Annick de Vries

Crucial role of transient receptor potential ankyrin 1 and mast cells in induction of nonallergic airway hyperreactivity in mice.

RATIONALE Airway hyperreactivity (AHR) is a key feature of bronchial asthma, and inhalation of irritants may facilitate development of nonallergic AHR. Swimmers exposed to hypochlorite (ClO(-))-containing water show a higher risk of developing AHR. We developed a mouse model in which instillation of ClO(-) before ovalbumin (OVA) induces AHR without bronchial inflammatory cells. OBJECTIVES To investigate the mechanisms of ClO(-)-OVA-induced nonallergic AHR. METHODS The involvement of the transient receptor potential ankyrin (TRPA)1 channel was checked in vivo by the use of TRPA1(-/-) mice and in vitro by Ca(2+) imaging experiments. The role of substance P (SP) was investigated by pretreating animals with the receptor antagonist RP67580, by replacing ClO(-) with SP in vivo, and by immunofluorescent staining of large airways of exposed mice. The role of mast cells was evaluated by exposing mast cell-deficient Kit(Wh)/Kit(Wsh) mice to ClO(-)-OVA with or without mast cell reconstitution. MEASUREMENTS AND MAIN RESULTS ClO(-)-OVA did not induce AHR in TRPA1(-/-) mice, and ClO(-) generates a Ca(2+) influx in TRPA1-transfected cells. Pretreatment with RP67580 reduces ClO(-)-OVA-induced AHR, although no increased SP expression was shown in the airways. SP-OVA exposure resulted in the same AHR as induced by ClO(-)-OVA. Kit(Wsh)/Kit(Wsh) mice did not develop AHR in response to ClO(-)-OVA unless they were reconstituted with bone marrow-derived mast cells. CONCLUSIONS Induction of AHR by exposure to ClO(-)-OVA depends on a neuroimmune interaction that involves TRPA1-dependent stimulation of sensory neurons and mast cell activation.

The role of sensory nerve endings in nerve growth factor-induced airway hyperresponsiveness to histamine in guinea-pigs

Nerve growth factor induces an airway hyperresponsiveness in vivo in guinea‐pigs, as we have shown previously. Since antagonizing the neurokinin‐1 (NK1) receptor can prevent this NGF‐induced airway hyperresponsiveness and since sensory nerves release tachykinins, we investigated the role of sensory nerves in the NGF‐induced airway hyperresponsiveness. We used isolated tracheal rings from guinea‐pigs to measure tracheal contractility. In these rings sensory nerve endings are present, but these endings lack any contact with their cell bodies. In this in vitro system, NGF dose‐dependently induced a tracheal hyperresponsiveness to histamine. The NK1 receptor antagonist SR140333 could block the induction of tracheal hyperresponsiveness. To further investigate the involvement of sensory nerve endings we used the cannabinoid receptor 1 (CB1) agonist R‐methanandamide to inhibit excitatory events at the nerve terminal. The CB1 receptor agonist was capable of blocking the tracheal hyperresponsiveness to NGF in the isolated system, as well as the airway hyperresponsiveness to NGF in vivo. This indicates that NGF can induce an increase in airway responsiveness in the absence of sensory nerve cell bodies. NGF may act by increasing substance P release from sensory nerve endings, without upregulation of substance P in the neurons. Substance P in its turn is responsible for the induction of the NGF‐induced airway hyperresponsiveness.

The majority of natalizumab-treated MS patients have high natalizumab concentrations at time of re-dosing:

Background: Natalizumab is efficacious in the treatment of relapsing-remitting multiple sclerosis. All patients receive the same treatment regimen of 300 mg every 4 weeks, despite differences in pharmacokinetics between individual patients. Objective: To give neurologists insight into natalizumab concentrations at time of re-dosing, we investigated longitudinal natalizumab concentrations in 80 patients in relation to disease activity, with possible influencing factors. Methods: In a prospective observational cohort study, natalizumab trough serum concentrations were measured in 80 patients. Data on demographics, duration of treatment, Expanded Disability Status Scale, clinical exacerbations, brain magnetic resonance imaging (MRI), and body weight were collected. Results: We measured high (≥10 µg/mL) natalizumab trough concentrations in 94% of patients. Intra-individual concentrations were stable. The spread in concentrations was substantial and did not correlate with disease activity. We found a negative association between natalizumab concentration and body weight (β = −0.30, p = 0.010). Interpretation: The majority of patients showed high natalizumab serum concentrations at time of re-dosing. Alternative treatment regimens could lead to more efficient use of natalizumab, but caution is warranted regarding the possibility of recurrence of disease activity. Prospective clinical trials are needed to establish the safety of extended dose intervals in natalizumab treatment.

Tu1730 Novel Transgenic Mast Cell-Deficient Mouse Model Reveals No Role for Mast Cells in the Pathogenesis of Postoperative Ileus

Background & Aims: The neuropeptide NT and its receptor NTR1 mediate intestinal inflammation and their expression is increased in IBD. Colitis and associated hypoxia through the activation of the transcription factor hypoxia-inducible factor HIF-1modulates the expression of miR-210. NT promotes the transcriptional activity of HIF-1α, an event associated with increased intestinal angiogenesis (DDW2013: #1738), and induces the expression of miR210 (Gastroenterology, 2011; 141:1749). Here we examined the implication of HIF-1α in miR-210 regulation by NT, and assessed the role of miR-210 in the development of colitis and intestinal angiogenesis. Methods: NCM460 cells overexpressing NTR1 (NCM460-NTR1) were exposed to NT (0.1 μM, 6 h), +/the HIF-1α inhibitor PX-478 (40 μM, 18 h); miR-210 expression was evaluated by qPCR. Acute colonic inflammation was induced by intracolonic administration of TNBS (5 mg/kg, 48 h) in wild type (WT) and NTR1 knockout (KO) mice. Colonic miR-210 was inhibited by intracolonic administration of locked nucleic acid anti-miR-210 (LNA-anti-miR-210). The degree of inflammation was evaluated on distal colon segments stained with H&E and angiogenesis was evaluated by von-Willebrand (vWB) staining. Results: NT treatment of NCM460-NTR1 cells induced miR-210 expression (p<0.006) that was attenuated (by ~65%, p<0.005) by treatment with the HIF-1α inhibitor, PX-478. TNBS treatment increased colonic miR-210 expression in WT mice, an effect suppressed in NTR1 KO mice (p=0.02, n=6/group). Moreover, miR-210 levels were elevated in tissue samples from patients with Ulcerative Colitis (UC) compared to controls (p=0.003, n=11/group). Intracolonic administration of LNA-anti-miR-210 in mice reduced endogenous colonic miR-210 levels by ~55% (p=0.01), without affecting expression of other miRs. LNAanti-miR-210 pre-treated colons (n=8/group) exhibited reduced levels of IL-6, Cxcl1, and TNFα (p<0.05 for all), diminished mucosal integrity score (p=0.0001), neutrophil infiltration (p=0.0004), total colitis score (p=0.0001), and vWB staining (p=0.008) compared to colons treated with TNBS alone. Conclusions: Our results demonstrate the importance of NT/miR210 axis in the pathophysiology of colitis and IBD. The successful intracolonic microRNA silencing indicates the utility of microRNA inhibitors as a novel therapeutic approach towards IBD. Supported by NIH grant DK60729 (CP), the Crohns and Colitis Foundation of America (KB) and the Blinder Research Foundation for Crohns Disease (IKML).