A month-of-birth effect in MS is unequivocal, with MS risk being increased for late spring birth and decreased for those in late autumn [171]. More

strikingly, in Scotland, which has the world’s highest MS rate, risk differences between April and Crizotinib nmr November birth reach an astonishing 50%, confirmed in three independent studies [171]. The mechanism by which gestational vitamin D deficiency contributes to increased MS risk later in life is not clear; however, animal model data suggest that developmental vitamin D deficiency may alter thymic development, impact T-cell selection, and disrupt T-cell homeostasis to favour a proinflammatory phenotype [172]. The neurodevelopmental impact of gestational vitamin D deficiency in relation to MS risk is not clear and warrants further study. A latitude

gradient has been noted in MS with the prevalence of the disease being minimal at the equator and increased in both Northern and Southern latitudes, observations that have been replicated in multiple cohorts [173] (reviewed in [174] and [175]). Further dissection of a BMN 673 supplier latitudinal gradient performed in the ethnically homogenous farmer population from France revealed that a north-east to south-west gradient in MS prevalence mirrored mean annual solar irradiation and mean regional serum vitamin D levels in normal adults [88, 173]. The relationship between latitude and MS disease prevalence is further illustrated by migration studies. Small but influential studies suggest that people younger than 15 years at the time of migration tend to adopt the MS risk of the country to which they migrate, whereas those older than 15 years carry the risk of MS of their country of origin [176]. The precise timing of this effect is unclear; however, the critical age of migration may extend into early adulthood [177]. Additional lines of evidence of hypovitaminosis D in MS risk come from serological

data click here of 25(OH)D levels and effect of vitamin D supplementation on MS disease risk and clinical activity. Hypovitaminosis D has been commonly found in MS patients, but the influence of increasing age, sensitivity to heat, and disability may all negatively influence serum 25(OH)D levels [178, 179]. A prospective longitudinal study of a large number of individuals serving in the US military implemented a nested case-control design comparing serum 25(OH)D levels collected before the date of onset of MS symptoms, and demonstrated an inverse correlation of MS risk with serum 25(OH)D levels, particularly before the age of 20 years [180]. Vitamin D supplementation has been suggested to reduce the risk of MS. A study that prospectively followed two cohorts of nurses within the USA found that vitamin D supplementation was inversely related to MS susceptibility in people who consumed at least 400 IU/day of vitamin D, which is considered a modest intake and only marginally increases serum 25(OH)D levels [181].

T helper-1 (Th1) cells are necessary for EAMG development and interferon-γ (IFN-γ) and interleukin-12 (IL-12; the major Th1 cytokines) play a critical role in EAMG development [[5-7]]. Th2 cells secrete a different cytokine profile that confers different effects on EAMG pathogenesis. On one hand, research describing the role of cytokines in the progression of MG and EAMG has revealed that the Th2 cell-related cytokine IL-4 (an efficient growth promoter for B-cell proliferation and differentiation) is important to the development of anti-AChR antibody production [[8]]. On the other hand, IL-4 appears to be involved in

the prevention of the development of EAMG [[9]]. Treg PD0332991 in vivo cells that secrete transforming growth factor-β (TGF-β) and down-regulate various T-cell-mediated responses are functionally defective in MG patients [[10, 11]]. Furthermore, the IL-17-producing Th17 Th-cell phenotype has been shown to play

a dominant role in promoting inflammation autoimmunity [[12, 13]] and EAMG [[14]]. Extracellular adenosine is considered to be an essential physiologically negative regulator Selleck LY2835219 of immune reactions [[15-17]] that initiates transmembrane signaling via 4 G protein-coupled adenosine receptor subtypes designated A1 receptor (R), A2AR, A2BR, and A3R [[18]] and the A2AR is predominantly expressed on T cells [[19, 20]]. The importance of A2AR in mediating adenosine-mediated negative regulation

has been clearly demonstrated in A2AR null mice [[15]] and increased awareness of the role played by both adenosine and A2AR in controlling immune function and inflammation has generated significant Glutathione peroxidase interest regarding the potential use of adenosine-receptor-based therapies in the treatment of autoimmune-based diseases [[18]]. In addition, recent reports have also indicated the development of A2AR-based treatments for autoimmune diseases such as systemic lupus erythematosus [[21]], Parkinson disease [[22]], and experimental autoimmune encephalomyelitis [[23, 24]]. Methotrexate, an antirheumatic drug, has been shown to modulate anti-inflammatory responses via interactions with the A2AR in vivo [[25]]. Besides, A2AR agonists have been reported to possess inhibitory effects in the context of alloantigen-induced immune responses associated with the attenuation of tissue rejection following skin transplantation [[26]] or hepatic ischemia/reperfusion injury [[27]]. However, the regulatory role of A2AR in mediating EAMG disease severity and progression has not been described. In this study, we investigated whether A2AR expression was functionally altered in rats presenting with EAMG and whether the administration of an A2AR agonist prevented EAMG induction or facilitated improvement of clinical symptoms associated with established disease.

Control mice received regular drinking water during the whole experiments. The antibiotic treatment protocol has been described previously and was started 13 days prior to start of DSS induction selleck chemical and continued to the end of the experiment [17]. After 13 and 27 days of antibiotic treatment, fecal samples were collected aseptically and cultivated on aerobic and anaerobic agar plates as previously described to verify successful depletion (def.: <1 CFU/mg feces) of cultivable microbes.

Only successfully depleted mice were included in subsequent data analyses. All use of laboratory animals was approved by the National Animal Research Authority (approval IDs: 48/05, 01/07, and 1468) and conducted SB203580 in accordance with the Norwegian Animal Welfare Act and the Norwegian Regulation on Animal Experimentation. We thank Linda Manley for helpful assistance with the laboratory animal experiments, Linda I. Solfjell for molecular biology work, Anne K. Axelssen for bacteriological work and Eric de Muinck for critical reading of the

manuscript. This study was supported by the Norwegian Cancer Association (DHR), Research Council of Norway (AE) and EU-FP7 Cross-Talk; Contract no. 215553 (RI). The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Table S1. Comparison Phosphatidylinositol diacylglycerol-lyase of global mRNA expression profiles in isolated colonic epithelial cells from conventional pIgR KO mice and WT mice. List of mRNAs more than twofold differentially regulated between pIgR KO-cvn versus WT-cvn. Symbol = gene symbol, ProbeID = Illumina probe ID, fold change > 1 indicates higher expression in pIgR-KO-cvn, while fold change < 1 indicates higher expression in WT-cvn. Table S2. Comparison of global mRNA expression profiles in isolated colonic epithelial cells from antibiotic

treated pIgR KO mice and WT mice. List of mRNAs more than twofold differentially regulated between pIgR KO-abx versus WT-abx. Symbol = gene symbol, ProbeID = Illumina probe ID, fold change > 1 indicates higher expression in pIgR KO-abx, while fold change < 1 indicates higher expression in WT-abx. Table S3. Comparison of global mRNA expression profiles in isolated colonic epithelial cells from antibiotic treated and conventional pIgR KO mice. List of mRNAs more than twofold differentially regulated between pIgR KO-cvn versus pIgR KO-abx. Symbol = gene symbol, ProbeID = Illumina probe ID, fold change > 1 indicates higher expression in pIgR KO-cvn, while fold change < 1 indicates higher expression in pIgR KO-abx. Table S4. List of gene symbols of intersection between the different groups (unique genes with q-value < 0.05 and fold-change > 2) shown in Venn diagram in Fig. 1. Table S5.

It includes an emphasis on the importance of providing informed consent, including expected survival times, for patients being offered dialysis as well as for those not receiving dialysis. The emphasis is on considering see more more than days survived on dialysis such as the likely QOL, the days survived outside of hospital, and the spiritual and cultural issues of the patient and their family that

will be critical to such discussions. The section on the law hopefully provides reassurance to nephrologists that well-based decision-making done as usual in the best interests of the patient is all that the law asks; the likelihood of being sued, an often stated reason for not suggesting a non-dialysis pathway, is very small indeed. We hope that readers of this document will (i) consider all this material in a new light; and (ii) recognize that it is not evidence free. The tools used in decision-making and management are imperfect both for selecting patients best suited to dialysis and for selecting

those best suited to a non-dialysis pathway; https://www.selleckchem.com/products/AZD1152-HQPA.html research strategies to improve these are outlined in this document. There is a strong emphasis in this document on the incorporation of key ethical principles into the process of decision-making regarding dialysis or non-dialysis management pathways, clear guidelines as to how to manage specific symptoms that accompany ESKD and guidelines for end of life care. It is imperative that patients and families are enrolled in such a programme long before the end stage of their CKD pathway so that they are aware of future clinical trajectories and feel supported throughout. A key message we hope to impart is that a well-structured Renal Supportive and Palliative Care programme without dialysis is a very positive part

of the management of ESKD for some patients and one that should not be overlooked. This document has been endorsed by Kidney Health Australia and the Australian and New Zealand Society Calpain of Nephrology. Nephrologists seek to provide dialysis to those who will benefit most. There are some who are unlikely to benefit or even be harmed by dialysis and it is important that we try to recognize such patients; these can be very difficult decisions. In older patients with co-morbidities the decision to initiate dialysis can be very difficult; helpful things to consider include: the number of cardiac or other co-morbidities, nutritional status, functional status, whether or not the patient is in a nursing home, and how the nephrologist responds to the question: ‘would you be surprised if your patient died within 12 months?’ Taken together, these issues help identify patients at risk of a poor outcome on dialysis.

Heat shock increased both HSP70 and IFNT expression. There was a significant correlation between HSP70 and IFNT transcript GSK458 solubility dmso levels irrespective of whether

a blastocyst had been exposed to heat shock or not. The increase in IFNT as a result of heat shock suggests that a proportion of the variation in IFNT expression observed in blastocyst-stage embryos is a response to stress. “
“The vaccine potential of meningococcal Omp85 was studied by comparing the immune responses of genetically modified deoxycholate-extracted outer membrane vesicles, expressing five-fold higher levels of Omp85, with wild-type vesicles. Groups (n = 6–12) of inbred and outbred mouse strains (Balb/c, C57BL/6, OFI and NMRI) were immunized with the two vaccines, and the induced antibody levels and bactericidal and opsonic activities measured. Except for Balb/c mice, which were low responders, the genetically modified vaccine raised high Omp85 antibody levels in all mouse strains. In comparison, the wild-type vaccine gave lower antibody levels, but NMRI mice responded to this vaccine with the same high levels as the modified vaccine in the other strains. Although the vaccines induced strain-dependent Omp85 antibody responses, the mouse strains showed high and similar serum bactericidal

titres. Titres were negligible with heterologous or PorA-negative meningococcal target strains, demonstrating the presence of the dominant bactericidal PorA antibodies. The two vaccines induced the same MLN0128 mw opsonic titres. Thus, the genetically modified vaccine with high Omp85

antibody levels and the wild-type vaccine induced the same levels of functional activities related to protection against meningococcal disease, suggesting that meningococcal Omp85 is a less attractive vaccine antigen. The meningococcal outer membrane protein Omp85 is one of the antigens in deoxycholate-extracted outer membrane vesicle (OMV) vaccines that have shown efficacy against serogroup B meningococcal disease in several countries [1-4]. With a rabbit antibody against denatured Omp85, this protein was found to be expressed by meningococcal strains of diverse serogroups and serotypes as well as by Neisseria gonorrhoeae, Neisseria lactamica and Neisseria Erastin polysaccharea [5]. Although it is present in only minor amounts in the OMVs, distinct levels of Omp85 antibodies were observed after vaccination of mice [1, 6, 7], in volunteers receiving different OMV vaccines and in patients recovering from meningococcal disease [8-13]. Bactericidal serum antibodies are known to correlate with protection against meningococcal disease [14, 15], and correlations between antibody levels to Omp85 and serum bactericidal activities indicated that Omp85 might induce bactericidal antibodies in humans [10, 12].

Robert Walker, Robert G Fassett and Rachael L Morton Concentration of research is recommended in the following areas: Prospective studies of the appropriateness, relevance, timing and sustainability of dialysis in elderly patients. Health-related quality of life (HRQoL) in older patients choosing not to dialyse and in those choosing to dialyse with comparison to a matched population without renal disease. Methods of communication of prognosis and factors affecting decision-making. Models of care – comparative studies to delineate how best to deliver renal supportive care. Treatment preferences amongst indigenous patients.

Symptom control, focussing on those areas specific to the needs of renal patients. There has been an increase of over 400% in the number of elderly and very elderly patients on dialysis in Australia and New Zealand (NZ) over the past two decades.[1] This rapid PD-0332991 mw increase has generated considerable debate resulting

in wide variation in attitude towards referral and acceptance of elderly patients for dialysis.[2-4] One major reason for this is that there is uncertainty about the outcome from dialysis treatment in this population.[5] If conservative management is shown to be an important and valid option with similar outcomes to dialysis, then this can be appropriately discussed with the individual and their family/whanau (Maori – extended family) without this being considered as rationing, or limiting health resources. Current studies suggest poor maintenance of GS-1101 price functional capacity and high mortality in nursing home patients accepted for dialysis

in the USA,[6] and a retrospective study suggests outcomes are much the same on dialysis or with conservative care if GBA3 aged >75 with greater than two comorbidities.[5] Prospective studies are required to address the appropriateness, relevance, timeliness, and the sustainability (both with respect to quality as well as quantity) of dialysis in the elderly. Providing information as to preferred options by this group related to their expectations and perceived quality of life will immediately influence delivery of healthcare. The provision of dialysis, preferably in a home setting or low level self care satellite units closer to the individuals’ residences, may allow better integration with primary and community care. Evidence is required to disentangle survival alone versus quality of life with respect to the provision of renal replacement therapy (RRT) and renal supportive care. Decision-making should, and clearly does, involve the patients and their carers, along with health service providers. However, there is currently a dearth of evidence related to such decision-making among dialysis patients in general, and elderly dialysis patients in particular.

RNA was reverse-transcribed using Moloney murine leukaemia virus reverse transcriptase (Invitrogen Corporation, Carlsbad, CA). Complementary DNA was amplified as follows: denaturation at 94° for

50 seconds, annealing at 57° for 50 seconds, and extension at 72° for 50 seconds. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control to ensure equal sample loading. Primers used were as follows: for IL-15Rα, 5′-GTCAAGAGCTACAGCTTGTAC-3′ and 5′-CATAGGTGGTGAGAGCAGTTTTC-3′; for IL-2Rα, 5′-AAGCTCTGCCACTCGGAACACAAC-3′ and 5′-TGATCAGCAGGAAAACACAGC-3′; for IL-2Rβ, 5′-ACCTCTTGGGCATCTGCAGC-3′ and 5′-CTCTCCAGCACTTCTAGTGG-3′; for IL-2Rγ, 5′-CCAGAAGTGCAGCCACTATC-3′ see more and 5′-GTGGATTGGGTGGCTCCAT-3′; PXD101 and for GAPDH, 5′-CCCTCCAAAATCAAGTGGGG-3′ and 5′-CGCCACAGTTTCCCGGAGGG-3′. For cell division experiments, FDCs (1 × 107 cells/ml) were labelled with carboxyfluorescein succinimidyl ester (CFSE; Sigma, 0·2 μm in phosphate-buffered saline) and incubated at 37° for 10 min. Cold

CFS was added to stop staining, and labelled cells were next washed twice with culture media. After 3 days of culture, CFSE intensity was measured using a FACSCalibur™ flow cytometer and analysed using flowjo software (Ashland, OR). The apoptosis assay employed staining with Annexin V and 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3); Molecular Probes, Eugene, OR]. The FDCs (1 × 106 cells/ml) suspended in 100 μl of Annexin V binding buffer [0·1 m HEPES/NaOH (pH 7·4), 1·4 m NaCl, 25 mm CaCl3] were stained with 5 μl Annexin V-APC second and 5 μl propidium iodide (BD Biosciences). Cells were incubated for 15 min at 25° in the dark. The same number of cells was employed for DiOC6(3) staining; 20 μl 8 μm DiOC6(3) was added, followed by incubation for 10 min. Samples were analysed on a FACSCalibur™ running cellquest-pro® programs (BD Biosciences). Follicular DCs at passages 4–9 were used in experiments. For FACS analysis, FDCs were collected using Enzyme-free Cell Dissociation Solution

(Specialty Media, Philipsburg, NJ). All FACS staining for surface CD14, CD44, CD54 and CD106 detection was performed as follows. Briefly, cells were washed in cold FACS buffer [0·05% (v/v) FCS, 0·01% (w/v) NaN3 in phosphate-buffered saline] and subsequently incubated with the appropriate concentration of anti-CD14, anti-CD44, anti-CD54 or anti-CD106 mAbs for 15 min at 4°. After washing with cold FACS buffer, cells were fixed in 1% (v/v) paraformaldehyde. Subsequently, samples were analysed on a FACSCalibur™ running cellquest-pro® program (BD Biosciences). Follicular DCs at passages 4–9 were seeded at 2 × 104 cells/well in 24-well plates. The next day, the medium was changed and a combination of reagents was added as indicated in the legend to Fig. 4. The concentration of each reagent was as follows: anti-IL-15 mAb (100 ng/ml), mouse IgG1 (100 ng/ml), GC-B cells (2 × 105 per well), TNF-α (10 ng/ml), IL-2 (30 U/ml), IL-4 (50 U/ml) and CD40L (100 ng/ml).

Overall, our results hint at the importance of monoubiquitination of AVM-associated proteins throughout the A. phagocytophilum infection cycle in promyelocytic HL-60 cells as well as endothelial cells, as a comparable degree of ubiquitination of the AVM was observed for infected RF/6A cells. Considerably, fewer ApVs of infected ISE6 cells exhibited ubiquitination than infected mammalian cells. Either AVM ubiquitination does not play a prominent role in A. phagocytophilum infection of ISE6 cells or association of ubiquitinated proteins with the AVM may be temporally regulated during infection of ISE6

cells. By accruing monoubiquitinated click here proteins that localize and direct traffic to endocytic compartments, A. phagocytophilum conceivably camouflages its vacuolar membrane as a means for avoiding lysosomal targeting. Support for this possibility comes from the precedent that the ApV selectively recruits Rab GTPases that are predominantly associated with recycling endosomes while concomitantly check details blocking recruitment of Rabs that are important for lysosomal delivery. Tetracycline treatment of infected cells culminates in the dissociation of recycling endosome-associated Rabs with the concomitant association of the lysosomal markers Rab7

and LAMP-1 (Huang et al., 2010a). Confocal microscopic analysis of fixed cells reveals that no more than 52.6% ± 4.2% or 61.0% ± 6.2% ApVs in HL-60 cells or RF/6A cells, respectively, are positive for ubiquitin at any time point examined. A highly similar trend occurs when one examines the percentages of ApVs to which GFP-tagged recycling endosome-associated Rab GTPases localize (Huang et al., 2010a). Ubiquitin machinery, like Rab GTPases, dynamically cycles on- and off-target organelle membranes (Grabbe et al., 2011; Segev, 2011). Thus, examining fixed A. phagocytophilum-infected cells provides a snapshot of the AVMs that are monoubiquitinated or have associated Rab GTPases at the instant at which preservative was added. Protein kinase N1 Several bacterial effectors have been shown to exploit the host cell’s ubiquitination system to diversify or regulate their biological functions. Several effectors secreted by intracellular bacterial pathogens

mimic the activities of E3 ubiquitin ligases to spatially or temporally regulate host or bacterial proteins (Kubori & Galan, 2003; Kubori et al., 2010). Alternatively, the ubiquitination of other bacterial effectors regulates their activities and subcellular localization rather than serve as a signal for their proteasomal degradation (Marcus et al., 2002; Knodler et al., 2009; Patel et al., 2009). As AVM monoubiquitination is bacterial protein synthesis-dependent, it is plausible that A. phagocytophilum encodes one or more effectors that either may recruit monoubiquitinated host proteins to the AVM or may be monoubiquitinated themselves. To date, only three A. phagocytophilum-encoded AVM proteins – APH_1387, APH_0032, and AptA – have been identified (Huang et al.

Networks are displayed graphically as gene/genes products (nodes) and the biological relationships between the nodes (edges). All edges are supported by at least one reference from

the literature, or from canonical information stored in the Ingenuity Pathways Knowledge Base. In addition, IPA computes a score for each network according to the fit of the user’s set of significant genes. The score, representing the – log(P-value), indicates the likelihood www.selleckchem.com/screening/gpcr-library.html of the Focus Genes in a network from Ingenuity Knowledge Database being found together randomly. We identified X chromosome sites that were consistently hypermethylated (n = 18, Table 1) and hypomethylated (n = 25, Table 2) in affected twins. Within the 5-kb window sampled for each X-linked gene, most of the differentially methylated regions (DMRs) were located in promoter regions or CpG islands while two hypermethylated (48980151–48980208 and 104355356–104355413) and six hypomethylated (103883076–103883125, selleckchem 47226194–47226247, 134532227–134532289, 134532327–134532376, 134532427–134532476, 134532627–134532676) DMRs were found downstream

of the transcription start sites. In all cases, DMRs were associated with known genes and we noticed that IL1RAPL2 was found in both lists (the two hypermethylated sites are downstream of the hypomethylated one). In some cases, multiple DMRs belong to the same gene (as in the case of hypomethylated peaks 2-hydroxyphytanoyl-CoA lyase 134532227–134532289, 134532327–134532376, 134532427–134532476 and 134532627–134532676 onto gene DDX26B) or a specific site is located in a CpG island of

a bidirectional promoter for two different genes (i.e. hypomethylated peak 152712287–152712338 for genes SSR4 and IDH3G). Three hypomethylated peaks are associated with intergenic single-nucleotide polymorphisms (SNP), with peak 13087308–13087357, including SNP rs61677044, peak 13087708–13087757 falling in a region 150 bp downstream from SNP rs16978681, peaks 126140539–126140588 and 126140739–126140788 mapping to a SNP-rich region. Genes identified by the hypermethylated and hypomethylated sites encode for proteins that are illustrated in Tables 3 and 4, respectively. The 26 proteins include transcription factors, membrane and soluble enzymes, surface antigens and translocation proteins while in some cases proteins are currently defined only structurally, but not functionally. We explored possible functional relationships between the 26 genes using the IPA Knowledge Database. Unsupervised IPA network analysis identified a single cluster of 25 genes that included seven of our 26 genes and 18 additional genes, which was unlikely to occur by chance (P = 10−13). The plausible biological network generated is shown in Fig.

cRNA preparation, purification and labelling, array hybridisation and scanning were performed Selleck Poziotinib according to the manufacturer’s protocol (Illumina). Gene expression was compared between (I) tumour spheroids, (II) tumour cells only sorted out from co-cultures

and (III) co-culture spheroids. Four biological replicates (monocytes from different donors) for co-culture spheroids and two replicates for tumour spheroids were studied using Illumina HumanRef-8 v.2.0 chips. Illumina BeadStudio was used for background correction and generating average signal intensity. R/Bioconductor 30 was used for quantile normalisation 31. Probes showing low variability were discarded by applying interquartile filter (IQR=0.25). A linear model 32 was employed by controlling the number of false positives by false discovery rate (FDR) with adjusted p-value of ≤0.05 33. A log2 fold change signal threshold (log2(FC)≥1.0) was applied for comparison of (I) and (II), and (log2(FC)≥1.5) for comparison of (II) and (III). MultiExperiment Viewer 34, 35 was used for hierarchical clustering, setting a Euclidean Ceritinib concentration distance as the measure of dissimilarity and average linkage as the linkage method. To identify biologically relevant regulatory processes and pathways, MetaCore with a FDR adjusted p-value of ≤0.05 was used. Primers were designed using Primer-3 (Supporting Information Table 3). Real-time

PCR was performed with Stratagene Mx3000P (Agilent). All gene expressions were normalised to Fossariinae housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT). Tumour cells were labelled with anti-EpCAM-FITC, stained with propidium iodide (15 μg/mL; Sigma) and analysed by flow cytometry. Pre-cleared cell culture supernatants

(days 5–8) were used for cytokine measurement (Bio-Plex Pro Human Cytokine Groups I and II, BioRad). Culture medium with 5% HS was used as blank and diluent. Detection was carried out with Luminex 200TM. Results were acquired using IS 2.3 software. CCL8 and CCL13 were detected using Duoset ELISA Development Systems (R&D Systems). Samples were diluted and prepared according to manufacturer’s protocol. Culture medium with 5% HS was used as the blank. Supernatants were incubated at 37°C, 5% CO2, 30 min to allow pH equilibration before assay. Total white blood cells (WBC) were obtained from buffy coats after red blood cell lysis. Totally, 7.5×105 WBC were seeded onto cell culture inserts (BD) with 8.0 μm pore sizes, incubated with supernatants in wells for 1 h. T cells (CD3, CD4, CD8) that trans-migrated through the inserts were distinguished by fluorescence labelling and analysed by flow cytometry (LSRII, BD). Countbright beads (Invitrogen) were used for cell number quantification. WBC from six donors and supernatants from three replicates (spheroid cultures) were used. Tumour cells from co-culture spheroids were labelled with anti-EpCAM-FITC followed by anti-FITC microbeads (Miltenyi) for magnetic sorting into tumour cells and TAMs.