The data also suggest R788 datasheet that the replication kinetics of PML-type JCV DNA differ among COS-tat cell clones. In the current study, we examined the propagation characteristics of PML-type JCV in COS-7 derived cell lines

expressing HIV-1 Tat protein. In COS-tat cells, production of virus progenies and replication of viral genomic DNA were increased compared to those in parental COS-7 cells, as judged by data from HA and real-time PCR assays. Based on the results obtained in the present and previous studies (8), we have demonstrated that stable expression of HIV-1 Tat facilitates propagation of, not only archetype, but also PML-type, JCV. In COS-tat cells, HIV-1 Tat-mediated JCV propagation can be examined without transfecting the cells with Tat PD0325901 in vivo expression plasmid or stimulating them with exogenous Tat. Thus, these cell lines may provide a useful model system for studying HIV-1 Tat-mediated propagation of

both archetype and PML-type JCV. When examining the characteristics of COS-tat cells, we found that stable expression of HIV-1 Tat resulted in down-regulation of cell proliferation. This reduction of the cell growth of COS-tat cells is consistent with earlier results indicating that Tat prevents proliferation of human intestinal epithelial cells (15). A growing body of evidence suggests that HIV-1 Tat regulates numerous cellular genes that are involved in cell signaling and translation, thereby controlling Atazanavir the proliferation of host cells (16). The precise mechanism by which Tat protein represses the proliferation of COS-tat cells is unclear; however, previous investigations suggest that HIV-1 Tat induces the expression of Purα, a single-stranded DNA binding protein which inhibits cell growth (16, 17). Therefore, it might be that the decreased proliferation of COS-tat cells is associated with Tat-induced expression of Purα. In our previous study, archetype JCV efficiently propagated in COS-tat7, COS-tat15, and COS-tat22 (8). Among the COS-tat cell clones tested, COS-tat22 cells exhibited a marked increase in the propagation of

archetype JCV at about 30 days after transfection with viral DNA (8). Consistent with earlier results, amounts of HA and viral DNA in COS-tat22 cells were greater than those in other COS-tat cell clones at 30 days following transfection with PML-type JCV DNA. It is likely that production of Tat protein leads to increased propagation of archetype and PML-type JCV in three COS-tat cell clones, although the extent of its expression varies between these clones (8). It has been reported by others that Tat protein can enhance late-promoter transcription of JCV through interaction with a sequence similar to TAR in the JCV control region (3, 4). It has also been demonstrated that Tat protein forms a complex with Purα, thereby stimulating viral DNA replication initiated at the JCV origin (5, 6).

SIEA flap’s region is innervated by the T12 nerve and the iliohypogastric nerve (IHN), but

no sensate SIEA flap has been reported so far. In this report, we present a case in which a sensate SIEA flap innervated by the IHN was used for reconstruction of a finger soft tissue defect. A 55-year-old male suffering from the volar skin necrosis of the right ring finger underwent the volar soft tissue reconstruction using a free sensate SIEA flap because of hypoplastic SCIA. The SIEA flap included the IHN anterior branch, and neuroraphy was performed between the IHN and the third common digital nerve in an end-to-side manner after vascular MK0683 order anastomoses. The reconstructed volar skin could sensate 14 weeks after the surgery. At postoperative 6 months, Semmes-Weinstein test and moving 2-point discrimination revealed

3.64 and 8 mm in the proximal portion of the SIEA flap where the IHN was supposed to innervate. see more The IHN may be included in a SIEA flap, and a sensate SIEA flap may be a useful option when a SCIP flap is not available. Further anatomical and clinical studies are required to clarify anatomy and clinical usefulness of the IHN. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“Background: Since the birth of reconstructive microvascular surgery, attempts have been made to shorten the operative time while maintaining patency and efficacy.

Several devices have been developed to aid microsurgical anastomoses. This article investigates each of the currently available technologies and attempts to provide objective evidence Telomerase supporting their use. Methods: Techniques of microvascular anastomosis were investigated by performing searches of the online databases Medline and Pubmed. Returned results were assessed according to the criteria for ranking medical evidence advocated by the Oxford Centre for Evidence Based Medicine. Emphasis was placed on publications with quantifiable endpoints such as unplanned return to theatre, flap salvage, and complication rates. Results: There is a relative paucity of high-level evidence supporting any form of assisted microvascular anastomosis. Specifically, there are no randomized prospective trials comparing outcomes using one method versus any other. However, comparative retrospective cohort studies do exist and have demonstrated convincing advantages of certain techniques. In particular, the Unilink™/3M™ coupler and the Autosuture™ Vessel Closure System® (VCS®) clip applicator have been shown to have level 2b evidence supporting their use, meaning that the body of evidence achieves a level of comparative cohort studies.

05) (Fig. 3C). IRF8 is a transcription factor that affects cytokine-mediated DC development of CD8+ DCs and pDCs. Since transcription of Irf8 mRNA is inhibited by GM-CSF

at early time points during development [20], and protease inhibitor Cystatin C is controlled by IRF8 in DCs [21], we proceeded to determine whether inhibition of Irf8 expression by GM-CSF at the BM precursor stages persisted with differentiated DCs. Purified BM-DCs cultured with different cytokines were lysed, and newly synthesized IRF8 and Cystatin C proteins after 30 min starvation were immunoprecipitated for quantitation. Addition of GM-CSF to the Flt3L culture inhibited the synthesis of IRF8 and its downstream product Cystatin

C in GMFL-DCs, which were knocked-down to the same levels as the DCs cultured with GM-CSF alone (Fig. 3D). These data suggest that restriction of IRF8 expression Selleck Ku-0059436 during the entire DC development period might Staurosporine nmr account for the resultant phenotypes. To investigate whether the dominant effect of GM-CSF over Flt3L in promoting DC differentiation was due to the high concentrations of GM-CSF used, we titrated the concentration GM-CSF in the presence of a constant amount of Flt3L in the culture. As the concentration of GM-CSF increased, CD8eDC and pDC subpopulations were reduced accordingly (Fig. 4A, top panel). Interestingly, cell size and granularity also changed, suggesting a new DC type had expanded (Fig. 4A, bottom panel). At 10 ng/mL GM-CSF, CD8eDCs, and pDCs are no longer detectable. At this dose, the cytometric profile (with dominance of Sirpα DCs) of cells cultured with both cytokines together looked almost identical to the DCs cultured with GM-CSF alone at the same concentration. When we examined the effect of GM-CSF alone on BM cells, we found that the concentration of GM-CSF that just began to be effective in promoting DC differentiation in the cultures with GM-CSF alone (2.5 ng/mL) corresponded

to the one that at which the new cell types appeared Adenosine triphosphate in the Flt3L culture. Moreover, 10 ng/mL of GM-CSF, the concentration at which the effect of Flt3L was abrogated in our system, was not the saturating concentration of GM-CSF in its effectiveness to drive DC differentiation (Fig. 4B). Collectively, these data suggest that the dominant effect of GM-CSF over Flt3L in redirecting DC development seen in previous experiments comes from its intrinsic ability rather than the high GM-CSF concentration used in these experiments. Since the precursor cells to FL-DCs and GM-DCs are different [4], and the lineage committed, immediate precursors for FL-DCs exist in fresh BM in vivo [22], we asked whether the FL-DC precursors expired or were diverted by GM-CSF into different lineage developmental pathways.

CNVs are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. CNVs are distributed widely in the genomes of apparently healthy individuals and thus constitute significant amounts of population-based genomic variation. Human CNV loci are Temsirolimus enriched for immune genes and one of the most striking examples of CNV in humans involves a genomic region containing the chemokine genes CCL3L and CCL4L. The CCL3L–CCL4L copy number

variable region (CNVR) shows extensive architectural complexity, with smaller CNVs within the larger ones and with interindividual variation in breakpoints. Furthermore, the individual genes embedded in this CNVR account for an additional level of genetic and mRNA complexity: CCL4L1 and X-396 datasheet CCL4L2 have identical exonic sequences but produce a different pattern of mRNAs. CCL3L2 was considered previously as a CCL3L1 pseudogene, but is actually transcribed. Since 2005, CCL3L-CCL4L CNV has been associated extensively with various human immunodeficiency virus-related outcomes, but some recent studies called these associations into question. This controversy may be due

in part to the differences in alternative methods for quantifying gene copy number and differentiating the individual genes. This review summarizes and discusses the current knowledge about CCL3L–CCL4L CNV and points out that elucidating their complete phenotypic impact requires dissecting Tau-protein kinase the combinatorial genomic complexity posed by various proportions of distinct CCL3L and CCL4L genes among individuals. In the last decade, many studies showed

that a major component of the differences between individuals is variation in the copy number of segments of the genome [copy number variation (CNV) or copy number polymorphism (CNP)]. CNVs are distributed widely in the genomes of healthy individuals and thus constitute significant amounts of population-based genomic variation [1–7]. CNV seems to be at least as important as single nucleotide polymorphisms (SNPs) in determining the differences between individual humans [8]. CNV also seems to be a major driving force in evolution, especially in the rapid evolution that has occurred, and continues to occur, within the human and great ape lineage. Compared with other mammals, the genomes of humans and other primates show an enrichment of CNVs. Primate lineage-specific gene CNV studies reveal that almost one-third of all human genes exhibit a copy-number change in one or more primate species [9–12]. To date, almost 58 000 human CNVs from approximately 14 500 regions (CNVRs) have been identified (data from Database of Genomic Variants, http://projects.tcag.ca/variation/). These CNVRs may cover 5–15% of the human genome and encompass hundreds of genes [4,13], and their abundance underscores their substantial contribution to genetic variation and genome evolution [14].

The most extensive inhibition of proliferation was observed at the highest concentrations (Fig. 4D and data not shown), indicating that the Treg are most potent suppressors at higher antigen dose. Notably, the amount of Treg in the bulk culture was insufficient to induce overt suppression, independent of antigen dose (Fig. 4D lower panels).

These data indicate that influenza-specific Treg are present in healthy donors, but the Treg do not dominate the M1-specific T-cell population expanded from PBMC in vitro. In order to test whether the Treg clones could also suppress when their cognate antigens are present in the natural context, we tested the suppressive capacity of D1.68 when stimulated by APC infected with live influenza virus (Fig. 5). Importantly, the proliferation of the responder cells was Talazoparib supplier not

influenced by the presence of influenza virus (Fig. 5A; upper panels and Fig. 5B left set of columns). Simply adding the Treg clone D1.68 did not result in substantial suppression of the responder cells either. However, in the presence of influenza virus-infected antigen presenting cells D1.68 Treg were activated and able to suppress the proliferation of the responder cells in a dose-dependent manner (Fig. 5A; middle panels and Fig. 5B middle set of columns). As a control, the non-suppressive T-cell clone D1.50 was added, but this clone was not able to suppress the responder cells. These data indicate that the influenza-specific Treg are able to suppress other T cells upon a challenge with virus-infected cells. Because the Treg clones were selected on the basis RGFP966 clinical trial of their IL-10 production we probed whether the suppressive capacity of Treg relied on IL-10. Treg were functionally tested in the presence of antibodies Thymidylate synthase against IL-10 and IL10R 5, 20 but this did not alleviate the suppression of proliferation and IFN-γ production of effector

cells in vitro (data not shown). Subsequently, we studied whether Treg interfered with the IL-2 pathway as IL-2 production by T-helper cells plays a critical role in the induction and sustainment of CTL 22 and can be suppressed by Treg 5, 20. To assess whether IL-2 production by influenza-specific T-helper cells was inhibited by influenza-specific Treg, a co-culture experiment was performed wherein the CFSE-labeled T-helper clone D1.50 started to produce IL-2 when APC presented the clone’s cognate antigen. Upon stimulation of the Treg clone (either FOXP3+ or FOXP3−), already present in the co-culture, the production of IL-2 by D1.50 was inhibited (Fig. 6A). This shows that IL-2 production by influenza-specific T-helper cells is inhibited by Treg specific for the same viral antigen. Quickly after activation CD8+ T cells start to upregulate the high-affinity chain of the IL-2 receptor (CD25) at their cell surface as this is critical for maintaining the CD8+ T-cell response 22.

Following stimulation in a 96-well

flat bottom plate, purified B cells were incubated with 4 μM DHE (Molecular Probes) as previously described by Laniewski and Grayson [45]. Surface staining was performed by incubating the cells in a 1:100 dilution of rat anti-mouse B220-allophycocyanin (BD Pharmingen) in 2% FACS Buffer (phosphate buffered saline plus 2% FCS) for 30 min on ice. Cells were washed three times and fixed in 2% paraformaldehyde (Sigma-Aldrich). Purified (1.5 × 106) find more B cells were seeded into wells containing an air-dried, poly-L-lysine (0.01% solution, Sigma-Aldrich)-coated coverslip for 30 min at room temperature. After washing with PBS, cells were stimulated in the presence or absence of 10 μg/mL anti-IgM or 0.2

mM hydrogen peroxide at 37°C. Additionally, one sample was pretreated with 20 mM NAC for 1 h prior to stimulation. At the end of each timepoint, samples were washed, incubated in vehicle or dimedone, and processed for confocal microscopy according to Seo and Carroll [25] using a 1:500 dilution of anti-dimedone antibody (Millipore) and a secondary goat anti-rabbit Alexa-Fluor 488 (Invitrogen). Following sulfenic acid staining, cells were stained with DRAQ5 (Cell Signaling) and mounted with INK 128 mouse ProLong Gold anti-fade reagent (Invitrogen) according to manufacturer’s protocol. 12-Bit images were acquired using a Zeiss LSM 510 confocal laser scanning microscope with a 63× magnification objective lens. For each experiment, exposure settings were determined to avoid saturation and were used for all samples in order to compare intensities. however The open source software ImageJ (National Institutes of Health) was used to quantify cysteine sulfenic acid levels within the nucleus and cytoplasm. The mean fluorescent intensity within the borders of the cell and nucleus was determined. To determine the cytoplasmic fluorescence, the nuclear value was subtracted from the whole cell value. Six fields of view were analyzed for each condition. Purified (2 × 106) B cells were stimulated with 10 μg/mL anti-IgM, washed one time with PBS, and lysed in the presence

of 50 mM Tris-HCl, 100 mM NaCl, 20 mM β-glycerophosphate, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal, 0.5% Triton X-100, 1 mM Na3VO4, 20 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM dimedone. After incubating on ice for 30 min, samples were stored at −80°C. For biotin-based affinity capture experiments, purified B cells (4 × 106) were stimulated with 10 μg/mL anti-IgM, washed one time with PBS, and lysed in the presence of 50 mM Tris-HCl, 100 mM NaCl, 100 μM DTPA, 20 mM β-glycerophosphate, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal, 0.5% Triton X-100, 1 mM Na3VO4, 20 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 200 units of catalase, 10 mM N-ethyl-maleimide, and 5 mM DCP-Bio1 [46].

However, a role of p53 in regulation of T-cell responses or apoptosis has been poorly selleck chemicals llc defined. TCR-mediated signaling in the absence of CD28 costimulation induces both apoptosis and proliferation of naïve T cells from WT mice. In this report we show that, in response to TCR stimulation, T cells from naïve p53-deficient mice exhibited higher proliferation and

drastically reduced apoptosis than WT T cells. CD28 costimulation enhanced the proliferation of TCR-stimulated WT and p53−/− T cells, suggesting that p53 uncouples CD28-mediated antiapoptotic and proliferative signals. To evaluate the physiological significance of these findings, we transplanted OVA expressing-EG.7 tumor cells into WT and p53−/− mice. Unlike WT mice, p53−/− mice exhibited a robust tumor-resistant phenotype and developed cytotoxic T-cell responses against OVA. Collectively, these data support the hypothesis that p53 is an essential factor in negative regulation of T-cell responses and have implication for immunomodulation during treatment of cancers and other inflammatory conditions. Transformation related protein 53 (Trp53 or p53) is a member of the p53 transcription factor family that regulates selleck inhibitor DNA repair,

genomic integrity, DNA replication, cell proliferation and apoptosis 1–3. It contains an N-terminal transactivation domain, a C-terminal tetramerization domain and a central DNA binding domain. Under normal conditions p53 is expressed at low levels in a variety of cell types. Exposure of cells to ionizing radiation, DNA damage, or certain cellular or physiological stresses leads Fenbendazole to stabilization and activation of p53 and its pathway 2. Once activated, p53 binds to target

DNA and initiates transcription of target genes that directly or indirectly inhibit the cell cycle or induce cell death 4, 5. Lack of p53 expression or function is related to development of a vast variety of tumor types and a role for p53 in apoptosis of cells has been the subject of numerous studies for many years. Traditionally, increased expression p53 has been reported in conditions that favor tumoroigenesis, e.g. ionizing radiations. However, p53 expression is also upregulated during inflammation and infections. Synovia from rheumatoid arthritis patients exhibit dominant negative mutations of p53 and expression of p53 is also upregulated in the joints of these patients 6. This increased level of p53 in arthritic synovium joints can be seen in the early stages of disease development 7. Further, lymphocytes from rheumatoid arthritis patients express lower levels of p53 mRNA and protein, and have an impaired ability to induce p53 expression after exposure to gamma radiation, which correlated with increased survival of CD4+ and CD8+ T cells after exposure to gamma radiation 8.

Since infections have drastically increased during the last decades, it is a major goal to investigate the mechanisms underlying pathogenicity of L. corymbifera. One of the first barriers, which the fungus needs to cope with in the lung tissue, is phagocytosis by alveolar macrophages. Here, we report on phagocytosis assays for murine alveolar Palbociclib clinical trial macrophages co-incubated with resting, swollen and opsonised spores of a virulent and an attenuated L. corymbifera strain. A major finding of this study is the significantly increased phagocytosis ratio of the virulent strain if compared to the attenuated strain.

We quantify the phagocytosis by performing automated analysis of fluorescence microscopy images and by computing ratios for (i) fungal phagocytosis, (ii) fungal adhesion to phagocytes and Selleckchem Kinase Inhibitor Library (iii) fungal aggregation and spore cluster distribution in space. Automation of the image analysis yields objective results that overcome the disadvantages of manual analyses being time consuming, error-prone and subjective.

Therefore, it can be expected that automated image analysis of confrontation assays will play a crucial role in future investigations of host–pathogen interactions. The genus Lichtheimia belongs to the Mucorales (subphylum: Mucoromycotina) which counted as the most prominent order of the Zygomycetes, a class of the phylum Zygomycota.[1] Traditionally, the Zygomycota, are known as the most basal terrestrial phylum of the kingdom of Fungi. The phylum formerly comprised two classes, the Zygomycetes and the Trichomycetes, which differed by the ecological niches they inhabit. Whilst Zygomycetes mainly

occur as saprobionts in soil or Sodium butyrate parasites and pathogens of plants, animals or other fungi, the Trichomycetes encompass phylogenetically diverse and unrelated groups of heterotrophic microorganisms which are united based on their ecological habitat and life style. They are typically endocommensals, particularly found in the digestive tract of the aquatic larvae of a number of insects or other arthropod host groups, including crustaceans and diplopods. The Zygomycota were eliminated as a coherent phylum because molecular phylogenetic analyses revealed its dispersal into five subphyla.[2-4] The phylogenetic relationships between these subphyla and their orders are not well resolved yet and are thus not well-understood so far. All five subphyla have the potential to form zygospores during conjugation of two yoke-shaped gametangia arising from compatible mating partners. The mucoralean genus Lichtheimia was formerly classified into the genus Absidia based on the Absidia-specific pyriform shaped collumellate sporangia but later designated to a separate phylogenetic lineage, which was designated into a separate family, the Lichtheimiaceae.[5, 6] Species within the genus Lichtheimia display features quite different from Absidia sensu stricto.

, 2004). Our observation suggests that this effect becomes more evident when the basal levels of EpoR expression are low and ARA290 is applied in nanomolar concentrations. Based on these initial results,

we chose an incubation time of 6 h and 10 nM or 100 nM ARA290 as an appropriate condition to prestimulate cells in further experiments. Epo and its analogues have been described to enhance Selleck MG132 proliferation in healthy tissue, tumors and cell lines (Kumar et al., 2005; Hardee et al., 2007). Such activity would clearly constitute a strong adverse effect for the usage of ARA290 in the urinary tract. In addition to its clinical relevance, pronounced differences in cell growth would also skew the results from in vitro assays. Therefore, we investigated the cell proliferation and viability of cells cultured in the presence of ARA290 for 24 h and performed an XTT assay. On applying the assay, EPZ 6438 we could not detect any significant difference in cell proliferation and viability between treated and control cells in concentrations used for further experiments (T24: 102.7±5.8% for 100 nM ARA290; 5637: 97.1±3.2% for 100 nM ARA290) nor at higher concentrations (for 1 μM ARA290, T24: 90.33±7.6%; 5637: 98.3±0.7%). No changes

were observed when cells were costimulated with inactivated bacteria (data not shown). The neutrophil-attractant chemokine IL-8 serves a crucial function during UTI in mediating the elimination Y-27632 2HCl of bacteria (Hedges et al., 1994; Agace, 1996). The treatment with recombinant Epo has repeatedly been demonstrated to reduce lipopolysaccharide-induced cytokine induction in leukocytes (Schultz et al., 2008; Strunk et al., 2008; Yazihan et al., 2008). To test whether ARA290 modulated this immune response, we costimulated bladder epithelial cell lines with E. coli NU14 and ARA290 in different concentrations. During the period corresponding to basal levels of EpoR expression, the additional presence of ARA290 enhanced IL-8 mRNA expression. At 3 h, an increase in the IL-8 mRNA levels was observed in T24 cells after costimulation with 100 nM ARA290, compared with

stimulation with bacteria alone (127% of 0 nM ARA290, P<0.05; Fig. 3a). This early proinflammatory effect was even stronger with 10 nM ARA290 (155% of 0 nM ARA290, P<0.05). Consequently, IL-8 protein levels were higher in cell culture supernatants 12 h after costimulation with 100 nM ARA290 (115% of 0 nM ARA290, Fig. 3b) or 10 nM ARA290 (125% of 0 nM ARA290, P<0.05). At later time points, when EpoR expression was upregulated, ARA290 costimulation did not further promote immune induction. In contrast, IL-8 levels were reduced on mRNA (61% of 0 nM ARA290, P<0.05; Fig. 3a) and protein levels (78% of 0 nM ARA290, P<0.05; Fig. 3b). This downregulation was also observed at 10 nM ARA290, even though not as pronounced (91% for mRNA and 81% of 0 nM ARA290 for protein, P<0.05).

2), indicating that cell identity was altered due to the action of GM-CSF. In other words, GM-CSF changed the DC progeny of Flt3L cultured

BM progenitors. It is well accepted that GM-CSF and Flt3L mobilize different find more precursor cells for DC differentiation. Ly6Chigh monocytes are the final precursor stage en route to the generation of GM-DCs from total BM [24]; but not FL-DCs, whose immediate precursors are Ly6C− pro-DCs [22]. Therefore, the dominant nature of GM-CSF over Flt3L in BM culture raises further issues. What is the developmental fate of precursor cells for FL-DCs in the dual cytokine culture? Do FL-DCs die of neglect or is their differentiation hijacked by GM-CSF to divert fate to GM-DCs? Data both already published and uncovered in the current study support the latter scenario. First, as the Flt3L level present in the dual cytokine cultures was the same as it was in the Flt3L single cultures, there should not have been a lack of Flt3L stimulation. Second, DC progenitors, including pro-DCs, express GM-CSF receptor [16], which makes it physically possible for these cells to receive GM-CSF signaling during maturation. Third, Flt3L signaling in BM

progenitor cells activates the transcription factor STAT3, whereas GM-CSF signaling activates STAT1, STAT3, and STAT5 [25]. Therefore, when DC precursors meet both cytokines, the signaling pathway of GM-CSF can subsume that of Flt3L. Fourth, some hematopoietic cytokines, such as M-CSF and G-CSF have already been reported to govern lineage choice [26, 27]. Finally, when purified and cultured in the presence of both Flt3L and LDK378 manufacturer GM-CSF, FL-DC-committed precursor cells were diverted toward GM-DCs in spite of the presence of Flt3L. This is direct in vitro evidence for a lineage diversion role of GM-CSF. Although the incidence of macrophage colony-forming 4-Aminobutyrate aminotransferase cells remained around 5% in these pro-DCs [22], this may have

been skewed by the collection of only cells loosely attached to the substrate at the end of the culture, thus omitting most of the strongly adherent macrophage population. However, phenotypic analysis consistently indicated that the harvested cells were predominantly CD11chi and MHCII+ DCs (Fig. 5) and thus unlikely to be macrophages. Furthermore, it would be difficult to explain the more than twofold expansion in cell numbers in the cultures with dual cytokines compared with that of Flt3L alone to be due to a minor macrophage population (Fig. 5). For the above reasons, we do not think that the altered outcomes from the combined GM-CSF and Flt3L additions were due to outgrowth from distinct precursors within the enriched pro-DC population. Nevertheless, we tried to perform limiting analysis studies using GFP+ pro-DCs from mice transgenic for GFP under the promiscuous UBC promoter [15]. We seeded pro-DCs at either one or ten cells per well with 200,000 BM feeder cells in 96-well microtitre trays.