As others have reported previously, this study suggested that fibrocyte generation from cultured peripheral blood mononuclear

cells (PBMCs) derived from donors without any known chronic diseases were vanishingly rare. In contrast, cultured PBMCs from many patients with Graves’ disease, regardless of duration, thyroidal status or treatment received, generated numerous fibrocytes that exhibited the expected CD34+Col1+CXCR4+ phenotype. Interestingly, the elevated frequency of fibrocyte generation was not universal among patients with the disease. Many of these individuals, even those with recent onset and clinically severe selleck chemicals llc disease, failed to generate fibrocytes at levels differing from those found in the control donors. The authors found relatively high levels of IGF-1R on fibrocytes, but the levels appear to be no different from those on fibrocytes donated by control subjects. The report by Douglas et al. [50] began to characterize the LY2109761 phenotypic attributes of

fibrocytes found in Graves’ disease. Those studies aimed at identification of those cellular features that might underlie their participation in TAO. The authors found that CD34+Col1+IGF-1R+ cells were relatively abundant in situ in orbital tissue from patients with TAO but were absent in those from healthy donors (Fig. 1). They were consistently CD31-, indicating that the putative fibrocytes were unrelated to endothelial cells. Surprisingly, high levels of TSHR were detected on the circulating fibrocyte surface. The levels of this protein appear equivalent to those found on thyroid epithelial cells, where they mediate thyroid

hormone production (Fig. 2). Even more surprising was their observation that the receptor is functional. When ligated with bovine thyroid-stimulating hormone (bTSH) or M22, an activating monoclonal antibody generated against TSHR, the production of inflammatory Liothyronine Sodium cytokines such as TNF-α and IL-6 is up-regulated dramatically (Fig. 3) [50]. When orbital fibroblasts from patients with TAO were subjected to flow cytometric analysis, a subpopulation of cells was found to exhibit the CD34+Col1+ phenotype. In contrast, CD34+ cells were uniformly absent among orbital fibroblasts from control donors. This phenotype was stable in culture over many serial passages. Moreover, it appears that the vast majority of CD34+ orbital fibroblasts are also CD90+ (Thy-1+).

The severe and uncontrolled inflammatory reactions observed in the TGF-β1 knock-out mouse attests to the physiological role of TGF-β as an endogenous anti-inflammatory cytokine [42]. Even though in this study Gram-negative

E. coli stimulated substantial amount of proinflammatory cytokines, the induction of pro- and anti-inflammatory cytokines with live Gram-positive bacteria (including GIT simulated bacteria), on average, was significantly higher. Hessle et al. [13] reported that Gram-positive bacteria appeared to stimulate IL-12 production and Gram-negative bacteria stimulate IL-10 production preferentially. However, concordant with observations reported in Berg et al. [43] and in our study, Gram-negative E. coli induced the secretion Ixazomib manufacturer of significant concentrations of proinflammatory cytokines by PBMCs and the CRL-9850 cell line. While the mechanisms by which some bacteria induce the production

of IL-10 are unclear, LPS of Gram-negative bacteria may stimulate this anti-inflammatory response [43]. Compounds other than LPS in lactobacilli probably contributed to the ability of these probiotic bacteria to stimulate an anti-inflammatory cytokine response. Probiotic Obeticholic Acid mouse LAVRI-A1, LGG, B94 and BL536 induced substantial amounts of pro-and anti-inflammatory cytokines in line with previous studies [44], with the balance skewed towards the anti-inflammatory response in our study. A demonstration of the utility of this response is the finding that LGG reduced inflammation in Crohn’s disease [45]. The human gut microbiota

has been estimated recently to consist of at least 400 different species [46], and it is likely that the potency of each of these species to influence immune homeostasis is different. Indeed, cytokine profiles in co-cultures of bacteria with PBMC show marked differences between strains [23]. In addition, the effects of lactobacilli supplementation on experimental autoimmune encephalomyelitis have been shown to be highly strain-dependent [47]. It is therefore conceivable that the contradicting results Lepirudin found in the human trials can be explained partly by differences in the immunomodulatory capacity of the strains used. The fact that the killed bacteria in our study were inefficient in inducing substantial amounts of pro- and anti-inflammatory cytokines compared to live bacteria suggests that extra- and intracellular bacterial components as well as metabolites probably contribute to cytokine production [48]. Conceivably, a combination of certain bacterial fragments, metabolites produced in situ and particular structural motifs may need to interact with receptors on monocytes to induce optimal cytokine synthesis [21,49]. Cross et al. [50] and Macpherson and Harris [51] reported that live lactobacilli were more potent inducers of cytokine production in mammalian leucocytes compared to killed bacteria, similar to our findings.

Intracellular staining was carried out using a cytofix/cytoperm kit according to the manufacturer’s instructions (BD Biosciences). Cell suspensions were acquired with an LSR-II flow cytometer (BD Cytometry Systems). Analysis was carried out using FlowJo software (TreeStar, San Carlos, CA). Using Prism 4 software (GraphPad Software Inc., San Diego, CA), comparisons of selleckchem statistical significance between groups were assessed using the Mann–Whitney U-test. In inflammatory environments, recruited leucocytes may have emergent properties that are dependent on multiple local interactions with many different soluble signalling molecules. In EAU, accumulating Mϕ, derived from BM cells, infiltrate inflammatory sites in large numbers

and perform as professional APCs. They interact with T cells, both enhancing and regulating immunity. We have demonstrated that the Mϕ that accumulate in the target organ modify T cell responses, suppressing T cell proliferation but preserving cytokine secretion.10 These Mϕ express cell surface markers such as Gr1 and CD31 that are associated

with immune regulation, and to investigate AZD0530 in vivo the function of such cells, we generated Mϕin vitro from BM cells cultured in an inert environment (hydrophobic PTFE-coated tissue culture bags). We compared the ability of these cells to present antigen with other APCs. The OVA323–339-specific TCR transgenic OT-II CD4+ T cells were co-cultured with different populations of professional APCs in the presence or absence of cognate OVA peptide. Wild-type (WT) splenocytes, B cells and dendritic cells stimulated peptide-specific T-cell proliferation, but BM-Mϕ did not (Fig. 1a). To address whether this was the result of a failure of Mϕ to interact with T cells, we analysed other markers of T-cell activation. Despite

the lack of proliferation, we observed that, following co-culture with BM-Mϕ, OT-II T cells adopted an activated cell surface Fenbendazole phenotype and expressed high levels of CD69, CD44 and CD25 (Fig. 1b). The OT-II T cells activated by Mϕ also produced high levels of IFN-γ, the production of which was shown to be independent of TNFR1 signalling as BM-Mϕ derived from TNFR1 knockout (TNFR1−/−) mice stimulated T cells to produce similar amounts of IFN-γ. Interferon-γ activates Mϕ, which in turn leads to autocrine TNF-α signalling that further mediates Mϕ activation.11 Blocking Mϕ activation by neutralizing IFN-γ or TNF-α by the addition of anti IFN-γ mAb or sTNFR1-immunoglobulin fusion protein restored peptide-dependent T-cell proliferation (Fig. 1d), supporting our previous data that the regulation of T-cell proliferation by myeloid cells in the target organ during autoimmunity is dependent on the activation of myeloid cells by IFN-γ and TNF-α.10 Consistent with these in vitro blocking studies, TNFR1−/− Mϕ stimulated T-cell proliferation across a range of peptide concentrations, whereas WT Mϕ stimulated little proliferation (Fig. 1e).

Ubiquitin-positive NCIs, which are evident in the degenerating lower motor neurons, have long been recognized as a characteristic feature of the cellular pathology. However, TDP-43 immunostaining may reveal positive neuronal and glial cytoplasmic inclusions (NCIs and GCIs) not only in the affected lower motor neuron nuclei but also in the other apparently normal non-motor neuron nuclei, indicating that SALS is a multisystem neuronal-glial proteinopathy of TDP-43 affecting a wide range of both neurons and glial cells in the CNS.[20] TDP-43

pathology is also evident in many patients with superoxide dismutase Adriamycin order 1 (SOD1)-unrelated familial ALS, in whom mutations within the TDP-43 gene (TARDBP) have been identified; interestingly, although

rare, TARDBP mutations have also been identified in patients with SALS.[21, 22] Based on these pathological and genetic findings, TDP-43 has been implicated as an important contributor to the pathogenesis of ALS. PolyQ diseases are inherited neurodegenerative disorders caused by expanded CAG repeats that encode abnormally long polyQ stretches in the disease proteins. The polyQ-positive NCIs and neuronal intranuclear inclusions (NIIs) are widespread in the CNS beyond the degenerative areas, indicating that the diseases are also multisystem neuronal proteinopathies.[23] TDP-43 pathology in the polyQ diseases was first reported in HD.[15] Unlike the neurodegenerative diseases showing TDP pathology mainly in the MI-503 in vivo limbic system, patients with HD have TDP-43-positive NCIs in the neocortex, where many polyQ-positive inclusions are also observed. More recently, intermediate-length polyQ expansions

(27–33 Qs) in ataxin 2 (ATX2), the causative gene of SCA2, were reported to be a genetic risk factor for SALS.[16] In cases of HD, Schwab et al. have reported that TDP-43 was frequently colocalized with huntingtin in dystrophic neuritis Ribonuclease T1 and various intracellular inclusions, but not in intranuclear inclusions.[15] Recently, Tada et al. examined autopsied patients with genetically confirmed HD with SALS, and found that two different proteinaceous inclusions coexisted in a small number of neurons in the affected brain. However, the two disease proteins, huntingtin and TDP-43, were not co-localized within the inclusions, although the regional distributions of TDP-43-positive inclusions and expanded polyQ (1C2)-positive inclusions partly overlapped.[19] Biochemically, TDP-43 isoforms similar to those seen in SALS were observed in one of the patients examined.[19] In these cases of HD with SALS, it seems that two distinct pathological pathways may each affect the brain. It is tempting to speculate that “aging” may promote the deleterious effect of mutant huntingtin on motor neurons and on TDP-43. We have previously reported the occurrence of TDP-43 pathology in SCA3/MJD.

In both analyses, the five strains

of S. dehoogii were grouped into four different clusters, underlining the pronounced degree of intraspecific variability found in this species. Pseudallescheria apiosperma was dispersed over three clusters. The ten investigated P. boydii strains were recovered in five different clusters using SJ and in four clusters using SSM. Reproducibility testing showed that the methods used were acceptable for analysing the physiological diversity in the Pseudallescheria/Scedosporium complex. Of a total https://www.selleckchem.com/products/AZD6244.html of 570 reactions available in the panel, 254 reactions were polymorphic (44.6%) (Table 2), while 271 reactions (47.5%) were invariant, and a total of 45 reactions (7.9%) were found to be unreliable and were this website removed from the data set. Reasons for removal were (i) instability or inconsistence (26 reactions; 4.6%) or (ii) turbidity of the medium

or too early colour change of the indicator, i.e. occurring immediately after inoculation (19 reactions; 3.3%). The variability of the test results may be caused by decomposition of the test compound (e.g. with thermolabile components, insufficient solubility or by deviations from optimal pH values). The same problems with these compounds had been encountered in the framework of characterisation of fermentative actinomycetes.23 Several compounds were tested at pH 4.0, pH 7.5 and pH 8.2, in most cases resulting in removal of the obtained results. In contrast, nearly half of the glucosidases and phosphatases reactions proved to show consistent responses

at pH 5.5. Acidification of the medium by Pseudallescheria and Scedosporium strains should be taken into account. Our test results essentially corresponded with those published by de Hoog et al. [1,5], for example, in positive reactions for d-galactose and negative for melezitose in the P. boydii complex, and negative results for creatinine, succinate and positive responses to l-arabinose, l-rhamnose, trehalose, cellobiose and salicin in S. prolificans. The taxonomic separation between purported Pseudallescheria and Petriella/Petriellopsis clades [24] is thus supported by physiological parameters. Within the Pseudallescheria clade, physiological data as assessed by the Taxa Profile system did not fully match with the subdivision of the group into at least five species, as proposed by DNA ligase Gilgado et al. [10,12]. Discrepancies were noted with maltose assimilation by P. minutispora and for l-arabinitol assimilation by two out of four S. prolificans strains. Particularly, our d-ribose results differed significantly, underpinning previous observations that testing pentose fermentation by assessment of acid production is highly liable to test errors.23 The results of Gilgado et al. [12] were obtained using macrodilution according to Yarrow [25]; it seems likely that results obtained with different techniques cannot be generalised.

However, further investigations are necessary to understand the biological significance of this finding. The nuclear nature of NFR-related 65- and 49-kDa antigens has been evidenced by cell fractionation experiments. In fact, sera collected from CD patients when NFR antibodies are observable show IgA reactivity in total cell protein extract and in its nuclear fraction that is absent in the cytosolic fraction. Serum IgA reactivity with 65- and 49-kDa antigens has been detected on lysates of the human Caco2 cell Ku 0059436 line, and is therefore definable as autoimmune. Moreover,

we also show that this autoreactivity is gluten-dependent, and therefore related strictly to CD. Indeed, it is present in CD patients’ sera up to NFR antibodies are observable and disappear on a GFD, with the clearance Torin 1 of NFR antibodies themselves. Circulating autoantibodies CD patients provide an important tool in screening, diagnosing and monitoring the disease. In detail, serum EMA and anti-tTG antibodies are used currently in clinical practice on account of their high sensitivity and specificity [16,17]. Furthermore, serum EMA disappear upon the mucosal healing subsequent to a GFD [21],

while after gluten reintroduction into the diet their reappearance may predict mucosal relapse [28]. The kinetics of EMA, however, is not well known and it is not investigated widely. In the present study, we show that EMA disappearance in sera from treated CD patients is complete within 76 ± 34 days after starting the GFD. At this time-point, serum NFR antibodies become observable and persist for a further 75 ± 41 days for a total of 151 ± 37 days from starting the GFD. Our data also show that, after the reintroduction of small amounts of gluten in the diet, NFR antibodies reappear within a few days, much 6-phosphogluconolactonase earlier than serum EMA. The biopsy culture study shows that NFR antibodies are produced early (4–6 h), while EMA appear after more than 12 h from starting the in vitro gliadin challenge. This in vitro finding is consistent with result of the in vivo gluten-induced reactivation of CD. Consequently, given that NFR seems

to be more sensitive than EMA as an early marker of CD reactivation, NFR antibody detection in serum from treated CD patients might become a valuable tool in monitoring adherence to GFD and identifying slight dietary transgressions. The appearance of serum NFR during gluten withdrawal, together with the persistence of symptoms when these antibodies are still positive but EMA are already negative, also suggest that NFR assessment could be an useful tool to determine the right time to perform a second duodenal biopsy. However, before applying these suggestions, our data need to be confirmed by large clinical trials. The presence of a serum NFR-like pattern in some healthy controls evaluated in this study could suggest a low specificity for NFR antibody detection in CD monitoring.

To confirm this, neutrophils were further identified as polymorphonuclear cells that express IL-8R (Fig. 5a–d). Furthermore, the results show an increased number of neutrophils in PC61-treated mice at 24 hr post-injection (Fig. 5d) reflecting the data on increased cellular mass in PC61-treated mice (Figs 1 and 3). As neutrophils were more abundant in the Treg-depleted animals, we examined relative levels of neutrophil chemoattractants, CXCL1 (KC) and CXCL2 (MIP-2), in the skin of Treg-reduced and control mice 24 hr post-inoculation with B16FasL cells. Elevated levels of both chemokines were observed in the skin of Treg-depleted

animals suggesting that Treg cells inhibit local neutrophil chemoattractant production (Fig. 5e). As detailed phenotypic characterization of neutrophils from tissue sections is difficult, cytospins were generated from the lavage fluid of mice receiving B16FasL Selleck Belnacasan cells i.p., enabling us to compare neutrophils isolated from PC61-treated and GL113-treated mice (Fig. 6). No differences were observed in expression of the neutrophil activation marker, CD11b or ROS (data not DNA Damage inhibitor shown). An effect of Treg cells on neutrophil activation cannot be ruled out, however, because it is possible that only activated

neutrophils would be recovered in the lavage fluid (and similarly the site of tumour cell inoculation) so any impact of Treg cells on neutrophil activation may be difficult to observe in vivo. However, differences were observed between neutrophils isolated from PC61-treated and GL113-treated mice (Fig. 6). Figure 6(a,b) shows examples of neutrophils isolated from GL113-treated and PC61-treated mice, respectively. Examples of segmented nuclei are given in Fig. 6(c), where segments are joined by thin strands of chromatin. Upon enumeration, it was evident that the proportion of neutrophils with a higher number of segments was increased 17-DMAG (Alvespimycin) HCl in PC61-treated mice (Fig. 6d,e), which results in an increase in the average number of segments per neutrophil (Fig. 6d,e). Hypersegmentation of nuclei in neutrophils has long been associated with more mature

neutrophils, and is an indicator of prolonged neutrophil survival.18 Collectively, these data support the premise that Treg cells affect neutrophil accumulation at the site of antigenic challenge not through inhibiting their activation but through influencing local chemokine production and by limiting their survival. To test the relevance of neutrophils in this model, we first determined, in an in vitro assay, whether neutrophils could impinge on tumour rejection through direct lysis of tumour cells. As shown in Fig. 7(a), neutrophils were capable of lysing both B16 and B16FasL cells. To test the hypothesis in vivo, mice were treated with both PC61 and RB6-8C5, to deplete CD25+ cells and neutrophils, respectively, followed by s.c. challenge with B16FasL (Fig. 7b).

3C), there was a significant decrease in the percentage of CD11b/CD11c+ DC (Fig. 3D and E). Notably, ER-β ligand treatment did not alter the percentage of CD4+CD25hiFoxp3+ T regulatory cells that could potentially suppress encephalitogenic TC in the CNS (not shown). Naïve mice did not show detectable levels of TC or DC in the CNS. Further analysis

of CD11b/CD11c+ DC in the CNS of EAE mice revealed that ER-β ligand treatment appeared to decrease MHCII expression when compared with vehicle-treated mice, but there were no differences in the level RG7204 solubility dmso of expression of the costimulatory molecules CD80 and CD86 on DC between treatment groups (Supporting Information Fig. 1). Altogether, these results showed that the cellular composition of CNS inflammation in EAE was affected by ER-β ligand treatment during the effector phase. Specifically, ER-β ligand treatment decreased the percentage of CD11b/CD11c+ DC in the CNS. We next asked whether ER-β ligand treatment might affect cytokine production

by DC in the target organ. We focused on TNF-α because TNF-α is known to mediate demyelination and axonal transection in EAE 24, 25, and we had observed protection of myelin and axons with ER-β ligand treatment (Fig. 2). DC were sorted ex vivo from the CNS of ER-β ligand and vehicle-treated mice at disease onset and TNF-α mRNA www.selleckchem.com/products/MG132.html levels were quantified by RT-PCR. TNF-α mRNA levels were reduced by 40% in CD11b/CD11c+ DC derived from ER-β ligand-treated EAE mice as compared with vehicle-treated (Fig. 4A). Together, these Sulfite dehydrogenase data showed that in addition to reducing the number of DC in the target organ (Fig. 3), ER-β ligand treatment also reduced their ability to make TNF-α. To further determine whether ER-β ligand treatment in vivo induced functional changes in CNS DC, we performed DC/TC co-cultures. DC were derived from the CNS of ER-β ligand or vehicle-treated EAE mice, whereas autoantigen-primed TC were obtained from LN of untreated mice immunized with autoantigen. Consistent with the previous studies using co-cultures 26, autoantigen stimulation

of co-cultures resulted in proliferation at DC/TC ratios of 1:5 and 1:20, but not at 1:50. Notably, there was no difference in this proliferation when comparing DC derived from ER-β ligand versus vehicle-treated mice (Fig. 4B). However, when TNF-α levels were examined in supernatants, decreased levels of TNF-α were found in cultures that contained DC derived from the CNS of ER-β ligand-treated, as compared with vehicle-treated mice (Fig. 4C). In this experiment, it is possible that the source of TNF-α may be DC and TC. As TNF-α can mediate demyelination and axonal transection in EAE 27, 28, effects on TNF-α production when DC were treated with ER-β ligand were consistent with reduced demyelination and axonal loss in ER-β ligand-treated EAE mice (Fig. 2).

Specific selleck chemical modulatory effects of MSCs from human and experimental animal sources have

been described for the differentiation, activation, proliferation and effector functions of multiple innate and adaptive immune cells 5–11. Among these, MSC-mediated inhibition of primary T-cell activation and proliferation, suppression of DC maturation and promotion of regulatory phenotypes in monocyte/macrophages and T cells have been most extensively characterised 7–9, 11, 12. In keeping with a paracrine or “trophic” model of MSC function in vivo 13, various MSC-produced soluble mediators have been implicated in these immunomodulatory effects including IL-10, IL-6, HGF, TGF-β, chemokine ligand-2 (CCL2), HLA-G, NO, tumor necrosis factor-inducible gene 6 protein (TSG-6), prostaglandin E2 (PGE2) and kyneurenine 1, 2, 7, 9, 12, 14–16. For some such mediators, expression by MSCs may be dependent on pre-exposure to exogenous factors (e.g. IFN-γ, TNF) or on contact-dependent MSC/target cell cross-talk 2, 7, 16–19. The potential for harnessing MSC immunomodulatory

properties has been highlighted by results in pre-clinical models of autoimmunity, allotransplantation, sepsis and acute ischemic injury 1, 4, 7, 14, 15 as well as by outcomes from clinical trials in inflammatory bowel disease, graft-versus-host disease and myocardial infarction 1, 20. T cells represent the primary effector cells for common autoimmune Baricitinib diseases and for rejection of transplanted organs and tissues 21. Furthermore, activated memory T cells have been implicated Liproxstatin-1 mw in non-antigen-specific forms of tissue injury such as ischemia-reperfusion 22, 23. In

addition to the investigation of mechanisms underlying MSC inhibition of T-cell activation, attention has also been directed toward their influence on specific T-cell effector phenotypes including CD8+ CTLs and the Th1, Th2 and Treg sub-types of CD4+ T cells which may be more or less prominent in individual immune-mediated diseases 12, 24–26. In vitro and in vivo experimental evidence would suggest that MSCs are consistently suppressive of CTL- and Th1-mediated immune responses while being less inhibitory toward Th2-type responses and actively promoting Treg survival and expansion 9, 12, 27. Less well understood for each of these subsets are the relative effects of MSCs on naïve T cells undergoing primary activation compared with previously activated, or memory-phenotype, T cells. The recent description of an additional CD4+ T-cell subset, termed Th17 cells, has added further complexity to our understanding of cellular adaptive immunity 28. The Th17 effector phenotype is characterised by synthesis of a signature cytokine, IL-17A, in addition to IL-17F, IL-21, IL-22 and CCL20 29.

The ability of MSC to induce apoptosis of T cells was investigated, both in vitro and in vivo. The induction of PBMC apoptosis in vitro by human MSC was examined using an MSC/PBMC co-culture model. A known inducer of PBMC apoptosis, cisplatin, caused significant apoptosis of PBMC (Fig. 4a), whereas allogeneic human MSC did not (P < 0·0001) (Fig. 4a). However, the lack of apoptosis in vitro might not reflect selleck chemicals the in vivo situation, therefore the NSG model was adapted to detect apoptotic cells. NSG mice were treated with PBS or PBMC, with or without MSCγ cell therapy on day 0. FLIVO (a reagent which detects active caspases of apoptotic cells

in vivo) was administered i.v. 12 days later and allowed to circulate for 1 h. After Obeticholic Acid cell line 1 h, the lungs (Fig. 4b) and livers (Fig. 4c) were harvested and analysed for FLIVO/CD4 staining by two-colour flow cytometry. Although CD4+ T cells were detected, there was no increase in apoptotic CD4+ T cells following MSCγ therapy in either organ sampled on day 12 (Fig. 4b,c) or at other times prior to day 12 (days 1 or 5, data not shown). These data suggested that MSC did not induce detectable apoptosis of donor human CD4+ T cells in vivo or in vitro and that this was unlikely to be the mechanism involved in the beneficial effect mediated by MSC in this

model. An alternative hypothesis for the beneficial effect of MSC cell therapy was formulated around the induction of donor

T cell anergy. To examine this, an in vitro two-step proliferation assay was designed which would closely mimic in vivo circumstances. First, murine DC isolated from the bone marrow of BALB/c mice were used to mimic the murine (host) antigen-presenting cell. These were matured using polyIC as a stimulus and co-cultured with human CD4+ T cells for 5 days in the presence or absence of MSC. After 5 days, the proliferation of human CD4+ T cells was analysed. Human CD4+ T cells proliferated strongly when cultured with mature murine Methane monooxygenase DC (P < 0·0001); however, allogeneic human MSC significantly reduced this effect (P < 0·05) (Fig. 5a). These data showed that MSC were capable of inhibiting T cell proliferation in a xenogeneic setting, analogous to that found in the aGVHD NSG model. To examine if the reduction in T cell proliferation by MSC was due to the induction of T cell anergy, a two-stage assay was then performed. Human CD4+ T cells were co-cultured with mature murine DC and/or MSC for 5 days; human CD4+ T cells were re-isolated from cultures by magnetic bead isolation. Re-isolated CD4+ T cells were allowed to rest overnight then cultured for a second time with irradiated BALB/c DC stimulated with or without polyIC/IL-2. Following the second-stage co-culture, human CD4+ T cells proliferated in response to irradiated mature DC (Fig. 5b).