Biology subjects

Huff, R. D.

Publications and source records attributed to Huff, R. D..

8 recordsLinked to original sources

bioRxiv2025

Engineering a Multilayer Microfluidic Airway-On-A-Chip with Tunable GelMA Hydrogel for Physiologically Relevant Aerosol Exposure Studies

Climate change-driven increases in forest fires pose a major global health risk due to exposure to smoke containing hazardous gases and fine particulates, emphasizing the need for physiologically relevant in vitro airway models for studying smoke-induced responses. Microfluidic lung-on-a-chip technologies provide a strong foundation for in vitro airway modeling and ongoing developments are expanding their ability to incorporate multicellular organization, extracellular matrix complexity, and physiologically relevant exposure methods. This work presents the optimization and integration of a photopolymerizable gelatin methacrylate (GelMA)-based hydrogel into a microfluidic airway-on-a-chip that models the human small conducting airways and supports controlled aerosol exposure to wood smoke. The GelMA hydrogel was optimized to support fibroblast encapsulation, endothelial, and epithelial adhesion and robust mechanical stability. The device combines the hydrogel with a compartmentalized microchannel layout, and sacrificial molding to create a 3D organotypic airway culture featuring a multilayer architecture, 3D stromal matrix, and a perfusable vasculature-like lumen. Coupling the platform with a custom aerosol exposure system enables precise, biomimetic exposure to whole wood smoke. Proof-of-concept studies using transforming growth factor beta1 (TGF-{beta}1) and whole wood smoke elicited expected inflammatory and fibrotic responses, validating the platforms physiological relevance for inhalation studies and investigating smoke-induced airway remodeling and inflammation.

bioengineering↗

bioRxiv2025

Woodsmoke and Diesel Exhaust: Distinct Transcriptomic Profiles in the Human Airway Epithelium

Climate change is increasing the frequency and severity of wildfires globally, causing significant woodsmoke (WS) emissions. Vehicles emit sizable amounts of toxic traffic-related air pollution (TRAP), for which diesel exhaust (DE) is a model. Both WS and DE contain particulate matter <2.5 microns (PM2.5) which deeply penetrates the lungs, causing respiratory epithelial inflammation that drives health effects. Regulations focus on PM2.5 concentration, despite emerging research that highlights how composition mediates health effects. As WS and DE are compositionally distinct, we conducted the first head-to-head comparison of effects on the transcriptomes of air-liquid interface cultured primary human bronchial epithelial cells (HBEC). Differentiated donor-matched HBEC transwells were exposed for 2-hours to filtered air (FA; control), or WS (furnace tube burning pine) or DE (Hatz 1B30E generator) both diluted to 300 {micro}g/m3 of PM2.5. WS had higher ultrafine PM, whereas DE exposure contained significantly higher NO2, CO, and O3. RNA sequencing showed that WS exposure resulted in 119 ({uparrow}41, {downarrow}78) differentially expressed genes, while DE modulated 399 ({uparrow}255, {downarrow}144) compared to FA exposure. WS was associated with small ribosomal subunit and cytochrome complex related genes, while DE exposure was associated with HIF-1 signaling, respiratory chain complex and interferon alpha/beta signaling/ISG15-protein conjugation, suggesting how TRAP exposure may enhance infection risk. We also analyzed exposure effects on protein immune-mediators. We demonstrate that two major air pollution sources modulate different genes and pathways in HBECs, with minimal overlap. This informs the debate regarding the regulatory focus on concentration and assumptions that similar concentrations of air pollution have indistinct effects. HighlightsO_LILung health effects of diesel exhaust (DE) and wood smoke (WS) are underexplored C_LIO_LIRNAseq of DE and WS-exposed primary lung epithelial cells revealed larger DE effects C_LIO_LIDE showed greater repression of host antiviral response-associated genes than WS C_LIO_LIA regulatory focus on PM concentration may miss composition-specific lung effects C_LI

bioinformatics↗

bioRxiv2024

Mapping in silico genetic networks of the KMT2D tumour suppressor gene to uncover novel functional associations and cancer cell vulnerabilities

Loss-of-function (LOF) alterations in tumour suppressor genes cannot be directly targeted. Approaches characterising gene function and vulnerabilities conferred by such mutations are required. Here, we computationally map genetic networks of KMT2D, a tumour suppressor gene frequently mutated in several cancer types. Using KMT2D loss-of-function (KMT2DLOF) mutations as a model, we illustrate the utility of in silico genetic networks in uncovering novel functional associations and vulnerabilities in cancer cells with LOF alterations affecting tumour suppressor genes. We revealed genetic interactors with functions in histone modification, metabolism, and immune response, and synthetic lethal (SL) candidates, including some encoding existing therapeutic targets. Analysing patient data from The Cancer Genome Atlas and the Personalized OncoGenomics Project, we showed, for example, elevated immune checkpoint response markers in KMT2DLOF cases, possibly supporting KMT2DLOF as an immune checkpoint inhibitor biomarker. Our study illustrates how tumour suppressor gene LOF alterations can be exploited to reveal potentially targetable cancer cell vulnerabilities.

bioinformatics↗

bioRxiv2023

Deep learning enables accurate soft tissue deformation estimation in vivo

Image-based deformation estimation is an important tool used in a variety of engineering problems, including crack propagation, fracture, and fatigue failure. These tools have been instrumental in biomechanics research where measuring in vitro and in vivo tissue deformations help evaluate tissue health and disease progression. However, accurately measuring tissue deformation in vivo is particularly challenging due to limited image signal-to-noise ratio. Therefore, we created a novel deep-learning approach for measuring deformation from a sequence of in vivo images called StrainNet. Utilizing a training dataset that incorporates image artifacts, StrainNet was designed to maximize performance in challenging in vivo settings. Artificially generated image sequences of human flexor tendons undergoing known deformations were used to compare StrainNet against two conventional image-based strain measurement techniques. StrainNet outperformed the traditional techniques by nearly 90%. High-frequency ultrasound imaging was then used to acquire images of the flexor tendons engaged during contraction. Only StrainNet was able to track tissue deformations under the in vivo test conditions. Findings revealed strong correlations between tendon deformation and contraction effort, highlighting the potential for StrainNet to be a valuable tool for assessing preventative care, rehabilitation strategies, or disease progression. Additionally, by using real-world data to train our model, StrainNet was able to generalize and reveal important relationships between the effort exerted by the participant and tendon mechanics. Overall, StrainNet demonstrated the effectiveness of using deep learning for image-based strain analysis in vivo.

bioengineering↗

bioRxiv2020

Potentiation of Long-Acting β2-Agonist and Glucocorticoid Responses in Human Airway Epithelial Cells by Modulation of Intracellular cAMP

IntroductionOver 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). HypothesisIncreasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. MethodsExpression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. ResultsUsing archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 was potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. ConclusionModulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition is able to potentiate LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggests further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

immunology↗

bioRxiv2020

Modulation of cAMP metabolism for CFTR potentiation in human airway epithelial cells

Cystic fibrosis (CF) is a genetic disease characterized by CF transmembrane regulator (CFTR) dysfunction. With over 2000 CFTR variants identified, in addition to known patient to patient variability, there is a need for personalized treatment. The discovery of CFTR modulators has shown efficacy in certain CF populations, however there are still CF populations without valid therapeutic options. With evidence suggesting that single drug therapeutics are insufficient for optimal management of CF disease, there has been an increased pursuit of combinatorial therapies. Our aim was to test cyclic AMP (cAMP) modulation, through ATP Binding Cassette Transporter C4 (ABCC4) and phosphodiesterase-4 (PDE-4) inhibition, as a potential add-on therapeutic to a clinically approved CFTR modulator, VX-770, as a method for increasing CFTR activity. Human airway epithelial cells (Calu-3) were used to test the efficacy of cAMP modulation by ABCC4 and PDE-4 inhibition through a series of concentration-response studies. Our results showed that cAMP modulation, in combination with VX-770, led to an increase in CFTR activity via an increase in sensitivity when compared to treatment of VX-770 alone. Our study suggests that cAMP modulation has potential to be pursued as an add-on therapy for the optimal management of CF disease.

cell biology↗

bioRxiv2019

A comparative analysis of cannabis and tobacco smoke exposure on human airway epithelial cell gene expression, immune phenotype, and response to formoterol and budesonide treatment

Global recreational cannabis use is a potentially important public health issue that would benefit from experimental evidence to inform policy, regulations, and individual user practices. Comparative analyses between cannabis and tobacco smoke, the latter long reported to have negative impacts on respiratory health, may help provide context and provide clinically relevant evidence.\n\nTo address this unmet need we performed a comparative study between cannabis and tobacco smoke exposure in the Calu-3 human airway epithelial cells using concentration-response and pharmacological intervention study designs with outcome measurements of cell viability, epithelial cell barrier function, cytokine profile, and transcriptomics.\n\nOur results demonstrate that cannabis smoke exposure reduces epithelial cell barrier function without impacting cell viability, accompanied by a cytokine profile associated with inflammation (elevated IL-6 and IL-8), barrier repair (elevated TGF- and PDGF-AA) and suppressed antiviral immunity (decreased IP-10 and RANTES). Transcriptomic analyses revealed a cannabis smoke induced signature associated with suppressed antiviral genes and induction of oncogenic and oxidative stress pathways. Similar trends were observed for tobacco smoke exposure. A formoterol/budesonide intervention was unable to prevent cannabis smoke-induced reductions in antiviral pathways or normalize induction of oncogenic and oxidative stress responses.\n\nOur results show striking similarities between cannabis and tobacco smoke exposure on impairing barrier function, suppressing antiviral pathways, potentiating of pro-inflammatory mediators, and inducing oncogenic and oxidative stress gene expression signatures. Furthermore, we demonstrate that an intervention with formoterol and budesonide is unable to completely normalized cannabisinduced responses. Collectively our data suggest that cannabis smoke exposure is not innocuous and may possess many of the deleterious properties of tobacco smoke, warranting additional studies to support public policy, government regulations, and individual user practices.

bioinformatics↗

bioRxiv2018

The impact of cigarette smoke exposure, and COPD or asthma status on ABC transporter gene expression in human airway epithelial cells

RationaleThe respiratory mucosa coordinates responses to infections, allergens, and exposures to air pollution. A relatively unexplored aspect of the respiratory mucosa are the expression and function of ATP Binding Cassette (ABC) transporters. ABC transporters are conserved in prokaryotes and eukaryotes, with humans expressing 48 transporters divided into 7 classes (ABCA, ABCB, ABCC, ABCD, ABDE, ABCF, and ABCG). Throughout the human body, ABC transporters regulate cAMP levels, chloride secretion, lipid transport, and anti-oxidant responses. A deeper exploration of the expression patterns of ABC transporters in the respiratory mucosa is warranted to determine their relevance in lung health and disease.\n\nMethodsWe used a bioinformatic approach complemented with in vitro experimental methods for validation of candidate ABC transporters. We analyzed the expression profiles of all 48 human ABC transporters in the respiratory mucosa using bronchial epithelial cell gene expression datasets available in NCBI GEO from well-characterized patient populations of healthy subjects and individuals that smoke cigarettes, or have been diagnosed with COPD or asthma. The Calu-3 airway epithelial cell line was used to interrogate selected results using a cigarette smoke extract exposure model.\n\nResultsUsing 9 distinct gene-expression datasets of primary human airway epithelial cells, we completed a focused analysis on 48 ABC transporters in samples from healthy subjects and individuals that smoke cigarettes, or have been diagnosed with COPD or asthma. In situ gene expression data demonstrate that ABC transporters are i) variably expressed in epithelial cells from different airway generations (top three expression levels - ABCA5, ABCA13, and ABCC5), ii) regulated by cigarette smoke exposure (ABCA13, ABCB6, ABCC1, and ABCC3), and iii) differentially expressed in individuals with COPD and asthma (ABCA13, ABCC1, ABCC2, ABCC9). An in vitro cell culture model of cigarette smoke exposure was able to recapitulate the in situ changes observed in cigarette smokers for ABCA13 and ABCC1.\n\nConclusionsOur in situ human gene expression data analysis reveals that ABC transporters are expressed throughout the airway generations in airway epithelial cells and can be modulated by environmental exposures important in chronic respiratory disease (e.g. cigarette smoking) and in individuals with chronic lung diseases (e.g. COPD or asthma). Our work highlights select ABC transporter candidates of interest and a relevant in vitro model that will enable a deeper understanding of the contribution of ABC transporters in the respiratory mucosa in lung health and disease.

bioinformatics↗