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The glucocorticoid receptor is present in a TCR-associated complex, which includes the Src family tyrosine kinase Lck. Glucocorticoids rapidly dissociate this complex, resulting in the inhibition of canonical Lck-phospholipase C (PLC)γ–dependent TCR signaling. The relative importance of this nongenomic role for the glucocorticoid receptor compared with its direct transcriptional effects is not known. Superantigens induce a state of steroid resistance in activated T cells. It was reported that, in addition to canonical Lck-PLCγ signaling, superantigens can activate a noncanonical G protein–PLCβ–dependent signaling pathway. In this study, we show that staphylococcal enterotoxin B activates a Gαq and PLCβ2–dependent pathway in human T cells. We find that this pathway bypasses the need for canonical Lck-PLCγ signaling in T cell activation and renders superantigen-stimulated T cells insensitive to glucocorticoids in vitro. We show that the PLCβ inhibitor U-73122 sensitizes staphylococcal enterotoxin B–treated mice to dexamethasone in vivo. In conclusion, we find that effects of glucocorticoids on TCR-induced T cell proliferation are mainly nongenomic and can be bypassed by the activation of an Lck-independent signaling pathway.

Background: The repertoire of immunomodulators that can be used for the treatment of inflammatory bowel disease is limited. The use of these drugs is further restricted by the occurrence of side effects in a proportion of patients. Miltefosine (hexadecylphosphocholine) is a lipid drug developed in the 1980s for the treatment of cancer but is nowadays best known for its application in the oral treatment of leishmaniasis. Although the exact mechanism of action of miltefosine has yet to be elucidated, the drug has previously been shown to inhibit phospholipases and protein kinase C, both key components of proproliferative signal transduction in T cells. Methods: Stimulated peripheral blood lymphocyte were treated with miltefosine, and proliferation was measured. We use the CD45RBhighT-cell transfer colitis model to investigate the effect of miltefosine treatment on intestinal inflammation. Effects on the severity of colitis were studied by histochemical and immunohistochemical staining, and cytokine levels were determined using a cytokine bead array. Results: Miltefosine inhibited T-cell proliferation in vitro. In the transfer model, miltefosine significantly ameliorated the severity of colitis as measured by clinical, (immuno)histochemical, and biochemical parameters. Conclusions: Miltefosine inhibits T-cell proliferation and effectively reduces inflammation in the T-cell transfer model. The drug may therefore be a candidate immunomodulator for inflammatory bowel disease. Copyright

Background and Aims: Anti-tumour necrosis factor [TNF] antibodies induce regulatory macrophages which display a phenotype resembling M2 type macrophages. Anti-TNF induced macrophages [Mϕind] have immunosuppressive and wound healing properties. The factors that contribute to the induction of Mϕind remain to be explored. Autophagy has been described as a factor that is important for the induction and function of M2 type macrophages. We studied the contribution of autophagy to the induction of Mϕind. Methods: We studied the effect of autophagy on Mϕind in vitro using peripheral blood mononuclear cells. Interferon gamma [IFN-γ] induced macrophages [Mφ1] were generated by culturing monocytes in the presence of IFN-γ. Mϕind were generated by performing mixed lymphocyte reactions [MLR] in the presence of anti-TNF antibodies; 28 healthy donors were genotyped for rs_2241880 [ATG16L1]. Cells were analysed by autophagy gene array, immunofluorescence, western blot, flowcytometry, 3H-thymidine incorporation and MTS assay. Results: Mϕind had a different expression profile of autophagy related transcripts with increased expression of 33/40 altered genes compared with Mφ1. In addition, autophagic activity was increased in Mϕind compared with Mφ1. Induction of Mϕind was positively correlated to the number of wild-type alleles for the ATG16L1 T300A risk allele present in the culture. Finally, the autophagy-related protein cathepsin S was highly expressed in Mφind and inhibition resulted in decreased viability as well as decreased expression of CD206. Conclusions: Mϕind have increased levels of autophagy compared with inflammatory Mφ1, and the induction of these macrophages is impaired in donors carrying the T300A risk allele for the ATG16L1 . Given the association between Mϕind and clinical response, this suggests that an intact autophagy pathway may be important for an optimal response to anti-TNF therapy in inflammatory bowel disease.

Background and aims Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I–III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays Methods The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied Results MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment Conclusions This study demonstrates that, in vitro , MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy.

Background and aim Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive effects both in vitro and in experimental colitis. Promising results of MSC therapy have been obtained in patients with severe graft versus host disease of the gut. Our objective was to determine the safety and feasibility of autologous bone marrow derived MSC therapy in patients with refractory Crohn9s disease. Patients and intervention 10 adult patients with refractory Crohn9s disease (eight females and two males) underwent bone marrow aspiration under local anaesthesia. Bone marrow MSCs were isolated and expanded ex vivo. MSCs were tested for phenotype and functionality in vitro. 9 patients received two doses of 1–2×10 6 cells/kg body weight, intravenously, 7 days apart. During follow-up, possible side effects and changes in patients9 Crohn9s disease activity index (CDAI) scores were monitored. Colonoscopies were performed at weeks 0 and 6, and mucosal inflammation was assessed by using the Crohn9s disease endoscopic index of severity. Results MSCs isolated from patients with Crohn9s disease showed similar morphology, phenotype and growth potential compared to MSCs from healthy donors. Importantly, immunomodulatory capacity was intact, as Crohn9s disease MSCs significantly reduced peripheral blood mononuclear cell proliferation in vitro. MSC infusion was without side effects, besides a mild allergic reaction probably due to the cryopreservant DMSO in one patient. Baseline median CDAI was 326 (224–378). Three patients showed clinical response (CDAI decrease ≥70 from baseline) 6 weeks post-treatment; conversely three patients required surgery due to disease worsening. Conclusions Administration of autologous bone marrow derived MSCs appears safe and feasible in the treatment of refractory Crohn9s disease. No serious adverse events were detected during bone marrow harvesting and administration.

Background: Regulatory macrophages play an important role in wound healing and gut homeostasis and have antiinflammatory properties. Induction of this cell type (Mψind) by the anti-tumor necrosis factor (TNF) antibodies, infliximab and adalimumab, has recently been shown in vitro. Also, the superiority of infliximab/azathioprine combination therapy over infliximab or azathioprine monotherapy has recently been established, but the mechanism behind this remains unclear. The aim of this study was to examine the induction of regulatory macrophages in patients with and without mucosal healing in response to infliximab. In addition, we studied the effect of infliximab/azathioprine combination treatment on the differentiation and function of regulatory macrophages. Methods: Inflammatory bowel disease (IBD) patients (n = 10) underwent endoscopy before and after first infliximab treatment. Immunohistochemical staining of CD68 and CD206 was performed in all patients. Mixed lymphocyte reactions (MLRs) were treated with infliximab, azathioprine, or both. Macrophage phenotype was evaluated by flow cytometry and inhibition of T-cell proliferation was measured in a secondary MLR containing macrophages and third-party lymphocytes. Results: A significant induction of regulatory macrophages was observed in patients with mucosal healing after treatment with infliximab; this induction was absent in patients without mucosal healing. In addition, Mψind have the ability to induce wound healing in an in vitro model, further suggesting a key role for infliximab-induced macrophages in mucosal healing. Upon infliximab/azathioprine combination treatment, an increased number of regulatory macrophages was observed. These macrophages also displayed stronger immunosuppressive properties than macrophages induced by infliximab monotherapy. Conclusions: These data show that regulatory macrophages may be involved in mucosal healing and provide a rationale for the superiority of infliximab/azathioprine combination treatment observed in the clinic. (Inflamm Bowel Dis 2012;)

During recent years, the treatment for patients with Crohn’s disease (CD) has changed substantially. With the introduction of anti-TNF alpha antibodies, the treatment goals have been more ambitious, for instance, with the introduction of mucosal healing. Today, upon diagnosing CD, clinicians are able to use predictors of a complicated disease course and act accordingly when designing an individual medical strategy.

OP03 Induction of autophagy upon DC-T cell contact is mediated by LKB1 AMPK mTOR signalling M.E. Wildenberg1 *, A.C. Vos2, M. Duijvestein2, A. Verhaar3, G. van den Brink4, D.W. Hommes5. 1Academic Medical Center, Tytgat Institute for Intestinal and Liver Research, Amsterdam, Netherlands, 2Leiden University Medical Center, Gastroenterology-Hepatalogy, Leiden, Netherlands, 3Leiden University Medical Center, Leiden, Netherlands, 4Academic Medical Center, Gastroenterology & Hepatology, Amsterdam, Netherlands, 5UCLA, Division of Digestive Diseases, Los Angeles, United States

primed, but not IL8 stimulated PMN from CD patients was observed. Conclusions: Although initiation of apoptosis in CD PMN is normal as determined by Annexin V staining, an enhanced survival signal provided by GM-CSF-induced PKB activation may lead to a decreased cleaving of caspases, thereby rescuing PMN from the apoptotic pathway. In toto, our results suggest an enhanced GM-CSF-mediated PMN survival, which may play a role in the chronic intestinal inflammation in CD.

stiffness-mediated induction of aSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression. In addition, CARD-024 partially stimulated members of the COX2/IL-1b inflammatory pathway. Conclusions: In summary, CARD-024 attenuated the pro-fibrotic response of colonic myofibroblasts to either TGFb stimulation or high matrix stiffness, suggesting that vitamin D analogs such as CARD-024 may ameliorate intestinal fibrosis.

food resuspended in physiological salt solution. Foods scoring positive in skin tests were tested. A drop was placed on the lower lip and left for 10 seconds to 2 minutes. The result was read 15 minutes late. Positive reactions in LFT can be divided into five types: (1) smoothing of the lower lip; (2) erythema under the lip; (3) contiguous rash of the cheek and chin; (4) edema of the cheek associated with rhinitis and watering eyes; and (5) systemic reaction associated with itching of the areas affected by eczema and coughing. Results: A total of 406 LFT were performed, 314 of which were positive and 92 negative, followed by positive food challenges. The foods provoking reactions were egg white (140 cases), peanut (80 cases), cow’s milk (79 cases), cod (8 cases), kiwi (4 cases), shrimp (3 cases). The LFT positivity was mostly (88.7% of cases) manifested as a labial edema with contiguous urticaria. There were systemic reactions in 4.6% of cases: generalized urticaria, hoarnseness and rapid-onset and generalized eczema. The 92 infants with negative labial food test results had positive food challenges. The reactions were in 66 cases generalized urticaria, 20 cases asthma attacks, 2 cases generalized eczema, and in 2 cases general anaphylaxis shock. The sensitivity of LFT was 78%. Conclusions: The LFT was easy to perform with children. Positive results indicate the presence of food allergy, but negative results require further investigations preferably double-blind, placebo-controlled food challenge. Further comparative analyses of the LFT and oral food challenge will better determine the value of the LFT for the exploration of cases of food allergy.