Administration of yokukansan ameliorated not only the TD-induced aggressive

behavior and neurological symptoms but also degeneration of the cerebral cells. NVP-BEZ235 ic50 These results suggest that the inhibitory effect of yokukansan on degeneration in various brain cells might be closely related to the amelioration of aggression and neurological symptoms in TD rats. “
“Neurogenesis and angiogenesis are two important processes that may contribute to the repair of brain injury after stroke. This study was designed to investigate whether transplantation of human embryonic neural stem cells (NSCs) into cortical peri-infarction 24 h after ischemia effects cell proliferation in the subventricular zone (SVZ) and angiogenesis in the peri-infarct zone. NSCs were prepared from embryonic human brains at 8 weeks gestation. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. Animals were randomly divided into two groups (n = 30, each) at 24 h after ischemia: NSC-grafted and medium-grafted groups. Angiogenesis inhibitor Toluidine blue staining and 5′-bromo-2′-deoxyuridine (BrdU) or von Willebrand factor (vWF) immunohistochemistry were performed at 7, 14 and 28 days after transplantation. NSC transplantation increased the number of BrdU-positive cells in the ischemic ipsilateral

SVZ compared with the medium control at 7 days (P < 0.01). Resminostat This difference in SVZ cell proliferation persisted at 14 days (P < 0.01), but was not significant at 28 days (P > 0.05). In addition, angiogenesis, as indicated by BrdU and vWF staining in cortical peri-infarct regions,

was augmented by 46% and 65% in NSC-grafted rats versus medium-grafted rats at 7 and 14 days, respectively (P < 0.05). However, this increase became non-significant at 28 days (P > 0.05). Our results indicate that NSC transplantation enhances endogenous cell proliferation in the SVZ and promotes angiogenesis in the peri-infarct zone, even if it is performed in the acute phase of ischemic injury. “
“K. Kemp, D. Gordon, D. C. Wraith, E. Mallam, E. Hartfield, J. Uney, A. Wilkins and N. Scolding (2011) Neuropathology and Applied Neurobiology37, 166–178 Fusion between human mesenchymal stem cells and rodent cerebellar Purkinje cells Aims: We explored whether cellular fusion and heterokaryon formation between human and rodent cells in the cerebellum of mice occurs after intravenous injection of human bone marrow-derived mesenchymal stem cells (MSCs). The influence of central nervous system inflammation on this process was also assessed. In addition, we examined whether tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, factors associated with inflammation, increase cellular fusion between human MSCs and rodent cerebellar neurons in vitro.

[85, 86] Compared with wild-type controls, osteopontin mRNA expression was greatly increased in the kidneys of homozygous Han:SPRD rats, and in heterozygous rats at later stages of disease.[35] In situ hybridization

localized osteopontin mRNA to the cortex and medulla of homozygous rats, and to focal areas of the CEC in heterozygous rats. In contrast, osteopontin was only localized to the medulla of wild-type rat kidneys.[35] Human ADPKD cyst fluid contains TNF-α, TNF-α converting enzyme (TACE), TNF-α receptor (TNFR)-I and TNFR-II.[87] In one study, TNF-α was identified in 72% of ADPKD cyst fluid samples.[88] Half of the positive samples had TNF-α concentrations exceeding 10 pg/mL,[88] a level comparable to that found in psoriatic arthritis synovial https://www.selleckchem.com/products/dinaciclib-sch727965.html Ku-0059436 order fluid.[89] Furthermore, the quantity (but not concentration) of intracystic TNF-α increases with increasing cyst size.[87] Compared with wild-type controls, cpk mice display an elevated level of TNF-α mRNA expression which increases with age.[24] This implies that TNF-α accumulates with disease progression in human and animal models of PKD. Importantly, Li et al. demonstrated that TNF-α contributes to cystogenesis.[87] TNF-α co-culture induced cystogenesis in Pkd2+/− and wild-type

embryonic kidney explants, and increased the expression of FIP2 (a TNF-α-induced protein), TNFR-I and TACE.[87] Since TNFR can stimulate TNF-α activity,[90] this may incite a vicious cycle of increasing inflammation. In bpk mice, TACE inhibition significantly reduced kidney-to-body weight ratio, and improved renal function (measured as BUN).[91] Since TACE catalyses the production of TNF-α, this result supports the theory that TNF-α is involved in cystogenesis in PKD. In an in vivo study, a higher incidence of cyst development was observed in Pkd2+/− mice treated with intraperitoneal TNF-α compared with untreated Pkd2+/− mice

at postnatal week 8.5 (approximately 40% vs 20% of animals).[87] In contrast, administration of the TNF-α-inhibitor etanercept to Pkd2+/− mice of the same age prevented cyst formation.[87] IL-1β is a cytokine that is produced by macrophages.[92] It mediates inflammation by upregulating the expression of adhesion molecules Vorinostat on endothelial cells, and by stimulating the release of prostaglandin E2 (PGE2, a prostanoid with pro- and anti-inflammatory actions).[92, 93] IL-1β was detected in approximately 70% of cyst fluid samples from symptomatic normal to end-stage ADPKD patients,[88] and was present in samples with higher concentrations of TNF-α, IL-2, and PGE2, suggesting that it was bioactive in vivo.[88] To date, no studies have conclusively delineated the source of pro-inflammatory chemokines in PKD. Gardner et al. identified several pro-inflammatory mediators (including TNF-α, IL-1β, IL-2 and PGE2) in the cyst fluids of ADPKD patients.[88] The authors proposed that the monokines (i.e.

Addition of 4AP, a relatively nonspecific KV channel blocker, significantly increased isolated arterial and venous basal tone and agonist-induced vasoconstriction [58, 69]. Chorionic plate arterial contraction has also been noted to be increased with more isoform-specific blockers margatoxin and stromatoxin-1, but only correolide increased contraction of chorionic plate veins [36]; basal

tone was unaffected. These data Z-VAD-FMK in vivo suggest KV1.2 and/or KV2.1 and KV1.5 in the control of agonist-induced contraction of human placental arteries and veins, respectively. Expression of other 4AP-insensitive KV7 channels has also been suggested; Mistry et al. noted low-level expression of KV7 channels in villus vascular tissues [47], and we have preliminary functional data demonstrating 4AP-insensitive KV7 channel activity in isolated chorionic plate arteries [45]. Endothelin-1 precontracted placental arterial relaxation to SNAP has been shown to be reduced in the presence of charybdotoxin, suggestive of functional BKCa and IKCa channels [58]. Agonist (U46619)-induced

contraction (but not basal tone) is increased by iberiotoxin in chorionic plate GSK-3 signaling pathway arteries but not veins; however, this finding was inconsistent with altered bath oxygenation [69]. Currently, the only functional evidence for twin-pore K+ channel GNAT2 activity has come from Wareing et al.; TASK-1 expression was noted (RT-PCR; Western blotting) and anandamide increased basal tone and agonist-induced contraction in isolated chorionic plate arteries [69]. These data do not represent a definitive proof of a role for TASK-1 channels in the control of fetoplacental vascular reactivity as anandamide has also been suggested to inhibit KV1.2 and KV1.5 channels (whose presence has also been suggested

using more specific blockers [36]). Taken together, these data suggest that a range of K+ channels are present in the fetoplacental vasculature and that they significantly contribute to normal vascular function (Table 2). However, these data are far from complete. The role of KCa channel subtypes requires further elucidation including an assessment of endothelial vs. smooth muscle cell reactivity using primary isolates or cultured cells. Future experiments with isolated smooth muscle and endothelial cells will also be key in determining if placental vascular K+ channels are the primary sensors of altered tissue oxygenation status. Altered K+ channel function has been suggested to induce increased vascular smooth muscle contractility in chronic hypertension [61]. Whether this occurs in FGR, where clinical umbilical arterial Doppler ultrasound waveform measurements suggest increased resistance to blood flow [59], remains unclear.

Therefore, the lipid backbone of BbGL-IIf is rotated 180° inside the CD1d groove relative to that of BbGL-IIc, which leads to a dramatic repositioning of the galactose of BbGL-IIf (51). These results show that the fatty acid moieties also play an important role in stimulating iNKT cell TCR by determining the orientation of the sugar. More recently, the crystal structures of two mouse ternary complexes were determined: CD1d-GalAGSL-iNKT TCR and CD1d-BbGL-IIc-iNKT TCR (53). These bacterial antigens and αGalCer bind to CD1d in

different ways, as explained above (53). Surprisingly, these glycolipids are orientated in almost the PD-0332991 supplier same position above the CD1d binding groove when the TCR is bound (53). These data demonstrate that the iNKT cell TCR induces conformational changes in both microbial antigens and CD1d to adopt a conserved binding mode. Natural killer T cells expressing an invariant T cell antigen receptor recognize a glycolipid from B. burgdorferi; however, do these cells play a protective role against B. burgdorferi infection? It was previously reported that CD1d deficient mice have increased bacterial burden and joint inflammation after syringe infection with B. burgdorferi (54). However, CD1d deficient mice lack not only iNKT cells, but also NKT cells

with diverse TCRs. Moreover, CD1d has been shown to GS 1101 have a signaling function independent of CD1d dependent NKT cells (55, 56). To determine if iNKT cells play a role in the response to B. burgdorferi, Jα18 deficient mice were infected using B. burgdorferi infected ticks, the natural route of infection. The Jα18 deficient mice exhibited more severe and prolonged joint inflammation compared to wild type mice (57). Jα18 deficient mice had a reduced ability to clear bacteria from infected tissues such as the bladder, ears, heart and joints (57). In the early phase of B. burgdorferi infection, iNKT GBA3 cells, but not conventional T cells, are activated and express intracellular cytokines including

IFNγ (57). iNKT cells inhibit carditis after B. burgdorferi infection by accumulating in the heart (58). After B. burgdorferi infection, IFNγ expression increases in wild type mice, but not in Jα18 deficient mice, and IFNγ receptor α chain deficient mice have higher bacterial burdens and increased inflammation in the heart compared to control mice (58). Furthermore, IFNγ treatment enhances B. burgdorferi uptake by macrophages (58). Collectively, these results show that iNKT cells play an important role in the clearance of bacteria and the prevention of chronic inflammation in the joints and heart in B. burgdorferi infection, suggesting that recognition of bacterial antigens by iNKT cell TCR contributes to the response to certain microbial pathogens. Natural killer T cells expressing an invariant T cell antigen receptor contribute to the clearance of bacteria after Sphingomonas infection. However, wild type mice, but not iNKT cell deficient mice, have been shown to die after S.

A statistical test based on measures of central tendency comparison was not applicable to the particular case of anti-IgM combined with IL-21. A P-value less than 0·05 was considered statistically significant. B cells die from apoptosis if maintained unstimulated in culture [31]. After 3 days, spontaneous apoptosis was higher in CD27+ than in CD27– B cells (79·2 versus 57·6%, P < 0·001) (Fig. 2a). When B cells are stimulated, they are rescued from apoptosis.

The effectiveness of the rescue depends upon both the kind of stimulus used and the subpopulation of B cells. For CD27– B cells, the strongest rescue effect was induced by anti-CD40 followed by CpG-ODN and to a lesser extent by anti-IgM, whereas for CD27+ B cells, CpG-ODN appeared to be the strongest rescue stimulus (Fig. 2b). Nevertheless, all the stimuli evaluated were more efficient in the CD27– than in the CD27+ Angiogenesis chemical population: anti-CD40 (77·9 versus 23·9%, P < 0·001), CpG-ODN (71·4 versus Crenolanib mw 57·3%, P < 0·01) and anti-IgM (52·7 versus 36·9%; P < 0·01) (Fig. 2b). Proliferation was evaluated simultaneously. Anti-CD40 and anti-IgM did not induce proliferation of either CD27– or CD27+ B cells while CpG-ODN induced proliferation of both subpopulations (Table 2). Although CpG-ODN

induced a lower level of proliferation on CD27– than CD27+ B cells (PI = 0·1 versus PI = 1·8, respectively; P < 0·001) (Table 2), it induced higher rescue from apoptosis in the CD27– population (Fig. 2b). These aforementioned results suggest that proliferation and rescue from apoptosis are two independent processes. CD27– B cells from CVID MB0 patients were less sensitive to rescue from apoptosis when stimulated with a T-dependent stimulus (anti-CD40) than control subjects (65·4 versus 77·9%, P < 0·05)

(Fig. 3a). They were also less sensitive to rescue from apoptosis when stimulated with a T-independent stimulus (CpG-ODN) than control subjects or CVID MB1 patients, although differences did not reach statistical significance (58·8 versus 71·4 and 63·0%, respectively, P = 0·075). CD27– B cells from CVID MB1 patients were rescued from apoptosis similarly to controls, regardless of the stimulus used (Fig. 3a). After BCR engagement with anti-IgM CD27– B cells from both CVID MB0 and MB1, patients old were rescued equally from apoptosis than healthy controls. CD27+ B cells from CVID MB0 patients, stimulated with either a T-dependent (anti-CD40) or a T-independent stimulus (CpG-ODN), were less sensitive to apoptosis rescue than control subjects (6·0 versus 23·9%, P < 0·01; and 23·2 versus 57·3%, P < 0·05, respectively) and CVID MB1 patients (6·0 versus 30·6%, P < 0·001; and 23·2 versus 65·7%, P < 0·01, respectively). They were also less sensitive to rescue from apoptosis after BCR engagement with anti-IgM than control subjects (19·2 versus 36·9%, P < 0·05) or CVID MB1 patients (19·2 versus 38·2%, P < 0·01) (Fig. 3b).

We investigated Aloxistatin in vitro the effect of parameters of classical indication for CRRT on mortality in patients on continuous renal replacement (CRRT) therapy. Methods: We prospectively and consecutively enrolled a total of 519 patients who stared renal replacement therapy. Results: Mean age was 63.4 ± 14.5 years old, and men were 59.5%

in all enrolled patients. Causes of acute kidney injury (AKI) were septic (46.4%), ischemic (19.5%), post-operation (9.1%), and nephrotoxic (6.2%) AKI. Level of pH (hazard ratio (HR) 1.403, 95% confidence interval (CI) 1.181–4.774, 7.20 < pH ≤ 7.25; OR 3.520, 95% CI 1.330–9.316, 7.15 < pH ≤ 7.20; HR 4.315, 95% CI 1.649–11.286, pH ≤ 7.15; P-for-trend 0.001, reference pH > 7.3), weight gain over 2 kg (HR 2.501, 95% CI 1.552–4.032), urine output (HR 2.190, 95% CI 1.408–3.406, urine output ≤ 0.3 ml/min/kg), and phosphorus level (HR 2.136, 95% CI 1.199–3.805, 5.5 < P ≤ 6.5; HR 4.737, 95% CI 2.613–8.590; P-for-trend < 0.001, reference P < 5.5). However, serum creatinine level (HR 0.892, 95% CI 0.824–0.966)

and increased amount of serum creatinine level (HR 1.083, 95% CI 0.930–1.260) were not associated with in-hospital mortality. Diagnostic values of composite of these factors (pH, weight gain, urine output, and phosphorus levels) (area under find more the curve (AUC) 0.7145, 95% CI 0.656–0.771) was higher than serum creatinine level (AUC 0.449, 95% CI 0.382–0.517), GFR (AUC 0.553, 95% CI 0.485–0.62), and AKIN stage (AUC 0.589, 95% CI 0.521–0.657). Conclusion: These data may suggest that classical indication should be considered for the optimal timing for initiation of CRRT in critically ill patients. HATTORI YUKA1, KIM HANGSOO2, TSUBOI NAOTAKE2, YAMAMOTO AKIHITO1, UEDA MINORU1, MATSUO SEIICHI2, MARUYAMA SHOICHI2 1Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine; 2Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine Introduction: Acute kidney injury (AKI) is a critical condition which is

associated with high mortality rates of 30 to 50%. Ischemia-reperfusion injury (IRI) is a major cause of AKI. However, available treatments for AKI are limited. Preclinical studies indicate that administered MSCs ameliorate Farnesyltransferase renal injury and accelerate kidney repair. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are medical waste, have received attention as a novel stem cell source. The purpose of this study is to clarify whether SHED have therapeutic effect on AKI induced by IRI. Methods: SHED were isolated from human exfoliated deciduous teeth as described previously. For all experiments 7- 8-wk-old male C57BL/6 mice weighing 18–22 g were used. Under anesthesia mice were subjected to right heminephrectomy.

Conclusion: These data confirm increased expression of IDO under hypoxic and inflammatory conditions, both of which are present within the diseased kidney environment. Blocking studies using the IDO inhibitor 1-MT are underway to determine LEE011 in vitro the functional role of IDO in PTEC immune-modulation. It is anticipated that results

from these experiments will help elucidate the mechanistic pathways of PTEC immune-modulation and may provide insights for novel therapy in the treatment of inflammatory kidney disease. 172 INTRARENAL INNERVATION IN HYPERTENSIVE AND DIABETIC RODENTS P DAVERN, K JANDELEIT-DAHM, G HEAD, A WATSON Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia Aim: To assess differences in intrarenal nerves in hypertensive and normotensive rodents with and without concomitant diabetes. Background: Hypertensive diabetic patients have increased renal sympathetic nerve activity and develop nephropathy at an accelerated rate however little is known of changes in renal sympathetic innervation in either hypertension or diabetes. Methods: Studies were carried out in hypertensive and diabetic rodents to assess differences in intrarenal innervation. Twenty-three week old hypertensive (BPH/2J) and normotensive (BPN/3J)

Schlager mice were killed and perfused with normal saline, cold 4% PFA and kidneys embedded in paraffin. Streptozotocin induced diabetic C57Bl6 and apolipoprotein E knockout (apoE KO) mice were killed after 20 weeks of diabetes and kidneys

fixed in 10% NBF before PFT�� datasheet being embedded in paraffin. Streptozotocin induced diabetic spontaneously hypertensive rats (SHRs) were killed after 32 weeks of diabetes and kidneys were similarly fixed and embedded. All kidneys were cut and stained with the neural marker tyrosine hydroxlyase (TH). Results: There was more staining for TH in cortical tubules of hypertensive mice compared with normotensive controls (26 ± 2% vs 19% ± 1% respectively, n = 4/group, P < 0.05). Diabetic C57Bl6 and apoE KO mice appeared to have a redistribution of staining with a greater staining intensity in the distal convoluted tubules. This pattern of staining was also seen in diabetic SHRs compared to non-diabetic SHRs. Conclusions: These results indicate that intrarenal innervation Masitinib (AB1010) is altered in the hypertensive and also the diabetic kidney, suggesting changes in the neural control of the kidney in such conditions. This has direct implications for the treatment of hypertension and renal disease, especially for renal nerve ablation. 173 DENOSUMAB CAUSES SEVERE HYPOCALCAEMIA AND HUNGRY BONE SYNDROME IN PATIENTS WITH ADVANCED CHRONIC KIDNEY DISEASE V DAVE, C CHIANG, J BOOTH, P MOUNT Austin Health, Victoria, Australia Aim: To study the risk of hypocalcaemia with denosumab in patients with stage IV and stage V chronic kidney disease (CKD).

Figure S1. Flow cytometric gates for the evaluation and collection of B lineage cells from the bone marrow of 8-week C57BL/6 mice. Table S1. CDR-H3 sequences obtained from wild-type C57BL/6 bone marrow B lineage cells Table S2. CDR-H3 sequences obtained from C57BL/6 IgHa.ΔD-iD congenic bone marrow mature, recirculating

B cells. “
“There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft-versus-host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue-derived mesenchymal Proteasome inhibitor stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-6] or under control conditions,

and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine-2,3-dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX-2, involved in prostaglandin E2 production. Both conditions had a stimulatory, but differential, BMN 673 mouse effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity

of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, Tobramycin in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy. Mesenchymal stem cells (MSC) are found in a variety of tissues, including bone marrow, skin and adipose tissue [1–3] and can be expanded easily in vitro. MSC are thought to have tissue regenerative properties, in the first place via their multi-lineage differentiation capacity [2] and, perhaps more importantly, via the secretion of trophic factors that may activate local progenitor cells [4]. In addition, MSC have potent immunomodulatory capacity. They inhibit the proliferation of T cells [5,6] and inhibit dendritic cell maturation [7,8]. These properties make MSC promising for a diversity of clinical applications; for example, for the prevention and treatment of autoimmune diseases and bone marrow rejection. Recently, interest has developed in the use of MSC in solid organ transplantation [9,10]. These conditions are associated with an inflammatory milieu.

Also during chronic LCMV infection, IL-6 has recently been identified to be a key molecule acting on CD4+ T cells during late stages of

chronic find more infection [[88]]. Signals via the IL-6 receptor on CD4+ T cells drove their differentiation into Tfh cells in a BCL-6 dependent manner. Furthermore, increased numbers of Tfh cells were essential for germinal center formation, LCMV-specific antibody production and subsequent viral control. Another CD4+ T-cell subset, which gains more and more interest in the context of chronic antigen exposure is the Treg cell subset. In particular, the ability of viruses to induce Treg cells, which subsequently suppress effector CD8+ T-cell responses appears to be a crucial viral escape mechanism [[89, 90]]. It was shown experimentally, that transient depletion of Treg cells during chronic Friend

retrovirus infection is sufficient to reinvigorate virus-specific CD8+ T-cell responses, thereby decreasing virus load [[91]]. For more detailed information on MK-1775 mw the role of Treg cells in the context of host-microorganism interactions we would like to refer to an excellent review by Belkaid and Tarbell [[92]]. Due to the complexity and the differences among the diverse immunization/infection models with respect to the antigen amounts, the nature of the inflammatory response present during the priming process of CD8+ T cells, the ability of the pathogen or adjuvant to induce DC maturation and the precursor frequencies of the responding CD8+ T cells, there are still unresolved controversies concerning the overall requirement of T-cell help, including the time points and mechanisms that are implicated Ribose-5-phosphate isomerase in the delivery of help for CD8+ T-cell responses. Hence, further studies are needed focusing in particular on the molecular differences between helped and “helpless” memory CD8+ T cells, as well as on the mechanisms employed by CD4+ T cells to impact on the generation of potent effector CD8+ T

cells and proliferation-competent memory CD8+ T cells, in the context of defined experimental models. In the future, such comparative studies are likely to reveal “public” and “private” patterns of the T-cell help (in-)dependence of CD8+ T-cell responses, which will be instrumental in tailoring T-cell based vaccines. “
“Traversal of pathogen across the blood–brain barrier (BBB) is an essential step for central nervous system (CNS) invasion. Pathogen traversal can occur paracellularly, transcellularly, and/or in infected phagocytes (Trojan horse mechanism). To trigger the translocation processes, mainly through paracellular and transcellular ways, interactions between protein molecules of pathogen and BBB are inevitable. Simply, it takes two to tango: both host receptors and pathogen ligands. Underlying molecular basis of BBB translocation of various pathogens has been revealed in the last decade, and a plethora of experimental data on protein–protein interactions has been created.

DCs were blocked with fetal bovine serum (FBS) 20% for 2 h. For indirect staining of Pgp in mDCs, 0·5 × 106 DCs were incubated overnight at 4°C with the primary anti-Pgp learn more monoclonal antibody (mAb) JSB1 (1/50 with

FBS 10%), anti-MRP1 mAb (4124) and DC LAMP antibody (1/50 with FBS 10%). Before incubation, cells were permeabilized to anti-Pgp mAb JSB1 incubation. After incubation, cells remained for 30 min at room temperature. The DCs were then incubated with the secondary antibodies Alexa 647 and Alexa 488 (1/100 with FBS 1%) for 45 min and washed. Finally, DCs were mounted in DAPI. Analysis of cell surface marker expression was performed using the dual-colour inmunofluorescence technique (Leica TCS-SL confocal espectral microscope, Mannheim, Germany) equipped with image analysis software (Leica confocal software). Distinguishing DCs from monocytes was also defined functionally by the ability to stimulate an allogeneic mixed leucocyte reaction (MLR) [20, 21]. Thus, we tested not only phenotypical changes, but also functionally tested CD3 proliferation. We performed a CFSE study to analyse the effector function of these DCs; the results supported the phenotypical changes and also emphasized the distinction from macrophages. Lymphocytes were stained with CFSE and exposed Wnt activity to mDCs (under hypoxia or LPS stimuli) with or without ABC transporter inhibitors. After 24 h,

medium was removed and co-culture was performed with fresh medium. Allogeneic CFSE-labelled PBMCs (2 × 105) were cultured alone (negative control) or in the presence of DCs collected Erastin nmr at the end of the 7-day culture after stimuli exposure (DC : T cell ratio 1:10; final volume 200 μl RPMI 10% FBS).

As positive control responder, PBMCs were stimulated with 1 μg/ml (PHA). After 5 days of culture (37°C, 5% CO2) the proliferation of responder cells was determined by flow cytometry after labelling with CD20, CD4 and CD8 antibody to exclude DCs and to define different B and T lymphocyte subpopulations. No ABC transporter inhibitors were used in T and DC co-cultures. In addition, MLR with purified T and B cells was performed with the RosetteSep human T cell enrichment cocktail and the RosetteSep human B cell enrichment cocktail, respectively (Stemcell Technology, Grenoble, France) After cell isolation the MLR technique was carried out as described. Flow cytometry analysis was performed using FACS Canto and diva software (Becton Dickinson). Interleukin-2, -4, -6, -10, -17a, TNF-α and interferon (IFN)-γ secretion protein levels from cell supernatant were measured quantitatively following cell stimulation by CBA (BD Biosciences). Cytokine quantification was performed on stimulated and non-stimulated, and treated and non-treated (with ABC inhibitors) DCs, and on lymphocytes after MLR. Each experiment was performed at least three times and representative data are shown.