cholerae O1/O139, which showed temperature-independent motility between 20 and 42°C, as shown in Figure 1. The difference between C. jejuni and H. pylori motility is clear at 42°C, at which temperature C. jejuni is still motile, but H. pylori motility has declined (Fig. 1). The swimming speed at 37°C was the fastest for C. jejuni (>100 μm/s); the swimming speeds of H. pylori, V. cholerae O1 and V. cholerae O139 were 55.4 ± 6.6, 80.2 ± 8.6 and 75.6 ± 8.9 μm/s,

respectively. The swimming speed of C. jejuni at 42°C was faster than at 37°C (>100 μm/s; the resolution limit of the assay system employed AZD4547 in vivo did not allow precise assessment of speed). The motility of C. coli was very similar to that of C. jejuni (Fig. 1); the swimming speeds at 37 or 42°C were >100 μm/s. Next, correlations between bacterial shape and motility were examined

for C. jejuni. C. jejuni enteritis strains (n = 30) and ATCC33560 all took the form of spiral rods with polar flagella at each selleck kinase inhibitor pole and were highly motile, as shown in Figures 1 and 2a, d. Five of the C. jejuni GBS strains (n = 7) strains took the form of motile spiral rods (as shown in Figs. 1 and 2a, d), whereas one strain (KB3439; belonging to ST22) took the form of a straight rod with polar flagella at each pole (Fig. 2b, e). Interestingly, strain KB3439 was highly motile (Fig. 1), its swimming speed of >100 μm/s (at 37–42°C) being similar to that of a spiral rod, indicating that a spiral body shape is not essential for motility. As expected, the remaining strain (KB3449; belonging to ST4051), which took the form of a straight rod without

flagella (Fig. 2c, f), showed no motility, (Fig. 1). As shown in Figure 3, all C. jejuni strains have cup-like structures (marked by closed arrowheads) at both ends of the bacterial spiral body, irrespective of their bacterial shapes or the presence or absence of flagella. These polar cup-like structures are located inside (and adjacent to) the inner membrane and are 33.8 ± 6.0 nm thick (including the inner membrane) and 206.4 ± 25.5 nm in length (n = 62), as shown in Figure 3a (inset panels in the right and middle lower corner). The space (cytoplasm) within the cup-like structures is less dense than the cytoplasm of the spiral (or straight) bodies (Fig. 3). Motile bacteria have a polar hollow for a flagellum Galeterone (Fig. 3a, b; indicated by arrows), in contrast to non-motile bacteria (Fig. 3c). Negative staining of C. jejuni cells (Fig. 4a, b) further demonstrated that inner tubular structures extend from the inner membrane and open into a funnel shape on the bacterial cell surface (diameter at the bacterial cell surface, 80.8 ± 10.1 nm [n = 31]); flagella expanding into a funnel shape toward the environment. The inner tubular structure is incompletely shown in Figure 4b, probably due to incomplete penetration of uranyl acetate into the tubular structure from the bacterial surface (funnel shape) side. The funnel shape was also confirmed by thin sections of C.

, 2010; Workentine et al., 2010). Phenotypic variants such as mucoid variants (Govan & Deretic, 1996), small colony variants (SCVs) (Häußler et al., 2003; Häußler, 2004) and quorum-sensing (QS) variants (D’Argenio

et al., 2007; Hoffman et al., 2009) are commonly check details isolated from chronic infections. It has therefore been suggested that long-term adaptation to the lung of the host results in reduced expression of acute virulence factors to develop a chronic infection type which may facilitate long-term, chronic infection (Proctor et al., 2006; Bragonzi et al., 2009; Hogardt & Heesemann, 2010). We have demonstrated that mucoid clinical strains of P. aeruginosa isolated from the sputa of chronically infected patients with CF exhibit seeding dispersal during in vitro biofilm growth, as do model laboratory strains. Intriguingly,

dispersal of clinical strains resulted in a higher frequency and colony diversity of dispersal variants than the dispersal population of the laboratory strain PAO1 (Kirov et al., 2005, 2007). Colony morphotype is only one indicator of variation, and therefore, this study characterised the biofilm dispersal population of a mucoid, chronic infection CF isolate of P. aeruginosa and the laboratory strain, PAO1, for a variety of functional traits to determine the extent of diversification that occurs during biofilm development. The traits examined included those that are likely to enhance the capacity of P. aeruginosa to establish chronic airway infection, such as the capacity to utilise different carbon sources, as this has been shown to be important selleck chemical for the growth of P. aeruginosa in artificial sputum

and is of functional significance for bacterial survival in the CF lung mucus (Sriramulu et al., 2005; Palmer et al., 2007; Starkey et al., 2009). Additionally, the dispersal population was tested for properties likely to contribute to niche colonisation and persistence in the CF airway, including attachment and biofilm formation, QS signal production and virulence factor production such as general protease and elastase production. The mutation frequencies of the parental strains were quantified under planktonic and biofilm growth to correlate mutation frequency with variant formation. Pseudomonas aeruginosa PAO1 (Holloway, 1955) and the clinical strain 18A, isolated from the sputum of a chronically infected patient Inositol monophosphatase 1 with CF in Tasmania, Australia, and characterised as previously described (O’May et al., 2006), were used here. The latter strain was selected for further study as it was representative of a number of clinical isolates that showed seeding dispersal and marked heterogeneity in the morphotypes of its dispersal cells (Kirov et al., 2005, 2007). Cultures were stored at −80 °C, and all strains and biofilm-derived isolates were routinely grown on LB10 agar [Luria–Bertani agar: 10 g L−1 of tryptone, 5 g L−1 of yeast extract, 10 g L−1 of NaCl and 15 g L−1 of agar (Research Organics Inc.)] at 37 °C.

However, this has not been shown in allogeneic immune systems. Here, in vitro, we analysed the effects of these five drugs on DC maturation and functions, click here including morphology, cytokine production, expressions of MHC class II, co-stimulatory molecules and Toll-like receptor (TLR)-4, and their allostimulatory capacity. We found that AZM significantly inhibited DC maturation and functions, including allogeneic responses. The present study suggests an attractive role for pharmacological therapy as a means of generating

DCs with tolerogenic/regulatory properties. AZM may have potential as a new therapeutic drug for controlling allograft immunity, such as acute graft-versus-host disease and graft rejection in organ transplantation. Female C57BL/6 (H-2 Kb) mice and BALB/c (H-2 Kd) mice aged 6–12 weeks were purchased from Japan SLC, Inc. (Shizuoka, Japan). Institutional approval was obtained for all animal experimentation. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) used to detect cell surface expression of CD3, CD4, CD11c, CD40, CD80, CD86, MHC class II, and TLR-4-message digest 2 (MD2) by flow cytometry, as well as isotype-matched

control mAbs, were purchased from BD Pharmingen and eBioscience (San Diego, CA, USA). RPMI-1640 supplemented with 10% fetal calf serum (FCS), 5 × 10−5 m 2-mercaptoethanol (ME) and 10 mm HEPES was used as the culture medium. Bone marrow (BM)-derived DCs

were generated as described elsewhere [23,24], with minor modifications. Briefly, BM cells flushed from tibias and femurs of BALB/c mice were seeded Selleck MG-132 at 2 × 106 cells onto a six-well culture plate in culture medium supplemented with 20 ng/ml recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF) (Kirin Brewery Co., Gunma, Japan). The culture medium was changed every 2 days. Loosely adherent clustered cells were used on day 6 as immature DCs (im-DCs). The purity of im-DCs was routinely Nintedanib (BIBF 1120) > 85%, as confirmed by dual positivity for MHC class II and CD11c. Vit. D3 (Sigma, St Louis, MO, USA), ACE inhibitor delapril (Takeda Co. Ltd, Osaka, Japan), PPAR-γ activator troglitazone (Sankyo Co., Tokyo, Japan), clarithromycin (CAM) (Taisho Pharmaceutical Co., Tokyo, Japan) or AZM (Pfizer Inc., Groton, CT, USA) as an NF-κB inhibitor was added to culture wells to the indicated final concentrations at various times. We tested these NF-κB inhibitors at several concentrations to generate BM-derived DCs. The final concentrations of NF-κB inhibitors, except Vit. D3, chosen for the study were 10 times their physiological concentrations shown to have therapeutic effects on several human diseases [25–28]. Vit. D3 (10 nm) [14] was added to culture wells on days 0, 2, 4 and 6. Troglitazone (10 µm), delapril (40 µg/ml) and CAM (20 µg/ml) were added every day (days 0–6).

A χ2 test was used to compare the incidence of adverse effects of

the two groups. All statistical tests were two-sided, with P-value less than 0.05 considered significant. A total of 75 patients with IgA nephropathy were initially screened from five centres. After screening, 69 of these patients were deemed eligible and 68 patients ultimately completed the study. Among these 68 patients, 42 were from one centre and 26 were from the other four centres. The 68 patients (27 males, CDK inhibitor 41 females) were randomly divided into two groups: the treatment group (probucol combined with valsartan, n = 33) and the control group (valsartan only, n = 35). Table 1 shows the baseline characteristics of the treatment and control groups. The median age was 34 (range 19–67) years in the treatment group and 34 (range 18–74) years in the control group. There were no significant differences between these two groups in blood pressure, Scr, 24-h urinary protein excretion, or any of the other parameters listed in Table 1. Table 2 shows the baseline Oxford classification scores of

IgA nephropathy (M/E/S/T) in the treatment and control groups. Again, there was no significant difference Selleckchem DAPT between the two groups (P > 0.05). All 68 patients were followed for 3 years and none developed ESRD. However, 43 patients (23 (69.7%) in the treatment group, 20 (57.1%) in the control Histamine H2 receptor group) had reductions of 24-h urinary protein by 50% or more relative to baseline levels (secondary endpoint). Kaplan–Meier analysis indicated that the time to 50% reduction in 24-h urinary protein was significantly shorter in the treatment group than in the control

group, the median of two groups was 8.13 months, 19.63 months, respectively (P = 0.019) (Fig. 2). At the 1-year follow-up, the level of 24-h urinary protein in the treatment group (995.49 ± 561.13 mg) and control group (1055.84 ± 761.09 mg) were reduced by 28.4% (P = 0.02) and 28.0% (P = 0.03) compared with baseline levels (Fig. 3). At the 2-year follow-up, the mean 24-h urinary protein in the treatment group (756.65 ± 475.21 mg) was markedly reduced compared with baseline (P < 0.01); but there was no significant difference compared to the control group (1432.33 ± 1135.33 mg, P = 0.056). The mean 24-h urinary protein in the control group (1432.33 ± 1135.33 mg) was higher than the level at the 1-year follow-up (1055.84 ± 761.09 mg), but not significantly different from the baseline level (P = 0.92) (Fig. 3). At the 3-year follow-up, the 24-h urinary protein in the treatment group (1385.32 ± 999.77 mg) and the control group (1343.31 ± 941.34 mg) were comparable to the baseline levels (P = 0.99 and P = 0.66, respectively) (Fig. 3). At the 1-year and 2-year follow-ups, the mean Scr in the treatment and control groups were comparable to the baseline levels (Table 3).

Th1-specific mRNA and protein expression in the nasal cavity of the controls was not different

from that in AR mice, but expression significantly increased with rhLF treatment. The mRNA and protein expression of endogenous LF in the nasal cavity was significantly downregulated in AR mice compared with the controls. However, after rhLF treatment, endogenous LF mRNA and protein expression was significantly upregulated. Exogenous rhLF inhibited allergic inflammation in AR mice, most likely by promoting the endogenous LF expression and skewing T cells to a Th1, but not a Th2 and Th17 phenotype in the nasal mucosa. Our findings suggest that rhLF treatment may be a novel therapeutic approach for prevention and treatment AR. Allergic rhinitis (AR) is one of the most prevalent airway diseases worldwide. AR exerts a heavy burden on society as it is an this website important risk factor for asthma and is associated with a high cost of treatment. Ivacaftor chemical structure Moreover, the worldwide prevalence of AR is increasing [1]. Thus, investigating the underlying mechanisms that cause the development of AR and further exploring novel therapies for AR treatment are crucial for the control of this global

disease. Allergic rhinitis is characterized by an imbalance of CD4+ T cell subsets and an accumulation of eosinophils and mast cells in the nasal mucosa. CD4+ T cell subsets can be classified into type 1 helper T (Th1), Th2, Th17 and regulatory T (Treg) cells based on the expression of specific cell surface markers, and the transcription factors T-bet (Th1), GATA-3 (Th2), ROR-C (Th17) and FOXP3 (Treg). These T cell subsets meditate various inflammations mainly through secreting all kinds of cytokines such as IFN-γ, IL-5, IL-17, IL-10, TGF-β1 and TNF-α [2-4]. In AR, the allergic response RAS p21 protein activator 1 observed predominantly involves Th2 cells, with a relative insufficiency of Th1 and Treg cells, This T cell subset skewing is considered as the classic Th1, Th2 and Treg paradigm in allergic diseases [5-9]. However, the discovery of a role for Th17 cells in the development of AR, including the secretion of pro-inflammatory cytokines such as TNF-α,

IL-β1 and IL-5, alters the classic T cell subset paradigm for AR. Although immunological imbalances in AR have been identified, treatments for AR are currently limited in their effectiveness. There are various therapeutic options for AR, including antihistamines, corticosteroids, anticholinergic agents, leukotriene inhibitors and immunotherapy. The most utilized is intranasal corticosteroids. Unfortunately, a significant number of AR patients have corticosteroid resistance and either cannot control their diseases or have many side effects after treatment [1]. The most encouraging treatment to date is specific immunotherapy, but its usefulness is greatly limited by efficacy, potential side effects, inconvenience and disease severity [10].

Concentrations have been examined at various times in the dosing interval, and the cervicovaginal concentrations vary significantly from drug to drug. One study examined how quickly each drug achieved concentrations in the genital tract compared to plasma at steady state in 27 women.57 They reported the median rank order of drugs with highest Palbociclib to lowest genital tract concentrations. As the authors anticipated, the commonly used nucleoside reverse transcriptase inhibitors tended to be high on the list while efavirenz was the lowest, with protease inhibitors (PIs) falling in the middle. This study confirmed findings from an earlier study of seven women.58 Another study with a larger sample

size (34) examined both drug concentrations as well as virologic response to drug.59 The use of ART in patients is an incredibly important factor in the determination of genital immunity. As these drugs appear in measurable concentrations in the genital fluids, it is also important to note that any in vitro models using live virus will not perform properly if using genital fluids from women taking ART. Although there is a strong correlation between plasma

viral load and genital tract viral load, there selleck is evidence of compartmentalization between the blood and genital tract in both men and women. Evidence of compartmentalization occurs in terms of resistance patterns.60–62 An interesting study examined the theory that virologic failure might occur in one compartment and not another. The authors examined 14 women with detectable HIV-1 in both plasma and genital tract despite antiretroviral therapy.63 Fifty-seven percent

of the patients exhibited Thiamet G mutations conferring high-level HIV-1 drug resistance. Interestingly, in one patient, resistance mutations appeared only in the plasma while all genital variants were susceptible. It has also been shown that resistance mutations detected in the genital tract can persist for years.64 Differences in resistance patterns as well as the possibility of resistance must be considered in studies including HIV-infected women. The HIV pandemic continues to result in millions of deaths annually on a global scale. Despite the advent of antiretroviral therapy, the spread of the infection has not been halted. The millions of dollars of research aimed at determining the pathogenesis of HIV spread have led to marked improvements in the understanding of disease. This has brought a change in life expectancy of those diagnosed with HIV in the United States from terminal to chronic illness. It has also caused a shift in attention from the blood compartment to the genital compartment as the major point-of-entry for HIV and thus for research endeavors. The many clinical characteristics that must be considered when studying the blood compartment must be expanded when considering research work on the genital compartment.

In contrast, treatment with LGG wild-type results in an up-regulation of TLR-1, -2 and -4 compared to the dltD-treated group, highlighting the impact of inactivating the dltD gene. It is known that LTA molecules of certain bacteria can induce

proinflammatory signalling in macrophages by interaction with TLR-2 [56]. The exact role of d-alanylation in interaction of LTA with specific TLRs (TLR-2, TLR-6) and co-receptors (CD14, CD36) is not yet well established. Based on the crystal structure of TLR-2, the two acyl chains of LTA are suggested to interact with the lipid binding pocket of TLR-2, while the hydrophilic Ixazomib in vitro glycerophosphate chain is thought to be exposed to solvent or to interact with TLR-6 or another co-receptor of TLR-2 [57–59]. However, as LTA is a major cell wall compound of lactobacilli, changing the structure

of LTA by removing d-alanine residues might as well effect the interactions with other surface molecules and therefore cause pleiotropic effects that can impact indirectly on the anti-inflammatory capacity of the lactobacilli. Nevertheless, our results with the dltD mutant compared to the wild-type probiotic strain are in line with those of the study by Grangette et al. [36], where a dltB mutant of L. plantarum NCIMB8826 also showed, compared to the wild-type strain, an enhanced anti-inflammatory capacity in vitro in monocytes and in a trinitrobenzene sulphonic acid (TNBS) colitis model [60]. Obeticholic Acid in vivo Although both experimental set-ups (probiotic strains and colitis models) differ significantly, the study by Grangette et al. [36] and this study both suggest a key role for LTA modification in pro-/anti-inflammatory

effects of probiotic lactobacilli. Finally, the data from our experiments with LGG in the DSS-induced murine colitis model cannot be translated easily to the clinical setting, as introducing bacterial mutants in humans is not straightforward. However, it is interesting to mention that we also performed a pilot study with LGG in patients with active pouchitis (unpublished). Lepirudin Two patients with acute pouchitis received daily 1011 CFU/ml of LGG (Valio, Helsinki, Finland) in capsules for 4 weeks in a randomized cross-over trial (4 weeks probiotics, 4 weeks placebo). In one of the patients, the symptoms of active pouchitis seemed to be exacerbated by the treatment. This study was discontinued and we decided to focus upon animal models, such as presented in this report, to understand more clearly the interaction of LGG with the intestinal mucosa. The data from our experiments, together with reports from other research groups on animal models [28,29] and Crohn’s disease patients [61], underline that caution should be taken when applying the wild-type strain of the well-known probiotic LGG in patients with active IBD.

At day 2, the well plates were centrifuged at 488 g for 10 min. Supernatants were collected for cytokine analysis (see below). For all cultures, the whole medium was then replaced. After 5 days of co-culture, supernatants

were again collected as described above and analysed for cytokines click here (see below). The cells were then resuspended in phosphate-buffered saline (PBS; Invitrogen) with 0·5% FCS (Biochrom) and 2 mM ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich). The lymphocytes were thus separated from the MSCs, washed and prepared for flow cytometry (see below). MSCs were detached with trypsin as described above, washed in whole medium and resuspended in PBS with 0·5% FCS and 2 mM EDTA. MSCs were then prepared for flow cytometry (see below). CD4+ Selleckchem Anti-infection Compound Library T cells enriched in Tregs were generated as described above by magnetic bead separation. The cells were resuspended in 48-well plates, each well containing 1 ml of medium (see above) and 50 000 T cells. In one group, the medium was supplemented with 5 ng/ml IL-6 (Miltenyi Biotec); in another, 10 ng/ml IL-6 was added to the medium. A third group was supplemented with supernatants from passage 2 bone marrow-derived MSCs cultured in DMEM-LG with 10% FCS and 1% penicillin/streptomycin. Cell cultures

without supplementation to the media were used as controls. At day 2, the 48-well plates were centrifuged at 488 g for 10 min. Supernatants were

collected and analysed for cytokines (see below). For all cultures, the whole medium was then replaced. After 5 days of culture, supernatants were collected as described above and analysed for cytokines (see below). The cells were then resuspended in PBS (Invitrogen) PtdIns(3,4)P2 with 0·5% FCS and 2 mM EDTA (Sigma-Aldrich) and prepared for flow cytometry. One-colour cytometry (MSCs) and three- and four-colour cytometry (T cells) was performed using a MACS QuantTM analyser and MACS Quantify version 2.1 software (Miltenyi Biotec). Positive fluorescence was defined as any event above the background fluorescence, which was defined by a line where 99·5% of the events in isotype antibody-labelled cells were considered negative. The following anti-human antibodies were used in the experiments: for T cell analysis, CD4 fluorescein isothiocyanate (FITC) mouse immunoglobulin (Ig)G1, CD25 phycoerythin (PE) or allophycocyanin (APC) mouse IgG2b (Miltenyi Biotec), CD127 APC or PE-Cy5 mouse IgG2a (BD Biosciences, Heidelberg, Germany). FoxP3 intracellular staining was performed with the FoxP3 staining buffer set and FoxP3-PE mouse IgG1 antibodies (BD Biosciences), according to the manufacturer’s protocol.

Loneliness, dementia, depression, Parkinson’s disease, mental stress and compromised gastrointestinal function may result in malnutrition, insufficient protein intake, vitamin deficiencies (especially vitamins A, C and E with antioxidative activities) and deficiencies in trace elements (especially zinc, which is crucial for lymphocyte check details proliferation); all of these factors can result in compromised immune functions [7–10]. In

addition, the elderly are more susceptible to malignancies, severe infections and long-term repeated chronic infections; they experience more trauma, have more major surgeries and have increased incidence of late-stage systemic diseases (renal dysfunction, liver failure and heart failure) and other critical illnesses, all of which may also significantly compromise immune function [11–14]. Moreover, those elderly people who take anti-inflammatory drugs, non-steroidal anti-inflammatory drugs, steroids, antibiotics, antidepressants, antihypertensives or allopurinol may also experience compromised immune function [15, 16]. Thus, even the SENIEUR protocol that has been accepted worldwide cannot meet all of the criteria necessary for selecting healthy Vemurafenib manufacturer subjects for ageing-related studies. Thus, the SENIEUR protocol was modified and improved with the aim of excluding those factors that could influence cellular immunity. In the present study, 28,376

subjects who were self-reported as healthy were reviewed over an 8-month period. From these, we enrolled 78 subjects aged ≥80 years, 128 subjects aged 60–80 years and 60 subjects aged 20–60 years. Although the number of older subjects, especially those aged ≥80 years, was small and may have

contributed to underestimating the extent of compromised immune function among the elderly, our findings may actually demonstrate the direct FER impact of ageing on cellular immunity. As is well known, antigen-presenting cells (APCs) may undergo differentiation and maturation following stimulation with antigens or other stimuli, after which they present antigens to naïve T cells, which become activated T cells. T cell-mediated specific immunity plays a central role in immune responses. T cell activation is primarily characterized by proliferation, and thus, T cell proliferation has been used as a marker of human immune potential. In addition, following treatment with multiple cytokines (recombinant human IL-2, IL-1, γ-INF and CD3 mAb), some PBMCs can become transformed into CD3- and CD56-positive CIK cells, which have both potent antitumour activities as T lymphocytes and non-MHC-restricted tumouricidal activities as NK cells. Thus, CIK tumouricidal activity can also be used as an indicator of human immune function [17, 18]. Our findings revealed that there were no marked differences in the number of peripheral blood total T cells, CD4+ cells, CD8+ cells or CD4+/CD8+ ratios among the subject groups of different ages.

Table 1 lists the primers that

were used for mRNA quantification. Samples were analysed using a Bio-Rad iCycler iQ (Bio-Rad, Hercules, CA). Changes in gene expression were determined by calculating the Δ cycle threshold (Ct) by subtracting the Ct for ribosomal protein L19 (RPL19) (reference gene) from the Ct of the gene of interest for each sample.26 The ΔCt of the control was subtracted from the corresponding treated sample giving rise to the ΔΔCt. The fold change was derived from the equation 2−[ΔΔ]Ct. To confirm that the reference gene ribosomal protein L19 was stably expressed in MoDCs and BDCs, a comparison was performed using either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or RPL19 as the GDC-0199 in vitro reference gene. Similar trends in fold change were observed. Complementary DNA was diluted to generate

a standard curve whose correlation coefficient was > 0·99. The efficiency of qPCR was determined from the slope using the equation (10[−1/M] − 1) × 100 and ranged between 90% and 110%. To evaluate changes in cytokine secretion, 1 × 106 MoDCs or BDCs were incubated in 1 ml culture medium for 24-hr in six-well plates (Corning) and culture supernatants were collected. Concentrations of IL-6, Ganetespib solubility dmso IL-8 and IL-10 were assayed using commercial kits as per the manufacturer’s instructions (R&D Systems, Minneapolis, MN). The ELISA for IFN-α, TNF-α and IL-12 were performed as previously described.27 Statistical analysis was performed by non-parametric Mann–Whitney U-tests (P-value < 0·05) using the statistical software programme graphpad prism 5 (GraphPad Software, Inc., La Jolla, CA). In this study, 800 ml of EDTA blood yielded approximately 2 × 109 PBMCs. Following CD14+ selection, an average of 2 × 108 monocytes were cultured in the presence of IL-4 and GM-CSF to Niclosamide generate MoDCs. On day 6, approximately 2 × 107 MoDCs were harvested and cultured for use. The CD14− population

was positively selected for cells expressing CD172, which equates to the BDC (CD14− CD172+) population. Approximately 3 × 107 BDCs were therefore isolated and rested overnight. In contrast to other studies, the protocol used in this study resulted in lower numbers of MoDCs compared with BDCs from an equal amount of blood.28 Dendritic cell morphology is characterized by a large cytoplasmic cell mass and extrusion of dendrites which increase the surface area available to sample and take up antigens. In this study, the morphologies of Giemsa-stained MoDCs (Fig. 1a) and BDCs (Fig. 1b) were compared. Both DC populations displayed a typical DC morphology, characterized by an irregular cell border with a large cytoplasmic cell mass. Expression of cell surface markers CD172, MHC II, CD16, CD1, CD80/86 and CD14 was assessed by flow cytometry in 6-day-old MoDCs and BDCs (Table 2). Both MoDCs and BDCs expressed all of these markers; however, BDCs showed similar expression of CD172 and MHC II, higher expression of CD16 and lower expression of CD80/86 and CD1.