Louis-Philippe Boulet - World Allergy Organization

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Louis-Philippe Boulet - World Allergy Organization

Strengths and limitations ofbronchoscopy (& related measures)- Role of the BALLouis-Philippe BouletMD FRCPC FCCPQuebec Heart & Lung InstituteQuebec City, Canada


Endoscopic sampling for airwayinflammatory diseasesSynopsis Endoscopic techniques for assessment ofairway features Indications and usefulness of thesetechniques Weaknesses of this type of sampling Conclusions


Flexible bronchoscopyInitially for assessment of endobronchialtumors/lesions/foreign bodies, etc…Development of BAL (+TBBx) for the assessment ofinterstitial lung diseases– Sarcoidosis– Allergic Alveolitis– Others…Standardization for assessment of airwayinflammatory disorders (e.g. Asthma…)– BAL– Bronchial biopsies


Airway inflammation andremodeling in asthma


Bronchial brushings Bronchial epithelial cellsamplingsampling (e.g. for cultures)


Bronchoalveolar lavage Cell differential Mediator & cytokines measurements


Bronchial biopsiesTissue samplingLight microscopy,Electron microscopyImmunohistochemical stains,fluorescence, in-situhybridization,or PCR for mRNA expression forcytokines and adhesion moleculesCell functionCell culturesGenes analysesBonchial biopsyForcepsInflammation and structural changes


Endoscopic techniques:limitationsShould be done in a research setting mostly, byexperienced endoscopists, , with all human andmaterial resources neededCannot usually be performed if airway obstructionis too severe or the patient unstablePotential side-effects effects of the technique ormedication used• Safety and good tolerance of these procedures inasthma patients of mild, moderate and severedisease have been well documented


BAL: advantages Samples obtained by BAL include luminalcells (predominantly macrophages) andsoluble factors coming from a relativelylarge airway surface area, including theperipheral airways With sequential lavage, , it is possible toobserve the results of allergen and placebochallenge in the same individual over time BAL includes both mediators and cellsreleased into the fluid phaseAdapted from Jeffery et al. 2003


BAL: strenghtsRecovery of biological samples in the form ofcellular and soluble materials from medium andsmall airways, and alveolar spaces.BAL has aided in the identification of manymediators currently thought to have a significantrole in the pathophysiology of asthma(e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, GM-CSF, TNF-a,ICAM-1)Murugan et al. 2009


BAL: limitationsNo spatial, structural, or histologic informationCellular signals may be affected by the method of collectionThe source of the cell sample is impreciseThe volume of sample recovered may relate to diseaseseverity - dilution factorsThe procedure itself or handling and processing of samplesmay introduce artifacts (e.g., lidocaine and bronchodilators may affect cell activity)The removal, washing, centrifuging, and resuspension ofmucus can activate cells and confound the cell and soluteinformation.BAL process may lead to transient fever within 24 hours afterthe lavage, , occasionally associated with lung infiltratesAdapted from Jeffery et al. 2003


BAL in asthmaIncreased numbers of eosinophils is suggestive ofasthma although neutrophils can be increased,particularly in severe asthmaVarious cytokines can be measured e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-10, IL-13, GM-CSF, TNF-a,ICAM-1)No consistent correlation with asthma severity


BAL in COPDThe predominant cell type in BAL of individuals with COPD isthe alveolar macrophageThe percentage of CD8 + T lymphocytes is significantly higher,and that of CD4+ T-cells Tsignificantly lower, in COPD (andhealthy smokers) compared with healthy non-smokersNeutrophils and eosinophils have generally been shown to beincreased in COPD BALMast cell numbers have also been reported to be increasedECP, myeloperoxidase, , and IL-8 8 are frequently increased inpatients with COPD and in healthy smokers.


BAL: limitationsThe clinical utility of BAL fluid biomarkers isquestioned by the lack of standardization fromlaboratory to laboratory in reporting the levels ofvarious markers, making comparisons betweenlaboratories difficult.As disease state changes, and volume recoverychanges, interpretation of serial evaluation in thesame individual is troublesome.Murugan et al. 2009


Endobronchial biopsies:- a useful research toolDirectly samples the resident cellsSpatial relationships of structural components maintainedAssesses the status of the airway mucosaIndividual structural components can be identifiedStructures can be removed and studied in isolation usinglaser capture dissectionInflammatory cell subtypes can be identified byimmunostainingBiomarkers of epithelial damage can be studiedAdapted from Jeffery et al. 2003


Endobronchial biopsies:- a useful research toolImmunologic cascades can be studied usingimmunohistochemistry, , ISH, and microarray analysis(for assessment of gene expression)Tissue explants and cells derived from biopsies can alsobe preserved in cultureResident cells, such as airway smooth muscle myocytes,have successfully been dissected from endobronchialbiopsies from subjects with asthma, allowing measuresof the proliferative responses of these cellsAdapted from Jeffery et al. 2003


Airway morphologyEpitheliumVesselsBasement membraneSmooth muscle


Bronchial biopsies of smoking asthmatic patientsCollagen ISquamous cell metaplasia


Inflammatory cells in Bronchial biopsies


Relationship between airways responsiveness tomethacholine and the number of (A) CD3 +, (B) CD4 +,(C) CD8+, and (D) CD45RO + in the bronchial laminapropria Sont et al. Thorax 1996


Biomarkers in severe asthmaBronchial biopsysections from patientswith severe asthmashowing MBP1eosinophils (a),CD681 macrophages(b), neutrophilelastase-positiveneutrophils (c)and CD31 T cells (d)Macedo et al. 2009


SwimmerAsthmaticCollagen 3Airway remodelingIn swimmers


Biomarkers in bronchial biopsies(asthma)• Epithelial shedding, goblet cell hyperplasia, thickenedlamina reticularis• Submucosal eosinophilic and lymphocytic infiltration• Increased granulation of mast cells and high affinity IgEreceptor bearing cells• Increased expression of IL-4, IL-5, endothelin, eotaxinand ICAM-1 enhanced IL-8 and IFN-g expression,also IL-5 and eotaxin expression and reduced IL-4expression of severe asthma compared with moderatedisease


Biomarkers in bronchial biopsies(COPD)Increase in macrophages, CD8 + T-cells +eosinophils+Increased expression of CCL5 and CXCL7 in the bronchialmucosaThe number of IL-17A+ , IL-22+ , IL-22+ and IL-23+immunoreactive cells is increased in the bronchialepitheliumThe number of cells expressing YKL-40, a chitin-bindingprotein that is elevated in patients with variousinflammatory conditionsAltered surfactant protein (SP)-A A expression in humanlung tissue samples obtained from patients with COPD


Genetic variation in immune signaling genes differentiallyexpressed in asthmatic lung tissues Tremblay et al. 2008LD pattern for SFRP1. A,SFRP1 LD pattern in theSLSJ familial sample. B,SFRP1 LD pattern in theCAPPS cohort. C, SFRP1LD pattern in the SAGEcohort. D, SFRP1 LDpattern in the Busseltoncohort. The location ofeach tested TagSNP alongthe chromosome isindicated in the upperpart of the figure.The numberin eachdiamond indicatesthe magnitude of LD (D9)as a percentage betweenrespective pairs of SNPs.The strength of LD isdepicted by progressionof color.


Bronchial biopsies for cell culturesFibroblasts Epithelial cellsBronchial BiopsiesReconstitution of bronchial mucosa


Endobronchial biopsies:limitationsSamples are limited in size to between 1 - 2 mm diameterThey may not be representative of the entire conductingairways (mainly from proximal airways and fromsubcarinae peribronchial tissue (deep to the mucosa)is not included in endobronchial samplesThe procedure itself may induce artifacts, especially dueto forceps damageBiopsy procedure itself creates scar tissue and caninitiate cellular processes that can proceed during theinterval (seconds) between sampling and fixationBiopsy provides only a snapshot in time of an ongoingdisease processAdapted Jeffery et al. 2003


Endobronchial biopsies:limitationsBronchial biopsies to quantify inflammation arelimited by normal anatomic variability, patchy focalinflammation, sample size, and number of biopsyspecimens and quantification of cellular infiltrate.Studies evaluating the variability and reproducibilityof data obtained from bronchial biopsies ofasthmatics suggest a sample size of 8–25individuals to study individual cell types and 13–48individuals per arm in parallel design drug studiesare required to establish adequate statistical power.Murigan et al 2009


Morphometric measurements


Bronchoscopy in the treatmentof asthma: thermoplasty !


Conclusions Endoscopic techniques may be veryhelpful in the study of thepathophysiology of airway diseases These techniques are invasive and notsuitable for clinical assessmentof asthma & COPD New developments can optimize theusefulness of these sampling

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