, CA, USA; DCs were subsorted by flow cytometry based on their expression of CD11c, CD11b, CD8α or PDCA-1 by flow cytometry to purity of >95% and viability >95% (7-AAD staining). OT-1 and OT-2

T cells were isolated using CD8 or CD4 microbeads buy LDE225 (Miltenyi Biotec, Auburn, CA, USA) and labelled with 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE) (Molecular Probes, Eugene, OR, USA) as described previously [38]. Purity of sorted cells was >98% and viability was >97% as determined by CD4/CD8/Vα2/Vβ5 expression and 7-AAD staining. Purified DCs (1 × 105) were cultured with irradiated splenocytes in a 1:3 ratio in 96-well U-bottomed plates. After 3, 6 and 16 h supernatant was analysed for type I IFN by reporter assay [39] and IL-10, tumour necrosis factor (TNF)-α and TGF-β by quantitative polymerase chain reaction (PCR) using SybrGreen and the following primers: ml32 forward 5-GAAACTGGCGGAAACCCA-3, ml32 reverse 5-GGATCTGGCCCTTGAACCTT-3, TNF-α forward 5-GTACTGGCATGTGTATGTCA-3, Exoribonuclease TNF-α reverse 5-TGGTTGAGGGAATCATT-3, IL-10 forward 5-GGTTGCCAAGCCTTATCGGA-3, IL-10 reverse 5-ACCTGCTCCACTGCCTTGCT-3, TGF-β forward 5-GACCGCAACAACGCCATCTA-3, TGF-β reverse 5-GGCGTATCAGTGGGGGTCAG-3. The fold increase of specific RNA (mRNA after apoptotic cells exposure/mRNA before apoptotic cells) was

determined after normalization to L32 for each sample. Purified DCs (1 × 105) were cultured with irradiated purified ActmOVA-Kbm1 T cells in a 1:3 ratio in 96-well U-bottomed plates. After 24 h, 1 × 105 CFSE-labelled OT-1 or OT-2 T cells were added to the wells. This experimental set-up allows us to study exclusively cross-priming by the DC subsets because the mutated peptide binding groove of Kbm1 cannot bind the OVA257–264 peptide [40] and the lack of MHC class II on the T cells prevents direct activation of the OT-2 T cells [41]. As positive control, DCs were pulsed with OVA peptides for 10 min and washed thoroughly. OT-1 and OT-2 T cell proliferation and survival were determined after 70 h by analysis of CFSE dilution together with staining for Vα2, CD4/CD8 and 7-AAD.