Asthma
Conditions
Brief summary
Being a key player in Asthma, eosinophils constitute a potential target to interfere with the series of biological events leading to Asthma pathogenesis. In this proposal, the investigators hypothesize that the interaction between eosinophils and airway smooth muscle cells (ASM) cells plays an important role in airway remodeling. Hence the investigators will investigate the role of eosinophils in enhancing ASM cells proliferation resulting in airways remodeling. The investigators will also investigate the mechanism behind this phenomenon. This will then pave the way for medical (drug) interference at one or several sites in order to prevent one of the main potential reasons behind airway remodeling, namely eosinophil-derived ASM proliferation.
Detailed description
Rational and hypothesis Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness (AHR). Its is well documented that the smooth muscle mass in asthmatic airways is increased due to hypertrophy and hyperplasia of the ASM cells. Moreover, eosinophils have been proposed in different studies to play a major role in airway remodeling. The association between increased eosinophil numbers and asthma severity has been well documented. Here, we hypothesized that the interaction between eosinophils and ASM cells plays an important role in airway remodeling and that eosinophils modulate the airways through enhancing ASM cell proliferation. Objectives of the study The aim of this study is to examine the effect of eosinophils on ASM cell proliferation using eosinophils isolated from asthmatic subjects. Specific aim 1. Effect of co-culture of ASM cells with eosinophils on ASM cell proliferation. In this specific aim, the effect of eosinophil on ASM cell proliferation will be studied by co-culturing eosinophils isolated from asthmatic and healthy patients with ASM cells in vitro. This will be done using non-stimulated eosinophils or eosinophils activated with Interleukin-5 (IL-5)and Interleukin-3 (IL-3). ASM proliferation will be monitored by determining the levels of the proliferation marker Ki-67 expression in ASM cells. Specific aim 2. Understanding the mechanism by which eosinophils enhance ASM proliferation. In this specific aim, we will study the mechanism by which eosinophils enhance the proliferation of ASM cells. To study if this effect of eosinophils is done through direct contact with ASM cells, we will co-culture ASM cells with eosinophils membrane fractions. Moreover, we will examine the effect of cytokines and extracellular matrix (ECM) proteins produced by eosinophils on smooth muscle cells
Interventions
None listed
Sponsors
King Saud University
Study design
Observational model
CASE_CONTROL
Eligibility
Sex/Gender
ALL
Age
14 Years to 35 Years
Healthy volunteers
Yes
Inclusion criteria
Asthma: Patient Selection (Inclusion Criteria) 1. Subjects with documented clinical history of asthma for a period of at least 6 months prior to study entry (and a minimum of one clinic follow-up visit since initial diagnosis) 2. Willing to provide written informed consent and in the judgment of the investigator, individuals who are able to understand the informed consent process. 3. Subjects with documented clinical history (in preceding 12 months) of airway reversibility of at least 12% based on Forced Expiratory Volume (FEV1), measured pre and post inhalation of a β-2 agonist (2 puffs of albuterol using a measured dose inhaler with spacer) OR 4. Individuals with strong history of asthma but with waning, or no current symptoms may be included in the study if their asthma was well controlled using an asthma medication. Principal investigator must verify or know the clinical history of an individual before accepting him as an asthmatic individual. 5. Able to perform Spirometry/FEV1 correctly.
Exclusion criteria
1. Age \< 5 years 2. Smoking for 20 years, 1 pack/day or more. 3. Congestive heart failure. 4. Chronic Obstructive Pulmonary Disease (COPD). 5. Chronic lung disease other than asthma and COPD. 6. Bronchiectasis.
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Fold Increase in ASM Proliferation Following Incubation With Eosinophils. | one day | Airway Smooth Muscle (ASM) cell proliferation was measured 24 hrs following their co-culture with eosinophil. This was determined using Ki-67 staining and flowcytometry. The fold increase in ASM proliferation was then determined. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Fold Increase in ASM Cells Proliferation Following Treatment With Cysteinyl Leukotrienes | one day | The effect of Eosinophil release of Cysteinyl Leukotrienes on ASM proliferation was determined using blocking agents. This was determined using Ki-67 staining and flowcytometry. |
Countries
Saudi Arabia
Participant flow
Recruitment details
recruitment started January 2010 and ended July 2011. Recruitment was all in King Khalid Hospital, Riyadh, Saudi Arabia
Participants by arm
| Arm | Count |
|---|---|
| Asthmatic Patients Patients were severe asthmatic with high peripheral blood eosinophil counts (\>5%). | 6 |
| Non Asthmatic Patients Non asthmatic patients with high eosinophil count (\>3%). | 6 |
| Total | 12 |
Baseline characteristics
| Characteristic | Non Asthmatic Patients | Asthmatic Patients | Total |
|---|---|---|---|
| Age, Categorical <=18 years | 1 Participants | 2 Participants | 3 Participants |
| Age, Categorical >=65 years | 0 Participants | 0 Participants | 0 Participants |
| Age, Categorical Between 18 and 65 years | 5 Participants | 4 Participants | 9 Participants |
| Age Continuous | 24 years STANDARD_DEVIATION 2.5 | 23 years STANDARD_DEVIATION 3 | 23.5 years STANDARD_DEVIATION 2.75 |
| Region of Enrollment Saudi Arabia | 6 participants | 6 participants | 12 participants |
| Sex: Female, Male Female | 1 Participants | 2 Participants | 3 Participants |
| Sex: Female, Male Male | 5 Participants | 4 Participants | 9 Participants |
Adverse events
| Event type | EG000 affected / at risk | EG001 affected / at risk |
|---|---|---|
| deaths Total, all-cause mortality | — / — | — / — |
| other Total, other adverse events | 0 / 0 | 0 / 0 |
| serious Total, serious adverse events | 0 / 0 | 0 / 0 |
Outcome results
Primary
Fold Increase in ASM Proliferation Following Incubation With Eosinophils.
Airway Smooth Muscle (ASM) cell proliferation was measured 24 hrs following their co-culture with eosinophil. This was determined using Ki-67 staining and flowcytometry. The fold increase in ASM proliferation was then determined.
Time frame: one day
Population: The number of participants for each group was determined to insure proper statistical significance when analysing the effect of isolated eosinophils on ASM cells proliferation
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Asthmatic Patients | Fold Increase in ASM Proliferation Following Incubation With Eosinophils. | 2 Fold increase in ASM cell proliferation | Standard Deviation 0.2 |
| Non Asthmatic Patients | Fold Increase in ASM Proliferation Following Incubation With Eosinophils. | 2 Fold increase in ASM cell proliferation | Standard Deviation 0.3 |
p-value: <0.05ANOVA
Secondary
Fold Increase in ASM Cells Proliferation Following Treatment With Cysteinyl Leukotrienes
The effect of Eosinophil release of Cysteinyl Leukotrienes on ASM proliferation was determined using blocking agents. This was determined using Ki-67 staining and flowcytometry.
Time frame: one day
Population: Eosinophils were collected from all the participants and used to trigger ASM proliferation.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Asthmatic Patients | Fold Increase in ASM Cells Proliferation Following Treatment With Cysteinyl Leukotrienes | 2.3 Fold increase in ASM proliferation | Standard Deviation 0.2 |
| Non Asthmatic Patients | Fold Increase in ASM Cells Proliferation Following Treatment With Cysteinyl Leukotrienes | 2.5 Fold increase in ASM proliferation | Standard Deviation 0.1 |