Nasal Brushing Research Articles

A new software for automated analysis of respiratory tract ciliary epithelium movement for the diagnosis of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare hereditary disease. In this ciliopathy, the disturbed structure and motility of the ciliary epithelium negatively affects the ciliary function and leads to prominent decrease or absence of mucociliary clearance. The European guidelines recommend analyzing the cilia beat frequency (СBF) in a native preparation or in ALI culture using light microscopy as one of the methods to confirm the diagnosis of PCD.The aim of this project was to create software for automated analysis of the movement/beating of the ciliary epithelium of the respiratory tract for the diagnosis of primary ciliary dyskinesia using digital high-speed video microscopy in vivo and in vitro.Methods. Five healthy donors and 10 patients with suspected PCD underwent nasal epithelial brush biopsy. The preparations were examined with a transmission electron microscope in vivo. Epithelial cells were also isolated from the nasal biopsy specimen, and ciliogenesis of these cells was performed by ALI-culturing, followed by digital high-speed video microscopy and assessment of the number of active cells and cilia beating frequency. The resulting video images were used to create the software.Results. Software for determination of ciliary epithelium beat frequency in primary ciliary dyskinesia (PCD HighSpeed Video Microscopy Analysis – PCD HSVMA) was created to optimize the diagnosis of PCD by light microscopy (software registration number 2023687245). The software is designed to count the number of active cells of ciliary epithelium and CBF (Hz) by digital high-speed video microscopy in vivo and in vitro in ALI-culture. PCD HSVMA software features storage of patient data, display of heat map, formation of a large server database of patients and video files, building of color and static histograms, processing of several areas in one video. Our software has a number of advantages over CiliarMove and Cilialyzer and has high correlation of CFB (Hz) estimation with these products.Conclusion. Our software can be used for improvement of PCD diagnostics in laboratories of healthcare institutions, in scientific institutions and can be included in specialist educational programs for laboratory doctors, pediatricians, general practitioners, pulmonologists, diagnosticians (endoscopists).

Differential modulation of allergic rhinitis nasal transcriptome by dupilumab and allergy immunotherapy.

Nasal epithelial cells are important regulators of barrier function and immune signaling; however, in allergic rhinitis (AR) these functions can be disrupted by inflammatory mediators. We aimed to better discern AR disease mechanismsusing transcriptome data from nasal brushing samples from individuals with and without AR. Data were drawn from a feasibility study of individuals with and without AR to Timothy grass and from a clinical trial evaluating 16 weeks of treatment with the following: dupilumab, a monoclonal antibody that binds interleukin (IL)-4Rα and inhibits type 2 inflammation by blocking signaling of both IL-4/IL-13; subcutaneous immunotherapy with Timothy grass (SCIT), which inhibits allergic responses through pleiotropic effects; SCIT + dupilumab; or placebo. Using nasal brushing samples from these studies, we defined distinct gene signatures in nasal tissue of AR disease and after nasal allergen challenge (NAC) and assessed how these signatures were modulated by study drug(s). Treatment with dupilumab (normalized enrichment score [NES] = -1.73, p = .002) or SCIT + dupilumab (NES = -2.55, p < .001), but not SCIT alone (NES = +1.16, p = .107), significantly repressed the AR disease signature. Dupilumab (NES = -2.55, p < .001), SCIT (NES = -2.99, p < .001), and SCIT + dupilumab (NES = -3.15, p < .001) all repressed the NAC gene signature. These results demonstrate type 2 inflammation is an important contributor to the pathophysiology of AR disease and that inhibition of the type 2 pathway with dupilumab may normalize nasal tissue gene expression.

The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression.

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

The IL-31/CysLT2R axis is associated with itching in patients with allergic rhinitis.

Itching is a troublesome symptom that disturbs patients with allergic rhinitis (AR). The molecular mechanisms underlying itching in AR need to be further illuminated. The aim of this study was to investigate the role of epithelial cell-derived interleukin-31 (IL-31) in nasal itching in AR. A total of 33 patients and 20 healthy control subjects were enrolled in this prospective study. The disease severity of patients with AR was assessed by the total visual analog scale score. The levels of IL-31, cysteinyl leukotriene receptor 1 (CysLT1R), and CysLT2R in the nasal brush specimens from the enrolled subjects were measured by quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemical staining. The expression of CysLT2R in a human nasal epithelial cell line (HNEpC) was assessed by immunofluorescence staining. Compared with the control subjects, the protein and mRNA levels of IL-31 and CysLT2R were significantly increased in patients with AR. Higher levels of IL-31 and CysLT2R in nasal epithelial cells were associated with itching but not nasal congestion, rhinorrhea, or sneezing in AR. A significant relationship was found between IL-31 and CysLT2R in nasal epithelial cells, with a correlation coefficient of 0.93. Furthermore, RT-PCR and immunofluorescence staining revealed that IL-31 directly induced CysLT2R expression in HNEpCs. Nasal steroid treatment inhibited IL-31 and CysLT2R expression in 13 patients with AR in vivo. Nasal epithelial cell-derived IL-31 might be associated with itching symptoms via CysLT2R in AR.

17q21 Variants Disturb Mucosal Host Defense in Childhood Asthma.

Rationale: The strongest genetic risk factor for childhood-onset asthma, the 17q21 locus, is associated with increased viral susceptibility and disease-promoting processes.Objectives: To identify biological targets underlying the escalated viral susceptibility associated with the clinical phenotype mediated by the 17q21 locus.Methods: Genome-wide transcriptome analysis of nasal brush samples from 261 children (78 healthy, 79 with wheezing at preschool age, 104 asthmatic) within the ALLIANCE (All-Age-Asthma) cohort, with a median age of 10.0 (range, 1.0-20.0) years, was conducted to explore the impact of their 17q21 genotype (SNP rs72163891). Concurrently, nasal secretions from the same patients and visits were collected, and high-sensitivity mesoscale technology was employed to measure IFN protein levels.Measurements and Main Results: This study revealed that the 17q21 risk allele induces a genotype- and asthma/wheeze phenotype-dependent enhancement of mucosal GSDMB expression as the only relevant 17q21-encoded gene in children with preschool wheeze. Increased GSDMB expression correlated with the activation of a type-1 proinflammatory, cell-lytic immune, and natural killer signature, encompassing key genes linked to an IFN type-2-signature (IFNG, CXCL9, CXCL10, KLRC1, CD8A, GZMA). Conversely, there was a reduction in IFN type 1 and type 3 expression signatures at the mRNA and protein levels.Conclusions: This study demonstrates a novel disease-driving mechanism induced by the 17q21 risk allele. Increased mucosal GSDMB expression is associated with a cell-lytic immune response coupled with compromised airway immunocompetence. These findings suggest that GSDMB-related airway cell death and perturbations in the mucosal IFN signature account for the increased vulnerability of 17q21 risk allele carriers to respiratory viral infections during early life, opening new options for future biological interventions.The All-Age-Asthma (ALLIANCE) cohort is registered at www.clinicaltrials.gov (pediatric arm, NCT02496468).

An epithelial gene signature of trans-IL-6 signaling defines a subgroup of type 2-low asthma

BackgroundAsthma is stratified into type 2-high and type 2-low inflammatory phenotypes. Limited success has been achieved in developing drugs that target type 2-low inflammation. Previous studies have linked IL-6 signaling to severe asthma. IL-6 cooperates with soluble-IL-6Rα to activate cell signaling in airway epithelium.ObjectiveWe sought to study the role of sIL-6Rα amplified IL-6 signaling in airway epithelium and to develop an IL-6+ sIL-6Rα gene signature that may be used to select asthma patients who potentially respond to anti-IL-6 therapy.MethodsHuman airway epithelial cells were stimulated with combinations of IL-6, sIL-6Rα, and inhibitors, sgp130 (Olamkicept), and anti-IL-6R (Tocilizumab), to assess effects on pathway activation, epithelial barrier integrity, and gene expression. A gene signature was generated to identify IL-6 high patients using bronchial biopsies and nasal brushes.ResultsSoluble-IL-6Rα amplified the activation of the IL-6 pathway, shown by the increase of STAT3 phosphorylation and stronger gene induction in airway epithelial cells compared to IL-6 alone. Olamkicept and Tocilizumab inhibited the effect of IL-6 + sIL-6Rα on gene expression. We developed an IL-6 + sIL-6Rα gene signature and observed enrichment of this signature in bronchial biopsies but not nasal brushes from asthma patients compared to healthy controls. An IL-6 + sIL-6Rα gene signature score was associated with lower levels of sputum eosinophils in asthma.ConclusionsIL-6Rα amplifies IL-6 signaling in bronchial epithelial cells. Higher local airway IL-6 + sIL-6Rα signaling is observed in asthma patients with low sputum eosinophils.

Outbreak of tauopathy in the olfactory system of PS19 mice: an early marker for Alzheimer’s disease diagnosis?

AbstractBackgroundClinical data indicate that impairment of olfactory function is an early indicator of neurodegenerative diseases, including Alzheimer’s disease (AD)(Attems et al., 2014). Our hypothesis is that typical AD lesions could appear in olfactory neurons from the olfactory epithelium (OE). The olfactory epithelium can be easily collected in human individuals (e.g., by nasal brushing), which provides an important tool to investigate the pathology in patients. Here, we address the question of the occurrence of tauopathy in the OE of transgenic mice and its correlation with pathology in the central nervous system (CNS). We use Tau P301S mice (PS19 mice), a tauopathy mouse model. These mice express human tau protein (1N4R) carrying the P301S mutation. They develop neurofibrillary tangle‐like inclusions in the cortex and hippocampus, leading to progressive neurodegeneration at eight months (Yoshiyama et al., 2007).MethodMouse samples were collected from heterozygous PS19 and wild‐type (WT) mice. Immunohistochemical staining (DAB) was used to demonstrate the presence of hyperphosphorylated human tau protein (hTau). We investigated regions of the olfactory system to correlate with data obtained in CNS regions (e.g., hippocampal formation, entorhinal cortex). Mouse heads decalcification was also implemented to preserve the olfactory epithelium structure. Western blots were performed to monitor tau levels and tau phosphorylation pattern.ResultThe hyperphosphorylated human tau protein (Ser202, Thr205) is only detected in PS19 mice sections while the endogenous murine tau protein is expressed both in WT and PS19 mice. The localization and expression of these proteins vary according to the region (OE or CNS subregions), the age (3, 6 or 9 months), and the genotype of the mice (WT or heterozygous PS19). It also seems that tau lesions may spread between brain regions that are directly connected, notably through the olfactory pathway.ConclusionIn PS19 mice, a pathological phosphorylation pattern peaked in the OE prior to vulnerable CNS regions or olfactory bulbs. We hypothesize that tau lesions appearing early in the OE may spread in the olfactory bulb and finally reach the entorhinal cortex and piriform cortex, highlighting the significance of the olfactory system investigation as an early diagnosis tool.

Effect of Body Weight on Response to Nasal Glucocorticoid Treatment in Allergic Rhinitis.

Allergic rhinitis is among the most common chronic diseases in the world. Obesity can lead to a chronic systemic inflammatory process. In this study, we evaluated the effects of body weight on the response to treatment of allergic rhinitis with nasal corticosteroids. Two groups of patients diagnosed with allergic rhinitis were compared: one composed of obese patients and one composed of normal weight patients. Nasal endoscopy, peak nasal inspiratory flow, quality of life, the VAS, SNOT22, and NOSE-5 questionnaires, and the concentration of nasal cytokines (INF-γ, TNF-ᾳ, IL-4, IL-5, IL-6, and IL-10) through nasal brushing were evaluated before and after treatment with 400 mcg/day nasal beclomethasone. No differences were identified between the groups in nasal endoscopy, peak nasal inspiratory flow, the VAS, SNOT22, and NOSE-5 questionnaires, or in the cytokines INF-γ, TNF-ᾳ, IL-4, IL-5, IL-6, and IL-10 prior to nasal corticosteroid treatment. Both groups showed improvement in the VAS, SNOT-22, and NOSE-5 questionnaires and an increase in peak nasal inspiratory volumes after treatment. In the eutrophic group, there was an increase in INF-γ and IL-5 after treatment. When comparing the variation in cytokines before and after treatment between groups, IL-10 was the cytokine that showed altered behavior dependent on weight. Obesity did not seem to impact nasal symptoms and physiology and presented a similar clinical response to treatment with nasal corticosteroids to normal weight patients. However, obese patients had an impaired anti-inflammatory response during treatment with nasal corticosteroids.

Investigation of nasal epithelial cells as a surrogate for bronchial epithelial cells in the research of equine asthma.

Equine asthma, previously known as Recurrent Airway Obstruction (RAO) or Inflammatory Airway Disease (IAD), is an often-debilitating condition that may severely affect both performance and quality of life. Research is hindered by the low sample numbers of subjects recruited to studies, a consequence in part of the invasive nature of the sampling methods of bronchial brushing and biopsy. We present an alternative method of sampling equine airway epithelial cells, the 'nasal brush method' (NBM). Obtained by light brushing of the ventral meatus whilst the horse is under standing sedation, these cells express the same markers of differentiation as their deeper counterparts. Grown as 3-D spheroids or as air-liquid interface cultures, nasal epithelial cells are responsive to the inflammatory cytokine interleukin-13. This may be attenuated by modulation of the Notch signalling pathway using the gamma-secretase inhibitor Semagecestat; a previously unreported finding that cements the link between equine and human asthma research and strengthens the case for a One Health approach in researching asthma pathophysiology and therapeutic intervention.

Mapping the Most Common Founder Variant in RSPH9 That Causes Primary Ciliary Dyskinesia in Multiple Consanguineous Families of Bedouin Arabs.

Primary ciliary dyskinesia (PCD) is a congenital thoracic disorder caused by dysfunction of motile cilia, resulting in insufficient mucociliary clearance of the lungs. The overall aim of this study is to identify causative defective genes in PCD-affected individuals in the Kuwaiti population. A cohort of multiple consanguineous PCD families was identified from Kuwaiti patients and genomic DNA from the family members was isolated using standard procedures. The DNA samples from all affected individuals were analyzed by whole exome sequencing (WES). Transmission electron microscopy (TEM) and immunofluorescent analysis (IF) were performed on samples obtained by nasal brushings to identify specific structural abnormalities within ciliated cells. Here, we present six multiplex families with 11 patients who all presented with typical PCD symptoms. Ten out of eleven patients inherited a 3 bp homozygous deletion of GAA in RSPH9, whereas the eleventh patients inherited this variant in trans with a frameshift deletion in RSPH9. Genetic results were confirmed by segregation analysis. The in-frame deletion of GAA in RSPH9 has previously been published as pathogenic in both annotated RSPH9 transcript variants (1 and 2). In contrast, the previously unpublished RSPH9 frameshift deletion identified in KU-15.IV2 impacts only RSPH9 transcript variant two. Regarding all 11 PCD individuals analyzed, IF results demonstrated absence of RSPH9 protein and TEM analysis showed the typical findings in RSPH9 mutant individuals. We present the largest cohort of PCD individuals affected by the founder in-frame deletion GAA in RSPH9. This founder variant is the most common PCD-causing variant in Bedouin Arabs in Kuwait.

Integrated study of systemic and local airway transcriptomes in asthma reveals causal mediation of systemic effects by airway key drivers

BackgroundSystemic and local profiles have each been associated with asthma, but parsing causal relationships between system-wide and airway-specific processes can be challenging. We sought to investigate systemic and airway processes in asthma and their causal relationships.MethodsThree hundred forty-one participants with persistent asthma and non-asthmatic controls were recruited and underwent peripheral blood mononuclear cell (PBMC) collection and nasal brushing. Transcriptome-wide RNA sequencing of the PBMC and nasal samples and a series of analyses were then performed using a discovery and independent test set approach at each step to ensure rigor. Analytic steps included differential expression analyses, coexpression and probabilistic causal (Bayesian) network constructions, key driver analyses, and causal mediation models.ResultsAmong the 341 participants, the median age was 13 years (IQR = 10–16), 164 (48%) were female, and 200 (58.7%) had persistent asthma with mean Asthma Control Test (ACT) score 16.6 (SD = 4.2). PBMC genes associated with asthma were enriched in co-expression modules for NK cell-mediated cytotoxicity (fold enrichment = 4.5, FDR = 6.47 × 10−32) and interleukin production (fold enrichment = 2.0, FDR = 1.01 × 10−15). Probabilistic causal network and key driver analyses identified NK cell granule protein (NKG7, fold change = 22.7, FDR = 1.02 × 10−31) and perforin (PRF1, fold change = 14.9, FDR = 1.31 × 10−22) as key drivers predicted to causally regulate PBMC asthma modules. Nasal genes associated with asthma were enriched in the tricarboxylic acid (TCA) cycle module (fold enrichment = 7.5 FDR = 5.09 × 10−107), with network analyses identifying G3BP stress granule assembly factor 1 (G3BP1, fold change = 9.1 FDR = 2.77 × 10−5) and InaD-like protein (INADL, fold change = 5.3 FDR = 2.98 × 10−9) as nasal key drivers. Causal mediation analyses revealed that associations between PBMC key drivers and asthma are causally mediated by nasal key drivers (FDR = 0.0076 to 0.015).ConclusionsIntegrated study of the systemic and airway transcriptomes in a well-phenotyped asthma cohort identified causal key drivers of asthma among PBMC and nasal transcripts. Associations between PBMC key drivers and asthma are causally mediated by nasal key drivers.

Temporal Stability of Ciliary Beating Post Nasal Brushing, Modulated by Storage Temperature.

Primary ciliary dyskinesia is a heterogeneous, inherited motile ciliopathy in which respiratory cilia beat abnormally, and some ultrastructural ciliary defects and specific genetic mutations have been associated with particular ciliary beating alterations. Ciliary beating can be evaluated using digital high-speed videomicroscopy (DHSV). However, normal reference values, essential to assess ciliary beating in patients referred for a PCD diagnostic, vary between centres, as minor variations in protocols might influence ciliary beating. Consequently, establishment of normal values is essential for each PCD diagnostic centre. We aimed to evaluate whether delay after sampling, and temperature for conservation of respiratory ciliated samples, might modify assessments of ciliary beating. In total, 37 healthy nasal brushing samples of respiratory ciliated epithelia were collected. Video sequences were recorded at 37 °C immediately using DHSV. Then, the samples were divided and conserved at 4 °C or at room temperature (RT). Ciliated beating edges were then recorded at 37 °C, at 3 h and at 9 h post sampling. In six samples, recordings were continued up to 72 h after sampling. Ciliary beating was assessed manually by ciliary beat frequency (CBFM) and ciliary beat pattern (CBP). A semi-automatic software was used for quantitative analysis. Both CBF and CBP evaluated manually and by a semi-automated method were stable 9 h after sampling. CBFM was higher when evaluated using samples stored at RT than at 4 °C. CBP and the semi-automated evaluation of ciliary beating were not affected by storage temperature. When establishing normal references values, ciliary beating can be evaluated at 37 °C up to 9 h after nasal brushing, but the storage temperature modifies ciliary beating and needs to be controlled.

Theratyping cystic fibrosis patients to guide elexacaftor/tezacaftor/ivacaftor out-of-label prescription.

Around 20% of people with cystic fibrosis (pwCF) do not have access to the triple combination elexacaftor/tezacaftor/ivacaftor (ETI) in Europe because they do not carry the F508del allele on the CF transmembrane conductance regulator (CFTR) gene. Considering that pwCF carrying rare variants may benefit from ETI, including variants already validated by the US Food and Drug Administration (FDA), a compassionate use programme was launched in France. PwCF were invited to undergo a nasal brushing to investigate whether the pharmacological rescue of CFTR activity by ETI in human nasal epithelial cell (HNEC) cultures was predictive of the clinical response. CFTR activity correction was studied by short-circuit current in HNEC cultures at basal state (dimethyl sulfoxide (DMSO)) and after ETI incubation and expressed as percentage of normal (wild-type (WT)) CFTR activity after sequential addition of forskolin and Inh-172 (ΔI ETI/DMSO%WT). 11 pwCF carried variants eligible for ETI according to the FDA label and 28 carried variants not listed by the FDA. ETI significantly increased CFTR activity of FDA-approved CFTR variants (I601F, G85E, S492F, M1101K, R347P, R74W;V201M;D1270N and H1085R). We point out ETI correction of non-FDA-approved variants, including N1303K, R334W, R1066C, Q552P and terminal splicing variants (4374+1G>A and 4096-3C>G). ΔI ETI/DMSO%WT was significantly correlated to change in percentage predicted forced expiratory volume in 1 s and sweat chloride concentration (p<0.0001 for both). G85E, R74W;V201M;D1270N, Q552P and M1101K were rescued more efficiently by other CFTR modulator combinations than ETI. Primary nasal epithelial cells hold promise for expanding the prescription of CFTR modulators in pwCF carrying rare mutants. Additional variants should be discussed for ETI indication.

A bronchial gene signature specific for severe COPD that is retained in the nose.

A subset of COPD patients develops advanced disease with severe airflow obstruction, hyperinflation and extensive emphysema. We propose that the pathogenesis in these patients differs from mild-moderate COPD and is reflected by bronchial gene expression. The aim of the present study was to identify a unique bronchial epithelial gene signature for severe COPD patients. We obtained RNA sequencing data from bronchial brushes from 123 ex-smokers with severe COPD, 23 with mild-moderate COPD and 23 non-COPD controls. We identified genes specific to severe COPD by comparing severe COPD to non-COPD controls, followed by removing genes that were also differentially expressed between mild-moderate COPD and non-COPD controls. Next, we performed a pathway analysis on these genes and evaluated whether this signature is retained in matched nasal brushings. We identified 219 genes uniquely differentially expressed in severe COPD. Interaction network analysis identified VEGFA and FN1 as the key genes with the most interactions. Genes were involved in extracellular matrix regulation, collagen binding and the immune response. Of interest were 10 genes (VEGFA, DCN, SPARC, COL6A2, MGP, CYR61, ANXA6, LGALS1, C1QA and C1QB) directly connected to fibronectin 1 (FN1). Most of these genes were lower expressed in severe COPD and showed the same effect in nasal brushings. We found a unique severe COPD bronchial gene signature with key roles for VEGFA and FN1, which was retained in the upper airways. This supports the hypothesis that severe COPD, at least partly, comprises a different pathology and supports the potential for biomarker development based on nasal brushes in COPD.

TP53 mutation prevalence in normal airway epithelium as a biomarker for lung cancer risk

BackgroundThere is a need for biomarkers that improve accuracy compared with current demographic risk indices to detect individuals at the highest lung cancer risk. Improved risk determination will enable more effective lung cancer screening and better stratification of lung nodules into high or low-risk category. We previously reported discovery of a biomarker for lung cancer risk characterized by increased prevalence of TP53 somatic mutations in airway epithelial cells (AEC). Here we present results from a validation study in an independent retrospective case–control cohort.MethodsTargeted next generation sequencing was used to identify mutations within three TP53 exons spanning 193 base pairs in AEC genomic DNA.ResultsTP53 mutation prevalence was associated with cancer status (P < 0.001). The lung cancer detection receiver operator characteristic (ROC) area under the curve (AUC) for the TP53 biomarker was 0.845 (95% confidence limits 0.749–0.942). In contrast, TP53 mutation prevalence was not significantly associated with age or smoking pack-years. The combination of TP53 mutation prevalence with PLCOM2012 risk score had an ROC AUC of 0.916 (0.846–0.986) and this was significantly higher than that for either factor alone (P < 0.03).ConclusionsThese results support the validity of the TP53 mutation prevalence biomarker and justify taking additional steps to assess this biomarker in AEC specimens from a prospective cohort and in matched nasal brushing specimens as a potential non-invasive surrogate specimen.

Ultrastructure for the diagnosis of primary ciliary dyskinesia in South Africa, a resource-limited setting.

International guidelines recommend a multi-faceted approach for successful diagnoses of primary ciliary dyskinesia (PCD). In the absence of a gold standard test, a combination of genetic testing/microscopic analysis of structure and function/nasal nitric oxide measurement is used. In resource-limited settings, often none of the above tests are available, and in South Africa, only transmission electron microscopy (TEM) is available in central anatomical pathology departments. The aim of this study was to describe the clinical and ultrastructural findings of suspected PCD cases managed by pediatric pulmonologists at a tertiary-level state funded hospital in Johannesburg. Nasal brushings were taken from 14 children with chronic respiratory symptoms in keeping with a PCD phenotype. Ultrastructural analysis in accordance with the international consensus guidelines for TEM-PCD diagnostic reporting was undertaken. TEM observations confirmed 43% (6) of the clinically-suspected cases (hallmark ultrastructural defects in the dynein arms of the outer doublets), whilst 57% (8) required another PCD testing modality to support ultrastructural observations. Of these, 25% (2) had neither ultrastructural defects nor did they present with bronchiectasis. Of the remaining cases, 83% (5) had very few ciliated cells (all of which were sparsely ciliated), together with goblet cell hyperplasia. There was the apparent absence of ciliary rootlets in 17% (1) case. In resource-limited settings in which TEM is the only available testing modality, confirmatory and probable diagnoses of PCD can be made to facilitate early initiation of treatment of children with chronic respiratory symptoms.

Nasal and blood transcriptomic pathways underpinning the clinical response to grass pollen immunotherapy

Allergen immunotherapy (AIT) is a well-established disease-modifying therapy for allergic rhinitis, yet the fundamental mechanisms underlying its clinical effect remain inadequately understood. Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy was a randomized, double-blind, placebo-controlled trial of individuals allergic to timothy grass who received 2 years of placebo (n= 30), subcutaneous immunotherapy (SCIT) (n= 27), or sublingual immunotherapy (SLIT) (n= 27) and were then followed for 1 additional year. We used yearly biospecimens from the Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy study to identify molecular mechanisms of response. We used longitudinal transcriptomic profiling of nasal brush and PBMC samples after allergen provocation to uncover airway and systemic expression pathways mediating responsiveness to AIT. ClinicalTrials.gov Identifier: NCT01335139, EudraCT Number: 2010-023536-16. SCIT and SLIT demonstrated similar changes in gene module expression over time. In nasal samples, alterations included downregulation of pathways of mucus hypersecretion, leukocyte migration/activation, and endoplasmic reticulum stress (log2 fold changes -0.133 to -0.640, false discovery rates [FDRs] <0.05). We observed upregulation of modules related to epithelial development, junction formation, and lipid metabolism (log2 fold changes 0.104 to 0.393, FDRs <0.05). In PBMCs, modules related to cellular stress response and type 2 cytokine signaling were reduced by immunotherapy (log2 fold changes -0.611 to -0.828, FDRs <0.05). Expression of these modules was also significantly associated with both Total Nasal Symptom Score and peak nasal inspiratory flow, indicating important links between treatment, module expression, and allergen response. Our results identify specific molecular responses of the nasal airway impacting barrier function, leukocyte migration activation, and mucus secretion that are affected by both SCIT and SLIT, offering potential targets to guide novel strategies for AIT.

Effect of Dupilumab on Type 2 Biomarkers in Chronic Rhinosinusitis With Nasal Polyps: SINUS-52 Study Results.

Chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and non-steroidal anti-inflammatory drug-exacerbated respiratory disease (NSAID-ERD) are frequent coexisting conditions and share type 2 inflammatory pathophysiology, with interleukin (IL)-4 and IL-13 as key cytokines. Dupilumab is a monoclonal antibody that blocks the shared receptor for IL-4 and IL-13. The objective of this analysis was to evaluate dupilumab's effect on type 2 inflammation biomarkers in patients with CRSwNP with/without coexisting asthma or NSAID-ERD from the SINUS-52 (NCT02898454) study. Patients received treatment with dupilumab or placebo for 52 weeks. Blood and urinary biomarkers were evaluated through 52 weeks, and nasal secretions and mucosa brushings through 24 weeks. Of 447 patients, 60% had coexisting asthma and 27% had coexisting NSAID-ERD. At baseline, blood eotaxin-3, eosinophils, and periostin, nasal secretion eotaxin-3, and urinary leukotriene E4 were significantly higher in patients with coexisting NSAID-ERD than without. Dupilumab reduced eotaxin-3, thymus and activation-regulated chemokine, periostin, and total immunoglobulin E in blood, eotaxin-3, periostin, IL-5, and eosinophil cationic protein in nasal secretions, and leukotriene E4 in urine. Reductions were generally similar or greater in the subgroups with asthma and NSAID-ERD than without. Dupilumab also reduced MUC5AC and mast cell counts in nasal mucosa brushings. Dupilumab reduced local and systemic type 2 inflammatory biomarkers in patients with CRSwNP, including mast cells in nasal mucosa and cysteinyl leukotrienes in urine. These findings provide insight into the processes driving CRSwNP and the mechanisms of dupilumab's therapeutic effects. SINUS-52 https://www.clinicaltrials.gov/ct2/show/NCT02898454. NCT02898454.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air-liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA.

Combining skin and olfactory α-synuclein seed amplification assays (SAA)—towards biomarker-driven phenotyping in synucleinopathies

Seed amplification assays (SAA) are becoming commonly used in synucleinopathies to detect α-synuclein aggregates. Studies in Parkinson’s disease (PD) and isolated REM-sleep behavior disorder (iRBD) have shown a considerably lower sensitivity in the olfactory epithelium than in CSF or skin. To get an insight into α-synuclein (α-syn) distribution within the nervous system and reasons for low sensitivity, we compared SAA assessment of nasal brushings and skin biopsies in PD (n = 27) and iRBD patients (n = 18) and unaffected controls (n = 30). α-syn misfolding was overall found less commonly in the olfactory epithelium than in the skin, which could be partially explained by the nasal brushing matrix exerting an inhibitory effect on aggregation. Importantly, the α-syn distribution was not uniform: there was a higher deposition of misfolded α-syn across all sampled tissues in the iRBD cohort compared to PD (supporting the notion of RBD as a marker of a more malignant subtype of synucleinopathy) and in a subgroup of PD patients, misfolded α-syn was detectable only in the olfactory epithelium, suggestive of the recently proposed brain-first PD subtype. Assaying α-syn of diverse origins, such as olfactory (part of the central nervous system) and skin (peripheral nervous system), could increase diagnostic accuracy and allow better stratification of patients.