Identification of Autoantigens in EGPA and Severe Eosinophilic Asthma
Identification of Autoantigens in EGPA and Severe Eosinophilic Asthma
In this project the investigators will look for auto-antibodies to relevant proteins both in
 native form and importantly in post-translationally modified forms. Potential modified
 auto-antigens are eosinophil proteins (analogous to the cytoplasmic neutrophil proteins
 identified in vasculitides such as Granulomatosis with Polyangiitis (formerly known as
 Wegener's granulomatosis) and alternatively structural proteins such as collagen V. As well
 as advancing the understanding of asthma pathology, identifying a serum auto-antibody that
 could then be used as a clinical blood test, analogous to anti-cyclic citrullinated peptide
 (CCP) antibodies in rheumatoid arthritis, may revolutionise diagnosis of severe eosinophilic
 asthma and Eosinophilic Granulomatosis with Polyangiitis (EGPA). There is a considerable
 burden of undiagnosed severe eosinophilic asthma in part due to difficulties in definitive
 diagnosis and a diagnostic blood test would help diagnose these patients, allowing them to
 receive necessary treatment.
The investigators will approach the research question with parallel agnostic and targeted
 approaches.

 In the agnostic approach the presence of auto-antibodies in patient serum and sputum to
 inactive and activated eosinophils, with and without post-translational modification, will be
 examined by indirect immunofluorescence.

 In the targeted approach the investigators will examine by enzyme-linked immunoassay (ELISA)
 the presence/absence of antibodies to pre-selected candidate eosinophil and base membrane
 proteins both in native form and post-translationally modified. Proteins to examine will be
 chosen based on literature review (e.g. eosinophil peroxidase and collagen V) and
 eosinophil-specific proteins identified by FANTOM5 (Functional Annotation of the
 Mouse/Mammalian Genome) geneset analysis (FANTOM Consortium et al. 2014).

 Both blood and sputum samples from highly-characterised patients with severe eosinophilic
 asthma and/or EGPA will be examined given the possibility of compartment-specific immune
 responses.

 Once candidate auto-antigens have been identified in the selected group of patients with
 severe eosinophilic asthma and EGPA, the investigators will then examine their prevalence in
 serum samples from a wider selection of patients with eosinophilic airways diseases including
 mild-to-moderate asthma, severe eosinophilic asthma, EGPA, nasal polyposis and eosinophilic
 chronic obstructive pulmonary disease (COPD) as well as healthy controls. Length of disease,
 atopy, presence/absence nasal polyps, gender, age will be examined as co-variates.
 Correlations with highest blood eosinophil counts, requirement for oral corticosteroids and
 presence of other auto-antibodies, e.g. anti-MPO (myeloperoxidase) ANCA (anti-neutrophil
 cytoplasmic antibody), will be examined. In particular the investigators will look for the
 presence of novel autoantibodies in specific patient subsets: i) ANCA negative, ii) ANCA
 positive by immunofluorescence but negative for anti-MPO and anti-PR3 (proteinase-3)
 antibodies, iii) ANA (anti-nuclear antibody) positive but ANCA and extractable nuclear
 antigen (ENA) negative; since patients in all three groups may have novel, as yet
 undetermined autoantibodies. ROC (receiver operator characteristic curve) AUC (area under
 curve) analyses will be conducted to ascertain the predictive value of blood auto-antibodies
 for diagnosis of eosinophilic airways disease.
Inclusion Criteria:

 -  Severe Eosinophilic Asthma (with multi-disciplinary diagnosis as per ERS/ATS Criteria,
 <20 pack year smoking history, blood eosinophils ≥ 0.3 x109/L on inhaled
 corticosteroids); or

 -  EGPA (as per American College of Rheumatology (ACR) Criteria); or

 -  Eosinophilic COPD (post-bronchodilator FEV1/FVC < 70% predicted, absence of
 bronchodilator reversibility, > 20 pack year smoking history, no history of asthma,
 blood eosinophils ≥ 0.3 x109/L); or

 -  Eosinophilic oesophagitis (with diagnostic histology); or

 -  Granulomatosis with Polyangiitis (GPA, formerly called Wegener's) (as per American
 College of Rheumatology (ACR) Criteria)

 Exclusion Criteria:

 -  Known Pregnancy

 -  Anaemia

 -  Hepatitis B Virus, Hepatitis C Virus or HIV infection

 -  Donation of more than 240mls blood in the last sixteen weeks (four months) to any
 other research study or as a donation to the National Blood Transfusion Service

 -  Rituximab, plasmapharesis or polyclonal immunoglobulin infusion (ever)