ASTHMA RESEARCH LAB NOTES
Blue = goblet cells
Yellow= region of airway lumen
Green= region airway inflammation
Purple= region of airway smooth muscle
Blue= smooth muscle
Green= intracellular eosinophils
Induction of Asthma
Acute exacerbations of asthma – pro inflammatory mechanisms involved in acute exacerbations of asthma (mediators)
· Induction phase of asthma involves IL-33 – ILC2
· Exacerbation of asthma IL-33
· Inter leukin 33 drives cytokine production responsible for producing IL33 in induction phase of asthma, involved in the development of exacerbations (features- inflammation, exaggerated, allergen or exposure-sever acute inflammation response, broncho-constriction reduces size of lumen) asthma
· Interleukin 33 induction of asthma and environment pollution- industrial activity and dust particulate pollutants
Can exposure to particulate pollutants drive exacerbations of asthma?
· Cell model biopsy tissue knock out mice produce IL33 test for IgE production
· Testing patient in country and city setting, noticing specific cytokine sin blood IL33 and ELIZA for production
· Animal model – mice & rate have IgE antibody or guinea pigs
· Ambient particulate pollutants - traffic related particulate pollutants
Modelling asthma exacerbations in mice study
· Mice are systematically sensitised to ovalbumin
· Inhale challenge with ovalbumin aerosol induces features of chronic asthma (4 weeks)
· Particulate pollutants administered intranasally to induce acute exacerbations
· Groups include; naïve, chronic challenged alone, chronic challenged alone + particulate pollutants, chronic challenge+ particulate pollutants + IL-33 neutralising antibody
· Airway inflammation is assessed through histology, TNF-alpha in bronchoalveolar lavage fluid assessed through ELISA
· Eosinophils were not increased in animals that received chronic challenge alone; they were increased in animals that received chronic ovalbumin following particulate pollutants. However neutralisation of IL-33 suppressed accumulation of eosinophils.
· ELIZA; utilises antibodies to identify specific protein in a liquid sample, an enzyme bound to antibody generates a coloured product and so protein concentration determined through a comparison of colour to a standard curve
· Useful fro measuring the concentration of a protein in liquid samples
· Capture antibody with sticks to plate in well, sample include mixture of protein, target protein captured, secondary antibody against target protein- biotin-detection reagent,
· Determining cellular source of a protein in a issue sample = Immunohistochemistry
· Characterising protein interactions= Pull down assay
· Identify protein by molecular weight = western blot
ELIZA measurement of TNF- alpha (analysis of data for TNF-alpha, graph of results)
QUESTIONS TO CONSIDER FOR REPORT
Can the same antibody clone be used for the capture antibody and the detection antibody?
Yes, the same antibody clone can be used
What factors determine how quickly the colour develops?
Based on these results, can exposure to environmental particulate pollutants promote exacerbations of asthma?
Yes, it can contribute to exacerbation this is seen in the data, lots of things may cause it, especially particulate may contribute
What is the role of IL-33 in this process?
IL-33 experimental groups neutralised- tumour necrosis T-alpha- anti IL-33, its reduced so take out 33 pr cytokine TNF-alpha reduce and so is inflammation
What are some of the limitations associated with this experiment?
Additional controls could be used, normal animals exposed to particulates, right controls
Do you think that antibody neutralisation of IL-33 might be a useful treatment for acute exacerbations of asthma?
MAYBE – experiment wasn’t designed to answer this question but could be a further experiment developed in the future
· Do these data suggest that environmental particulate pollutants can trigger exacerbations of asthma?
· What do these results suggest about the role of IL-33 in the induction of an acute exacerbation of asthma?
· Do you think that antibody neutralisation of IL-33 might be a useful treatment for acute exacerbations of asthma in patients?
· Are there any additional control groups that would improve the validity of these data?
· additional marks - determine whether the differences between the groups are statistically significant.
Concentration of Groups
Chronic challenge + pollution
Chronic Challenge + pollution + anti-IL-3 Ab
Concentration of TNF-a (pg/ml)
Standard curve for absorbance of TNF-a
Absorbtion @ 650nm