Osteopontin is expressed and functional in human eosinophils.

BACKGROUND: Eosinophils are critically involved in allergic inflammation and tissue remodeling. Osteopontin (OPN) is a glycoprotein molecule which exhibits pro-fibrogenic and pro-angiogenic properties and has recently also been implicated in allergic diseases. In this study, we investigated the expression and function of OPN in human eosinophils. METHODS: Osteopontin mRNA (RT-PCR) and protein (immunofluorescence) expression in peripheral blood eosinophils from atopic human subjects were evaluated. Soluble OPN release was determined in resting and activated eosinophils. The contribution of OPN to eosinophil-induced angiogenesis was determined using the chick embryo chorio- allantoic membrane (CAM) assay and OPN-induced eosinophil chemotaxis was determined (ChemoTx System microplate wells). Finally, OPN expression in bronchoalveolar lavage (BAL) fluids from mild asthmatic and normal control subjects was determined. RESULTS: Osteopontin is expressed in human eosinophils and is increased following GM-CSF and IL-5 activation. Eosinophil-derived OPN contributes to eosinophil-induced angiogenesis. Recombinant OPN promotes eosinophil chemotaxis in vitro and this effect is mediated by alpha(4)beta(1) integrin binding. Soluble OPN is increased in the bronchoalveolar lavage fluid from mild asthmatic subjects and correlates with eosinophil counts. CONCLUSIONS: We therefore conclude that OPN is likely to contribute to the process of angiogenesis observed in the airways in asthma.

Frmd7 expression in developing mouse brain.

AIMS: Mutations in the FERM domain containing 7 (FRMD7) genes are known to cause a significant number of cases of congenital idiopathic nystagmus (CIN). Only limited expression data exist suggesting low levels of expression in all tissues. In this study, we assess the expression profile of the murine homologue of FRMD7 (Frmd7) in tissue from three murine organs during development. METHODS: cDNA was extracted from heart, lung, and brain tissues of MF-1 mice at 12 developmental time points, embryonic days 11-19, postnatal days 1 and 8, and from adult mice. Relative expression of Frmd7 mRNA was calculated using quantitative real-time PCR techniques with two normalising genes (Gapdh and Actb). RESULTS: Expression of Frmd7 was low in all tissues consistent with earlier reports. In heart and lung tissues, expression remained very low with an increase only in adult samples. In brain tissue, expression levels were higher at all time points with a significant increase at embryonic day 18, with no gender-specific influence on Frmd7 expression. CONCLUSIONS: Frmd7 is expressed at low levels in all tissues studied suggesting a role in many tissue types. However, higher overall expression and a sharp increase at ED18 in the murine brain suggest a different role in this tissue.Earlier studies have shown that genes expressed in the murine brain during development exhibit temporal functional clustering. The temporal pattern of Frmd7 expression found in this study mirrors that of genes involved in synapse formation/function, and genes related to axon growth/guidance. This suggests a role for Frmd7 in these processes and should direct further expression studies.

Chronological expression of Ciliated Bronchial Epithelium 1 during pulmonary development.

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

Local genetic and environmental factors in asthma disease pathogenesis: chronicity and persistence mechanisms.

While asthma is an inflammatory disorder of the airways usually associated with atopy, an important additional component is involvement of the epithelium and underlying mesenchyme acting as a trophic unit (EMTU). In addition to allergens, a wide range of environmental factors interact with the EMTU, such as virus infections, environmental tobacco smoke and pollutants, to initiate tissue damage and aberrant repair responses that are translated into remodelling of the airways. While candidate gene association studies have revealed polymorphic variants that influence asthmatic inflammation, positional cloning of previously unknown genes is identifying a high proportion of novel genes in the EMTU. Dipeptidyl peptidase (DPP) 10 and disintegrin and metalloproteinase (ADAM)33 are newly identified genes strongly associated with asthma that are preferentially expressed in the airway epithelium and underlying mesenchyme, respectively. Also of increasing importance is the recognition that genes associated with asthma and atopy have important interactions with the environment through epigenetic mechanisms that influence their expression. This type of research will not only identify biomarkers of different types of asthma across the full range of phenotypic expression, but will also identify novel therapeutic targets that could influence the natural history of the heterogenes lung disease.

Enhanced upregulation of smooth muscle related transcripts by TGF beta2 in asthmatic (myo) fibroblasts.

BACKGROUND: Transforming growth factor beta (TGF beta) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFbeta(2) favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype. METHODS: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGF beta2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 "housekeeping" genes. Expression of alpha-smooth muscle actin (alpha SMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and gamma-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting. RESULTS: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGF beta2 induced mRNA expression for all five smooth muscle related transcripts; alpha SMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively). CONCLUSIONS: Although TGF beta2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGF beta2 is unable to induce a bona fide smooth muscle cell phenotype, it may "prime" (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.

The role of ADAM33 in the pathogenesis of asthma.

While asthma is a disorder of the conducting airways characterised by Th2-directed inflammation, a second set of mechanisms is being increasingly recognised as fundamental to disease chronicity and severity, for which the term "remodelling" has been used. The cellular and mediator responses underpinning airway remodelling involve aberrant communication between the airway epithelium and underlying mesenchyme, involving the generation of growth factors that lead to proliferation of fibroblasts and smooth muscle and the deposition of matrix proteins to cause airway wall thickening linked to bronchial hyperresponsiveness and fixed airflow obstruction. The identification of ADAM33 on chromosome 20p13 from positional cloning as a novel candidate gene involved in the pathogenesis of these structural and functional changes has opened the way to further insight into these processes that contribute to corticosteroid refractoriness. The preferential expression of ADAM33 in mesenchymal cells and its multiple molecular actions provide ample opportunity for incriminating this molecule in chronic asthma. Its association with progressive asthma and in predicting reduced lung function in young children suggest that ADAM33 has an important role in the natural history and possibly the origins of asthma, a disease unique to humans.