Azithromycin is used in long-term, low-dose treatment of airway diseases where airway wall remodelling is present. Since it improves total score symptom and respiratory function of such patients, we hypothesise that azithromycin's additional clinical benefits are due to an inhibition of airway smooth muscle cell (SMC) proliferation.Rabbit tracheal SMCs were treated with azithromycin (10 -5 to 10 -6 M) in the presence or absence of 10% fetal bovine serum (FBS). The proliferation was estimated using the Cell Titer 961 AQ ueous One Solution Assay (Promega, Madison, WI, USA). Cell viability was assessed with Trypan blue staining and flow cytometry after 7-aminoactinomycin D (7-AAD) staining. Induction of autophagy was studied by indirect immmunofluorescence and/or Western blotting with antibodies against human smooth muscle a-actin, beclin 1, light chain 3 and caspase 3. The involvement of the phosphoinositide 3-kinase pathway was investigated with the inhibitors LY294002 and wortmannin. Incubation with azithromycin for 72 h in the presence of FBS reduced SMC proliferation and viability in a dose-dependent manner. Azithromycin treatment was accompanied by the formation of cytoplasmic vacuoles, characteristic of autophagy. All these effects were reversible after azithromycin removal and prevented by the autophagy inhibitor, 3-methyladenine, or LY294002, but not by wortmannin.In conclusion, azithromycin reduces proliferation and causes autophagy of airway SMCs.KEYWORDS: Airway smooth muscle cells, autophagy, azithromycin, proliferation M acrolide antibiotics are widely used for the treatment of airway diseases. Lowdose, long-term macrolide therapy has been reported to be very effective in patients with chronic airway diseases, such as diffuse panbronchiolitis, chronic bronchitis and bronchial asthma [1][2][3][4][5]. In many of these diseases, airway wall remodelling is present. Airway wall remodelling includes thickening of the reticular membrane, proliferation of the smooth muscle cells (SMCs) and increase in both number and size of vessels [6,7].The improvement of pulmonary function, total score symptom and quality of life with macrolides is mainly attributed to their antimicrobial and anti-inflammatory activity. However, data available from studies on airway smooth muscle show an effect of macrolides on the contractility of airway smooth muscle. Specifically, erythromycin inhibits cholinergic neuroeffector transmission in the airways [8,9] and azithromycin has a direct relaxant effect on precontracted rabbit airway smooth muscle [10].It is possible that azithromycin has an additional effect in airway SMC proliferation. As has been shown previously, erythromycin inhibits hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium [11], roxithromycin inhibits proliferation of human coronary artery SMCs [12], and rapamycin and its analogue SAR943 inhibit proliferation of human epithelial and SMCs [13], while clarithromycin and azithromycin induce apoptosis of activated lymphocytes [14]. In o...

Clinical Microbiology and Infection xxx (xxxx) xxx Please cite this article as: Ptasinska A et al., Diagnostic accuracy of loop-mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations, Clinical Microbiology and Infection,

Conclusion: This study provides evidence that, in Caucasian patients with NSCLC, highly prevalent expression patterns of TAA genes, predominantly of overexpressed TAAs, do exist. This result implies that the combined use of these TAA could help in designing more effective NSCLC immunotherapeutic protocols.

Background Inflammatory bowel disease may arise with inadequate immune response to intestinal bacteria. NOD2 is an established gene in Crohn’s disease pathogenesis, with deleterious variation associated with reduced NFKB signaling. We hypothesized that deleterious variation across the NOD2 signaling pathway impacts on transcription. Methods Treatment-naïve pediatric inflammatory bowel disease patients had ileal biopsies for targeted autoimmune RNA-sequencing and blood for whole exome sequencing collected at diagnostic endoscopy. Utilizing GenePy, a per-individual, per-gene score, genes within the NOD signaling pathway were assigned a quantitative score representing total variant burden. Where multiple genes formed complexes, GenePy scores were summed to create a “complex” score. Normalized transcript expression of 95 genes within this pathway was retrieved. Regression analysis was performed to determine the impact of genomic variation on gene transcription. Results Thirty-nine patients were included. Limited clustering of patients based on NOD signaling transcripts was related to underlying genomic variation. Patients harboring deleterious variation in NOD2 had reduced NOD2 (β = -0.702, P = 4.3 × 10-5) and increased NFKBIA (β = 0.486, P = .001), reflecting reduced NFKB signal activation. Deleterious variation in the NOD2-RIPK2 complex was associated with increased NLRP3 (β = 0.8, P = 3.1475 × 10-8) and TXN (β = -0.417, P = 8.4 × 10-5) transcription, components of the NLRP3 inflammasome. Deleterious variation in the TAK1-TAB complex resulted in reduced MAPK14 transcription (β = -0.677, P = 1.7 × 10-5), a key signal transduction protein in the NOD2 signaling cascade and increased IFNA1 (β = 0.479, P = .001), indicating reduced transcription of NFKB activators and alternative interferon transcription in these patients. Conclusions Data integration identified perturbation of NOD2 signaling transcription correlated with genomic variation. A hypoimmune NFKB signaling transcription response was observed. Alternative inflammatory pathways were activated and may represent therapeutic targets in specific patients.

The macrolide antibiotic azithromycin has an antiproliferative and autophagic effect on rabbit tracheal smooth muscle cells (SMCs). The purpose of this study is to investigate the effect of azithromycin on human bronchial SMCs. Human bronchial SMCs were treated with azithromycin (10 À5 M) in the presence or absence of 10% fetal bovine serum (FBS). Cell number was estimated using the Cell Titer 96 AQ ueous One Solution Assay. Induction of autophagy was studied by observation of cell morphology in cells treated or not with the autophagy inhibitor, 3-methyladenine (3-MA), as well as by Lysotracker Red staining of lysosomes. Activation of apoptosis was assessed with flow cytometry after annexin staining. Incubation with azithromycin for 24, 48 or 72 h reduced viability in FBS-deprived cells, as well as cells cultured in FBS-containing medium. Azithromycin treatment resulted in the formation of cytoplasmic vacuoles that could not be prevented by 3-MA. Furthermore, 3-MA did not reverse the effect of azithromycin on the viability of SMCs. There was an increase in the number of lysosomes in cells treated with azithromycin. Finally, azithromycin increased the percentage of early apoptotic cells. In conclusion, azithromycin reduces the viability of human bronchial SMCs possibly by leading to apoptotic cell death.