Such a finding may enhance the negative and/or the positive predictive value of a chemical biomarker. Previous study demonstrated that sonographic measurement of fetal membrane thickness could be helpful in the prediction of preterm delivery.[16] Using the amniotic fluid and cervical length data from the randomized trials noted above, we examined the relationship

between cervical length and levels of inflammatory mediators in amniotic fluid.[17] Pembrolizumab Spearman correlations were used to determine which cytokines correlate with cervical length. Stepwise regression identified the most significant cytokine predictive of early delivery, and a ROC curve determined the cervical length cutoff predictive of intra-amniotic inflammation. Our results indicate that cervical length ≤5 mm is associated with significant

increases in amniotic fluid inflammatory cytokines, even in the absence of infection or labor. A cervical length of ≤5 mm was associated with significant increases in inflammatory mediators (Interleukin (IL)-1β, IL-2, IL-6, IL-8, and MCP-1), which have been previously shown to be associated with preterm labor.[18, 19] Unfortunately, the dataset was too small to allow a multivariable analysis including both cervical length and mediator levels in predicting outcome. While a very short cervical length is a good indicator of intrauterine inflammation, it represents the final common pathway of multiple inciting events that can result in preterm labor. As Quizartinib ic50 such, it is not an ideal biomarker when utilized alone. Many of these patients will go on to delivery prematurely despite intervention. It is likely that markers which identify earlier in-utero events will allow more effective therapies Cytidine deaminase to be designed to stop the preterm labor cascade before the cervix becomes shortened. It appears that the intrauterine compartments are mostly immunologically distinct, and the expression of inflammatory markers in various maternal-fetal compartments will

be differentially expressed in non-invasive sampling sites. Because the etiology of preterm labor is multifactorial, using multiple biomarkers from distinct biologic pathways will better predict the risk of preterm labor. Furthermore, combining non-invasive tools such as a physical or ultrasound finding physical finding may improve the ability of specific biomarker in predicting outcome. Platforms to measure for example the levels of inflammatory mediator are commercially available and can easily be incorporated into ongoing trials looking at interventions to treat preterm labor. Initially, data can be collected in an observational manner and correlated with outcomes.

The mean fraction of lymphocytes migrating on the EC surface or undergoing TEM among the treatment versus control groups among several experiments was calculated and tested for statistical significance (p<0.05) by paired Student's t-test (SPSS, Chicago, IL, USA). All the data are shown as mean±SEM. To evaluate the position of lymphocytes at the interendothelial junctions, data from four

independent experiments Sirolimus were pooled and tested for significance using Chi square analysis (SPSS). This work was supported by operating grants from the Heart & Stroke Foundation of Canada and the CIHR to A.G.M. M.N. is the recipient of a University of Alberta 75th anniversary studentship award and Queen Elizabeth II Graduate

Scholarship. Conflict of interest: The authors declare no financial or commercial conflict on interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Citation Aldo PB, Mulla MJ, Romero R, Mor G, Abrahams VM. Viral ssRNA induces first-trimester trophoblast apoptosis through an inflammatory mechanism. Am J Reprod Immunol 2010; 64: 27–37 Problem  Infection during pregnancy represents a significant cause of mobility and PLX3397 purchase mortality. While viruses pose a major threat, little is

known about their effect on early pregnancy, or the mechanisms involved. The objective of this study was to characterize the trophoblast response following exposure to viral ssRNA. Method of study  First trimester trophoblast cells were treated with or without viral ssRNA. Cytokine production was measured using multiplex analysis and ELISA. Apoptosis was determined using Hoechst staining, cell viability, and caspase activity assays. Results  Treatment of trophoblasts fantofarone with viral ssRNA increased their secretion of IL-8, IL-6, and IFNβ. However, the ssRNA also induced trophoblast apoptosis. To test whether the viral ssRNA-induced inflammatory response was responsible for this induction of apoptosis, conditioned media (CM) from trophoblasts were added to a fresh culture of cells. The CM from viral ssRNA-treated induced higher levels of trophoblast apoptosis than the control CM. Moreover, recombinant IFNβ induced trophoblast apoptosis. Conclusion  We demonstrate that viral ssRNA induces a pro-inflammatory and type I interferon response in the trophoblast and this inflammatory process may indirectly induce trophoblast apoptosis. These results provide a novel mechanism by which certain viral infections might compromise placental integrity and function, and therefore, pregnancy outcome.

As shown in Fig. 4, co-culture of both naïve- and memory-phenotype CD4+ T cells with a low ratio of MSCs was associated with a moderate anti-proliferative

effect under Th17-skewing conditions using CFSE labelling (Fig. 4A) and a reduced proportion of IL-17A+ cells within each generation of cell division using intracellular staining for IL-17A (Fig. 4B and C). It was concluded that the presence of low numbers of MSCs during a Th17-biased activation culture of either naïve or memory CD4+ T cells resulted in separate effects on T-cell proliferation and on induction of high-level IL-17A production. In additional experiments the specificity and direct nature of MSC suppression of Th17 differentiation was demonstrated. Inhibition of IL-17A secretion upon re-stimulation of Th17-skewed check details naïve- and memory-phenotype CD4+ cells was not apparent following co-culture with primary fibroblasts (Supplemental Fig. S4A). The possibility that monocyte/macrophages or DCs were responsible for indirectly mediating MSC suppressive buy Gemcitabine effects on T-cell responders was eliminated by experiments in which primary CD4+ T-cell/MSC co-cultures were initiated with anti-CD3/anti-CD28-coated beads rather than splenic APCs. In this case, the Th-17-suppressive effect of MSCs for both naïve

and memory CD4+T cells persisted (Supplemental Fig. S4B). In order to identify potential mediators DOK2 of MSC-induced Th17 suppression, experiments were carried out in which FACS-purified naïve CD4+ T cells were Th17-skewed in APC-free culture (anti-CD3/anti-CD28 beads) in the presence or absence of MSCs (1:200 ratio) with or without blocking/inhibiting factors for candidate mediators. The primary experimental read-out was secretion of IL-17A following overnight stimulation of re-purified CD4+ T cells. As shown in Fig. 5A, the non-specific COX

inhibitor indomethacin reversed the MSC suppressive effect and, in some experiments, was associated with a paradoxical increase. The observation was consistent with induction, via T-cell–MSC contact, of a COX-dependent soluble mediator. To test this further, culture supernatants were removed from 4-day, APC-free Th17 cultures generated with and without indomethacin in the presence or absence of MSCs. These supernatants were applied to newly initiated Th17 cultures along with unconditioned medium and MSC-conditioned medium containing equivalent concentrations of Th17 inducing factors with and without indomethacin (Fig. 5B). CD4+ T cells were then re-purified from each culture and stimulated overnight, after which IL-17A production was measured. As shown, MSC-conditioned medium was associated with a modest reduction in IL-17A compared with unconditioned medium.

We found that IL-1Ra levels in BALF

of IPF patients were increased, but this was not enough to equal the vast increase in local IL-1β. Galunisertib supplier Altogether, this resulted in a 3·5-fold decrease in the IL-1Ra/IL-1β ratio in IPF patients compared to healthy controls. In animal studies it has been shown that alterations in the balance between IL-1β and IL-1Ra cause the development of lung fibrosis. Mice with bleomycin-induced fibrosis have an up-regulated expression of IL-1β mRNA after instillation of bleomycin [20], and addition of recombinant IL-1β induces fibrotic remodelling [8]. Overexpression of IL-1β in rat lungs after intratracheal administration of bleomycin was associated with severe progressive tissue fibrosis

in the lung, characterized by the presence of myofibroblasts, fibroblast foci and significant extracellular accumulations of collagen and fibronectin [4]. Other studies showed that administration of exogenous IL-1Ra prevented or even reversed the generation of pulmonary and synovial fibrosis [21–23]. The pathogenetic processes in bleomycin-induced fibrosis are simply a model for IPF and results cannot be extrapolated to human IPF. However, in patients with acute myocardial infarction, there is evidence that IL-1 blockade with IL-1Ra Protease Inhibitor Library cell assay suppresses the inflammatory response and positively affects tissue remodelling [24]. IL-1 ligands such as IL-1α, IL-1β and IL-1Ra all bind to the IL-1 receptor (IL-1R1). Mice lacking the IL-1R1 receptor showed significantly reduced cellular infiltrates, alveolar wall destruction and collagen deposition.

Moreover, blockade of the IL-1R1 receptor by exogenous IL-Ra (anakinra) dramatically reduced neutrophil influx and the formation of bleomycin-induced fibrosis in mice [8]. Altogether, IL-1 seems to be a critical cytokine and may possibly be a therapeutic target in IPF. There are different hypotheses Ibrutinib research buy about the role of inflammation and thus proinflammatory cytokines such as IL-1β in the role of pulmonary fibrosis. Historically, the hypothesis was that inflammation in response to an unknown agent was the key process in IPF, ultimately resulting in fibrosis. The current concept is that IPF is a result of repeated episodes of lung injury, with a minor role for inflammation. This concept states that inflammation in IPF could be a consequence of the architectural remodelling, rather than a cause. The increased parameters of inflammation such as neutrophilia in BALF may be a reflection of remodelling and traction bronchiectasis due to fibrosis [25]. However, this does not exclude a role for inflammation in an earlier stage of the disease. An interesting paper in this context is the study by Flaherty et al.

, 2005). The influence of lactic acid on cytokine production by peripheral blood mononuclear cells (PBMCs) has not Protein Tyrosine Kinase inhibitor been determined previously, and is the subject of this communication. The findings have biological relevance for an enhanced understanding of infection-related immune mechanisms operative in the lactic acid-dominated female lower genital tract. Venous blood was obtained from 10 healthy female and male volunteers and PBMCs isolated by Ficoll-Hypaque (GE Healthcare Biosciences, Piscataway, NJ) gradient centrifugation. The mononuclear

cell band was recovered, the cells were washed twice in RPMI 1640 culture medium (Invitrogen, Carlsbad, CA) and resuspended in RPMI to a final viable concentration of 1 × 106 cells mL−1. Viability was determined by trypan blue exclusion. The PBMCs were added to the wells of a sterile microtiter plate (1 × 105 cells per well) that contained RPMI medium±various concentrations

of l-lactic acid (Sigma-Aldrich, St. Louis, MO) or l-lactic acid that had been neutralized with sodium hydroxide to the pH of RPMI medium. In other experiments, hydrochloric acid (HCl) was added to RPMI medium to match the pH obtained by lactic acid addition. After incubation for 24 h in a 37 °C, 5% CO2 incubator, either lipopolysaccharide (50 ng mL−1Escherichia coli serotype 0111:B4, Sigma-Aldrich) or an equivalent volume of RPMI was added to quadruplicate wells and incubation Stem Cell Compound Library was continued for another 24 h. The culture supernatants were then collected by centrifugation and stored at −80 °C until assayed for cytokines. Cell viability as well as the pH in each well were checked at the conclusion of the experiment. All reagents were filter sterilized before use and a sterile technique was used throughout. The study was approved by

the institutional review board of the Weill Cornell Medical Center–New York Presbyterian Hospital and written informed consent was obtained from all participants. The culture supernatants were tested in duplicate for IL-23, IL-12, IL-10, IL-6 and tumor necrosis factor-α (TNF-α) using commercial enzyme-linked immunosorbent IKBKE assay kits (ebioscience, San Diego, CA for IL-23 and IL-12; Invitrogen for IL-10 and TNF-α; R&D Systems, Minneapolis, MN for IL-6). Experimental values were averaged and converted to pg mL−1 by reference to a standard curve that was generated in parallel to the test samples. The lower limits of sensitivity were 15 pg mL−1 for IL-23, 4 pg mL−1 for IL-12, 0.2 pg mL−1 for IL-10, 9.4 pg mL−1 for IL-6 and 1.7 pg mL−1 for TNF-α. The associations between cytokine levels and incubation condition were analyzed using the Mann–Whitney test. A P value of<0.05 was considered significant. graph pad instat (Graft Pad Software, San Diego, CA) was utilized for the analysis. The addition of lactic acid to PBMCs incubated with lipopolysaccharide resulted in a marked increase in IL-23 secretion over that released in the presence of lipopolysaccharide alone (P=0.0068).

In addition, this NFR-like pattern is

never associated with EMA and, in the controls treated with 12 months of gluten withdrawal, it did not disappear, showing the absence of a gluten dependency. On the other hand, as only two of 20 CD patients evaluated in this study show serum ANA-positive results, it is possible to conclude that NFR antibodies are different Selleck Y 27632 from the classics ANA. Incidentally, the ANA prevalence observed in our CD patients does not exceed the frequency reported currently for different classes of healthy individuals [35]. In conclusion, this is an early translational study describing a new autoantibody named NFR related to CD. In fact, the presence of NFR antibodies in CD patients’ serum is gluten-dependent and, accordingly, they could be considered to be CD-specific. The identity of NFR-related 65- and 49-kDa autoantigens is yet unknown, and therefore further investigations should be addressed to either obtain new knowledge on the humoral response of CD or to facilitate the development of a novel and promising serological test. In this regard, if our data are confirmed by large clinical trials, serum NFR antibody detection might to become a useful tool to monitor treated CD patients. The present study was supported by research funds assigned to Antonio Picarelli MD from the Sapienza University, Rome, GSK2126458 supplier Italy. Authors declare that there

are no financial or other relationships that might lead to conflicts of interest. “
“This Viewpoint series provides authoritative and detailed outlines of exciting areas of DC research. Some of the subjects that frequently come up include development of DC; distribution of DC in lymphoid and non-lymphoid tissues such as skin, intestine and lung; different

forms or subsets of DC; and the role of DC in initiating tolerance and immunity. In this Preface, I will introduce the Viewpoints and consider some future challenges as well as the medical relevance of DC research. The development of DC, at least in mice, can be described with increasing precision because of discoveries summarized in the Viewpoint by Liu and stiripentol Nussenzweig 1: (i) in the steady state, DC arise from a bone marrow progenitor that is shared with monocytes and macrophages 2; (ii) this progenitor gives rise to two cell types in the steady-state bone marrow: monocytes and a common DC progenitor 3–5; (iii) the latter gives rise to committed preDC that express some MHC II and CD11c, leave the marrow and circulate briefly in the blood before populating lymphoid and non-lymphoid organs 6, 7; (iv) Flt3 ligand (Flt3L) drives DC development 8, so that Flt3 knockout mice have a DC deficit 9, while administration of Flt3L expands DC numbers at least ten-fold in mice 10 and in humans 11. The discovery of distinct steps in DC development should make it possible to identify the relevant transcription factors and, in turn, new markers to improve the definition and understanding of the DC lineage.

4 peptides act as targets for CD8+ T cells in PBMCs from patients with pulmonary TB, we performed tetramer-guided analysis of 13 peptides identified by peptide binding. Sixteen tetramers were constructed: four tetramers covering A*0201, three tetramers covering A*2402 and B*0702, and two tetramers covering B*1501 and A*1101; B*0801 and A*0301 were

covered with a single tetramer (Table 2). No tetramers were constructed for HLA-A*0101 as the MHC class I–peptide complexes did not exhibit sufficient stability. PBMCs from 14 MHC class I typed patients were analysed for epitope-specific T cells using MHC allele-matched tetramers. We identified three patterns: (i) some of the tetramers showed no T-cell binding compared with the Hydroxychloroquine mw negative control tetramer, for example A*2402 GYAGTLQSL (TB10.420–28);

(ii) other tetramers showed T-cell binding in PBMCs from some patients but not in others, for example B*1501 WQAQWNQAM (TB10.454–62); (iii) and other tetramers identified peptide-specific T cells in all patients with matching MHC alleles, for example B*0702 MAMMARDTA (TB10.481–89). This epitope exhibited the most frequent T-cell population; up to 2% of all CD8+ T cells recognized this peptide in one patient (Table 3). In general, and as validated by the negative control tetramer-binding data, the frequencies of tetramer-binding T cells for HLA-A*0201 and A*2402 were relatively low, while Palbociclib datasheet the opposite was found to be true for HLA-B*0702 and B*0801. For the peptides IMYNYPAML (TB10.44–12) and MMARDTAEA (TB10.483–91) several different tetramers were constructed; for example, Cediranib (AZD2171) the peptide IMYNYPAML was used for HLA-A*0201, A*2402 and B*0702. This peptide was strongly recognized if presented by the HLA-B allele but not as strongly if presented by HLA-A alleles. The other ‘cross-presented’ peptides showed a similar recognition pattern. Identification of novel MHC class I-presented peptides is useful for the development of TB diagnostics and to gauge TB vaccine-take. TB10.4 is present in M. tuberculosis and environmental mycobacterial

species, including the vaccine strain BCG. The value of testing TB10.4 CD8+ T-cell responses lies in the gauging of vaccines containing TB10.4 antigens. We confirmed the previous identification of some TB10.4 peptides, i.e. QIMYNYPAM (TB10.43–11) (H-2kd), IMYNYPAML (TB10.44–12) (HLA-A*0201) and GYAGTLQSL (TB10.420–28) (HLA-A*2402 and H-2kd),13,16,17,23 but the majority of TB10.4 peptides identified have not previously been reported or were previously identified as peptides binding to an ‘unknown allele’. Binding peptides were found for all the investigated alleles, and yet the frequency of peptide binding was different among the alleles. For instance, A*0201 showed a very high number of binding peptides (20%) while the opposite was true for A*0101 and B*0801.

Total RNA was extracted from acinar cells or macrophages with Trizol (Gibco, Carlsbad, CA, USA), as described [16,24].

Reverse-transcribed cDNAs were amplified using specific primers for VIP, VPAC1, VPAC2, bax, TNF-α and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and conditions as stated previously [16,24–27]. The following sequences were used for forward and reverse primers. Bax: 5′-GGAATTCCAAGAAGCTGAGCGAGTGT-3′ and 5′-GGAATTCTTCTTCCA GATGGTGAGCGAG-3′; VPAC1: 5′-GTGAAGACCGGCTACACCAT-3′ and 5′-TGAAGAGGGCCATATCCTTG-3′; VPAC2: 5′-CCAAGTCCACACTGCTGCTA-3′ and 5′-CTCGCCATCTTCTTTTCAG-3′; VIP: 5′-TTCACCAGCGATTACAGCAG-3′ and 5′-TCACAGCCATTTGCTTTCTG-3′; TNF-α: 5′-CCTTGTTCGGCTCTCTT TTGC-3′ and 5′-AGTGATGTAGCGACAGCCTGG-3′ GAPDH: 5′-TGATGACAT CAAGAAGGTGGTGAAG-3′ PD0325901 solubility dmso and 5′-TCCTTGGAGGCCATGTAGGCCAT-3′. PCR products were size-fractionated on 2% agarose gels and visualized by staining with ethidium bromide using a size molecular marker. For real-time experiments, VIP and TNF-α expression were determined as described [26,27]. Western blot (WB) assays and confocal microscopy were used to analyse NF-κB activation in acinar cells or macrophages. For WB assays, both cytosolic and nuclear fractions were analysed independently after cell isolation. Isolated cells were washed gently and homogenized in 10 mm HEPES pH 7·9; 1 mm ethylenediamine

tetraacetic acid (EDTA); 1 mm ethylene glycol tetraacetic acid (EGTA), 5 mm sodium fluoride (NaF), 1 mm NaVO4, 1 mm dithiothreitol (DTT), 10 mm KCl, 0·5% NP-40

with protease inhibitors, as described BMS-354825 clinical trial [16,24]. After 15 min on ice, samples were centrifuged at 8000 g for 15 min. Supernatants (cytosolic extracts) were fractionated in 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and immunoblotted with rabbit polyclonal anti-I-κB-α or goat polyclonal anti-actin (Santa Cruz Biotechnology, CA, USA) [24]. Nuclear extracts were obtained by resuspending pellets in 10 mm HEPES pH 7·9, 1 mm EDTA, 1 mm EGTA, 5 mm NaF, 1 mm NaVO4, 10 mm Na2MO4, 1 mm DTT and 0·4 m KCl, 20% glycerol. Proteins were fractioned on 10% SDS-PAGE gels and immunoblotted with anti-p65 or goat polyclonal anti-actin (Santa Cruz Biotechnology) Bands were revealed with peroxidase-conjugated antibodies and enhanced chemiluminescence detection system (Pierce, Etofibrate Rockford, IL, USA). Densitometry analysis of proteins was performed with ImageQuant®. For confocal microscopy studies, acini or macrophages were fixed and permeabilized with methanol at −20°C, incubated with mouse p65 antibody (Santa Cruz Biotechnology) and FITC-conjugated anti-mouse antibody (BD Pharmingen, San Diego, CA, USA), washed and stained with 0·5 µg/ml propidium iodide (PI) and observed at confocal microscope Olympus FV 300 coupled to Olympus BX61. To study apoptosis of acinar cells WB, RT–PCR and annexin V/propidium iodide staining and cytometric detection were used.

Infection is a leading complication of immunosuppressive treatment and an important cause of mortality in Asian LN patients.[65] Management of patients would need to take into consideration infections that are prevalent or endemic in some Asian countries, such

as hepatitis selleck screening library B and tuberculosis, since prophylaxis or pre-emptive treatment may be indicated.[66, 67] The risk of infective complications influences the dose of immunosuppressive agents. The starting dose of prednisolone is usually in the range of 0.8–1 mg/kg body weight daily for the initial treatment of severe proliferative LN. The use of pulse methylprednisolone varies, but many would use intravenous pulse methylprednisolone at 0.5–1 g daily for three days in patients who show extensive crescents on renal biopsy or rapidly progressive renal impairment. Also there is variation on the rate of corticosteroid dose tapering. The target dose of MMF for induction therapy in severe LN is mostly in the range of 1.5–2 g/day, and a high tolerance to MMF is generally observed. The choice and duration of MMF treatment are often dependent on financial Epacadostat considerations, since MMF or mycophenolic acid sodium is either a self-financed item or second-line (to CYC) immunosuppressive agent in many Asian countries, although health insurance reimbursement is available in some countries with specified criteria. In this context, quality-of-life scores were higher

during MMF treatment compared with scores associated with CYC induction in patients who had experience with both treatments, while the treatment cost associated with MMF could be partially offset by savings from the reduced incidence of complications.[34, C-X-C chemokine receptor type 7 (CXCR-7) 68] Also, the data from a recent

report showed that in patients who had been treated with prednisolone and MMF as continuous induction-maintenance immunosuppression the risk of disease flare was lower when MMF was given for at least 24 months compared with shorter treatment durations[35] In general, treatment is guided by disease severity, which is informed by histological data indicating the class, severity, and reversibility of nephritis, and clinical data which include the change in proteinuria, renal function, serology and extra-renal lupus manifestations. A summary of the consensus recommendations by ALNN members is presented (Table 3). Initial (Induction) immunosuppression in the form of combination therapy with corticosteroids (e.g. prednisolone 0.8 mg/kg/day) and either MMF or CYC (Level 1b) Pulse corticosteroid (e.g. methylprednisolone 0.5 to 1.0 g/day for 3 days) advisable when renal biopsy shows crescentic involvement >10% or evidence of deteriorating renal function. (Level 5) Tapering of corticosteroids to begin after 2 weeks except in patients with no sign of improvement, aiming to reach <20 mg/day after 3 months and ≤7.5 mg/day after 6 months.

Antigens consisted of mumps virus see more (Whittaker Bioproducts, Walkersville, MD, USA), Candida albicans (Greer Laboratories, Lenoir, NC, USA) and tetanus toxoid (Connaught Laboratories Ltd, Swiftwater, PA, USA). Serum immunoglobulin levels and IgG subclasses were measured by rate nephelometry. Pneumococcal and tetanus antibody titres were measured by multi-analyte fluorescence detection (Arup Laboratories, Salt Lake City, UT, USA). Pneumococcal antibody titres against 14 serotypes (1, 3, 4, 5, 6B,

7F, 8, 9N, 9V, 12F, 14, 18C, 19F, 23F) were obtained prior to and 4 weeks after administration of the 23-valent polysaccharide Pneumovax-23 vaccine (Merck, Whitehouse Station, NJ, USA). Protective pneumococcal antibody titres were defined as IgG

> 1 µg/ml, or a greater than fourfold increase of titres after vaccination with Pneumovax-23. Protective antibody titres to tetanus were defined as anti-tetanus toxoid IgG > 0·10 IU/ml. Lymphocyte subsets were measured in whole blood. One hundred µl blood was mixed with 25 µl of fluorochrome-conjugated antibodies and isotype controls for 30 min at room temperature followed by lysis by lysing Pritelivir price buffer (Becton Dickinson). Cells were centrifuged and then washed 1× with phosphate-buffered saline (PBS), acquired by fluorescence activated cell sorter (FACS)Calibur and analysed by Simultest (Becton Dickinson). Lymphocyte subsets and TLR-4 expression on CD14+ macrophages were determined by multi-colour flow cytometry (FACScalibur) with FITC- and PE-conjugated monoclonal antibodies and isotype controls, using Simulset software (Becton Dickinson). Peripheral

blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, and lymphocyte proliferation in response to mitogens (PHA, ConA, PWM) and antigens (mumps, C. albicans, tetanus toxoid) were measured by [3H]-thymidine incorporation. Data were analysed as net counts/min after subtracting background counts. C59 chemical structure Natural killer (NK) cell-mediated cytotoxicity was determined by a non-radioactive cytotoxicity assay kit (ACT1; Cell Technology Inc., Mountain View, CA, USA), using flow cytometry according to the manufacturer’s instructions. Briefly, human erythroleukaemic tumour cells K562 (target cells) were labelled with the cell-tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and cultured with PBMCs (2·5 × 105 cells) at effector : target ratios of 12·5:1, 25:1, 50:1 and 100:1. After 6-h incubation at 37°C, 7-amino-actinomycin D (7AAD) stain was added to measure cell death. Data from 1 × 104 cells were collected and analysed by FACScalibur flow cytometer. To measure neutrophil oxidative burst, 1 µl of 5 mM dihydrorhodamine and 1 µl of dimethyl sulphoxide were added to 100 µl of heparinized blood.