Abhinav Kumar Checkervarty
Doctor of Philosophy in Experimental Medicine (PhD)
Multi-OMIC biomarkers to predict neonatal vaccine response
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Allergic rhinitis (AR) is the most prevalent allergic disease worldwide, affecting up to 40% of the global population. AR is a symptomatic disorder of the nose induced by IgE-mediated allergic inflammation of the nasal mucosa. Many studies have focused on the local inflammatory site contributing to an understanding of the pathophysiology of AR. The systemic immune responses of AR, however, have not been well investigated, and approaches for such investigation are scant. In order to identify systemic immune response signatures, we used peripheral blood collected from subjects with AR following nasal allergen challenge (NAC). The response signatures we identified consist of immune gene clusters associated with frequencies of corresponding immune cells that reflect dynamic immune responses after NAC. In particular, we found individual clusters associated with neutrophils, neutrophil/lymphocyte ratio (NLR), and lymphocytes, which demonstrated significantly different patterns between allergic and non-allergic subjects. The NLR-associated cluster was also moderately associated with clinical symptoms at 6 h post-NAC in cat allergic subjects.We tested how the identified signatures in cat allergy are expressed in birch and ragweed allergies, which are seasonal/intermittent allergies, using the NAC and Environmental Exposure Unit models. In the NAC model, while the birch allergic subjects did not demonstrate significantly (p > 0.05) different total nasal symptom score (TNSS) from the cat allergic subjects, there were fewer significantly (BH-FDR
Individuals with allergic asthma respond differently, but reproducibly, to allergen inhalation challenge. Some individuals develop an early response only (isolated early responders, ERs) while others also go on to develop a late response (dual responders, DRs). The early asthmatic response is characterized by acute airway contraction immediately following allergen inhalation and resolves within 1-3 hours. 60% of asthmatic individuals go on to develop a late asthmatic response that occurs 3-4 hours after allergen inhalation and is characterized by prolonged airway contraction, cellular infiltration of the airways, chronic inflammation, and airway remodeling. It is not understood why late responses do not develop in all sensitized individuals.In this thesis, I combined the power of the allergen inhalation challenge model to study airway responses with unbiased high throughput technologies and data driven computational methods to delineate molecular differences between ERs and DRs using peripheral blood. I identified many markers that discriminated ERs and DRs such as fibronectin, many pro-inflammatory, anti-inflammatory and cell-specific genes, lipid metabolites and amino acids and the T helper type 17 to T regulatory cell (Th17/Treg) ratio. Transcriptional biomarker panels performed well (AUC ~70%) in predicting at risk/susceptible individuals for the late phase asthmatic response prior to allergen challenge. These panels depicted a heightened pro-inflammatory (activation of nuclear factor (NF)-B signaling) and dampened anti-inflammatory (reduced expression of decoy and formyl peptide receptors which are involved in host response to pathogens) phenotype in DRs compared to ERs. I developed a statistical method which I used to identify multi-omic biomarker panels (cells, gene transcripts and metabolites). The identified panels achieved a systems view of the underlying molecular interactions highlighting common pathways such as lipid metabolism and valine, leucine and isoleucine metabolism across different biological compartments.I demonstrated that inherent molecular differences in blood exist between ERs and DRs suggesting that some asthmatic individuals display early systemic indications of chronic asthma. These findings may be used to develop blood tests to risk-stratify subjects to improve response to therapies, and may lead to earlier and accurate diagnoses.
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Allergic rhinitis (AR) is a heterogeneous disorder that is associated with inflammation of the upper airways. The prevalence of AR has increased rapidly in recent years, and currently affects 10 - 40% of the global population. Common examples of symptoms experienced after allergen exposure include nasal congestion, rhinorrhea, sneezing, and nasal itching. Additional symptoms include conjunctivitis and exacerbation of comorbid asthma. AR is characterized by an early phase response (EPR) and, in some individuals, a subsequent late-phase response (LPR). The induction of allergic responses can be studied using controlled allergen challenge facilities (CACF). Multiple CACFs have identified three response phenotypes in AR: early responders, protracted early responders, and dual responders. Molecular and genetic differences between AR phenotypes have not been well investigated. In order to identify molecular differences between phenotypes, we used baseline peripheral blood collected from individuals with AR. Blood samples from discovery and validation cohorts were profiled for biomarker candidates using a custom gene expression assay. Using univariate and multivariate analyses, we were unable to identify and validate a clear discriminatory signal between AR phenotypes. Next, we investigated the relationship between single nucleotide polymorphisms (SNPs) in cholinergic synapse pathway genes and the development of the LPR. We specifically looked at the cholinergic synapse pathway because polymorphisms in these genes have previously been associated with late asthmatic responses. Participants were split into two categories based on late-onset congestion, which is the predominant nasal symptom experienced during the LPR: low congestion (LC) and high congestion (HC). Allele frequencies of 25 SNPs located in cholinergic synapse pathway genes (ADCY3, AKT3, CACNA1S, CHRM3, CHRNB2, GNG4, and KCNQ4), were found to be significantly different between HC and LC subgroups. Additionally, we identified that the minor allele content of the HC subgroup was significantly higher than that of the LC subgroup. The cholinergic system may be a potential therapeutic target for the LPR.
Rationale: Acute exacerbations of chronic obstructive pulmonary disease(AECOPD) are caused by a variety of different etiologic agents. Our aim was to phenotype COPD exacerbations using imaging(chest x-ray[CXR] and computed tomography[CT]), blood tests(C-reactive protein [CRP] and the N-terminal of the prohormone brain natriuretic peptide [NT-proBNP]), and a molecular pathogen detection method.Methods: Subjects who were hospitalized with a primary diagnosis of AECOPD were enrolled in the Rapid Transition Program(RTP). We examined a subset of subjects who had had CXRs, CT scans, and blood collected for CRP and NT-proBNP. A radiologist blinded to the clinical and laboratory characteristics of the subjects interpreted the CXRs and CT images. Logistic regression models were used to assess the performance of these biomarkers in predicting the radiological parameters. Sputum samples in a subset of subjects were tested by a molecular pathogen detection method to phenotype AECOPD into non-infectious, bacterial, and virally-associated phenotypes. Differences between the phenotypes in terms of clinical features, CRP and NT-proBNP concentrations, complete blood counts, and 1-year mortality rate were examined.Results: NT-proBNP was associated with cardiac enlargement, pulmonary edema, and pleural effusion on CXR, whereas on CT images, NT-proBNP was associated with pleural effusion. CRP, on the other hand, was associated with consolidation, ground glass opacities, and pleural effusion on CT images. A CRP sensitivity-oriented cut-point of 11.5 mg/L was reached by setting a minimum sensitivity of 90% and applying the Youden index, for the presence of consolidation on CT images in subjects admitted as cases of AECOPD, which had a sensitivity of 91% and a specificity of 53% (P
Aspergillus fumigatus (A. fumigatus) is an opportunistic fungal pathogen that is widely distributed in nature through the release of conidiospores (conidia). Upon inhalation, fungal conidia (2-3 μm) are capable of reaching the bronchial and alveolar epithelia. This interaction between conidia and airway epithelial cells may result in the development of allergic, chronic or invasive aspergillosis in susceptible hosts. Characterization of the early molecular response of host using a multi-OMICs molecular approach is an important first step for better understanding the host-pathogen interaction. The aim of my research was to investigate the early molecular response of host upon interaction with A. fumigatus using an in-vitro model that closely recapitulates the in-vivo bronchial epithelium, and assess the applicability of this model to study host-pathogen interactions. A multi-OMICs approach utilizing NanoString and shotgun proteomics was applied to primary human bronchial epithelial cells (HBECs) grown for 21-28 days as differentiated air-liquid interface (ALI) cultures. Comparative analyses were conducted to compare the gene expression profiles of ALI cultures to submerged monolayer cultures of human airway epithelial cell line (1HAEo-) upon conidial exposure. In addition, transcriptional profiles of ALI cultures upon exposure to wild-type (WT) conidia of A. fumigatus were compared to Kdnase mutant strain (Δkdnase) of A. fumigatus and to Respiratory Syncytial Virus (RSV).Unlike submerged monolayer cultures, ALI cultures of primary HBECs internalized less than 1% of bound conidia 6 hours post-exposure. Transcriptomic and proteomic analyses of primary HBECs in ALI revealed that exposure to the fungus enriched the expression of genes related to cell cycle regulation, apoptosis/autophagy, iron homeostasis, calcium metabolism, complement and coagulation cascades, endoplasmic stress and the unfolded protein response. Comparative analyses to submerged monolayer cultures of 1HAEs indicated that the host molecular response in each model is different. The immune response in differentiated ALI cultures upon exposure to Δkdnase A. fumigatus conidia and RSV was pathogen-specific. Hence, ALI cultures of primary HBECs can provide novel insights into the mechanisms involved in the early molecular response associated with this opportunistic fungal pathogen.
Asthma is a chronic inflammatory airway disorder characterized by reversible airway obstruction and hyperresponsiveness. It affects more than 300 million people worldwide but remains poorly understood. Asthma can be induced by a variety triggers, including allergens, substances found in the workplace, cold air and exercises, and greatly diminishes the life quality of patients. In this thesis, I investigated two major categories of asthma: allergic asthma and occupational asthma. Two types of response are involved in allergic asthma. While 50% of the affected individuals only develop an acute early asthmatic response (EAR), the other individuals develop both the EAR and a chronic late asthmatic response (LAR). Those individuals who present an isolated EAR are classified as early responders (ERs) and individuals who present both the EAR and the LAR are classified as dual responders (DRs). In our study, patients with mild asthma were challenged with specific allergens and their blood was collected prior to the challenge. By measuring the gene expression and the protein levels of complement and coagulation molecules, I demonstrated that the complement and coagulation system may play a role in the LAR of allergic asthma. Occupational asthma is caused by sensitivity to specific molecules found in the working environment. Western red cedar asthma (WRCA) is the most common form of occupational asthma in the Pacific Northwest region of North America, including British Columbia. It is due to sensitivity to a low molecular weight molecule, plicatic acid (PA), found in the dust of western red cedar wood. The current diagnosis of WRCA is through multiple bronchial challenges, which are time-consuming, complicated and expensive. Blood samples were collected from individuals who were suspected to have WRCA prior to the bronchial challenges. Gene expression was measured using the NanoString platform. Using a pathway-directed approach of random forest and leave-one-out cross-validation, I identified and validated a blood-based two-gene biomarker panel which may help distinguish patients with WRCA from those with asthma due to non-western red cedar causes at baseline. Having such a biomarker panel may greatly simplify the current diagnosis of WRCA by way of a simple blood test.
Allergen inhalation challenge (AIC) triggers biphasic responses in allergic asthmatic individuals. Airway narrowing represents the early phase response, which typically occurs within 30 minutes of allergen inhalation. In 50-60% of allergic asthmatic adults, the early response is followed by the late phase response, usually starting around 3 hours after AIC, and characterized by cellular inflammation of the airway, increased lung tissue permeability, and mucus secretion. The pathways leading to the late response are not completely understood. The purpose of this thesis is to investigate the mechanisms behind the allergic asthmatic response profiles using peripheral blood samples obtained from asthmatics prior to and 2 hours following AIC. Subjects exhibited either an isolated early response of ≥20% fall in FEV₁ (isolated early responder – ER), or an early response followed by a late phase response of ≥15% fall in FEV₁ (dual responder – DR). Genome-wide transcriptional profiling using microarrays indicated significant perturbations in the Nrf2 (NF-E2-related factor 2)-mediated oxidative stress response pathway following allergen inhalation. Notably, the ABCC1 (ATP-binding cassette, sub-family C (CFTR/MRP), member 1) gene within the pathway showed a decreased expression post-challenge, as validated through RT-qPCR. Furthermore, a significant decrease in the level of plasma chemokine (C-C motif) ligand 2 (CCL2) was evident, which was replicated using immunoassays in additional cohorts of allergic rhinitis and individuals with occupational asthma. However, this may be attributable to inherent fluctuations, based on similar results from control subjects. The comparison of transcriptomic response profiles between ERs and DRs undergoing cat allergen inhalation challenge revealed linoleic acid metabolism as the most significant pathway. Separation of whole-blood gene expression profiles into cell-specific signals using the csSAM algorithm suggested that key transcriptomic differences lie in eosinophils and lymphocytes when comparing between ERs and DRs at the post-challenge time point. These findings are in support of the current model of asthma pathophysiology and provide valuable insights into molecular changes occurring as early as 2 hours after allergen inhalation. Further study into the underlying mechanisms leading to the different response patterns may expose new therapeutic targets effective in minimizing the late response, which is associated with chronic asthma.
The cells of the airway epithelium play critical roles in host defense to inhaled irritants, and in asthma pathogenesis. These cells are constantly exposed to environmental factors, including the conidia of the ubiquitous mould Aspergillus fumigatus, which are small enough to reach the alveoli. A. fumigatus is associated with a spectrum of diseases ranging from asthma and allergic bronchopulmonary aspergillosis to aspergilloma and invasive aspergillosis. Airway epithelial cells have been shown to internalize A. fumigatus conidia in vitro, but the implications of this process for pathogenesis remain unclear. We have developed a cell culture model for this interaction using the human bronchial epithelium cell line 16HBE and a transgenic A. fumigatus strain expressing green fluorescent protein (GFP). Immunofluorescent staining and nystatin protection assays indicated that cells internalized upwards of 50% of bound conidia. Using fluorescence-activated cell sorting (FACS), cells directly interacting with conidia and cells not associated with any conidia were sorted into separate samples, with an overall accuracy of 75%. Genome-wide transcriptional profiling using microarrays revealed significant responses of 16HBE cells and conidia to each other. Significant changes in gene expression were identified between cells and conidia incubated alone versus together, as well as between GFP positive and negative sorted cells. The identification of biologically relevant responses in both species validates this methodology, and motivates further work to characterize the interactions between A. fumigatus conidia and primary airway epithelial cells obtained from normal and asthmatic patients.