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P187 Matrix metalloproteinases and their inhibitors in sputum of asthmatics
  1. P Sankaran,
  2. O Jupp,
  3. M Jefferson,
  4. D Sexton,
  5. C Brockwell,
  6. I Clark,
  7. AM Wilson
  1. Univeristy of East Anglia, Norwich, Norfolk

Abstract

Introduction and Objectives Metalloproteinases are implicated in the development of airway remodelling in asthma due to their ability to cleave collagen and elastin with extracellular matrix. We optimised a method to purify messenger Ribonucleic acid (mRNA) sputum samples.

Methods The mRNA expression of a wide range of pertinent Matrix Metalloproteinases (MMP), A Disintegrin And Metalloproteinases (ADAM), A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS) and Tissue Inhibitors of Metalloproteinases (TIMP) was measured from induced sputum with hypertonic saline using Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) in 17 (11 male) non-smoking adults with steroid naive asthma and 12 (6 male) healthy controls. Ten patients with asthma completed open labelled montelukast therapy 10mg per day for 8 weeks. Total mRNA was extracted from the cellular content of the induced sputum plug using a combination of Trizol extraction and Qiagen RNeasy spin columns. To each 0.5ml Trizol extract, 300µl of chloroform was added. The aqueous layer was recovered into a fresh tube and mixed with a half volume of 100% ethanol. Samples were applied to RNeasy Mini spin columns. RT-PCR, using Taqman low-density arrays, was used to determine gene expression.

Results The mean (SD) age and forced expiratory volume in 1 second of asthmatics was 36 (13.4) years and 101.16 (15.47)% predicted respectively and for healthy volunteers was 36 (7.2) years and 92.16 (17.43)% predicted. MMP25 expression was significantly (p = 0.04) higher and MMP15 expression was significantly (p = 0.04) lower is asthmatics compared to healthy volunteers. ADAM28 was significantly (p = 0.03) higher and ADAM17 and ADAMTS15 expression were significantly lower in asthma (p = 0.007 & 0.008 respectively). TIMP2 expression was significantly (p = 0.007) lower in the asthma. There were no significant changes in expression of any of the metalloproteinases or their inhibitors after montelukast therapy.

Conclusion We have studied a wide range of known MMPs, ADAMs, ADAMTSs and inhibitors with a refined technique and successfully increased the yield from induced sputum samples. Significant differences were found between healthy volunteers and asthmatic patients for gene expression of some metalloproteinases/TIMPs. This technique could be used in the future when evaluating gene expression in asthma.

Abstract P187 Table 1.

Changes in m-RNA levels for metalloproteinases and their inhibitors.

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