Effect of Mepolizumab on the Phenotype/Proteome/Transcriptome of Eosinophils in Severe Eosinophilic Asthma
试验速览
- 阶段
- 不适用
- 状态
- 招募中
- 入组人数
- 30
- 试验地点
- 5
- 主要终点
- Measure changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques
研究概览
简要总结
Two parts: A:Case-control study including 15 healthy adult donors and 15 severe adult eosinophilic asthmatics selected for treatment with mepolizumab. B: A longitudinal cohort study,where the same patients once on mepolizumab treatment are followed over time (0, 4, 16 and 32 weeks). SCOPE: response to mepolizumab in severe adult eosinophilic asthma.
INCLUSION CRITERIA: Male or female, 18-75 years-old, with severe eosinophilic asthma. EXCLUSION CRITERIA: Smoking history, recent exacerbations, other pulmonary or systemic disease with eosinophilia, malignancy, pregnancy, obesity (BMI >35). OBJECTIVES: General objective: Discovery of predictive/prognostic biomarkers of response to mepolizumab using flow cytometry, transcriptomic, and proteomic technologies. OTHER OBJECTIVES: 1.-To identify changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques. 2.-Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab.3.-Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab.4.-Check whether late-onset severe eosinophilic asthmatics display elevated levels of IGF-1, IGF-BP3, IGF-ALS in serum samples, if the response of mepolizumab depends on the levels of this markers, and if treatment with this biological reduces the concentration in serum of these IGF-family members. 5.-Identify proteins with differential abundance within the deep serum proteome of patients with SEA in response to treatment with mepolizumab by means of non-targeted proteomic analysis.
MEASUREMENTS: Flow cytometry assays with multimarker panels 1 (regulatory), 2 (activation), and 3 eosinophil subsets. Clinical, hematological, biochemical and flow cytometry data generated at times T4, T16 and T32. Total RNA extraction from eosinophil lysates, assay of quality and quantity of RNA, and storage at -80ºC. Evaluation of the levels of 770 human protein-coding mRNAs linked to the recruitment, activation, and effector functions of myeloid cells by means of a direct multiplexed molecular measurement platform named nCounter® NanoString) in combination with a pre-made "nCounter® Human Myeloid Innate Immunity Panel (v2)". Perform retrotranscription and qPCR analyses of those mRNAs in eosinophils displaying the greatest abundance changes in response to mepolizumab treatment according to the nCounter® study. In addition, some additional mRNAs not included in the "nanoString Myeloid Innate Immunity" panel, such as FOXP3 (regulatory function), CRLF2, ST2, or IL-7R (cytokine receptors; activation), will be analysed. HPRT1 gene will be used as a house-keeping gene in this set of RTqPCR experiments. Perform SWATH-MS analysis in samples from 15 healthy donors and 15 patients (T0, T4, T16, T32) ("information-dependent acquisition" method or IDA; "Targeted label-free proteomics") in eosinophil homogenates. High abundant serum protein depletion using two protocols (P1: affinity chromatography, and P2: DTT precipitation) and SWATH-MS analysis of medium-low abundant serum proteome in samples from 15 healthy donors and 15 patients (T0, T4, T16, T32) ("information-dependent acquisition" method or IDA; "Targeted label-free proteomics").
详细描述
Hypotheses
Hº1. The levels of certain surface molecules on eosinophils or the presence or absence of certain proteins in the proteome of this leukocyte subset prior mepolizumab treatment can be used as predictive/prognostic markers of response to this biological.
Hº2. Mepolizumab alters the abundance of several surface or intracellular proteins in eosinophils as an outcome related to changes in their activation status, migratory ability, regulatory/effector function, or subset composition.
Hº3. Late-onset severe eosinophilic asthmatics have elevations in the serum concentration of different members of the IGF family (IGF-1, IGF-BP3, IGF-ALS) and mepolizumab treatment reduces these levels and behaves as a response-biomarker along with the number of eosinophils and clinical exacerbations.
H°4. Mepolizumab alters the abundance of several proteins in the medium-low abundance serum proteome of patients with SEA. Therefore, these proteins could be used as predictive/prognostic markers of response to this biological and could provide a better understanding of both eosinophilic and non-eosinophilic-related biological functions of IL-5 in SEA.
研究设计
- 研究类型
- Observational
- 观察模型
- Case Control
- 时间视角
- Prospective
入排标准
- 年龄范围
- 18 Years 至 75 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •Diagnosis of severe uncontrolled asthma according to ERS/ATS criteria
- •Persistent eosinophilia in blood (>300 cells/μL)
- •Frequent exacerbations (≥ two per year)
- •Signature of informed consent and agree to comply with all the visits of the study and all the procedures that this entails.
排除标准
- •Smoking history: Current smokers or former smokers with a smoking history of ≥10 pack-years
- •Clinically important pulmonary disease other than asthma (e.g. active lung infection, COPD, bronchiectasis, pulmonary fibrosis, cystic fibrosis, hypoventilation syndrome associated with obesity, lung cancer, alpha 1 anti-trypsin deficiency, and primary ciliary dyskinesia) or ever been diagnosed with pulmonary or systemic disease, other than asthma, that are associated with elevated peripheral eosinophil counts
- •• Any disorder, including, but not limited to, cardiovascular, gastrointestinal, hepatic, renal, neurological, musculoskeletal, infectious, endocrine, metabolic, haematological, psychiatric, or major physical impairment that is not stable in the opinion of the Investigator.
- •Malignancy: A current malignancy or previous history of cancer in remission.
- •Acute upper or lower respiratory infections requiring antibiotics or antiviral medication within 30 days prior to the Visit
- •Xolair: Participants who have received omalizumab (Xolair) or another monoclonal antibody previously.
- •Participants who have received systemic corticosteroids within 30 days before Visit 1 [53].
- •Pregnancy: Participants who are pregnant or breastfeeding.
- •Obesity class 2 or higher (BMI≥ 35 kg/m2)
研究组 & 干预措施
Severe eosinophilic asthma
Severe uncontrolled asthma according to ERS/ATS criteria and persistent eosinophilia in blood (>300 cells/μL)
干预措施: Mepolizumab 100 MG (Drug)
结局指标
主要结局
Measure changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques
时间窗: 32 weeks
Flow cytometry assays with multimarker panels 1 (regulatory), 2 (activation), and 3 (eosinophil subsets)
Measure changes in medium-low abundant serum proteins in response to treatment with mepolizumab using LC-MS/MS
时间窗: 32 weeks
Changes in the levels of multiple proteins within the low abundant serum proteome from patients, measured as the ratio to baseline (T=0) at weeks 4 and 32 (SWATH-MS), and ratio to healthy controls (T=0) in patients at T=0.
次要结局
- Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab.(32 weeks)
研究者
Francisco Javier González Barcala
SPECIALIST RESPIRATORY MEDICINE
Hospital Clinico Universitario de Santiago