β-hexosaminidase release was determined after incubating HMC-1 for 1 h with live trichomonads (2·5 × 106, 5 × 106), CM or TCM. As a positive control, mast cells were incubated for 1 h in PMA (100 nm) plus A23187 (10 μm). HMC-1 cells (5 × 105) were incubated with live T. vaginalis, CM or TCM. After 1 h, 50-μL aliquots of culture supernatants of the mast cells or the cell find more pellet after lysis with 1% Triton X-100 were added to 200 μL of 2 mmp-nitrophenyl-N-acetyl-d-glucosamine in 0·2 m citrate buffer (pH 4·5) as substrate. After

1 h at 37°C, the reaction was stopped with 500 μL of 0·05 m sodium carbonate buffer (pH 10). Absorbance was measured with an ELISA reader at 405 nm. The percentage β-hexosaminidase release was calculated from the

equation: [β-hexosaminidase release (%) = (absorbance of supernatant)/(absorbance of supernatant + absorbance of pellet) × 100]. For measurement of IL-8 production by MS-74 FDA-approved Drug Library VEC, 3 × 105 VEC/well were cultivated for 2 days and then incubated with live T. vaginalis (0·3 × 106, 1·5 × 106, 3 × 106) in a 24-well microtitre plate at 37°C for various times. To measure IL-6 production, VEC were incubated with live T. vaginalis (3 × 106) for 6 h at 37°C. Also, to observe cytokine release by mast cells, HMC-1 cells (1 × 106) were incubated with CM or TCM at 37°C for 6 h. IL-8 and TNF-α proteins were measured by ELISA using a commercial kit (BD Bioscience, San Diego, CA, USA). To examine MCP-1 expression by MS-74 VEC stimulated with T. vaginalis, 3 × 105 VEC/well were cultivated for 2 days and then incubated with live T. vaginalis (3 × 106 cells/well) in 24-well microplates for various times. To examine cytokine

expression by HMC-1 mast cells, HMC-1 cells (2 × 106 cells) were stimulated with CM or TCM or with PMA (25 nm) plus A23187 (1 μm) for 30 min. Total RNA was extracted from the cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as described previously (13). Primer sequences and PCR conditions used for amplification of β-actin, MCP-1, TNF-α and IL-8 were as follows: MG-132 manufacturer β-actin (5′-CCA GAG CAA GAG AGG TAT CC-3′ and 5′-CTG TGG TGG TGA AGC TGT AG-3′), human MCP-1 (5′-TCC TGT GCC TGC TGC TCA TAG-3′ and 5′-TTC TGA ACC CAC TTC TGC TTG G-3′), TNF-α (5′-ACT CTT CTG CCT GCT GCA CTT TGG-3′ and 5′-GTT GAC CTT TGT CTG GTA GGA GAC GG-3′) and IL-8 (5′-GCC AAG AGA ATA TCC GAA CT-3′ and 5′–AAA GTG CAA CCA CAT GTC CT-3′). PCR conditions were as follows: initial DNA denaturation at 94°C for 5 min and 35 rounds of denaturation (98°C for 15 s), annealing (55°C for MCP-1 and TNF-α, 56°C for IL-8 and 58°C for β-actin, for 30 s in each case) and extension (72°C for 35 s). PCR products were electrophoresed on 2% agarose gels containing 0·5 μL/mL ethidium bromide and photographed under ultraviolet light.

Control antibodies included Rat IgG2a isotype control mAb (eBioscience), mouse anti-Border disease virus p125/p80 mAb VPM21 and purified rabbit immunoglobulin (Sigma-Aldrich, St. Louis, MO, USA), for rat, mouse and rabbit primary antibodies, respectively. All antibodies were diluted in PBS/T80 containing 10% NGS. Slides see more were washed twice in PBS, and the appropriate secondary antibody (peroxidase-labelled anti-mouse or anti-rabbit EnVision™+ reagent, Dako) was applied to sections for 30 min at RT. After a final PBS wash, sections were incubated with 3,3′-diaminobenzidine (DAB) for 7·5 min at RT, washed in distilled water, counterstained

with haematoxylin, dehydrated and mounted in Shandon synthetic mountant (Thermo Scientific). Each nodule was scanned under the light microscope. The initial scanning was performed with a wide-angle lens at low power (×20), and the following data were recorded: the predominant inflammatory cell type, the distribution of the cell infiltrate (diffuse or focal/multifocal) and the location of the infiltrate within the nodule (peripheral, central

or both). CD3+ and Pax5+ cells tended to occur in a focal/multifocal distribution pattern in the sections, and the foci of CD3+ and Pax5+ cells were counted in the most active ×20 field (the field with the highest number of foci). CD3+ and Pax5+ infiltrates were subjectively scored 0–3 (Table 1). MAC387+ infiltrates

were also scored 0–3; however, MAC387+ cells occurred more diffusely in sections, either evenly distributed or in patches, and therefore, the scoring system was slightly different MK-8669 research buy (Table 2). Numbers of FoxP3+ cells were counted in 10 nonoverlapping ×400 fields (five peripheral and five central fields per oesophageal nodule using a 0·0625 mm2 graticule). In the normal oesophagus control group and lymph nodes, five nonoverlapping ×400 fields were counted. Counting was confined to CD3+ areas. Statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc. CA, USA). The difference in prevalence and distribution Casein kinase 1 of the different proportions of cell types was tested using the chi-square test. The differences between the scores of the different types of infiltrate were tested for significance between all groups using a Kruskal–Wallis test, followed by Dunn’s post hoc test. P values of <0·05 were considered significant. Myeloid cells predominated in 70% of cases, while T cells predominated in 23% of cases. In the remaining 7% of cases, the number of T cells and myeloid cells was approximately equal. There was no difference in the proportion of myeloid and T cells between the neoplastic and non-neoplastic groups (P = 0·27). When cells were present in normal oesophageal sections, they were diffusely scattered and myeloid and T cells tended to occur in equal proportions (Table 3).

It seemed that the confusion could arise from the variety of growth conditions and purification methods used by different research groups working mainly with two model strains: S. epidermidis RP62A and S. aureus MN8m. In order to clarify this ambiguity, a direct comparative study of ‘PS/A’ and PIA has been carried out in our group. As a first step, we established a simple protocol for a large-scale biofilm culture S1P Receptor inhibitor and a mild method of extraction and separation of components of the biofilm matrix for a model biofilm-forming strain S.

epidermidis RP62A (Sadovskaya et al., 2005). We then compared the chromatographic elution profiles and the chemical structure of PNAG, prepared from two model strains, S. epidermidis RP62A and S. aureus MN8m, grown

under identical conditions and using the same method of extraction and purification as the GlcNAc-containing polysaccharides. In agreement with the literature data (Mack et al., 1996; Joyce et al., 2003), the PNAG obtained of both strains represented a β(1,6)-linked N-acetylglucosaminoglycan, with a part of the GlcNAc residues deacetylated and partially O-succinylated. The molecular CHIR 99021 weights (MWs) of the two polymers were close, and their chemical structure was identical, except for the degree of partial N-deacetylation and O-succinylation (Sadovskaya et al., 2005). The PNAG from S. epidermidis RP62A did not contain any phosphate substitution; the presence of phosphate demonstrated by Mack Quisqualic acid et al. (1996) was probably due to the contamination by the phosphate buffer used during purification. Therefore, our data confirmed that, as stated in Maira-Litran et al. (2004), ‘PIA and PS/A are the same chemical entity – PNAG’. The chemical structure of PNAG from a number of strains of CoNS from our collection was also investigated. We have shown that the PNAG of all

strains studied had the same structural features as the one from model staphylococcal strains, with the difference in the quantities produced and the degree in substitution with charged groups (Sadovskaya et al., 2006). A genetic locus pgaABCD, promoting surface binding, intercellular adhesion, and biofilm formation, has been identified recently in a number of Gram-negative bacteria. Genetic and biochemical studies demonstrated that, despite a very limited homology of pga and ica at the nucleotide or the amino acid level, a pga-dependent polysaccharide in Escherichia coli was a poly-β-(1,6)-GlcNAc (PGA), a polymer with a structure close to staphylococcal PNAG (Wang et al., 2004). Later, we have isolated a pga-dependent polysaccharide from the biofilms of a swine pathogen Actinobacillus pleuropneumoniae (Izano et al., 2007) and a human periodontal pathogen Aggregatibacter actinomycetemcomitans (Izano et al., 2008). We have shown that polysaccharides of the two strains were β(1,6)-linked poly-GlcNAc. Depending on the strain and the preparation, some of the GlcNAc residues (1–15%) were N-deacetylated.

Blood was taken from the mice and the percentage of CFSE-positive erythrocytes estimated by flow cytometry. In some experiments, mice were injected intraperitoneally (i.p.) with 500 μL of CGN at a

concentration of 2 mg/mL in PBS once every 2 days. This treatment was started 1 day before infection and continued until the end of each experiment. Suspensions of Py-infected erythrocytes were stained with APC-annexin (BD biosciences) Staurosporine cell line and the DNA dye Syto 16 (Invitrogen, Carlsbad, CA, USA) to detect PS and parasite DNA, respectively. For annexin V binding, erythrocytes were incubated with annexin V for 20 min at room temperature in annexin-binding buffer (140 mM NaCl, 10 mM HEPES, 5 mM glucose, 5 mM CaCl2, pH 7.4). Syto16 (final concentration of 20 nM) was then added to the suspensions followed by incubation for 20 min at room temperature. Cells were

then analyzed by flow cytometry. Intracellular Ca2+ measurement was performed as previously described 35 with minor modifications. Packed erythrocytes (2 μL in 2 mL of Ringer’s solution (1% Hct)) were loaded with Fluo-4/AM (Invitrogen) by addition of 2 μL of a Fluo-4/AM stock solution (2.0 mM in DMSO). The cells were incubated at 37°C for 15 min with vigorous shaking in a dark room followed by incubation with an additional 2 μM of Fluo-4/AM and 0.2 μg/mL Hoechst 33342 (Molecular Probes) for another Selleckchem Doxorubicin 25 min. Cells were then washed twice with Ringer’s solution containing 0.5% BSA (Sigma) and once with Ringer’s solution alone. As a positive control, erythrocytes were stimulated with 1 μM ionomycin for 3 min prior to analysis to increase intracellular

Ca2+ activity. Thin blood films were prepared and slides were analyzed using a fluorescence microscope (Keyence, Osaka, Japan). Differences between groups were analyzed for statistical significance using the Mann–Whitney or Wilcoxon tests. For the survival curves, Kaplan–Meier plots and χ2 tests were used. A p value<0.05 was considered to be statistically significant. The authors thank Mr. T. Matsumoto, (Keyence Co. Ltd., Osaka, Japan) for technical support in using fluorescence microscopy and the members of the Department of Parasitology, Institutes of Tropical Medicine, Nagasaki University, for support in completing additional experiments. This work was supported Lck by the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Grants 21022036, 20390121). Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA.

Histone acetylation is induced in response to TLR stimulation in macrophages, and is involved in the expression of multiple proinflammatory cytokine genes. Acetylated histones are recognized by the bromodomain and extra terminal domain (BET) family of proteins (Fig. 1). Among the BET proteins, Brd4 is known to associate with P-TEFb, a Cdk9-cyclin T heterodimer that stimulates transcriptional elongation by RNA polymerase II 28, 29. A small compound (I-BET) interacting with the bromodomain has been identified and this compound was shown to suppress

inflammatory gene expression in TLR-stimulated Silmitasertib supplier macrophages by disrupting chromatin complexes 30. Treatment with I-BET rendered mice resistant to endotoxin shock and bacteria-induced sepsis, suggesting that inflammatory responses can be controlled by regulating epigenetic changes on proinflammatory gene promoters. Furthermore, trimethylation of H3K4 on cytokine gene promoters was also shown to be induced in M1 macrophages in response to TLR stimulation, indicating that a change in histone modification is induced in the course of M1 macrophage activation leading to chromatin remodeling and inflammatory gene expression 19. The methylation of H3K27 is mediated by the Polycomb repressive complex 2 (PRC2) composed of Ezh2, selleck Suz12 and

Eed 31. Proteins harboring a Jumonji-C (JmjC) domain, Jmjd3 (also known as Kdm6b), UTX and UTY, are known to act as H3K27 demethylases catalyzing trimethyl H3K27me3 to monomethyl H3K27me1 32–34. Among these enzymes, the expression of Jmjd3 is TLR-inducible in macrophages via an NF-κB-dependent pathway. Since H3K27 trimethylation is implicated in the silencing of gene expression, it has been postulated that Jmjd3 is involved in the fine-tuning of macrophage activation toward M1 by regulating a set of genes such as Bmp2 and Hox34, 35. However, production of proinflammatory cytokines in response to TLR ligand stimulation was not impaired in macrophages from Jmjd3-deificient mice,

and cytokine production in response to Listeria monocytogenes Bupivacaine infection was unaffected by Jmjd3 deficiency 36. Thus, Jmjd3 is dispensable for M1 macrophage polarization. In contrast, Jmjd3 is essential for M2 macrophage polarization to helminth infection and chitin administration in mice. Chitin is a polymerized sugar and a structural component of helminths, arthropods and fungi 37. Chitin administration recruits macrophages with M2 character to the site of administration, which is important for subsequent recruitment of eosinophils 38, 39. Jmdj3-deficient BM chimeric mice were defective in the expression of M2 macrophage markers in F4/80+CD11b+ macrophages and eosinophil recruitment in response to chitin administration. Furthermore, activation of M2 macrophages to Nippostrongylus brasiliensis infection was severely impaired in the absence of Jmjd3.

Knocking-down of the E-cadherin expression on the surface by specific siRNA, resulted in cells that still formed a monolayer, which, however, tended to disperse spontaneously. PMNs or elastase increase dyshesion, most likely by cleaving the residual E-cadherin molecules. Nevertheless, participation of adhesion molecules other than E-cadherin cannot be ruled out. Of interest were the functional consequences of the loss of E-cadherin. We observed an enhanced migratory capacity PLX4032 of the elastase-treated tumor cells in both an in vitro invasion assay and a scratch “wound healing” assay. Enhanced migration

is most likely due to the loss of E-cadherin, as we found that under our experimental conditions that T3M4 with siRNA-silenced E-cadherin expression also showed enhanced migration. While our data clearly showed

dispersal and enhanced migratory activity of the pancreas tumor cells, questions remain about the underlying molecular mechanisms and even more importantly on a possible relevance for the in vivo situation. With regard to the former, a mere mechanical interpretation would be that dispersed, single cells migrate more readily compared to cells attached within a monolayer [25]. On the other hand, there is evidence that elastase-mediated loss of E-cadherin initiates the transcription of a number of target genes, which might be responsible for an altered phenotype [26, 27]. First evidence that neutrophil ID-8 elastase-mediated cleavage of E-cadherin induces such an altered phenotype also under our experimental condition is the translocation of β-catenin into the nucleus after Cabozantinib mouse the treatment of cancer cells with elastase. This interpretation is in line with data by others, who described an enhanced migratory activity of esophageal cancer cells after treatment with PMN elastase [28]. Furthermore, “abnormal” nuclear β-catenin expression in

PDAC correlates with increased lymph node or liver metastases [29]. The question of the in vivo relevance is more difficult to assess. Infiltration of PMNs into tumors has been described in pancreatic cancer and tumors of the periampullary region revealing a “micropapillary” growth pattern [6, 7], but overall it was concluded that intratumor PMN infiltration is an uncommon phenomenon in PDAC. In contrast to these studies, in which only PMNs in the direct vicinity to tumor cells were counted, we also included PMNs in the desmoplastic tumor stroma, because the latter are prominent in PDAC [3], and may play an essential role in tumor progression [30, 31]. To take all tumor associated PMN into account — the intratumor and the stroma infiltrating PMN as well — was proposed before in a study with gastric adenocarcinoma, which is also associated with a desmoplastic tumor stroma [32] and explains why we have a higher incidence of neutrophils in our study.

The rather large nucleus and very narrow cytoplasm of Treg cells makes it difficult to discriminate Inhibitor Library chemical structure clearly the brown from the red staining. After trying different combinations for the cell surface and nuclear staining, we settled for the combination of brown (DAB) for surface staining and red (AEC) for nuclear staining which gave the best color discrimination. Each decidual tissue sample was macroscopically separated from the chorionic villi and washed thoroughly in Hank’s solution to get rid of debris and contaminating

blood. Decidual mononuclear cells (DMC) were isolated as previously described.36 Briefly, decidual tissue was cut into small pieces and filtered through a 60-μm stainless steel mesh to make

single cell suspension. The resultant suspension was subjected to Percoll (Pharmacia) density gradient centrifugation. this website The interface between 40 and 80% Percoll, containing mononuclear cells and epithelial cells, was collected. Contaminating epithelial cells were removed by incubation with mAb BerEP4 and goat anti-mouse magnetic beads (Dynabeads M-450; Dynal, Oslo, Norway), followed by magnet treatment. Peripheral blood mononuclear cells (PBMC) from pregnant and non-pregnant donors were isolated by Lymphoprep (Nycomed, Norway) gradient centrifugation according to the manufacturer’s instructions. Immunoflorescence staining and three color FACS analyses were performed in 9 consecutive paired DMC and PBMC samples from pregnant women and 9 PBMC from non-pregnant women. For immunofluorescence staining, we used Human Regulatory T cell Staining kit (eBioscience) according to the manufacturer’s instructions. In brief, one million (1 × 106) DMC or PBMC per tube were incubated with CD4-FITC/CD25-APC cocktail for 30 min in dark at 4°C. After

washing with cold FC staining buffer, the cells were permeabilized with Fix/Perm buffer for 30 min in dark at 4°C, and non-specific binding was blocked by incubation with 2% normal rat serum for 15 min. Then, Pregnenolone the cells were incubated with Foxp3-PE Ab for 30 min, in dark, at 4°C. Control stains were included – positive control staining with CD45-FITC/CD14-PE (Dako) and isotype control staining with mouse IgG2a. In addition, PBMC and DMC suspensions were double stained with Foxp3 and CD56 (MY31), CD8-FITC (DK25), pan-γδ-FITC (5A6.E9) and Vδ1-FITC (TS8.2) mAbs, and goat anti-mouse IgG Fab-FITC (F0479). Percentages of Foxp3-positive cells were calculated within the CD4+ CD25−, CD4+ CD25+, and CD4+ CD25++ cell fractions. Data were acquired on FACS Calibur instrument (Becton Dickinson, San Jose, CA, USA) and analyzed using cellquest software (BD).

DECTIN-1 and LOX-1 act as pattern recognition receptors on innate immune cells by binding to β-glucans and bacterial surface molecules, respectively [15, 16], whereas CLEC-2 has been reported to have an internal ligand and mediate platelet activation [17]. The very recently identified orphan receptors CLEC12B and CLEC9A [18, 19] are also located within the myeloid cluster of the NK gene complex. selleck chemicals Given the importance of the encoded receptors, it was of interest to further investigate this genomic region to potentially identify additional genes and to unravel its evolutionary development by comparing this gene cluster in different species. This study will therefore reveal the arrangement of genes within

the myeloid cluster of the NK gene complex and help to better understand the evolutionary processes that lead to its current conformation. Bioinformatics.  Novel genes were searched for by comparing sequences available from the UCSC Genome Browser (available at: http://genome.ucsc.edu/) and the NCBI Map Viewer (available at: http://www.ncbi.nlm.nih.gov/mapview). CAL-101 nmr The human reference sequence (hg18) is based on NCBI Build 36.1 and was produced

by the International Human Genome Sequencing Consortium. The mouse genome data (mm9) was obtained from the build 37 assembly by NCBI. Sequences of other species (chimp, rhesus, cow and dog) were also obtained from UCSC Genome Browser and NCBI Map Viewer. For the search of genes already known in one species (e.g. NKG2i in

mice), the NCBI BLAST (blastn) algorithm was used (available at: http://www.ncbi.nlm.nih.gov/BLAST/) to find possibly existing novel mRNA or EST of a potential homologue of the already known gene. Accession numbers of the sequences used: human: CLEC12B NM_oo1129998, CLEC9A NM_207345, CLEC1 NM_016511, DECTIN-1 NM_022570, LOX-1 NM_002543, FLJ31166NM_153022, Gabarapl1 NM_031412. mouse: CLEC12b NM_027709AK016908, CLEC2 NM_019985, CLEC9a NM_172732, CLEC1 NM_175526, DECTIN-1 NM_020008, LOX-1 NM_138648, ‘mouse FLJ31166’ NM_001081186, Gabarapl1 NM_020590, NKG2i NM_153590. chimp: CLEC2 XM_520735, CLEC9a XM_001143778, CLEC1 XM_520737, DECTIN-1 XM_528732, LOX-1 XM_528733, ‘FLJ31166’ (included Gabarapl1 sequence) XM_520738. dog: CLEC12b XM_849067, CLEC2 XM_543823, CLEC9a XM_849058, CLEC1 XM_543822, DECTIN-1 XM_849050, Urocanase LOX-1 XM_543821, ‘FLJ31166’XM_849040, Gabarapl1 XM_848051. Sequence alignments and detection of homologies.  Sequence alignments were performed using different programs depending on the particular requirements. For alignments of shorter DNA and protein sequences, we used the MacVector7.0 software for bigger alignments and alignments that should make genomic rearrangements detectable, the Shuffle LAGAN tool (available at: http://lagan.stanford.edu/lagan_web/index.shtml) was used. Homologies of large genomic sequences were detected and plotted by the mVista Browser (available at: http://genome.lbl.gov/vista/index.

The urinary NGF levels of OAB, IC/PBS and controls from previous studies were used for comparison. NGF levels were compared among subgroups and between urinary tract diseases with or without associated OAB symptoms. The urinary NGF levels

Protease Inhibitor Library ic50 were also compared among natural filling, after normal saline filling and after potassium chloride test in a group of OAB and IC/PBS patients. Results: Patients with acute bacterial cystitis, urinary tract stones or urothelial cell carcinoma had elevated NGF levels that were not associated with the presence of OAB symptoms. Symptomatic cystitis patients who had resolved OAB symptoms after antibiotic treatment had a significant decrease in urinary NGF levels. The urinary NGF levels decreased significantly in OAB patients with effective antimuscarinic treatment for 6 months, but remained stationary and higher than the controls for up to 12 months after treatment. Conclusion: Urinary NGF is not produced solely in patients with OAB or IC/PBS. Acute bacterial cystitis, urinary tract stones and urothelial cell carcinoma can have high selleckchem urinary NGF production. “
“Overactive bladder syndrome (OAB), characterized by urinary frequency, nocturia and urgency with or without incontinence, is a widespread medical condition

with significant impact on quality of life. Three main factors have been proposed regarding the cause of OAB: myogenic, neurogenic and urotheliogenic. Disturbance of any of the three factors or a combination of these factors can attribute to OAB. Metabolic derangement, bladder outlet obstruction and inflammation can increase the excitability of nerve, detrusor muscle and alter the sensory Erlotinib manufacturer and barrier functions of the urothelium. The detection of proteins in the urine such as NGF, PGE2, and proinflammatory chemokines may advance our understanding of the pathophysiology of OAB and offer novel

diagnostic biomarkers of OAB. Overactive bladder syndrome (OAB) is a common medical condition with significant impact on quality of life across the world. It is characterized by urinary frequency, nocturia and urgency with or without incontinence.1 It has been estimated that the prevalence of OAB was 10.7% in the worldwide population in 2008, and will increase to 20.1% in 2018.2 It occurs more frequently in women than in men, and its incidence increases with age.3 Although many basic and clinical studies have been performed, the cause of OAB remains to be established.4 The mainstay of current pharmacological treatment involves the use of muscarinic antagonists, but their therapeutic effectiveness is limited by a combination of limited efficacy and troublesome side-effects.5,6 Therefore, finding the etiology of OAB is important for developing effective treatments. Here we review recent research in the pathophysiology of OAB and focus on bladder outlet obstruction (BOO), metabolic syndrome and inflammation (Fig.

For the detection of homologies between multiple short DNA and protein sequences, the ClustalW algorithm of the MacVector7.0 software or the BLAST 2 SEQUENCES selleck inhibitor Version of the NCBI BLAST algorithm was used. Construction of phylogenetic trees.  Phylogenetic trees were constructed with the EBI ClustalW tool (available at: http://www.ebi.ac.uk/clustalw/) using the CTLD sequences of the lectin-like genes starting from the first highly conserved cysteine

residue. Scanning of UTR sequences.  The investigation into the human CLEC9A UTR was performed using UTRScan, UTRdb and UTRblast (all available at: http://utrsite.ba.itb.cnr.it/). Cells.  Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described [13]. In short, cells were grown in M199 medium (Lonza, Basel, Switzerland) with 20% FCS, 2 ml/500 ml endothelial cell growth supplement (PromoCell, Heidelberg, Germany), 2 U/ml heparin (Roche, Mannheim, Germany) and 10 ml/500 ml PSFG (penicillin 10,000 U/ml, streptomycin 10 mg/ml, fungizon, find more 200 mmol glutamin (Lonza) in a 5% CO2 atmosphere at 37 °C. Venous peripheral blood of healthy volunteers was obtained from Red Cross (Vienna, Austria), and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque™ PLUS (GE Healthcare, Freiburg, Germany) gradient centrifugation according to the manufacturer’s

instructions. Cord blood dendritic cells (CBDC) were kindly provided by Dr. Frank Kalthoff (Novartis, Vienna, Austria). Suspension cell lines used: 721.221, Mono-Mac-6, K-562, Jurkat, U-937, CCRF-CEM, P815, NK-92 and

RPMI-8866 were all grown in RPMI1640 medium (Life Technologies Ltd., Paisley, UK) containing 10% FCS and 10 mm l-glutamine (Lonza) in a 5% CO2 atmosphere at 37 °C. NK-92 cultures were supplemented in addition with 1 mm sodium pyruvate, 50 mmβ-mercaptoethanol (both Sigma-Aldrich, Gillingham, UK) and human rIL-2 (R&D Systems, Wiesbaden, Germany) at a final concentration of 20 IU/ml. very Stimulation of cells.  CBDC were stimulated for maturation with 100 ng/ml LPS (Sigma-Aldrich), 4 μg/ml of anti-CD40 mAb (mAb clone 626.2) cross-linked in solution by the addition of 2 μg/ml of F(ab’)2-fragments of goat anti-mouse IgG (Pierce Chemical Corp, Rockford, IL, USA), 25 μg/ml Zymosan A (Sigma-Aldrich) or 10 ng/ml IFN-γ for 6 h. Stimulation of the cells was verified by real-time RT-PCR showing the upregulation of E-Selectin mRNA in HUVEC and CCL22 (chemokine (C-C motif) ligand 22) mRNA in dendritic cells by real-time RT-PCR. RNA isolation and real-time RT-PCR.  Total cellular RNA was isolated following cell lysis in Trizol (Invitrogen, Groningen, The Netherlands) by chloroform extraction and precipitation of the RNA using isopropanol. RNA were reverse transcribed into cDNA (SuperscriptTM II RT, Invitrogen) using oligo-dT primers, and real-time RT-PCR was used to monitor gene expression using a Light Cycler instrument (Roche Diagnostics GmbH, Mannheim, Germany) according to established procedures [20].