One candidate upstream component is the leucine-rich repeat (LRR)-containing G-protein-coupled receptor (GPCR) follicle-stimulating hormone receptor (FSHR-1), which was identified in a limited reverse Anti-infection Compound Library mw genetic screen of 14 candidate transmembrane LRR receptors in C. elegans. RNAi directed against fshr-1 results in a high degree of susceptibility to killing by P. aeruginosa, Staphylococcus aureus and Enterococcus faecalis, but not in a reduced lifespan during infection by non-pathogenic E. coli[24]. Expression of FSHR-1 in intestinal cells is necessary and sufficient for its role in innate immunity. Genetic analysis

indicates that FSHR-1 functions in the intestine in a separate pathway from PMK-1 and DAF-2, the worm insulin receptor that is involved in stress responses (see below) [24]. Further, qRT–PCR analysis shows that FSHR-1 and the PMK-1/p38 MAPK cassette regulate the induction of overlapping, but non-identical, sets of P. aeruginosa-induced genes. Although transcriptional profiling data suggest that FSHR-1 regulates host response genes independently of PMK-1, it is unclear whether the PMK-1/p38 MAPK cassette may be involved partially in signal transduction downstream of FSHR-1 [24].

It is also possible that the FSHR-1 and PMK-1/p38 MAPK pathways function Carnitine palmitoyltransferase II in parallel but converge on common sets of target genes in response to pathogen infection. How is FSHR-1 involved in mediating the C. elegans host response? Doxorubicin concentration We currently lack evidence that FSHR-1 can sense infection directly, for instance by binding pathogen-associated molecular patterns (PAMPs). FSHR-1 is the sole C. elegans LGR-type

GPCR. In mammals the heterodimeric glycopeptide hormone FSHα/β is the canonical ligand for this class of GPCR. Worms do not have an identifiable FSHα subunit and the endogenous ligands, if any, have not been identified. As an LGR-type receptor, one might expect FSHR-1 to transduce signals through heterotrimeric G-proteins in the intestinal cell. Recent findings implicate at least one heterotrimeric G-protein in signal transduction events upstream of PMK-1 in a different tissue, the hypodermis (see below). Whether this or other G-proteins mediate FSHR-1 signal transduction in the intestine remains unknown. Recent findings show that the protein kinase Cδ (PKCδ) TPA-1 activates the protein kinase D DKF-2 upstream of PMK-1/p38 in the intestine [20]. The upstream signals that control TPA-1 activity remain unknown, although by analogy with other systems, a likely candidate is diacylglycerol (DAG, produced by phospholipase C).

Table 3 shows the results in the 14 patients without acute mast cell mediator release or evidence of mastocytosis from the Sheffield Allergy Clinic. Three of 14 were falsely elevated and had evidence of RF and some HAMA activity. Eleven of 14 samples with undetectable IgM RF levels had tryptase concentrations which were not affected by the action of the HBT tubes. This suggests a lack of heterophile interference and demonstrates the existence

of a cohort of patients in whom unexpectedly raised tryptase levels appear to be real. Care should Venetoclax be exercised in the interpretation of MCT results due to the significant potential for interference by heterophilic antibodies including RF. This study shows that eight

of 56 sera (14%) with MCT > 14 µg/l were confirmed as having falsely elevated MCT. Five of 51 (10%) with MCT > 20 µg/l (WHO minor criteria for SM) were falsely elevated. All false positives had raised levels of IgM RF. Of the cohort with unexplained raised MCT, 20% were false positives due to assay interference but 80% were not, and had truly elevated stable increases of uncertain clinical significance. None of these patients had evidence of mastocytosis on extensive investigation. The persistently raised tryptase in this cohort of patients who do not have any clinical features of mastocytosis is interesting, but any attempts to explain it are speculative. Three of these were false positive elevations due to heterophilic interference from rheumatoid selleck compound factor activity. There do not appear to be any obvious clinical differences that would distinguish these patients from most of our cohort with idiopathic urticaria and angioedema. Longer-term follow-up may be

revealing. MCT is Unoprostone an important marker of acute mast cell mediator release in severe allergic reactions or mastocytosis [9]. It is recognized increasingly that there are some individuals who have persistently elevated tryptase using the current assay but in whom no evidence of either disorder can be found, leading to suspicion of assay interference [1,2,6,10]. However, the manufacturer states that the assay is not affected significantly by heterophile interference. We confirm that the presence of IgM RF correlates with interference in the Phadia tryptase assay and results in overestimation of tryptase or false positivity. This study demonstrates that IgM RF or a HAMA-like activity associated with IgM RF interferes with the assay and leads usually to overestimation of the true MCT value. We confirm that there are patients with persistently raised MCT who appear to be unaffected by HAMA or RF blocking, and these cases are not rare. It is important to note that the values produced following HBT treatment must be interpreted with caution, as this may not remove all the interfering heterophile activity and still give a misleading raised value for the analyte being measured [3].

The animals were sacrificed Selleckchem SCH 900776 after 7 days (n = 7), 6 weeks (n = 6) or 10 months (n = 9) after the transplantation. MRI demonstrated that BMSCs migrated to the damage area through the corpus callosum. Histological analysis showed that activated microglia were present around the bolus of donor cells 7 days after the allogeneic cell transplantation, although an immunosuppressive

drug was administered. The SPIO-labeled BMSCs resided and started to proliferate around the route of the cell transplantation. Within 6 weeks, large numbers of SPIO-labeled BMSCs reached the lacunar infarction area from the transplantation region through the corpus callosum. Some SPIO nanoparticles were phagocytized by microglia. After 10 months, the number of SPIO-positive cells was lower compared with the 7-day and 6-week groups. There was no tumorigenesis or severe injury observed in any of the animals. These findings suggest that BMSCs are safe www.selleckchem.com/products/gsk126.html after cell transplantation for the treatment of stroke. “
“The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2)

has been related to the aberrant splicing of several genes, including those encoding for ryanodine receptor 1 (RYR1), sarcoplasmatic/endoplasmatic Ca2+-ATPase (SERCA) and α1S subunit of voltage-gated Ca2+ channels (Cav1.1). The aim of this study is to determine whether alterations of these genes are associated with changes in the regulation of intracellular Ca2+ homeostasis and signalling. We analysed the expression of RYR1, SERCA and Cav1.1 and the intracellular Ca2+ handling in cultured myotubes isolated from DM1, DM2 and control muscle biopsies by semiquantitative RT-PCR and confocal Ca2+ imaging respectively. (i) The alternative

splicing of RYR1, SERCA and Cav1.1 was more severely affected in DM1 than in DM2 myotubes; (ii) DM1 myotubes exhibited higher resting intracellular Ca2+ levels than DM2; (iii) the amplitude of intracellular Ca2+ transients induced by sustained membrane depolarization was higher in DM1 myotubes than in controls, whereas DM2 showed opposite behaviour; and (iv) in both DM myotubes, Ca2+ release from sarcoplasmic reticulum through RYR1 was lower than in controls. The aberrant splicing of RYR1, SERCA1 and Cav1.1 may alter intracellular Ca2+ signalling in DM1 and DM2 myotubes. The differing dysregulation of intracellular Ca2+ handling in DM1 Dimethyl sulfoxide and DM2 may explain their distinct sarcolemmal hyperexcitabilities. “
“Glioblastoma (GBM), the most frequent and aggressive brain tumor, is characterized by marked angiogenesis directly related to invasiveness and poor prognosis. Hypoxia is considered to be an important stimulus for angiogenesis by inducing hypoxia-inducible factor 1-alpha (HIF-1α) overexpression that activates platelet-derived growth factor (PDGF) and VEGF. The aim of this study is to analyze the expression of PDGF-C, VEGF in endothelial and tumor cells of GBM and their relation to HIF-1α expression.

Another DC subset, the plasmacytoid DCs, induces peripheral tolerance under non-inflammatory conditions in the spleen and lymph nodes [12]. Further studies on DC subsets in the lungs are necessary to distinguish the role of DCs in asthma and design more effective preventative or therapeutic strategies for asthma [12]. Both DCs and FcγR are implicated in the development of allergic airway inflammation in bronchial asthma. FcRs on APCs and DCs and their signalling also play important roles in the development and control of the pathogenesis of asthma. The present report demonstrates that manipulation of the inhibitory FcR pathway is

a practical therapeutic means for controlling allergic airway inflammation. Targeting IgG-Fc and FcγRIIb https://www.selleckchem.com/products/c646.html on CD11c+ DC is a promising therapeutic strategy in allergic asthma. We appreciate the advice and expertise of Drs Tetsuya Takagawa and Kentarou Minagawa. We would also like to thank Drs Kazumi Kaneshiro, Haruko Shinke, Emi Kuramoto, Yuko Kono, Akihiro Sakashita, Natsumi Hara, Nobuko Hazeki, Keiko Okuno, Suya Okamoto and Daisuke Tamura for their helpful discussions. Smoothened antagonist This study was supported by KAKENHI (19790557). M. Yoshida was supported, in part, by grants for the Global Center of Excellence (COE) Program ‘Global Center of Excellence for Education

and Research on Signal Transduction Medicine in the Coming Generation’ from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, The Mother and Child Health Foundation and the Long-range Research Initiative of Japan Chemical Industry Association. The authors declare no conflicts of interest. IMP dehydrogenase “
“Accumulating evidence shows that galectins play roles in the initiation and resolution phases of inflammatory responses by promoting anti-

or proinflammatory effects. This study investigated the presence of three members of the galectin family (galectin-1, -3 and -9) in induced sputum samples of asthma patients, as well as their possible implication in the immunopathogenesis of human asthma. Levels of interleukin (IL)-5, IL-13, and galectins were determined in leucocytes isolated from induced sputum samples by reverse transcription–polymerase chain reaction (RT–PCR) immunofluorescence and flow cytometry. High levels of IL-5 and IL-13 mRNA were detected in sputum cells from asthma patients. In parallel, immunoregulatory proteins galectin-1 and galectin-9 showed a reduced expression on macrophages from sputum samples compared with cells from healthy donors. In-vitro immunoassays showed that galectin-1 and galectin-9, but not galectin-3, are able to induce the production of IL-10 by peripheral blood mononuclear cells from healthy donors. These findings indicate that macrophages from sputum samples of asthma patients express low levels of galectin-1 and galectin-9, favouring the exacerbated immune response observed in this disease.

This review highlights recent work concerned with the precise mapping

(localization) of brain activation in human infants, providing evidence that prefrontal cortex exhibits functional activation much earlier than previously thought. A systematic evaluation of the activation patterns in these neuroimaging studies mainly based on functional near-infrared spectroscopy reveals that prefrontal cortex function can be broadly divided into two distinct anatomical clusters with different functional properties. One cluster of activations falls within the region of the medial prefrontal cortex and is mainly involved in affective processes; another cluster is located in lateral aspects of the prefrontal cortex and shows sensitivity to cognitive processes such as memory and attention.

selleck This distinction is in line with adult data and evolutionary models and may represent a developmentally continuous organization principle of prefrontal cortex function. All in all, this review is aimed at providing a synthesis of new findings that are emerging from the use of neuroimaging techniques with infants as well as at encouraging further theory-driven research to understand the developmental origins of prefrontal cortex function. “
“We investigated the emergence in infancy of a preference to imitate individuals who display confidence over lack of confidence. Eighteen- Regorafenib chemical structure and 24-month-olds (N = 70) were presented with an experimenter who demonstrated the use of several objects accompanied by either nonverbal expressions of confidence or lack of confidence. At 24 months, infants were more likely to imitate the actions when demonstrated by a confident experimenter than by an unconfident experimenter; 18-month-olds showed no such preference. The experimenter

then presented an additional imitation trial and a word-learning trial while displaying a neutral expression. Twenty-four-month-olds persisted in preferentially imitating a previously confident experimenter, but prior confidence had no effect on their word learning. These Megestrol Acetate findings demonstrate a developmental increase in infants’ use of confidence cues toward the end of the second year of life. “
“This study examined infants’ sensitivity to a speaker’s verbal accuracy and whether the reliability of the speaker had an effect on their selective trust. Forty-nine 18-month-old infants were exposed to a speaker who either accurately or inaccurately labeled familiar objects. Subsequently, the speaker administered a series of tasks in which infants had an opportunity to: learn a novel word, imitate the speaker’s “irrational” actions, and help the speaker obtain an out-of-reach object. In contrast to infants in the accurate (reliable) condition, those in the inaccurate (unreliable) condition performed more poorly on a word-learning task and were less likely to imitate.

Detection was performed by western blot analysis using anti-SphK1 antibodies. Monocytes (1×106 cells/mL) were preincubated this website for 20 min at 37°C in the presence or absence of inhibitors as indicated in the text and subsequently cultured for up to 24 h in the presence or absence of 4 μM CXCL4. Amounts of CCL2, TNF, and IL-6 were determined in cell culture supernatants by Beadlyte® Human Multi-Cytokine Detection System 4 (Millipore GmbH, Schwalbach, Germany), while S1P levels were analyzed using a S1P competitive ELISA kit (Echelon

Biosciences, Salt Lake City, UT) according to the manufacturer’s recommendations. Activation of caspase-9 and caspase-3 was determined in lysates from CXCL4 or S1P stimulated cells in the presence or absence of SKI or PD098059, and in unstimulated cells exactly following the manufacturer’s instructions. In brief, cell lysates containing 10 μg total protein were incubated for 60 min at room temperature or at 37°C, in the presence of caspase-9 or caspase-3 substrate peptide, respectively. Caspase-9 activation was determined in a microplate

luminometer (LB 96V; Berthold) by measurement of chemiluminescence in the presence of Ac-LEHD-pNA (Caspase-Glo® 9 Assay; Promega (Mannheim, Germany)), and caspase-3 activity was tested in the presence of DEVD-AFC (Caspase-3 apoptosis detection kit; Santa Cruz Biotechnology) using a PTI-RF-M2001 spectrofluorometer (Photon Technology International, Wedel, Germany) at an exitation Selleck C59 wnt wavelength of Bacterial neuraminidase 400 nm and an emission wavelength of 505 nm. Data are presented as x-fold induction of the corresponding control (freshly isolated monocytes). Data are presented as mean±SD for the number of experiments indicated in the figure legends. Statistically significant (p<0.05) differences among the treatment groups were calculated using repeated measures (paired) one-way ANOVA test, followed by Tukey–Kramer multiple comparisons test for more than two treatment groups, and Student's paired t-test or Wilcoxon matched-pairs test for two treatment

groups. The authors thank Christine Engellenner, and Diana Heinrich for excellent technical assistance. This work was supported in part by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 415, Projekt B6 (F. P.) and Projekt A11 (S. S.), and by the DFG priority program 1267, grant SCHU-733/9-1 (S. S.). Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Epidermal Langerhans cells (LCs) are dendritic APCs that play an important role in cutaneous immune responses. LCs are associated with epidermal nerves and the neuropeptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) inhibit LC Ag presentation for Th1-type immune responses.

All experiments were approved by the VAMC-Institutional Animal Care and Use Committee. Bone marrow (BM)-derived DCs (BMDCs) were generated from the femurs, tibias and pelvic bones of euthanized mice. The bones were cleaned with sterile Kim Wipes, both ends of each bone were cut, and the bone marrow was flushed out. Contaminating erythrocytes were lysed using ACK lysis buffer for 5 min at room temperature. Cells (1 × 106/mL/well) were cultured in 24-well plates using RPMI 1640 basal medium supplemented with 10% foetal bovine serum, 1% penicillin/streptomycin solution (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA),

50 μm 2-mercaptoethanol (SIGMA, St Louis, MO, USA), 10 mm HEPES (Hyclone) and 20 ng/mL murine GM-CSF (R&D Systems, Minneapolis, MN, USA). The culture medium was changed completely every 2–3 days Dabrafenib molecular weight with Deforolimus price fresh medium containing GM-CSF. The subset of DCs thus generated is referred to as myeloid-derived DCs (12). The cells were cultured and tested for the expression of DC markers at days 7, 10 and 14. Dendritic cell phenotyping targeted loosely adherent cells, collected by gentle pipetting, for the expression of DC markers. Day 14 was determined to be the most optimal time for maximal generation of DCs, because >95% cells expressed the DC differentiation markers. Cell viability was also determined by

the trypan blue exclusion test. In all the batches tested, the viability was >95%. The cells harvested from the BM or spleens were considered immature DCs. The immature DCs were exposed to various antigens for 18 h, whereupon the conditioned media (CM) and cells were harvested. Lipopolysaccharide (LPS) (SIGMA) was

dissolved as per the manufacturer’s instructions and used as a positive control at a concentration of 1 μg/mL. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of heparinized blood from anonymized healthy volunteer donor pools, using centrifugation on Ficoll–Hypaque gradients (SIGMA). Monocytes were isolated from PBMCs by positive selection using CD14+ Tolmetin beads (Miltenyi Biotech, Boston, MA, USA). The CD14+ cells were cultured in RPMI 1640 with 10% FBS and 1% penicillin/streptomycin solution containing hGMCSF and hIL-4 (R&D systems), 50 and 14 ng/mL, respectively, for 5 days, until the cells were expressing >90% CD11c, CD11b and <5% CD14+. An increase in the appearance of other DC markers, such as CD86 and HLA-DR, was noted. Before specific antibody labelling, DCs were incubated with normal mouse and human IgG to block Fc receptors. Cells were then incubated with 200 μg/mL of antibody solution for 30 min in the dark at 4°C. The labelling buffer consisted of PBS with 1% FBS (21). The cells were washed and fixed with 1% paraformaldehyde and analysed using a BD Aria II cytometer using FACSDiva 6.1.1 software (Becton Dickinson, San Jose, CA, USA).

Cross-linking was performed as described

previously. Cells were incubated for 72 h at 37°C and 5% CO2 and pulsed with radioactive [3H]-thymidine for the last 18 h to assess proliferation. The BIACore 2000, sensor chip CM5, surfactant P-20, HBS-EP [10 mM HEPES, 0·15 M NaCl, 3·4 mM ethylendiamine tetraacetic acid (EDTA), 0·005% P-20, pH 7·4], amine coupling kit and 10 mM acetate pH 4·5 were from BIACore, Inc. (Piscataway, NJ, USA). Immobilization of antibodies to the sensor chip surface was performed according to the Target Selective Inhibitor Library manufacturer’s instructions, using a continuous flow of 10 mM HEPES, 0·15 M NaCl, 3·4 mM EDTA and 0·005% P-20, pH 7·4 (HBS-EP buffer). Briefly, carboxyl groups on the sensor chip surfaces were activated by injecting 60 µl of a mixture containing 0·2 M N-ethyl-N′ (dimethylaminopropyl)carbodiimide (EDC) and 0·05 M N-hydroxysuccinimide (NHS). Specific surfaces were obtained by injecting antibody diluted in 10 mM acetate, pH 4·5 at a concentration of 30 µg/ml. Excess reactive groups on the surfaces were deactivated

Selleckchem Trichostatin A by injecting 60 µl of 1 M ethanolamine. Final immobilized levels were ∼9000–12 000 resonance units (RU) for the antibodies. A blank, mock-coupled reference surface was also prepared on the sensor chip. To perform a competition binding analysis of the anti-mBTLA mAbs by BIACore, each antibody was immobilized to a different flow cell of a CM5 sensor chip. Murine BTLA-mFc was captured on the antibody surfaces and then either the immobilized antibody or a different antibody was injected over the captured mBTLA-mFc. DO11.10 splenocytes, 20 × 106, were adoptively transferred into BALB/c recipients. The next day mice were treated intraperitoneally with 15 mg/kg 4��8C of anti-BTLA reagent or control reagent. Three h after protein treatment animals were administered 10 µg of biotin-labelled rat

anti-mIL-2 (clone JES6-5 H4) to capture secreted IL-2 as described previously. Mice were then injected in the footpad with 100 µg of OVA protein to activate the monoclonal population of transferred DO11.10 T cells. The mice were rested for 18 h before exsanguination and then serum IL-2 was detected using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Studies that benchmarked the effect of CTLA4-Fc in this model were performed in a similar manner. Figure 1 shows the effect of anti-BTLA reagents on the anti-CD3ε-induced proliferation of murine spleen-derived T cells in vitro. We have shown previously that the mHVEM-mFc ligand and all the putative anti-BTLA mAb-stained T and B cells by fluorescence activated cell sorter analysis (FACS) and that the staining could be reversed specifically with soluble mBTLA-mFc (data not shown).

778, P < 0.01). Infusion of vasopressin reversed all of above parameters. Conclusion: Our observations suggest that long-term treatment of CsA inhibits BDNF and its receptor expression in the kidney, and that this may be associated with impairment of urine concentration ability. Enhanced apoptotic cell death at least partially accounts for the CsA-induced urinary concentration defect

in a rat model of chronic CsA nephropathy. FUJIMOTO KEIJI, MUKAI KIYOTAKA, OKUSHI YUKI, OKINO KAZUAKI, SHOJIMA KIYO, MATSUI YUKI, ATSUMI HIROKATSU, ADACHI HIROKI, OKUYAMA HIROSHI, YAMAYA HIDEKI, YOKOYAMA CCI-779 HITOSHI Division of Nephrology, Kanazawa Medical University School of Medicine Introduction: The activation of β3 integrin on glomerular podocytes by soluble urokinase receptor (suPAR) might be the cause of primary focal segmental glomerulosclerosis (FSGS). However, the clinical significance of serum suPAR and activated β3 integrin in untreated nephrotic diseases MI-503 price is still unclear. Methods: This single-center cohort study assessed the association of serum suPAR and renal expression of activated β3 integrin in Japanese primary nephrotic syndrome (NS).

Serum suPAR was measured in sera frozen at −80°C using a commercial ELISA kit, Quantikine Human suPAR Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol. Frozen sections from untreated 6 primary NS and 5 normal tissues of renal resection at the time of surgery were examined by immune-fluorescence (IF) methods using anti- activated β3 integrin (AP-5). Results: We investigated serum suPAR level in 31 NS patients [7 FSGS, 11 minimal change NS (MCNS), 11 membranous nephropathy (MN), and 2 membranoproliferative glomerulonephritis (MPGN)] and 20 healthy control subjects.

The pretreatment serum suPAR level in the primary NS was higher than that in the Progesterone controls (P < 0.01), but no difference among the pathological types. An inverse correlation between the pretreatment serum suPAR level and eGFR was noted in all primary NS, and each of the FSGS, MN patients [all primary NS (n = 31, r = −0.55, P = 0.001); FSGS (n = 7, r = −0.80, P = 0.03); MN (n = 11, r = −0.63, P = 0.036)]. Furthermore, time-course changes in the serum suPAR level over 2 months after therapy were associated with the therapeutic responsiveness of primary NS, particularly the differentiation of MCNS from FSGS (cut-off value: −251 pg/ml, AUC = 0.933, P = 0.018). Otherwise, activated β3 integrin was mainly detected on proximal tubular epithelium, but not glomerular capillaries in primary NS. The intensity of IF on proximal tubular epithelium was much higher in primary NS than that in normal controls.

5). To evaluate further whether inhibition of signalling pathways modulate TG2 expression at the protein level, Caco-2 cells were incubated with TNF-α + IFN-γ in the presence of inhibitors. Western blot analysis revealed that TG2 protein induction was inhibited when treatment with TNF-α + IFN-γ was performed in the presence of sulphasalazine or wortmannin. The intensity of protein bands from TNF-α + IFN-γ-treated samples obtained in the presence of inhibitors was similar to that obtained from untreated cells. In order to evaluate further whether TG2 produced in TNF-α + IFN-γ-treated cells is correctly folded and located at the cellular membrane, flow cytometric

analysis was Tanespimycin manufacturer performed on THP-1 cells stimulated with TNF-α + IFN-γ for 20 h. A panel of four anti-TG2 monoclonal antibodies (named 5G7G6, 2G3H8, 4E1G9 and 1H7H9), recognizing different MAPK inhibitor epitopes, was used to evaluate the surface expression of TG2. The four

anti-TG2 antibodies detected TG2 on the cell surface [16]. Flow cytometric analysis, using the 1H7H9 monoclonal antibody, showed that treatment of THP-1 cells with TNF-α + IFN-γ for 20 h increased TG2 protein at the cellular membrane [mean fluorescence intensity (MFI) = 30,78 in treated cells compared with MFI = 16·41 for unstimulated cells (Fig. 6). Similar results were obtained when flow cytometric analysis was performed using the anti-TG2 monoclonal antibodies 4E1G9, 5G6G7 and 2G3H8 (not shown). To evaluate whether inhibition of signalling pathways modulate the density of TG2 molecules at the cell surface, flow cytometry was performed on THP-1 cells incubated for 20h with TNF-α + IFN-γ in the presence of inhibitors. Interestingly, the induction of TG2 protein produced by the

double stimulus with TNF-α + IFN-γ was blocked completely in the presence of sulphasalazine. When the other inhibitors (Ly294002, SB203580, SP600125 and wortmannin) were tested, the expression of surface TG2 was only partially inhibited. These results are in accordance with those obtained by qRT–PCR, Western blot and luciferase activity analysis, and highlight the central role of NF-κB activity on TG2 expression. To investigate whether the synergistic induction of TG2 by TNF-α + IFN-γ ADAM7 in cell lines also occurred in intestinal tissue, biopsy samples from the duodenum of untreated CD patients and controls were incubated with the combination of TNF-α + IFN-γ for 24 h. Under basal conditions, intestinal mucosa of untreated CD patients had a higher TG2 mRNA content (9·8-fold increase in comparison with the housekeeping gene β-actin) than control samples (5·1-fold increase) (Fig. 7a). Intestinal tissues from untreated CD patients as well as controls showed up-regulation of TG2 mRNA (8·5- and 14·8-fold increase, respectively) when compared to unstimulated samples.