Circulating calprotectin as a potential biomarker of persistent olfactory dysfunctions in Post-COVID-19 patients.

BACKGROUND: This longitudinal prospective study aims to investigate the potential of circulating calprotectin (cCLP) as a biomarker in persistent olfactory dysfunctions following COVID-19 infection. METHODS: Thirty-six patients with persistent hyposmia or anosmia post COVID-19 were enrolled (HT0) and re-evaluated after three months of olfactory training (HT1). Two control groups included 18 subjects without olfactory defects post COVID-19 (CG1) and 18 healthy individuals (CG2). Nasal brushing of the olfactory cleft and blood collection were performed to assess circulating calprotectin levels. RESULTS: Higher calprotectin levels were observed in serum and nasal supernatant of hyposmic patients (HT0) compared to control groups (CG1 and CG2). Post-olfactory training (HT1), olfactory function improved significantly, paralleled by decreased calprotectin levels in serum and nasal samples. Circulating calprotectin holds potential as a biomarker in persistent olfactory dysfunctions after COVID-19. The decrease in calprotectin levels post-olfactory training implies a role in monitoring and evaluating treatment responses. DISCUSSION AND CONCLUSIONS: These findings contribute to the growing literature on potential biomarkers in post-COVID-19 olfactory dysfunctions and underscore the importance of investigating novel biomarkers for personalized patient management. Nevertheless, further studies are needed to validate the application of calprotectin assay in nasal diseases and its correlation with nasal cytology.

Culture expansion of primary human nasal epithelial cells (NEC) isolated with a nasal scraping spoon.

OBJECTIVE: To obtain high-purity nasal epithelial cells (NEC) while avoiding the irritation experienced by patients during nasal biopsies. METHODS: This prospective, observational study enrolled patients undergoing surgical treatment for nasal septum deviation. After general anaesthesia, a novel nasal scraping spoon was used to collect epithelial cells from the mid-part of the inferior turbinate. The cells were evenly plated on six-well plates coated with rat tail collagen. The morphology and growth of the cells were observed at different time-points using an inverted phase-contrast microscope. Immunofluorescent staining of cytokeratin 18 was used to identify NEC. Ki67 staining was used to check cell viability. RESULTS: This study collected samples from 19 patients during a short procedure. No postoperative complications were observed. Cell samples ranging from 8.31 × 105 to 2.04 × 106 cells/sample were obtained. The culture model was suitable for primary NEC culture as demonstrated by the faster proliferation (5-7 days). There was no fungal or bacterial contamination. Immunofluorescent staining confirmed the presence and proliferative activity of NEC in the cultures. CONCLUSION: A novel nasal scraping spoon provided an easy sampling method, avoided nasal injuries and psychological barriers to sampling and sufficient viable NEC to establish primary cultures.

Isolation of Nasal Brush Cells for Single-cell Preparations.

Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and ex vivo assays. Graphic abstract: Workflow of nasal digestion for brush cell isolation.

Optimizing Protein Harvest From Nasal Brushings for Determining Local Allergy Responses.

Background Rhinitis is a highly prevalent yet often misdiagnosed condition. Patients who have local allergic rhinitis are regularly mislabeled as having a nonallergic etiology. Thus, a highly accurate, reproducible, and noninvasive assessment, which can be performed quickly and with minimal discomfort to the patient, is required. Objective The aim of this research was to identify the efficiency of various nasal brushes as tools for harvest and collection of epithelial proteins and its suitability for identification of rhinitis. Methods Nasal epithelial mucosa samples were taken from patients undergoing turbinate surgery using a cytology brush, a dental brush, and a nasal curette in random order. After washing in phosphate-buffered saline, the suspended cells were sonicated. Total protein content was assessed for all samples by bicinchoninic acid assay measured using a Nanodrop machine. Identification of nasal-specific immunoglobulin E (spIgE) was then assessed using immunoassay and compared to the patient's allergic status from epicutaneous and serum testing. The lower threshold limit for the spIgE in nasal brushings was determined using the results of serum spIgE tests as the reference. The diagnostic accuracy of this new established cutoff value was determined. Results The cytology brush was found to be the optimal tool for maximal nasal mucosa protein collection followed by dental brush and nasal curette (0.75 ± 0.45 mg/mL vs 0.43 ± 0.24 mg/mL vs 0.071 ± 0.55 mg/mL, respectively; P < .01). The optimal cutoff value of nasal spIgE from the cytology nasal brushings was 0.14 kUA/L to predict allergic status from serum testing. This gave a sensitivity of 75%, specificity of 86%, positive predictive value of 74%, likelihood ration positive of 5.40, and diagnostic odds ratio of 18.62. Conclusion The cytology brush is the optimal tool for protein collection. This is an easy and direct method to sample the nasal mucosa for assessment of nasal allergy or future biomarkers.