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Asthma is a complex disease involving gene and environment interactions (Holgate). Although atopy is a strong predisposing Rationale risk factor for asthma, local tissue susceptibilities also contribute to disease expression (Moffitt). The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction complexes (Swindle et al, 2009). To explain the link between environment exposures and airway vulnerability, our previous studies based on a small number of volunteers have shown that tight junctions are abnormal in asthma. To confirm the previous findings and strengthen the concept of defective epithelial barrier function in asthma, we studied a much bigger population of asthmatics to assess their epithelial barrier integrity and permeability.Non-asthmatic normal and asthmatic subjects were recruited and clinically characterised in accordance with GINA guidelines. Methods:Bronchial brushings were obtained by fibreoptic bronchoscopy following ethical approval and informed consent. Primary bronchial epithelial cells (pBECs) were expanded from brushings then seeded onto collagen I coated porous transwells and differentiated at in vitro an air-liquid interface (ALI) for 3-4 weeks. Transepithelial electrical resistance (TER) was monitored as an indication of tight junction formation and epithelial integrity using a set of chopstick electrodes and a voltohmmeter. Paracellular permeability was quantified by FITC-dextran flux assay.We studied differentiated bronchial epithelial cultures (n=83) grown from bronchial brushings obtained from normal (40) Results:in vitro or asthmatic (n=43) volunteers. Tight junction function assessed by TER measurement was significantly lower in cultures from asthmatic donors compared to normals (median (interquartile range) = 346 (248-536) Ω·cm 428 (340-593) -analysis of the data revealed that 2 versus TER was significantly decreased as a function of disease severity (p<0.05). TER measurements were inversely correlated with permeability to fluorescently conjugated 20 or 4 kDa dextrans indicating alterations to both ionic and macromolecular permeability in asthma.Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of low Conclusions: molecular weight allergens and other agents into the airway tissue leading to immune activation and may thus contribute to the end organ expression of asthma. This work suggests that addressing the barrier defect in asthma may offer a novel therapeutic approach for difficult-to-treat asthmatic subjects who fail to respond to conventional therapy. This abstract is funded by:

Summary The chimaeric anti‐CD20 antibody rituximab (Rituxan) sensitises lymphoma cells to small molecule cytotoxic drugs and to protein toxins. We have explored the augmentive effect of rituximab on the anti‐CD19 immunotoxin BU12‐SAPORIN in a model of human lymphoma. Intact rituximab and its F(ab)2 derivative both augmented the immunospecific protein synthesis inhibitory effects of BU12‐SAPORIN in a complement‐independent manner. A combination of rituximab + BU12‐SAPORIN completely abolished the proliferation of Ramos cells in vitro and also induced a significantly greater degree of apoptosis in these cells. Treatment with rituximab, BU12‐SAPORIN or a combination of both induced poly(ADPribose) polymerase and caspase 3 cleavage, although this was always consistently greater in combination‐treated cells. zVAD almost completely inhibited apoptosis in rituximab‐ or BU12‐SAPORIN‐treated cells but only partially in combination‐treated cells. In severe combined immunodeficient (SCID)‐Ramos mice the combination of rituximab + BU12‐SAPORIN was significantly better therapeutically than either single agent. The immunological fidelity of the therapeutic effect because of combination treatment was demonstrated through the failure of rituximab to augment an irrelevant anti‐CD7 immunotoxin. The therapeutic efficacy of rituximab and combination treatment was reduced in SCID‐Ramos mice depleted of serum complement while natural killer cell depletion failed to show any convincing role for antibody‐dependent cellular cytotoxicity. This study shows a clear therapeutic advantage from using rituximab to immunospecifically augment immunotoxin cytotoxicity warranting further investigation.