One of the most prominent differences between the CD56bright and the CD56dim NK subsets is their intrinsic cytotoxic capabilities. As mentioned above, resting CD56dim NK cells are much more cytotoxic than resting CD56bright NK cells.7 The molecular mechanisms responsible for this are not fully understood. CD56dim NK cells are more granular than CD56bright NK

cells13 and differences in see more the intracellular signaling pathways between the two subsets may also account for their cytotoxic capabilities. Indeed, it was demonstrated by gene expression profiling that compared with CD56dim NK cells, CD56bright NK cells express lower levels of the CD3ζ adaptor molecule, which mediates some of the natural cytotoxicity receptor signaling.14 Importantly, CD56dim NK cells exhibit high

expression levels of FcγRIII (CD16), whereas CD56bright NK cells do not express CD16 or express only low levels of it and therefore, cannot perform antibody-dependent cellular cytotoxicity (ADCC). CD16 is a unique receptor not only check details because of its late function when the adaptive immune response is already activated, but also because among almost all NK cell receptors tested, it is the only receptor that could function independently without the help of other NK cell receptors.8 It is now well established that NK cells can act as major regulators of the immune response, in addition to their ‘classical’ role of killing much hazardous cells. The CD56bright CD16− NK subset is considered as the regulatory subset and a prominent example for its regulatory role is the function of these NK cells in the uterine mucosa prior to and during pregnancy, in the endometrium and decidua tissues, respectively. The data on mouse endometrial NK (eNK) cells are quite limited. It is known that mouse eNK cells

are first found in 2-week-old mice as small and agranular cells.15 Recently, it has been suggested that B220+CD11c+NK1.1+ cells may be analogous to human CD56bright NK cells16 and a recent study indeed identified these cells in the uterus of virgin mice.17 In this study, the phenotype of mouse eNK cells was examined and it was demonstrated that eNK cells are B220+CD11c+NK1.1+ DX5+ (a phenotype that is similar to that of mouse peripheral blood and spleen NK cells18). These eNK cells also express CD122 (the IL-2/IL-15 receptor common β subunit), NKp46 (which is considered the most specific NK marker across species), CD11b (an integrin subunit), CD27 (TNF receptor family member), and CD69 (an activation marker which is also expressed on human eNK cells). It is important to note that mouse eNK cells do not stain for DBA,17–19 which binds N-acetyl-d-glalctosamine conjugates and is considered a selective marker of mouse uterine NK cells.

gingivalis was inserted into the p-MAL plasmid pMD157, followed by transfection to E. coli and incubation. After 1 or 2 days of incubation, the E. coli suspension was centrifuged and the pellet was homogenized. The homogenized suspension

was subjected to the dialysis treatment, gel-filtration chromatography, and ion-exchange chromatography. Finally, isolation I-BET-762 order of the antigen was performed using amylose resin column affinity chromatography, and 25k-hagA was obtained via cleavage treatment of 25k-hagA-MBP using Factor Xa (New England BioLabs, Ipswich, MA). For sublingual immunization on days 0, 7, and 14, mice were anesthetized with pentobarbital, and 30 μL of phosphate-buffered saline (PBS) containing 50 μg of 25k-hagA-MBP was delivered with a micropipette applied against the ventral side of the tongue while directed toward the floor of the mouth. Mice were immunized with 7.5 μL of antigen four times (total volume = 30 μL). Ten minutes of interval were set between each administrations. A nonimmunized

group was PBS treated. Animals were maintained with their heads placed in ante flexion for 30 s during each delivery. Serum and saliva were collected from each group to examine the 25k-hagA-MBP-specific Ab responses. Ab titers were detected using an enzyme-linked immunosorbent assay (ELISA) as described previously (Maeba et al., 2005). Briefly, plates were coated with 25k-hagA-MBP (5 μg mL−1). After TGF-beta inhibitor washing with PBS containing 0.05% Tween 20, plates were blocked with PBS containing 1% bovine serum albumin. Next, serial dilutions of serum or saliva samples were added in duplicate. The starting dilution of the serum was 1 : 26, while that of the saliva was 1 : 22. The plates were incubated for 5 h at room temperature, washed, and then incubated with horseradish peroxidase-labeled goat anti-mouse heavy chain γ, γ1, γ2a, γ2b, γ3, or α-specific antibodies (Southern Biotechnology Associates, Birmingham,

AL) at 4 °C for 20 h. Finally, 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic Nitroxoline acid) (ABTS) with H2O2 (Moss, Inc., Pasadena, MD) was added for color development. Endpoint titers were expressed as the reciprocal log2 of the last dilution, which gave an optical density at 415 nm of 0.1 greater than that of nonimmunized control samples after 15 min of incubation. Single-cell suspensions were obtained from the salivary gland 7 days after the last immunization. Briefly, salivary glands were carefully extracted, teased apart, and dissociated using 0.3 mg mL−1 collagenase (Nitta Gelatin Co. Ltd, Osaka, Japan) in RPMI-1640 (Wako Pure Chemical Industries Ltd, Osaka Japan). Mononuclear cells were obtained at the interface of the 50% and 75% layers of a discontinuous Percoll gradient (GE Healthcare UK, Ltd, Little Chalfont, Buckinghamshire, UK) (Maeba et al., 2005). To assess the numbers of antigen-specific AFCs, an enzyme-linked immunospot (ELISPOT) assay was performed as described previously (Yamamoto et al., 1997).

1A and B). Of note, although at low frequencies, IFNAR−/− P14 cells were still detectable at day 37 post-infection in the blood, indicating that memory T cells developed and were maintained over a long time period, as also observed for single LCMV infection 19. This finding

could be confirmed by monitoring the total number of IFNAR−/− P14 selleck cells in spleen and LNs 45 days post-infection (Figs. 1B and 6). For further functional analyses we focused on day 3 and day 6 post-infection, as at these time points the numbers of IFNAR−/− P14 cells were sufficient for detailed analysis. To determine whether impaired expansion of IFNAR−/− P14 cells was accompanied by altered effector functions, we measured LY2157299 molecular weight their capacity to secrete IFN-γ upon in vitro peptide restimulation. In accordance with our recent studies 17, we found that cells lacking type-I IFN signaling showed less capacity to secrete IFN-γ as well as to degranulate (measured by cell surface CD107a mobilization) compared with WT P14 cells (Fig. 1C and D) while expressing comparable levels of perforin and granzyme B (Fig.

1D). Thus, although IFNAR−/− CD8+ T cells initially expanded and gained effector functions, albeit at reduced levels, type-I IFN signaling was a major promoter of their expansion, survival and effector differentiation under inflammatory conditions

of an LCMV infection. It is well established that type-I IFN and IL-12 have redundant functions in their role as a third signal during CD8+ T-cell activation; both pro-inflammatory cytokines can promote expansion as well as survival of activated CD8+ T cells in vivo 13, 18–20. Additionally, there is abundant evidence that IL-12 signaling during CD8+ T-cell priming promotes the terminal differentiation of short-lived effector cells 3–5. However, a direct role of type-I IFN in SLEC formation in vivo has not been cAMP studied to date. Thus, we examined in vivo the expression of cell surface markers which have been described to identify SLECs (CD44high, CD127low, KLRG1high) and MPECs (CD44high, CD127high, KLRG1low) 3 and 6 days post co-infection. Notably, WT and IFNAR−/− P14 cells showed comparable naïve phenotypes (CD44low, CD25low, CD127high, KLRG1low and CD62Lhigh) (Fig. 2A and data not shown). WT P14 cells exhibited a pronounced upregulation of CD25 as early as day 3 post-infection (Fig. 2A and B), whereas IFNAR−/− P14 cells in the same recipients only slightly increased CD25 expression. By day 3 post-infection, WT P14 cells could be divided into two populations with respect to CD62L expression (CD62Lhigh and CD62Llow) and by day 6 the majority of the WT P14 cells showed low expression of CD62L.

However, primary renal diseases for ESRD are different by race and area and the incidence, prevalence and mortality of CKD vary accordingly.14 Consequently, the CKD screening and prevention programs requires different approaches depending on the patient’s race, habitual and socioeconomic status and be modified in response learn more to the situations where they would be conducted. The authors thank Dr Hung-Chun Chen and the organizing committee for providing this opportunity to share experience on prevention and management of CKD. Dr Nan Chen’s work was supported in part by grants from the Leading Academic Discipline Project of Shanghai Health

Bureau (05III001), the Shanghai Leading Academic Discipline Project (T0201) and the Science and Technology Commission of Shanghai Municipality (08dz1900502). The Authors state that there is no conflict of interest regarding the material discussed in the manuscript. “
“Date written: July 2008 Final submission: October 2008 No recommendations possible based on Level I or II evidence (Suggestions

are based on Level III and IV evidence) check details As dialysis is an accepted and available mode of treatment for end-stage kidney disease (ESKD) in Australia and New Zealand, the decision concerning acceptance onto a dialysis programme should be made on the basis of the patient’s need. The cardinal factor for acceptance onto dialysis or continuation Rolziracetam of dialysis is whether dialysis is likely to be of benefit to the patient.* *Additional notes: 1 Lack of certainty about whether the treatment will be of benefit to the patient may suggest the use of temporary dialysis or a ‘trial’ so

that dialysis as a treatment option can be evaluated. Survey individual unit documentation of implementation of the above ‘Suggestions for Clinical Care’ and rates of insertion and completion of the checklist titled ‘Approaching ESKD’ (Appendix) in patient notes. These draft guidelines do not refer to temporary dialysis, but expressly consider acceptance onto long-term dialysis, which would be terminated only by the death of the patient, successful renal transplantation, inability to maintain successful dialysis or elective withdrawal of dialysis by the patient. There is broad consensus in Australia and New Zealand that people in our society regardless of age, race, gender, religion and underlying disease have equal rights to access health facilities. Unless the patient has chosen to accept only supportive treatment, individuals and society at large expect that ESKD should not, except in unusual circumstances, be the primary cause of death.

There were Dabrafenib nmr 22 nails biopsies from onychomycosis patients taken for the research of morphopathological changes in the thickened nail plate affected by onychomycosis. Samples of cadaverous’ nails were used as a control material. The material was stained with haematoxylin and eosin and immunohistochemical methods. Terminal deoxynucleotidyl transferase dUTP nick end labelling reaction and periodic acid-Schiff reaction were also performed. We found patchy hypertrophy in the granulose layer of the epidermis,

with focal acanthosis. In the horn layer, we identified nests of parakeratosis of various sizes, with incorporations of homogenous and eosinophil masses. We found high levels of interleukin 6 and interleukin 10 positive cells in the nail bed and in the bloodstream. Interleukin 1, however, was not a part of any of the functional units of any of the nails. Significant amount of fibres containing human selleck products beta defensin-2 were found in the bed and plate of the nail. Therefore one can conclude that as regards the nails affected by onychomycosis, the most effective morphopathogenical processes include cytokine and defensin excretion occurrence in the nail bed. “
“Descriptive values were determined for eight

antifungal agents within the course of a multi-centre study encompassing 1062 German and Austrian clinical yeast isolates. Candida albicans (54%) was the predominant species isolated followed by Candida glabrata (22%), Candida parapsilosis (6%), Candida tropicalis (5.7%), Candida krusei (4.3%), as well as eleven further candidal and four non-Candida yeast species. While 519 (48.9%) isolates

were tested susceptible to all antifungals tested, no isolate was found to exhibit complete cross resistance. For C. albicans, the proportions of susceptible isolates were 93.2% (amphotericin B), 95.6% (flucytosine), 84.3% (fluconazole), 83.8% (posaconazole), 91.8% (voriconazole), 96.5% (anidulafungin), 96.2% (caspofungin) and 97.6% (micafungin). Patterns of complete parallel resistances were observed within azoles (8.8%) and echinocandins (1.7%). While a decreased susceptibility was found infrequently for echinocandins and flucytosine, Nintedanib (BIBF 1120) it was more common for azoles with highest proportions for isolates of C. glabrata (fluconazole, 40.6%; posaconazole, 37.2%), Candida guilliermondii (fluconazole and posaconazole, each 25.0%), C. krusei (posaconazole, 28.3%; voriconazole, 60%), C. parapsilosis (fluconazole, 70.3%) and C. tropicalis (fluconazole, 62.3%). The descriptive values obtained in this study represent a valid basis for the comparison of recent and future epidemiological surveys to analyse the susceptibility of yeast isolates towards major antifungal substances. “
“Malaria is the most important parasitic infection in people, affecting 5–10% of the world’s population with more than two million deaths a year.

In the following we will discuss the relevance that neurogenesis may play in the aetiology and/or maintenance of two selected diseases, major depression and epilepsy (for an extended review please refer to [62,63]). One of the hallmarks in the aetiology of affective disorders such as major depression is stress, which is among the most powerful negative regulators of hippocampal neurogenesis. Together with the findings that a number of clinically used antidepressants (ADs) such as fluoxetine strongly enhance neurogenesis, the idea was proposed that new neurones may be critically involved

in the disease process of depression and/or represent a potential treatment target [64–66]. This was supported by the clinical observation that a number of ADs require chronic treatment to become effective which may be due to the need for www.selleckchem.com/products/Rapamycin.html AD-induced neurogenesis, which would take several weeks before drug-induced neurones become functionally integrated. An important milestone supporting the relevance of neurogenesis in major depression was a study showing that irradiation-mediated this website inhibition of neurogenesis substantially reduced the ability

of fluoxetine (and other ADs) to affect mood-related behaviour in rodents [67]. However, it became also evident over the last years that not all drugs with AD efficacy require proper neurogenesis to be effective (at least in rodent models of major depression) [68]. Similarly, genetically enhanced neurogenesis by itself does not have mood-manipulating effects under physiological conditions even though fantofarone this genetic, neurogenesis-enhancing approach still needs to be tested in disease models [59]. Mechanistically, the role of new neurones in the context of affective disorders may be twofold. One obvious role of neurones in the context of depressive disease lies in their function in cognitive processes that may amplify/induce disease symptoms. In addition, recent data suggest that new neurones may also directly

serve as a buffer for stress response by having a substantial impact on the hypothalamic–pituitary–adrenal (HPA) axis [69]. Even though it is clear that altered or failing hippocampal neurogenesis is certainly not the only cause of affective disorders, current efforts aim to develop novel strategies to pharmacologically enhance neurogenesis that may help treat depression or ameliorate disease symptoms [66]. In contrast to affective disorders, the key alteration in hippocampal neurogenesis after epileptic seizures is not manifested by a reduction in newborn neurone numbers but rather by an initial increase in newborn neurone numbers followed by aberrant maturation and ectopic migration within the dentate circuitry [70–74].

These data suggested that exogenous administration of CGS21680 could prevent early events associated with the induction of EAMG, for example, events linked to the T-cell compartment (Ag recognition, epitope spreading, and T-cell expansion) [[2]]. However, in established EAMG, once damage to the neuromuscular junction occurred as a consequence of auto-immune memory,

T- and B-cell responses (in combination with complement activation) directed against the AChR, treatment with CGS21680 was much GSK-3 inhibitor review less effective. A2AR, similar to other Gs-protein-coupled receptors, signals mainly via the adenylate cyclase–cAMP–PKA canonical pathway [[31]]. Recent data have further explained how the A2AR-mediated increase of cAMP may inhibit general T-cell responses such as proliferation [[32]] and cytokine production [[28, 33]]. Therefore the PKA inhibitor (H-89) was included in this assay to verify whether suppression of inflammation mediated by A2AR depended on the cAMP pathway. Furthermore, whether

A2AR-mediated inhibition occurred only during the presence of the A2AR agonist or selleck chemicals if it conferred a permanent alteration to T-cell function was also examined. These results provided evidence that A2AR agonists persistently inhibited the production of anti-AChR IgG antibodies mediated partly as a result of the inhibition of PKA activation (Fig. 4). We next determined the nature of the B cells or CD4+ T cells impacted by CGS21680. First, both proliferation and anti-AChR IgG secretion by B cells was assessed, demonstrating that CGS21680 neither altered the anti-AChR IgG secretion profile nor interfered with B-cell proliferation (Fig. 5). These results were similar to previously published reports [[34]] that demonstrated that B cells responded poorly to A2AR stimulation (determined Oxymatrine by measuring cAMP levels in CD4+ T cells, CD8+ T, cells and B cells) following incubation with an A2AR agonist. This led us next to focus on the effect of CGS21680 on CD4+ T-cell function. Although the symptoms

of MG and EAMG are the result of auto-antibodies, CD4+ T cells specific for the target antigen (along with the cytokines secreted) have an important role in the disease development and progression. CD4+ T cells play a role in pathogenesis by driving the synthesis of high-affinity anti-AChR antibodies, as well as secreting proinflammatory cytokines [[6, 8, 9]]. Binding of those antibody subclasses to AChR at the neuromuscular junction triggers complement-mediated destruction of the postsynaptic membrane [[9]]. Here, we demonstrated that the number of Th1 cells and Th2 cells were decreased following A2AR activation (Fig. 6 and 9). This result challenged the hypothesis that lymphocyte-expressed A2AR might shift the Th-cell responses from a Th1 toward a Th2 response.

Pipette up glomeruli by lifting the sieves and washing down glomeruli to one side of the wall of the 125 µM sieve (for an adult kidney) or 125 µM and 90 µM sieves (for a young child’s kidney). Transfer glomeruli to culture treated flasks or Petri dishes (IWAKI 3123-75 or 4020-010) and place into 37°C incubator. Only change the medium when some of the glomeruli are firmly attached find more (3–5 days). Usually cellular outgrowth starts in 7–10 days, at which time the majority of cells are podocytes. At this stage podocytes grow rapidly and predominate; after 2 weeks other cells such as mesangial cells may appear and

would eventually take over, so it is important to harvest podocytes within 2 weeks to avoid contamination with other cell types. Occasionally, contamination

with non-podocytes may necessitate subcloning (see Subcloning of immortalized podocytes). Trypsinize cells (Sigma T3924 which is 0.05% trypsin; Sigma-Aldrich, Dorset, UK) and separate single cells away from the glomeruli using a 40 µM cell Autophagy activator strainer when patches of podocytes reach confluence. Re-plate cells in T75 or T25 culture treated flask with less than 40% density overnight. These are primary culture podocytes, ready to be transduced with the immortalizing transgene on the following day (Fig. 2). Primary cells are infected with tsSV40T and hTERT vectors9 containing respectively G418 and hygromycin resistance genes, over 18 h with Polybrene 10 µg/mL (Sigma H-9268). Then subconfluent cells are transferred from 37°C to 33°C for selection

using G418 (400 µg/mL; Sigma-Aldrich) and hygromycin (25 µg/mL; Sigma-Aldrich) for 2 weeks (Fig. 3). Currently we use a bicistronic vector containing tsSV40T and hTERT, which has a single resistance cassette to G418. Keep in culture until new immortalized cells grow, taking at least 1 month (Fig. 3). To obtain a homogenous cell culture derived from single cell clones, cells are subcloned using treated NIH 3T3 fibroblasts as non-dividing feeder cells. Grow NIH 3T3 fibroblast cells at 37°C till confluent then treat with 0.25 µg/mL Hydroxychloroquine mitomycin C overnight. Change the medium after treatment and trypsinize cells on the following day and reseed NIH 3T3 cells in 4 × 75 cm2 flasks or 5–6 Petri dishes containing ∼105 cells or ∼5 × 104 cells in each dish. Count podocytes before trypsinizing, then dilute the cell suspension to the desired seeding concentration into each NIH 3T3 flask or Petri dish, for example 100 cells, 300 cells, 500 cells and 1000 cells. Leave cells at 33°C for another 5–7 days and then change the medium as necessary. After about 5 weeks, single clonal cells grow out visibly which are picked by cloning rings or cloning discs (both from Sigma-Aldrich). Cut off the top of a flask with an electrically heated scalpel, and using sterile forceps dab cloning rings with silicone grease (Fisher scientific laboratory – autoclave before use) or discs with 0.25% trypsin-EDTA.

However, these findings were not exclusive to the MS brain, as EBER+ cells were also found in cases of stroke. We proposed a more indirect mechanism by which latent EBV infection could contribute to neuroinflammation:

that these small RNAs bind to Toll-like receptor 3 and potentially other intracellular receptors such as retinoic acid-inducible gene 1 (RIG-I) and thus stimulate IFN-α production in active MS lesions (Fig. 2). A recent study showed that EBERs were indeed released from EBV-infected cells and acted as local immunomodulators [48]. Could innate activation triggered by latent EBV infection be part of the game? Perhaps we have to think differently – EBV might be more subtle than we anticipated. After all, it is a persistent virus selected to co-exist with the host rather than endanger it. In a small Phase Atezolizumab II trial with rituximab (anti-CD20), there was a dramatic reduction of disease activity in RRMS patients within 48 weeks [49]. Rituximab is a genetically engineered

www.selleckchem.com/products/Maraviroc.html chimeric ‘humanized’ molecule that targets CD20+ B cells and is used for treating B cell lymphoma. CD20 is present on B cells and pre-B cells but lost upon plasma cell differentiation [50, 51]. The primary end-point of this trial was mean gadolinium (Gd)-enhancing lesions (inflammatory activity) assessed by MRI from baseline to week 48. A decrease in disease activity was already noted at week 4 and most pronounced at week 12. Such very early treatment responses suggest that rituximab treatment Fludarabine may act directly via B cell lysis – or, indeed, on the inflammatory mechanisms – rather than by reducing pathogenic autoantibody levels. Indeed, rituximab does not affect serum and CSF antibody levels [52]. Interestingly, in a trial on PPMS, the primary

end-point was not reached; however, there was a suggestion of an effect in subjects with evidence of active inflammation [53]. Treatment with rituximab led to predominance of circulating naive and immature B cells. In the CSF, T and B cell numbers were decreased, and resting B cells predominated. Two additional humanized antibodies targeting different epitopes on CD20 are now being trialled in MS: ofatumumab and ocrelizumab [54]. Ocrelizumab appears to target mature B cells. It has reached Phase III for several autoimmune diseases, e.g. rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and Phase II for MS. Those for RA and SLE were halted in May 2010 because of occasional serious/fatal opportunistic infections in high-dose arms, especially in subjects with Asian ancestry. The Phase II study in RRMS in October 2010 showed statistically significant reductions at week 24 in both lesion load (as measured by MRI activity) and relapse rate, compared to placebo, both doses (200 mg and 600 mg) being well tolerated.

Furthermore, S1pr5−/− mice constitute an interesting model to study the role of Ly6C− monocytes in immunity, a point that remains unclear. WT C57BL/6 mice were purchased from Charles River Laboratories (L’Arbresle, France). S1pr5−/− mice [18], Ccr2−/− [30], and Cx3cr1gfp/gfp mice [31] have been previously described. In

some experiments, we also used C57BL/6 CD45.1 mice or C57BL/6 CD45.1 × CD45.2 mice that were bred in our animal house. Female mice 8–24 week-old were used unless specified. DOP (Sigma, St. Louis, MO, USA) was provided in the drinking water (30 μg/mL) supplemented with glucose. Experimental procedures and mice housing were approved by the local Ethics Committee and carried out according to the French and European laws. C57BL/6 CD45.1 × CD45.2 mice were irradiated twice BVD-523 manufacturer at 450 rad within a 4-h interval. Four hours RXDX-106 purchase after the last irradiation, they received an intravenous injection of a 1:1 mixture of BM cells from WT CD45.1 and S1pr5−/− CD45.2 mice. BM chimeras were analyzed 6–12 weeks after reconstitution. This technique was previously described [32]. Briefly, mice were injected intravenously with 1 μg anti-CD45 Mab (30F11) coupled to phycoerythrin (PE) or PE-cyanin-5 (BD Biosciences, San Jose, USA). Mice were sacrificed 2 min after antibody injection. BM was

then collected and analyzed by flow cytometry. BM cells from WT CD45.1 and S1pr5−/− or Cx3cr1gfp/gfp (CD45.2) mice were prepared and mixed at a 1:1 ratio before intravenous injection (1 × 107 cells of each genotype in PBS) into anesthetized CD45.1 × CD45.2 C57BL/6 mice. Sixteen hours later, mice were sacrificed, blood and bone marrow was collected and the percentage of monocyte subsets of each

donor mice was measured by flow cytometry after staining for CD45.1 and CD45.2 expression. Cell viability was measured in ex vivo isolated cell suspensions using Annexin V and 7-AAD staining (BD Biosciences) and flow cytometry. BM, spleen, lung, lymph node, kidney, and blood cells were isolated and stained as previously described [33]. Cell counts were determined using an accuri C6 flow cytometer (BD Accuri Cytometers, Ann Arbor, MI, USA). Monocytes were identified as CD115+ in the Thalidomide blood or as CD11b+CD11clowNK1.1−CD19−Ly6G− in the BM and spleen. The following Mabs from eBioscience (San Diego, CA, USA) or BD Biosciences (Becton Dickinson, San Jose, USA) were used: anti-CD115 (AFS98), anti-Ly6C (HK1.4), anti-Ly6G (1A8), anti-CD19 (ebio1D3), anti-CD3 (145–2C11), anti-NK1.1 (PK136), anti NKp46 (29A1.4), anti-CD11b (M1/70), anti-CD45.1 (A20), anti CD45.2 (104), and relevant isotype controls. Bcl2 expression was measured using a commercial kit (BD Biosciences) according to the manufacturer’s instructions. Flow cytometry was carried out on a FACS Canto, a FACS Canto II or a FACS LSR II (Becton Dickinson). For S1P migration assays, monocytes were purified from BM cells using a negative selection procedure.