June Giddings

Essential role for the p110δ phosphoinositide 3-kinase in the allergic response

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcɛRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110δ isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen–IgE-induced degranulation and cytokine release. Inactivation of p110δ protects mice against anaphylactic allergic responses. These results identify p110δ as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

Pulmonary Bioavailability of a Phosphorothioate Oligonucleotide (CGP 64128A): Comparison with Other Delivery Routes

AbstractPurpose. Phosphorothioate antisense oligodeoxynucleotides are promising therapeutic candidates. When given systemically in clinical trials they are administered via slow intravenous infusion to avoid their putative plasma concentration-dependent haemodynamic side-effects. In this study, we have evaluated alternative parenteral and non-parenteral administration routes which have the potential to enhance the therapeutic and commercial potential of these agents. Methods. The delivery of CGP 64128A by intravenous, subcutaneous, intra-peritoneal, oral and intra-tracheal (pulmonary) routes was investigated in rats using radiolabelled compound and supported by more specific capillary gel electrophoretic analyses. Results. Intravenously administered CGP 64128A exhibited the rapid blood clearance and distinctive tissue distribution which are typical for phosphorothioate oligodeoxynucleotides. Subcutaneous and intra-peritoneal administration resulted in significant bioavailabilities (30.9% and 28.1% over 360 min, respectively) and reduced peak plasma levels when compared with intravenous dosing. Administration via the gastrointestinal tract gave negligible bioavailability (<2%). Intra-tracheal administration resulted in significant but dose-dependent bioavailabilities of 3.2, 16.5 and 39.8% at 0.06, 0.6 and 6.0 mg/kg, respectively. Conclusions. Significant bioavailabilities of CGP 64128A were achieved following subcutaneous, intra-peritoneal and intra-tracheal administration. Pulmonary delivery represents a promising mode of non-parenteral dosing for antisense oligonucleotides. The dose-dependent increase in pulmonary bioavailability suggests that low doses may be retained in the lungs for local effects whereas higher doses may be suitable for the treatment of a broader spectrum of systemic diseases.

Goblet Cells Are Derived from a FOXJ1-Expressing Progenitor in a Human Airway Epithelium

The overproduction of mucus is a key pathology associated with respiratory diseases, such as asthma and chronic obstructive pulmonary disease. These conditions are characterized by an increase in the number of mucus-producing goblet cells in the airways. We have studied the cellular origins of goblet cells using primary human bronchial epithelial cells (HBECs), which can be differentiated to form a stratified epithelium containing ciliated, basal and goblet cells. Treatment of differentiated HBEC cultures with the cytokine IL-13, an important mediator in asthma, increased the numbers of goblet cells and decreased the numbers of ciliated cells. To determine whether ciliated cells act as goblet cell progenitors, ciliated cells in HBEC cultures were hereditably labeled with enhanced green fluorescent protein (EGFP) using two lentiviral vectors, one which contained Cre recombinase under the control of a FOXJ1 promoter and a second Cytomegalovirus (CMV)-floxed-EGFP construct. The fate of the EGFP-labeled ciliated cells was tracked in HBEC cultures. Treatment with IL-13 reduced the numbers of EGFP-labeled ciliated cells compared with untreated cultures. In contrast, IL-13 treatment significantly increased the numbers of EGFP-labeled goblet cells. This study demonstrates that goblet cells formed in response to IL-13 treatment are in part or wholly derived from progenitors that express the ciliated cell marker, FOXJ1.

Regulation of Neuregulin 1β1–Induced MUC5AC and MUC5B Expression in Human Airway Epithelium

Excessive mucus production has been linked to many of the pathologic features of respiratory diseases, including obstruction of the airways, decline in lung function, increased rates of mortality, and increased infections. The mucins, MUC5AC and MUC5B, contribute to the viscoelastic properties of mucus, and are found at elevated levels in the airways of individuals with chronic respiratory diseases. The T helper type 2 cell cytokine, IL-13, is known to regulate MUC5AC expression in goblet cells of the airways, although much less is known about the regulation of MUC5B expression. In a study to further understand the mediators of MUC5AC and MUC5B expression, neuregulin (NRG) 1beta1 was identified as novel regulator of goblet cell formation in primary cultures of human bronchial epithelial cells (HBECs). NRG1beta1 increased expression of MUCAC and MUC5B proteins in a time- and dose-dependent fashion in HBEC cultures. NRG1beta1-induced expression of MU5AC and MUC5B was shown to involve v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB) and ErbB3 receptors, but not ErbB4 receptors. Treatment of HBECs with inhibitors of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, and phosphatidylinositol 3-kinase indicated that these kinases were involved in NRG1beta1-induced MUC5AC and MUC5B expression. Additionally, NRG1beta1 was shown to induce the phosphorylation of the ErbB2 receptor, AKT, and extracellular signal-regulated kinase 1/2. NRG1beta1 protein was found increased in the airways of antigen-challenged mice, together with increases in MUC5AC and MUC5B message. Together, these data indicate that NRG1beta1 is a novel mediator of MUC5AC and MUC5B expression in HBECs, and may represent a novel therapeutic target for mucus hypersecretion in respiratory diseases.

Distribution of a 20-mer phosphorothioate oligonucleotide, CGP69846A (ISIS 5132), into airway leukocytes and epithelial cells following intratracheal delivery to brown-norway rats.

AbstractPurpose. To evaluate the pulmonary distribution of CGP69846A (ISIS 5132), a phosphorothioate oligonucleotide, following intra-tracheal (i.t.) instillation into Brown-Norway rats. Methods. The pharmacokinetic profile of [3H]-CGP69846A was investigated following i.t. instillation into both naïve and inflamed airways of Brown-Norway rats. The cellular distribution was determined using autoradiography, immunohistochemistry and flow cytometry/fluorescence microscopy, in inflamed airways. Results. CGP69846A displayed a dose-dependent lung retention following i.t. administration which was unaffected by local inflammation. Autoradiography and immunohistochemistry showed distribution to alveolar macrophages, eosinophils, bronchial and tracheal epithelium and alveolar cells. Studies with [FITCJ-CGP69846A demonstrated a preferential association of oligonucleotide with leukocytes in bronchial lavage fluid of: macrophages > eosinophils = neutrophils > > lymphocytes. Conclusions. The dose-dependency of lung retention together with cell-specific uptake suggests that the lung can be used as a local target for antisense molecules with potentially minimal systemic effects. Furthermore, the preferential targeting of macrophages and the airway epithelium by oligonucleotides may represent rational cellular targets for antisense therapeutics.