The Airway & Asthma Translational Research Unit (AATRU) laboratory is focused on understanding mechanisms of airway remodeling in lung disease, particularly in severe asthma, using molecular, biochemical, and transcriptional approaches to improve treatment using individualized medicine.
The Airway & Asthma Translational Research Unit (AATRU) laboratory was established in 1995 to acquire, securely store and utilize integrated human airway biospecimens and clinical data to better understand the biochemical, molecular and transcriptional basis for the structural and physiological consequences of airway remodeling in lung diseases, specifically severe asthma. The laboratory hopes to advance our understanding of the mechanisms that underline the pathophysiology of asthma leading to new strategies for trial designs and therapeutic interventions.
One of the main functions of the laboratory is in supporting the >25 clinical trials run by Dr Castro at KUMC. Biological specimens are procured using standardized, validated procedures that allows clinical data to be referenced in coordination with structural, mechanistic as well as biochemical for genomic, metabolomic, proteomic, microbiome analyses in unraveling asthma endotypes.
AATRU also provides consultation and collaborative services to investigators wishing to study human airways in their research projects. Adult and Pediatric biospecimens facilitate understanding the underlying pathophysiology of asthma endotypes. Biospecimens consist of study participant blood products, including serum, plasma, as well as peripheral blood mononuclear cells (PBMCs); urine, feces, induced and spontaneous sputum, nasal fluid and tissue, exhaled breath condensate (EBC) and bronchial alveolar lavage fluid and endobronchial tissue biopsies.
The laboratory supports the coordinators in Biospecimen processing, aliquoting and storage as well as shipping these specimens to trial centers and collaborators throughout the world. As part of Biospecimen processing, the laboratory has established an Airway Biobank which houses >850 unique subjects and >3500 biospecimens over the past 25+ years of human subjects' research. Please contact Dr. Castro or the AATRU laboratory for biospecimen availability and potential collaborations.
A RedCap based secure electronic database that captures subject clinical data, run by Project Design and Analysis - Enterprise Sciences at KUMC. Clinical data consists of clinical laboratory data, medications, lung function testing, CT/MRI imaging as well as subject demographics enabling research to be undertaken on well-characterized subject populations.
AATRU Biospecimen Database
An electronic database built on the open-source platform Open Specimen run by the Research Institute of Technology Biostatistics and Data Science at KUMC. Open Specimen is a laboratory information system (LIS) for cataloging, storage, and distribution of biospecimens for future and collaborative research.
Airway remodeling in lung diseases including asthma results in measurable quantitative alterations of the structure of the lung. A bronchoscopist uses a bronchoscope to visualize the airways and forceps to collect microscopic pieces of tissue. This tissue is fixed, embedded and sectioned then processed for Histology stains (H&E) or Immuno-histochemistry (IHC) procedures.
Histology stains and IHC allows for the visualization of specific proteins using light microscopy. The stained or labeled sectioned tissue is captured on a tiling microscope as a colored image. This image is loaded into Image Analysis Software (Image Pro 10, Mediacy) where additional digital processing and analysis occurs. In severe asthma the epithelium and laminar reticularis becomes thicker and an increase in smooth muscle actin is observed (Figure 1).
In addition, the presence of goblet cells can be identified in the epithelium which produce mucus as a pathological feature of asthma. This technique is one of the few recognized measures of airway remodeling in biopsy tissue. The laboratory has ongoing Image Analysis projects analyzing biopsy tissue from ongoing NIH funded studies including SARPIII and IV.
Airway remodeling can be visualized at the cellular level using high-image resolution techniques like immune-fluorescent confocal microscopy. Cellular markers including receptors, proteins, transcription factors and nuclei can be labeled with specific antibodies or dyes conjugated to a fluorophore with specific excitation and emission wavelengths. The fluorescent microscope captures these fluorophores in x-y and z planes to generate a 3D image. Images can be reconstructed in a 3D space using Image Analysis Software (Image Pro 10) to recapitulate the architecture of the epithelium.
In Figure 2, subjects with severe asthma had increased levels of Mucin in the airways (Muc5AC labeling), decreased presence of the transcription factor for Cilia (Foxj1 labeling) compared to healthy subjects' biopsies. Ongoing projects in the lab will determine the cell types present in the airway, the 3D architecture and orientation of these cells types in the airway and the expression of proteins (mucins, cilia, IL-13/EGF) and receptors (EGFR, IL-13R, Chemokines) important in epithelial differentiation and inflammation to understand how the airway remodels during the pathophysiology of severe asthma.
Transcriptomics is routinely used to profile diseases for gene related production of pathology inducing proteins, biological pathways and for novel biomarkers. The sputum as well as epithelium of the lung has been used to transcriptionally profile the genes involved in severe asthma in the NIH funded SARP cohort.
Our group has identified the upregulation of Th2 inflammatory related genes in pediatric and adult severe asthma from the sputum and endobronchial brushes of the lung epithelium (Peters MC. 2019. J Allergy Clin Immunology).
In Figure 3, RNA-sequencing identified the downregulation of cilia related genes (FOXJ1, TEK2, SPAG6) and increase of the mucin gene (Muc5AC) in severe asthma when compared to healthy individuals in the lung epithelium. Additional Research opportunities are available for RNA-sequencing of sputum, nasal and lung epithelium as well as whole blood and feces for identifying novel genes involved in severe asthma from NIH and Pharma funded clinical trials.
The AATRU takes advantage of an in vitro cell culture model that replicates the lung to perform mechanistic studies on airway epithelium to understand the endotypes of asthma and test novel biologics. Air liquid interface (ALI) cultures are when epithelial cells are isolated and cultured from endobronchial brushes obtained from the human airway. Epithelial cells are first expanded in submerged cultures then transferred to porous inserts until confluent.
Once confluent, the apical fluid is removed exposing the epithelium to 'air', like the environment within the lung, and the basal medium is changed to induce epithelial cell differentiation. A pseudostratified epithelium contains the cell types that make up the airways -- ciliated, basal as well as mucus producing cells after ~30 days of culture (Figure 4 A to B).
Figure 5 illustrates the differences in cilia production from a healthy individual (A; wavelike motion) to that of an individual with severe asthma (B; illustrates the lack of beating cilia).
Figure 5: Lung Epithelial ALI cultures after 30 days of differentiation. Healthy pseudostratified epithelium with beating cilia (A) and pseudostratified epithelium lacking cilia in severe asthma (B) from individuals enrolled in the Severe Asthma Research Program (SARP III, videos at 20X).
Current research projects focus on understanding
- the cellular differences of the pseudostratified epithelium of epithelial cells grown from healthy vs individuals with different endotypes of asthma,
- differentiation and inflammatory factors including epidermal growth factor (EGF) and inflammatory cytokines (IL-13) that potentiate these asthma endotypes and
- the cellular mechanisms of biologics including dupilumab an IL-13 Receptor blocker used to treat severe asthma in the hopes of improving treatments and outcomes for asthma.
Additionally, the AATRU lab always has short term focused research projects for interested summer students, fellows and collaborators. Research projects can be either pre-specified or individually developed depending on one's research interests.
- Azithromycin to Prevent Wheezing following severe RSV bronchiolitis-II (APW-II) - This study (Bacharier LB. 2015. JAMA) administers to Azithromycin to infants diagnosed with RSV infection to decrease the risk of developing asthma later in life. Current projects are available to perform ELISA's on nasal washes obtained overtime of the infection to study novel biomarkers including MMP-9 and IL-8.
- Quantification of Lung tissue biopsies and ALI membranes for NIH and Pharma Studies - The laboratory houses a brand-new Nikon Eclipse Ni with a DS-2 Camera Tiling microscope to capture light microscopy images for IHC and pathology stains like H&E and muci-carmen and a shared Nikon Eclipse Ti Confocal Microscope used to capture high resolution immunofluorescent images.
The laboratory also has multiple copies of Image Pro 10 (Mediacy) for the quantification and manipulation of light and fluorescent images. Image Pro 10 can 1) quantitate airway remodeling in tissue using morphometric measures including epithelial area, basement membrane length, laminar reticularis area and smooth muscle area, 1) quantitate the expression of proteins (including mucin) or transcription factors (Foxj1 a regulator of cilia) in the epithelium and 3) render high resolution images produced by confocal microscopy to generate 3D images to quantify and determine spacial relationships of cells and proteins in the lung epithelium. Current projects are underway and can be expanded to 1) quantitate and 2) visualize airway remodeling in various asthma endotypes.