Sara Nullens

Basophilic histamine content and release during venom immunotherapy: insights by flow cytometry.

Despite the efficiency of venom immunotherapy, the effects on basophils and mast cells remain incompletely understood and probably vary according to the treatment phase.

STAT5 in human basophils: IL-3 is required for its FcεRI-mediated phosphorylation†

Basophils are key effector cells in allergic inflammatory reactions. However, the mechanisms of FcεRI‐induced degranulation are complex and remain to be disentangled. Signal transducer and activator of transcription (STAT) molecules modulate various cell functions. STAT5 appears to be essential for IgE‐mediated mast cell function, but its role in human basophils after cross‐linking FcεRI is unknown. In this study, STAT5 phosphorylation was investigated by flow cytometry, and combined with analyses of the degranulation marker CD63 at single cell level. Kinetics of STAT5 phosphorylation were studied in basophils of birch pollen allergic patients and showed a fast phosphorylation induced by interleukin (IL)‐3, but not with antigen alone. Stimulating basophils with a mixture of allergen and IL‐3 resulted in a two to three fold higher phosphorylation of STAT5 than induced by IL‐3 alone. In the presence of IL‐3, antigen elicited a dose‐dependent STAT5 response. In conclusion, this study demonstrates that STAT5 in human basophils is activated through both the IL‐3 and the FcεRI signaling pathway.

EFFECT OF GTS-21, AN ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR AGONIST, ON CLP-INDUCED INFLAMMATORY, GASTROINTESTINAL MOTILITY, AND COLONIC PERMEABILITY CHANGES IN MICE.

Background: During abdominal sepsis, the inhibition of gastrointestinal (GI) motility together with mucosal barrier dysfunction will lead to increased bacterial translocation and maintenance of sepsis. The activation of the vagal anti-inflammatory pathway remains an appealing therapeutic strategy in sepsis. In this respect, selective alpha7 nicotinic acetylcholine receptor (&agr;7nAChR) agonists have shown anti-inflammatory properties in several animal models of inflammation. Methods: Sepsis was induced in OF-1 mice by cecal ligation and puncture (CLP). GI transit was quantified, and cytokine levels were determined in serum and colon. Colonic permeability was assessed by means of Evans blue injection. We studied the effect of GTS-21, an &agr;7nAChR agonist, on the aforementioned parameters. Splenectomized animals as well as &agr;7nAChR-knock-out animals (Chrna7−/−) were included to study the role of splenic macrophages and the &agr;7nAChR during polymicrobial abdominal sepsis. Results: In septic animals, GTS-21 significantly ameliorated GI motility, lowered systemic and colonic levels of IL-6, decreased colonic permeability, and decreased the number of positive cultures obtained from blood and mesenteric lymph nodes. Splenectomy prevented animals from developing sepsis-induced ileus. Chrna7−/− mice displayed a more severe septic phenotype, whereas GTS-21 remarkably was also beneficial in these animals. Conclusion: Our results show that peripheral targeting of the vagal anti-inflammatory pathway proves beneficial in an animal model of polymicrobial abdominal sepsis. A major role is allocated to splenic immune cells in the development of sepsis, as preventive splenectomy was protective for the development of sepsis. Data on the Chrna7−/− mice suggest that the beneficial effects mediated by GTS-21 on inflammation and motility might be related to activation of other receptors besides the &agr;7nAChR.

Treatment with egg antigens of Schistosoma mansoni ameliorates experimental colitis in mice through a colonic T-cell-dependent mechanism

Background:Helminth-derived molecules are being identified as a new therapeutic approach for immune-mediated diseases. We investigated the anti-inflammatory effect and the immunological mechanisms of Schistosoma mansoni soluble egg antigens (SmSEA) in a mouse model of chronic colitis. Methods:Colitis was induced in immunocompromised severe combined immunodeficiency mice by the adoptive transfer of CD4+CD25−CD62L+ T cells. Two weeks post-transfer, SmSEA treatments were started (study 1: 1 × 20 &mgr;g SmSEA per week 5 times; study 2: 2 × 20 &mgr;g SmSEA per week 3 times). From the start of the treatment (week 2), the clinical outcome and colonic inflammation were assessed at different time points by a clinical disease score and colonoscopy, respectively. At the end of the studies, the colons were harvested for macroscopic examination, and colonic lamina propria mononuclear cells were isolated for flow cytometric T-cell characterization. Results:In both studies, administration of SmSEA in colitis mice improved all the inflammatory parameters studied. However in study 1, this beneficial effect on inflammation diminished with time, and the T-cell characterization of the lamina propria mononuclear cells, performed at week 6, revealed no immunological effects of the SmSEA treatment. In study 2, mice were killed earlier (week 4) and at that time point, we found a significant downregulation of the number of interleukin-17A–producing T cells and a significant upregulation of the number of interleukin-4–producing T cells in the colon of the SmSEA-treated colitis mice. Conclusions:Our results demonstrated that the administration of SmSEA reduces the severity of colitis in the adoptive transfer mouse model characterized by an increased Th2 response and a suppressed Th17 response in the colon.

Role of tachykinin receptors in the modulation of colonic peristaltic activity in mice.

Tachykinins are important mediators of neuroneuronal and neuromuscular transmission in the gastrointestinal tract, however their contribution to colonic peristalsis in mice remains unclear. Therefore, our aim was to characterise the functional role of tachykinins in mediating peristalsis by evaluating the effect of selective tachykinin NK(1), NK(2) and NK(3) receptor agonists and antagonists on in vitro colonic peristaltic activity in mice. Using a modified Trendelenburg set-up, gradual distension of proximal and distal colonic segments evoked rhythmic, aborally migrating contractions. Peristaltic activity was assessed by quantifying the amplitude and interval of the corresponding pressure waves. Stimulation of NK(1) receptors showed regional differences as both the pressure amplitude and interval were enhanced in the distal colon without affecting peristalsis proximally. Blockade of NK(1) receptors reduced the peristaltic pressure amplitude in the proximal and distal colon while the interval was not significantly altered. NK(2) receptor stimulation resulted in a modest enhancement of the amplitude in proximal and distal segments and a slightly prolonged interval distally. Blockade of NK(2) receptors reduced the peristaltic pressure amplitude and interval in the distal colon. NK(3) receptor stimulation significantly augmented the amplitude in both segments and prolonged the interval distally. However, NK(3) receptor blockade had no effect on peristaltic activity. In conclusion, tachykinins contribute to colonic peristalsis in mice by acting mainly on NK(1) and NK(2) receptors and their effects show a proximal-to-distal gradient. NK(3) receptors might play a role in conditions of excess tachykinin release but appear not to be involved under the conditions of the present study.

Beneficial Effects of Anti-Interleukin-6 Antibodies on Impaired Gastrointestinal Motility, Inflammation and Increased Colonic Permeability in a Murine Model of Sepsis Are Most Pronounced When Administered in a Preventive Setup

Background and Objectives During sepsis, gastrointestinal ileus, mucosal barrier dysfunction and bacterial translocation are accepted to be important triggers that can maintain or exacerbate the septic state. In the caecal ligation and puncture animal model of sepsis, we demonstrated that systemic and colonic interleukin-6 levels are significantly increased coinciding with an impaired colonic barrier function. We therefore aimed to study the effect of therapeutic or curative administration of anti-IL6 antibodies on overall GI motility, colonic permeability and translocation of intestinal bacteria in blood and mesenteric lymph nodes in the mouse caecal ligation and puncture model. Methods OF-1 mice were randomized to either the preventive or curative protocol, in which they received 1 mg/kg of antibodies to interleukin-6, or its IgG isotype control solution. They subsequently underwent either the caecal ligation and puncture procedure, or sham-surgery. GI motility was assessed 48h following the procedure, as well as colonic permeability, serum and colon cytokines, colonic tight junction proteins at the mRNA level; cultures of blood and mesenteric lymph nodes were performed. Results Preventive administration of anti-interleukin-6 antibodies successfully counteracted the gastrointestinal motility disturbances and impaired colonic barrier function that could be observed in vehicle-treated septic animals. Serum and colonic levels of proinflammatory cytokines were significantly lower when animals were preventively treated with anti-interleukin-6 antibodies. A repetitive injection 24h later resulted in the most pronounced effects. Curative treatment significantly lowered systemic and colonic inflammation markers while the effects on transit and permeability were unfortunately no longer significant. Conclusions Caecal ligation and puncture resulted in septic ileus with an increased colonic permeability. Antibodies to interleukin-6 were able to ameliorate gastro-intestinal motility, suppress inflammation and normalize the permeability of the colonic wall, with the preventive administration combined with a repeat injection being far more efficacious than the sole preventive or curative one.

Mo1703 Schistosoma Mansoni Helminth Egg Antigens (SEA) and Their Protective Effect on Colitis in a Murine Transfer Model

DSS in drinking water in WT mice for 5 days. After this time, DSS was replaced with regular water and treated intrarectally with or without IBD98E for 6 days. Body weight, disease activity index were monitored daily, while endoscopic analysis was performed at day 0, 5, 9 and 11. At the sacrifice, a histological analysis was evaluated. Caco-2 cell line was used to test in vitro the effects of IBD98E. The cell viability and proliferation after treatment with of IBD98E were evaluated respectively by XTT test and BrdU incorporation. Wound scratch was made in the monolayers of cells incubated for 24h with epidermal growth factor (EGF) 100 ng/ml or IBD98E with or without mitomycin C treatment to inhibit cell proliferation. Photographs of the wounded area were taken at time 0 and after 24 h of the stimulation, and the percentage wound closure was calculated. Results. Colitic mice treated with IBD98E showed faster clinical recovery, significant reduced endoscopic signs of mucosal inflammation (p<0,01) with mild bleeding after 4 and 6 days of the treatment compared to those receiving saline. Furthermore, the histological analysis revealed a minor ulceration score in mice treated with IBD98E. In vitro, IBD98E treatment enhanced cell viability promoting proliferation. Indeed, a significant increased of BrdU incorporation (p<0,05) as well as a wound closure (p<0,05) was found after 24 h in the cells stimulated with IBD98E compared to untreated cells, while no significant differences were observed between IBD98E and EGF. Interestingly, no wound closure was found in response to IBD98E after pre-treatment with mitomycin C, indicating that IBD98E promotes proliferation but does not affect the migration of epithelial cells. Conclusions. Overall these data demonstrate that sodium hyaluronate gel application is safe and favors the clinical and endoscopic outcomes of colitis reducing mucosal bleeding and promoting mucosal wound healing through proliferation of epithelial cells. Therefore IBD98E could represent a new potential treatment to promote mucosal repair in UC patients.

Identifying Therapeutic Targets for Sepsis Research: A Characterization Study of the Inflammatory Players in the Cecal Ligation and Puncture Model

During sepsis, disturbed gastrointestinal motility and increased mucosal permeability can aggravate sepsis due to the increased risk of bacterial translocation. To help identify new therapeutic targets, there is a need for animal models that mimic the immunological changes in the gastrointestinal tract as observed during human sepsis. We therefore characterized in detail the gastrointestinal neuroimmune environment in the cecal ligation and puncture (CLP) model, which is the gold standard animal model of microbial sepsis. Mice were sacrificed at day 2 and day 7, during which gastrointestinal motility was assessed and cytokines were measured in the serum and the colon. In the spleen, lymph nodes, ileum, and colon, subsets of leukocyte populations were identified by flow cytometry. Septic animals displayed an impaired gastrointestinal motility at day 2 and day 7. Two days post-CLP, increased serum and colonic levels of proinflammatory cytokines were measured. Flow cytometry revealed an influx of neutrophils in the colon and ileum, increased numbers of macrophages in the spleen and mesenteric lymph nodes, and an enhanced number of mast cells in all tissues. At day 7 post-CLP, lymphocyte depletion was observed in all tissues coinciding with increased IL-10 and TGF-β levels, as well as increased colonic levels of IL-17A and IFN-γ. Thus, CLP-induced sepsis in mice results in simultaneous activation of pro- and anti-inflammatory players at day 2 and day 7 in different tissues, mimicking human sepsis.