Of note is the fact that this natural anti-NeuGcGM3 antibody

response decreases with age and is absent in most of the NSCLC patients assessed. Healthy human sera were tested by ELISA for the recognition of NeuGcGM3 and NeuAcGM3 gangliosides. In 65 out of 100 donors tested, anti-NeuGcGM3 antibodies of IgM and/or IgG isotype were detected. Only four donors showed a low reactivity against NeuAcGM3 (Fig. 1A). There were no differences between male and female anti-NeuGcGM3 antibody levels (Supporting Information Fig. 1). Previous studies about antibodies against common neuronal gangliosides showed that their levels significantly decreased with age [19]. In order to determine if the natural antibody levels against NeuGcGM3 are affected by age, the antibody response in donors of different ages was compared by ELISA. As shown in Figure 1B, there was a negative correlation between the level click here of the anti-NeuGcGM3 response and the increase of the donors’ age. Not only was the level of the anti-NeuGcGM3 response lower, but also the percentage of healthy donors with positive anti-NeuGcGM3 response decreased with age (Fig. 1C). Next, Navitoclax supplier we determined whether the lower content of anti-NeuGcM3 anti-bodies in elderly healthy donors was a consequence of a decrease in the concentration of IgM and IgG immunoglobulins. Total IgM

and IgG antibody levels did not decrease with the age of the healthy donors (Supporting

Information Fig. 2). Having evaluated the capacity of healthy human FAD antibodies to bind the ganglioside NeuGcGM3 by ELISA, we tested whether these antibodies are able to recognize the ganglioside in a natural context, exposed on the cytoplasmic membrane of tumor cells. To do this, the 100 human serum samples were incubated with the murine lymphocytic leukemia cell line L1210, which expresses NeuGcGM3 ganglioside [20]. NeuGcGM3 ganglioside expression on this cell line was confirmed by TLC-immunostaining (Supporting Information Fig. 3), and the antibody binding was measured by flow cytometry. Sera from 40 of the 65 healthy donors with a positive anti-NeuGcGM3 response by ELISA showed binding to L1210 cell line. Five of the sera that did not recognize NeuGcGM3 when tested by ELISA bound to this tumor cell line, presumably by binding to a different antigen. Figure 2A shows the results obtained with sera from three representative healthy donors with different levels of recognition of L1210 cells. To confirm that human serum antibodies recognize NeuGcGM3 ganglioside on the cell surface, we compared binding to L1210 with binding to cells that do not express this ganglioside. NeuGcGM3-negative cells were healthy human PBMCs and L1210 cmah-kd cells, which do not express the enzyme that catalyzes the conversion of N-acetyl to N-glycolyl sialic acid.

Alveolar epithelial type II cells (AECII) and mixed alveolar epithelial cells (mAEC) were stimulated with 20 µg/ml lipopolysaccharide (LPS) and co-exposed to sevoflurane for 8 h. In-vitro active sodium transport via ENaC and Na+/K+-ATPase was determined, assessing 22sodium and 86rubidium influx, respectively. Intratracheally applied LPS (150 µg) was used for the ALI in rats under sevoflurane or propofol anaesthesia (8 h). Oxygenation index (PaO2/FiO2) was calculated

and lung oedema assessed determining lung wet/dry ratio. In AECII LPS decreased activity of ENaC and Na+/K+-ATPase by 17·4% ± 13·3% standard deviation and 16·2% ± 13·1%, respectively. These effects were reversible in the presence of sevoflurane. Significant

better oxygenation was observed with an increase of PaO2/FiO2 from 189 ± 142 mmHg to 454 ± 25 mmHg after 8 h in the sevoflurane/LPS compared to the propofol/LPS group. The wet/dry ratio in sevoflurane/LPS Selleck Fostamatinib was reduced by 21·6% ± 2·3% Buparlisib research buy in comparison to propofol/LPS-treated animals. Sevoflurane has a stimulating effect on ENaC and Na+/K+-ATPase in vitro in LPS-injured AECII. In-vivo experiments, however, give strong evidence that sevoflurane does not affect water reabsorption and oedema resolution, but possibly oedema formation. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are a major cause of acute respiratory failure in critically ill patients [1]. The mortality of ARDS has remained high since its first description by Ashbaugh and colleagues [2], although lung protective ventilatory strategies have reduced mortality from 60–70% to 35–40% [3,4]. While drug treatment is investigated intensively,

no pharmacological approach has yet been established [5–8]. ALI/ARDS is characterized by capillary leak and reduced fluid reabsorption, resulting in lung oedema. The level of decreased rate of fluid clearance has significant prognostic value for morbidity and mortality [9]. In addition to reduced fluid reabsorption, protein clearance is also impaired. As demonstrated in patients with ARDS, non-survivors have three Baricitinib times higher alveolar protein concentrations than survivors [10,11]. Several studies have tried to detect the underlying mechanism of impairment of alveolar fluid clearance in ALI/ARDS and various pathways have been suggested [12–14]. According to experimental evidence, the active sodium (Na+) transport is thereby the most important ion transport mechanism involved in fluid reabsorption out of the alveolar space [15,16]. The broadly accepted paradigm for Na+ transport in the alveoli is a two-step process: Na+ enters the cell by epithelial amiloride-sensitive Na+-channels (ENaC) located at the apical surface and is extruded by basolaterally located sodium–potassium–adenosine–triphosphatase pumps (Na+/K+-ATPases) [17,18].

detected circulating T cells specific to gTG in CD patients without a gluten challenge [14]. These cells were detectable in the peripheral blood of more than half of adult CD patients on a gluten-free diet, but not detectable in healthy controls. Importantly, all the studies outlined above have analysed T cell responses in adult CD patients, whereas gliadin-specific SB203580 T cell responses in children with CD are explored far less widely. One study analysing intestinal CD4+ T cell responses suggested that the responsiveness to gliadin epitopes in paediatric CD patients differs from that found in adults [15]. Currently, it is unknown whether gliadin-specific T cells

are also detectable in the peripheral blood of children with

newly diagnosed CD. However, it is conceivable that the immune response in children at diagnosis represents an earlier and more active form of the disease, as responsiveness has not waned due to antigen elimination associated with a gluten elimination diet. In the present study, we used the CFSE dilution method selleck chemical to detect peripheral blood gliadin-specific T cells in children undergoing diagnostic small intestine biopsy for the diagnosis or exclusion of CD. In recent years, there has been increased debate on whether diagnostic biopsy is warranted in symptomatic children, and in some cases diagnostic criteria have been suggested based solely on antibody findings [16]. Therefore, our aim was to clarify the potential value of the detection gliadin-specific T cells in the periphery in supporting the diagnosis of CD. For this, we analysed proliferative

responses to both native gliadin and gTG as well as two synthetic peptides containing previously reported immunodominat epitopes of α-gliadin. We also characterized the memory phenotype and the expression of β7 integrin, a gut-homing molecule, on gliadin-specific T cells. Twenty Finnish children (10 girls and 10 boys) with newly diagnosed CD were included into this study. Blood samples were taken during the clinical visit where the CD diagnosis was confirmed with capsular Tyrosine-protein kinase BLK endoscopy, before the child was started on a gluten-free diet. In total, 19 of these 20 children were tested positive for tissue transglutaminase antibodies (TGA) (Celikey; Phadia, Freiburg, Germany); one of the children was not tested for TGA but was highly positive for endomysial antibody. The diagnosis of CD was set based on histological findings in the duodenal biopsy. Sixteen of the children (80%) were HLA-DQ2-positive, three were HLA-DQ8-positive (15%) and the HLA typing was not carried out on one of the children. The median age of children with CD was 8·3 years (range 3·6–14·8). The control group comprised 64 healthy children (27 girls and 37 boys) carrying the CD-associated HLA alleles.

Non-specific binding was blocked using 10% goat serum in TBST (0·1 m Tris–HCl, pH 7·5; 0·15 m NaCl; 0·1% Tween-20) for 30 min. Sections were then incubated for 60 min with the following primary antibodies: CD3e-biotin, CD11b, CD11c-allophycocyanin (APC), CD103-phycoerythrin

(PE), CD11c-biotin (BD Biosciences, Stockholm, Sweden) and with IgD (Biolegend, San Diego, CA), diluted in TBST. Unlabelled Pexidartinib mw antibodies were detected using Cy5-conjugated anti-rat IgG (Jackson ImmunoResearch, West Grove, PA), and biotinylated antibodies were detected using fluorophore tyramide (PerkinElmer, Waltham, MA). Tissue sections were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA), and analysed using laser scanning confocal microscopy (Leica TSP-2; Leica, Heidelberg, Germany). Images were analysed using leica lcs software (Leica, San Jose, CA) and Adobe Photoshop CS3. Intracellular staining for Foxp3 was carried out using a Mouse Regulatory T Cell Staining kit (eBioscience, San Diego, CA). 7-Amino-actinomycin D (7AAD) was used to exclude dead cells. The following conjugated antibodies were used for surface staining: CD3e-APC, CD4-Alexa-700, CD8a-PE-Cy7, CD11b-APC-Cy7,

CD11c-Pacific blue, CD45R-Pacific blue, CD45R-Alexa Fluor 488, MHC-II-Alexa-700, Aurora Kinase inhibitor KJ1-26-PE and Foxp3-PE (eBioscience), CD19-APC, CD25-APC-Cy7, CD62L-APC, CD103-PE (BD Bioscience), and streptavidin-Qdot 605 (Invitrogen). CD172a antibody was provided by Dr Karl Lagenaur and biotinylated in-house. Flow cytometry was performed on an LSR:II (BD Bioscience) and results were analysed using flowjo software (Tree Star, Ashland, OR). CD4+ T cells were enriched from spleens and LN of DO11.10 mice by positive selection magnetic separation using a MACS LS-column (Miltenyi Biotec, BergischGladbach, Germany). CD4+ cells were stained with 2·5 μm 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) and 2·5 × 106 to 5 × 106 cells were selleck compound injected intravenously into recipient CD47−/− and WT mice. The following day, mice were fed 10 mg OVA (grade V; Sigma, Stockholm, Sweden) in the presence or absence of 10 μg CT (Sigma) in 3% NaHCO3, or injected with 100 μg OVA intravenously. After 3 days, organs

were harvested and CD4+ T-cell proliferation was analysed by CFSE profiling. CD47−/− and WT mice were fed PBS or OVA (5 or 50 mg). Ten days later, all mice were challenged subcutaneously with 100 μg OVA in incomplete Freund’s adjuvant (IFA). Draining LN (inguinal) were harvested 1 week later and cells were re-stimulated with low-endotoxin OVA. Three days later, [3H]thymidine was added for 6 hr, then cells were harvested, and thymidine incorporation was measured using a β-counter. The stimulation index was defined as cellular proliferation in the OVA-fed group in relation to the PBS-fed group normalized to 0%. Wild-type mice that received PBS were used as reference for OVA-fed WT mice, and PBS-fed CD47−/− mice were reference for OVA-fed CD47−/− mice.

Whether or

not this is due to an intrinsic defect in the immune system of DS individuals or mainly secondary to the various DS-associated characteristics needs to be investigated further. Chromosome 21 genes that may influence the immune response include SOD1 and RCAN1. Selleck SAHA HDAC Several components of the immune system are variably affected in DS subjects, from which the most consistently reported are defective neutrophil chemotaxis and low humoral immune responses, associated with infections being predominantly of the respiratory tract. Factors that may induce immunodeficiency have been postulated, such as zinc deficiency and accelerated immunosenescence, although their clinical significances have not been established. Common anatomical defects of DS disturb natural barriers and facilitate the infectious disease process and need be considered in the management of infections in these patients. We recommend investigation of DS children who present with increased frequency of infections for immunological and non-immunological factors that increase the risk of infection. In this evaluation, low specific antibody titres to routine childhood vaccines would suggest the need for additional booster immunization doses. The authors thank Dr Carla Davis and Dr Kathlyn

Ostermaier for critical review of this manuscript. The authors have nothing to disclose. “
“Macrophages altered by various Th2-associated and anti-inflammatory mediators – including Omipalisib ic50 IL-4 and IL-13 [inducing alternatively activated macrophages (AAMs)], IL-10 and TGF-β– were generically termed M2. However, markers that discriminate between AAMs and other M2 remain scarce. We previously described E-cadherin as a marker for AAMs, permitting

these macrophages to fuse upon IL-4 stimulation. To identify novel potential contributors to macrophage fusion, we assessed the effect of IL-4 on other adherens and tight junction–associated components. We observed an induction of claudin-1 (Cldn1), Cldn2 and Cldn11 genes by IL-4 in different mouse macrophage populations. Extending our findings to other stimuli revealed Cldn1 as a mainly TGF-β-induced gene and showed that Cldn11 is predominantly associated with IL-4-induced AAMs. Cldn2 is upregulated by diverse stimuli and is not associated with a specific macrophage Bumetanide activation state in vitro. Interestingly, different claudin genes preferentially associate with M2 from distinct diseases. While Cldn11 is predominantly expressed in AAMs from helminth-infected mice, Cldn1 is the major macrophage claudin during chronic trypanosomiasis and Cldn2 dominates in tumour-associated macrophages. Overall, we identified Cldn1, Cldn2 and Cldn11 as genes that discriminate between diverse types of M2. Macrophages are very versatile innate immune cells that adopt various activation states depending on the environment.

One of the foremost mysteries about iNKT cells is how they are able to mediate such contrasting immunological effects

as CAL-101 price promoting tumour rejection or clearance of microbial infections, and preventing or ameliorating autoimmune diseases. Previous studies have established that the iNKT cell population contains functionally distinct subsets; for example, CD4− iNKT cells appear to be biased towards production of Th1 cytokines and expression of perforin, whereas CD4+ iNKT cells produce both Th1 and Th2 cytokines and are more notable for up-regulating FAS-ligand after stimulation.37 Thus, it is possible that different iNKT cell subsets become activated in different situations, and mediate distinct effects. This could be a result of differential anatomical localization of iNKT subsets, or of different costimulation requirements. However, as described in the next paragraph, it is not clear that different iNKT cell subsets recognize distinct antigens. Because of their canonical TCR rearrangements, all iNKT cells share the ability to recognize a specific molecular ‘pattern’ in which a galactose or glucose sugar is attached in an α-anomeric conformation to the polar head group of a lipid.38,39 The prototypical synthetic lipid of this type, α-galactosylceramide

(α-GalCer), is a highly potent agonist for iNKT cells.15 Lipids with structural similarity to α-GalCer have been identified from several microbial sources, including a pathogenic Borrelia species.40–43 However, these microbial analogues check details of α-GalCer generally appear to be substantially weaker TCR agonists than α-GalCer

itself. Importantly, mammalian cells do not seem to produce glycolipids in which the first sugar is attached to the lipid via an α-linkage, and thus the self antigens Selleckchem Palbociclib recognized by iNKT cells apparently do not contain this molecular pattern. The nature of the self antigens recognized by iNKT cells will be discussed at the end of the review; suffice it to note here that there is also as yet no clear evidence that iNKT self-antigen specificities differ according to subset. Another possibility (not mutually exclusive with the subset model) is that the same iNKT cell can mediate distinct functional effects as a result of variations in the activation stimuli in different contexts. We have recently shown that iNKT cells produce cytokines hierarchically in response to increasing TCR signal strength: granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-13 are activated by exposure to low doses of α-GalCer, higher levels of α-GalCer increase secretion of these cytokines and also induce IFN-γ and IL-4, and production of IL-2 requires the highest amounts of antigen.

3 (IV.3) and (3) medium containing F(ab)’2 goat anti-mouse IgG (GAM). Alternatively, cells were treated with anti-dinitrophenol (DNP) IgE (Sigma-Aldrich) and incubated on ice for 30 min followed by addition of DNP-BSA (Invitrogen, selleck chemicals Carlsbad, CA, USA) to stimulate serotonin release. Following stimulation, cells were placed at 37 °C for 30 min to allow secretion to proceed. Secretion was terminated by addition of 100 μl ice-cold medium and placement of cells on ice. After supernatants were removed from each well, cells were lysed by addition of phosphate-buffered saline (PBS) containing 1% sodium dodecyl sulphate (SDS). The 3H-serotonin in

supernatants and lysates was determined by liquid scintillation counting. Serotonin release is calculated as the percent of the total serotonin available for secretion (serotonin release mediated by the calcium ionophore A23187). MDV3100 order For inhibition assays, cells were preincubated with medium containing either 25 μg/ml piceatannol or 10 nm wortmannin (Sigma) for 15 min at 37 °C. These specific Syk and PI3K inhibitors were chosen for consistency with previous observations. Phagocytosis assay.  5 × 105 cells were plated per well in 6-well tissue culture dishes. The following day, the medium was replaced with fresh complete medium containing 1 × 108 IgG-opsonized sheep red blood cells (EA). After 30 min at 37 °C, externally bound EA were lysed by exposure

for 1 min to cold hypotonic saline. Washed cells were fixed in Wright-Giemsa stain, and phagocytosis of EA was assessed in at least 300 cells by light microscopy. For inhibition studies, cells were preincubated for 15 min at 37 °C with either 25 μg/ml piceatannol or 10 nm wortmannin. Statistical analysis.  Statistics were performed using Students t-test. To create a model system to investigate FcγRIIA tirggered serotonin secretion, wildtype FcγRIIA or mutants of FcγRIIA were stably transfected into RBL-2H3 cells. FACS analysis with anti-FcγRII D-malate dehydrogenase monoclonal antibody (IV.3) and FITC-conjugated goat anti-mouse

secondary antibody demonstrated that the selected cell lines transfected with FcγRIIA-wt or the various mutant FcγRIIA plasmids expressed quantitatively equivalent levels of FcγRII on the surface (Fig. 1). When stimulated with FcγRII-specific mAb IV.3 F(ab)’2/GAM F(ab)’2 (IV.3 + GAM), FcγRIIA-transfected RBL cells preloaded with 3H-serotonin released an average of 21% of total available radioactive serotonin (Fig. 2A). This release represents an almost 7-fold increase over what is observed for RBL-2H3 cells into which FcγRIIA had not been transfected (<4%, Fig. 2A). Less than 4% of total available serotonin is also released after mock stimulation of WT RBL-2H3 cells. This baseline release is considered due to general cell “leakiness”. Mock-stimulated FcγRIIA transfected RBL-2H3 cells also released baseline levels of serotonin (∼3%), indicating that cell surface expression of FcγRIIA by itself does not increase release of serotonin (Fig.

Conflict of interest: see more A. V. S. H. and H. M. are named inventors on a composition of matter patent for MVA85A filed by the University of Oxford, and are shareholders in a Joint Venture formed for the

further development of this vaccine. 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. “
“The development of clinical therapeutics that interfere with the migration of leukocytes has revolutionized the treatment of multiple sclerosis and holds great promise for the treatment of a wide range of inflammatory diseases. As the molecules essential for the multi-step adhesion cascade that mediates cellular migration have been elucidated, the number of potential targets available to modulate leukocyte trafficking

has increased exponentially. In this Viewpoint, we briefly review our current understanding of these mole-cular targets and how these targets vary by tissue and leukocyte subset with emphasis on T cells. We then describe the two currently approved therapeutics that target cell migration, natalizumab and fingolimod, and discuss how an improved understanding of their function could pave the way for the development of safer and more efficacious therapies for inflammatory and autoimmune diseases. Nearly 50 years ago, Gowans and Knight published a seminal study demonstrating that labeled lymphocytes injected into rats migrated 5-Fluoracil clinical trial from the blood into secondary lymphoid organs (SLOs) and then returned to the circulation via the thoracic duct [1]. In Phenylethanolamine N-methyltransferase an accompanying paper by Marchesi and Gowans, lymphocytes were observed to adhere to what are now called high endothelial venules and pass through the endothelial layer in a directed migration into the lymph node [2]. This process was hypothesized to be selective, as only small lymphocytes emigrated from the venules while larger lymphocytes were excluded. In the time since these first observations were made, knowledge of the molecular mechanisms

that underpin lymphocyte trafficking has exploded. The selective migration observed by Marchesi and Gowans is now understood to be a tightly orchestrated multistep adhesion cascade, regulated by selectins, integrins, chemokines, and chemoattractant lipids, that specifically directs the trafficking of leukocytes into sites essential for their function. Such an improved understanding of the underlying mechanisms involved has resulted in the identification of an array of potential drug targets aimed at modulating cell migration in order to treat a broad range of autoimmune and inflammatory diseases. Today, two drugs targeting cell migration are approved for clinical use in multiple sclerosis, one of which is also approved for Crohn’s disease; and many more are currently in clinical trial for these and other inflammatory diseases.

The role of plasmids in antibiotic resistance was evaluated by plasmid curing and gene transfer experiments. The genetic and molecular analysis of these factors could explain the resistance Cyclopamine concentration and survival of this opportunistic pathogen under adverse conditions such as those found in patients and nosocomial environments and could prove the

significance of biofilm formation and antibiotic resistance in UTI-associated Acinetobacter isolates. Urine samples and urinary catheters from patients with UTI were collected from two hospitals in Pune, India using standard procedures. The samples were collected aseptically and isolation was performed on selective Acinetobacter minimal medium (Juni, 1972), cystein lactose electrolyte deficient agar (HiMedia, Mumbai), Holton’s medium (Holton, 1983) and violet red bile agar (HiMedia). Pritelivir ic50 UTI samples were suitably diluted and plated onto the selective agar media, while the urinary catheter surfaces were scraped aseptically and transferred to sterile medium. The biomass was mixed using a vortex mixer, diluted and plated onto the selective agar medium. The plates

were incubated at 37 °C for 24 h. Fifty strains of Acinetobacter spp. were identified at genus level based on their morphological characteristics and modified chromosomal DNA transformation assay (Yavankar et al., 2007). The biochemical characterization and identification of these isolates at genus and species levels was confirmed using the analytical profile index (API) assays (BioMerieux, Marcy l’Etoile, France). API ID32GN is a standard system equipped with 32 miniaturized assimilation tests with a computerized database for Gram-negative bacteria and different clinical Acinetobacter

isolates (Towner & Chopade, 1987). The identified isolates were stored in glycerol stock at −80 °C. The bacterial isolates were inoculated in Luria–Bertani (LB) broth, incubated at 37 °C for 24 h and used for further experimentation. CSH was determined by the affinity test to xylene (Teixeira et al., 1993). The hydrophobicity index (HI) was C59 order calculated using the following equation: The biofilm-forming isolates of A. baumannii were grown in LB at 30 °C for 24 h. The bacterial suspension was centrifuged at 6000 g at 4 °C for 40 min. Fresh human blood was washed three times with sterile normal saline. Saline and 3% v/v human erythrocytes (50 μL each) were added to 100 μL of bacterial supernatant in each well of the microtiter plate and mixed by rotation for 5 min. Normal saline and uninoculated LB were used as the negative controls and phytohemagglutinin was used as the positive control. Agglutination of RBCs was determined within 30 min to 1 h (Patil & Chopade, 2001). The agglutinated cells were scored as positive for the presence of lectin. Acinetobacter isolates were inoculated in LB broth and incubated overnight at 37 °C. After the incubation period, 0.1 mL of the culture was added to 10 mL LB (0.5 ×) and dispensed in 20-mL polypropylene centrifuge tubes.

This is of interest for diseases, such as systemic infections, rheumatoid arthritis and osteoarthritis, which are associated with an increased activation of coagulation and the presence of physiological concentrations

of coagulation proteases, which may contribute to pro- or anti-inflammatory responses in a PAR-dependent manner. Therefore, in this study, it was investigated whether coagulation proteases (FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with free FX, free FX, free FXa and thrombin) in physiological concentrations can elicit pro- or anti-inflammatory responses in a PAR-dependent manner in naïve (non-preactivated) human monocytes and PBMCs. Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden) and CD14 microbeads from Miltenyi Biotec (Bergisch Gladbach, Selleck Wnt inhibitor Germany). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Invitrogen (Carlsbad, CA, USA). Heat-inactivated human male AB serum was from

Sigma-Aldrich (St. Louis, MO, USA). Allophycocyanin (APC)-conjugated monoclonal mouse anti-human CD14 antibody and APC-conjugated isotype control antibody were from BD Biosciences (Franklin Lakes, NJ, USA). Phycoerythrin (PE)-conjugated monoclonal mouse anti-human PAR-1 (ATAP2) antibody, FITC-conjugated monoclonal mouse anti-human PAR-2 (SAM11) antibody, JNK inhibitor chemical structure PE-conjugated monoclonal mouse anti-human PAR-3 (8E8) antibody, and APC-, PE- and FITC-conjugated isotype control antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel). PE-conjugated monoclonal mouse anti-human TF (HTF-1) antibody and PE-conjugated isotype control antibody were from BD Biosciences. Recombinant human FVIIa was kindly provided by Novo Nordisk A/S (Maaloev, Denmark). Recombinant human tissue factor (4500L), human factor X (527) and human activated factor X (526) were purchased from American Diagnostica

Inc. (Stamford, CT, USA). Human alpha thrombin factor IIa (IHT; activity ≥2700 NIH units/mg) was obtained from Innovative Research (Novi, USA). The of activity of the purchased coagulation proteases was tested positive in coagulation assays before use. Purified LPS was purchased from Sigma-Aldrich. PAR-1 antagonist FR171113 was obtained from Tocris Bioscience (Bristol, UK). FR171113 is a highly purified (>98%) specific PAR-1 antagonist which is able to inhibit thrombin-induced platelet aggregation. Interleukin-1β (IL-1β), Interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were from Invitrogen. Interleukin-6 and IL-8 ELISA kits were obtained from eBioscience (San Diego, CA, USA). All other chemicals were from Sigma-Aldrich. Peripheral blood was obtained from five different healthy donors after informed consent (age 37.2 ± 4.9 years; 2 males and 3 females). PBMCs were isolated by Ficoll-Paque (Pharmacia) according to standard procedures.