Single-cell RNA sequencing of bronchoscopy specimens: development of a rapid minimal handling protocol.

Single-cell RNA sequencing (scRNA-seq) is an important tool for understanding disease pathophysiology, including airway diseases. Currently, the majority of scRNA-seq studies in airway diseases have used invasive methods (airway biopsy, surgical resection), which carry inherent risks and thus present a major limitation to scRNA-seq investigation of airway pathobiology. Bronchial brushing, where the airway mucosa is sampled using a cytological brush, is a viable, less invasive method of obtaining airway cells for scRNA-seq. Here we describe the development of a rapid and minimal handling protocol for preparing single-cell suspensions from bronchial brush specimens for scRNA-seq. Our optimized protocol maximizes cell recovery and cell quality and facilitates large-scale profiling of the airway transcriptome at single-cell resolution.

Post-COVID-19 dyspnoea and pulmonary imaging: a systematic review and meta-analysis.

BACKGROUND: A proportion of coronavirus disease 2019 (COVID-19) survivors experience persistent dyspnoea without measurable impairments in lung function. We performed a systematic review and meta-analysis to determine relationships between dyspnoea and imaging abnormalities over time in post-COVID-19 patients. METHODS: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we analysed studies published prior to 15 September 2022 and indexed by Google Scholar, PubMed and LitCOVID which assessed chest imaging in adults ≥3 months after COVID-19. Demographic, chest imaging, spirometric and post-COVID-19 symptom data were extracted. The relationships between imaging abnormalities and dyspnoea, sex and age were determined using a random effects model and meta-regression. RESULTS: 47 studies were included in the meta-analysis (n=3557). The most prevalent computed tomography (CT) imaging abnormality was ground-glass opacities (GGOs) (44.9% (95% CI 37.0-52.9%) at any follow-up time-point). Occurrence of reticulations significantly decreased between early and late follow-up (p=0.01). The prevalence of imaging abnormalities was related to the proportion of patients with dyspnoea (p=0.012). The proportion of females was negatively correlated with the presence of reticulations (p=0.001), bronchiectasis (p=0.001) and consolidations (p=0.025). Age was positively correlated with imaging abnormalities across all modalities (p=0.002) and imaging abnormalities present only on CT (p=0.001) (GGOs (p=0.004) and reticulations (p=0.001)). Spirometric values improved during follow-up but remained within the normal range at all time-points. CONCLUSIONS: Imaging abnormalities were common 3 months after COVID-19 and their occurrence was significantly related to the presence of dyspnoea. This suggests that CT imaging is a sensitive tool for detecting pulmonary abnormalities in patients with dyspnoea, even in the presence of normal spirometric measurements.

Dysbiosis of the Female Murine Gut Microbiome Exacerbates Neutrophil-mediated Vascular Allograft Injury by Affecting Immunoregulation by Acetate.

BACKGROUND: The gut microbiota affects immune responses that cause organ transplant rejection, but the mechanisms by which this occurs remain poorly understood. METHODS: We have examined, in a murine model, how disruption of the gut microbiota with antibiotics early in life alters this microbial community later in life to affect immune responses that injure vascular allografts. RESULTS: Analysis of 16S rRNA and whole genome sequencing of the gut microbiota demonstrated that early life disruption of this microbial community with antibiotics caused a reduction in taxa and enzymatic genes involved in the synthesis of acetate, an immunoregulatory metabolite in mice and humans. When allograft vascular injury was examined, early life disruption of the gut microbiota increased neutrophil accumulation and related medial injury of transplanted arteries. Normalizing the gut microbiota by co-housing and oral administration of acetate prevented neutrophil-mediated vascular allograft injury. CONCLUSIONS: Dysbiosis of the gut microbiome that reduces its production of the immunoregulatory metabolite acetate exacerbates neutrophil-mediated allograft vascular injury.