Identification of Autoantigens in EGPA and Severe Eosinophilic Asthma

Study Overview
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Brief Summary
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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.

Detailed Description
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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.

Official Title
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Identification of Autoantigens and Their Potential Post-translational Modification in EGPA and Severe Eosinophilic Asthma

Conditions
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Churg-Strauss Syndrome, Eosinophilic Asthma, Eosinophilic Esophagitis, Eosinophilic Pneumonia

Intervention / Treatment
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* Diagnostic Test: Autoantibody ELISA


Participation Criteria
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Eligibility Criteria
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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)

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Severe eosinophilic asthma and/or EGPA<br> | Diagnostic Test: Autoantibody ELISA<br>* Serum will be tested for novel autoantibodies against eosinophil proteins by ELISA, such as those previously identified as of importance (e.g. eosinophil-peroxidase, EPX) and also novel candidate proteins specific to eosinophils (including ARFIP1, BCL2A1, PPCDC, SLC12A6 etc) as identified by FANTOM5 geneset analysis.<br>|
| Other respiratory conditions<br>Milder asthma, other vasculitides, eosinophilic COPD, and/or eosinophilic oesophagitis | Diagnostic Test: Autoantibody ELISA<br>* Serum will be tested for novel autoantibodies against eosinophil proteins by ELISA, such as those previously identified as of importance (e.g. eosinophil-peroxidase, EPX) and also novel candidate proteins specific to eosinophils (including ARFIP1, BCL2A1, PPCDC, SLC12A6 etc) as identified by FANTOM5 geneset analysis.<br>|
| Healthy controls<br> | Diagnostic Test: Autoantibody ELISA<br>* Serum will be tested for novel autoantibodies against eosinophil proteins by ELISA, such as those previously identified as of importance (e.g. eosinophil-peroxidase, EPX) and also novel candidate proteins specific to eosinophils (including ARFIP1, BCL2A1, PPCDC, SLC12A6 etc) as identified by FANTOM5 geneset analysis.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Autoantibody positivity | Positive OD by ELISA | Baseline, at study entry |