Comparing Cytology Brushes for Optimal Human Nasal Epithelial Cell Collection: Implications for Airway Disease Diagnosis and Research.

Primary nasal epithelial cells and culture models are used as important diagnostic, research and drug development tools for several airway diseases. Various instruments have been used for the collection of human nasal epithelial (HNE) cells but no global consensus yet exists regarding the optimal tool. This study compares the efficiency of two cytology brushes (Olympus (2 mm diameter) and Endoscan (8 mm diameter)) in collecting HNE cells. The study involved two phases, with phase one comparing the yield, morphology and cilia beat frequency (CBF) of cells collected from paediatric participants using each of the two brushes. Phase two compared nasal brushing under general anaesthetic and in the awake state, across a wide age range, via the retrospective audit of the use of the Endoscan brush in 145 participants. Results indicated no significant difference in CBF measurements between the two brushes, suggesting that the choice of brush does not compromise diagnostic accuracy. However, the Endoscan brush collected significantly more total and live cells than the Olympus brush, making it a more efficient option. Importantly, the Endoscan brush is more cost-effective, with a notable price difference between the two brushes.

Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.

BACKGROUND: Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation. METHODS: Nasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free "dual-SMAD inhibition" (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult™-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALI and SMADiALI under basal and CFTR corrector treated (VX-809) conditions. RESULTS: No significant difference in the structural morphology or baseline global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALI compare to their CRCALI counterparts. CONCLUSION: Alteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges.

Lack of association of the CD14/C -- 159T polymorphism with susceptibility and progression parameters in Turkish multiple sclerosis patients.

Soluble (s) CD14, being a receptor for lipopolysaccharides (LPSs) may inhibit LPS-triggered apoptosis and T lymphocyte proliferation. C to T exchange at position -159 in the promoter region of the CD14 gene might lead to higher sCD14 levels. Limited number of groups have studied whether these polymorphisms might influence the development of organ specific autoimmunity and whether higher CD14 levels are associated with increased levels of cytokines trigerring inflammatory processes. However their data contradict each other. In this study serum levels of sCD14 based on ELISA were measured in 77 treatment-naive patients and in 67 healthy controls. As the C-159T proximal promoter region regulates sCD14 levels, we investigated whether C-159T polymorphism is related to progression index in 250 MS patients vs. 183 healthy controls. CD14 polymorphism frequency between the healthy controls and the MS patients were not significantly different. While TT genotype of MS patients demonstrated significantly lower sCD14 levels compared to CC genotype; this difference was not reflected on the disease progression index. Our study that extends the prior data of previous studies reflects that sCD14 do not appear to be a solely prominent element of innate immunity in MS.

The role of SPINK5 in asthma related physiological events in the airway epithelium.

BACKGROUND: Genetic studies have shown that variants in SPINK5 may be associated with atopic diseases and asthma. However, the functional role of SPINK5 protein in asthma has not been elucidated. OBJECTIVES: To determine the effects of SPINK5 on asthma related physiological events such as apoptosis, mucus and cytokine production by epithelial cells. METHODS: A549 cells were transfected with SPINK5 expression vector and stimulated with increasing doses of hydrogen peroxide and neutrophil elastase (NE) for measurement of cell viability or apoptosis and analysis of mucus production. Cell viability was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide) assay and apoptosis by Annexin V/PI staining. Levels of IL-4, IL-6, IL-8, IL-12, IL-13, IFNγ, IL-1ß and RANTES were determined by ELISA in cell culture supernatants. Mucus production was determined by RT-PCR of the MUC5AC gene and PAS staining in NE treated cells. RESULTS: Epithelial cells transfected with SPINK5 expression vector produced more IL-6, IL-8 and RANTES compared to non-transfected cells (p < 0.001, p = 0.003, p < 0.001, respectively). Even though cells transfected with SPINK5 vector displayed significantly higher cell death, we have not observed any clear effect of SPINK5 on apoptosis. PAS staining showed that SPINK5 slightly decreased the mucin production induced by neutrophil elastase in A549 cells. However, SPINK5 had no effect on MUC5AC transcription. CONCLUSION: SPINK5 is an important molecule in asthma. Its role extends beyond its well known protease inhibitor properties.