Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.

BACKGROUND: Aberrant airway epithelial remodeling is one of the cardinal histopathologic features of inflammatory airway diseases, but whether it alters the mucociliary apparatus remains unknown. OBJECTIVE: We sought to investigate the morphologic pattern of motile cilia and ciliogenesis-associated makers in hyperplastic nasal epithelium from nasal polyps (NPs) both in vivo and in vitro. METHODS: Biopsy specimens obtained from patients with NPs (n = 44) and inferior turbinate from healthy control subjects (n = 38) were analyzed by using scanning electron microscopy, immunofluorescence staining, single-cell (cytospin) staining, quantitative real-time PCR, and human nasal epithelial stem/progenitor cell culture and differentiation. RESULTS: Abnormal cilia architecture (untidy, overly dense, and lengthened) was more commonly observed in patients with NPs by using scanning electron microscopy. Ectopic lengthened cilia were visualized by means of immunofluorescence (patients with NPs: 6.33 µm [5.51-7.43 µm] vs control subjects: 3.73 µm [3.50-4.27 µm], P < .0001), at the site of epithelial hyperplasia in isolated single cells (patients with NPs: 6.55 ± 0.23 µm vs control subjects 4.89 ± 0.24 µm, P < .0001), and in differentiated ciliated cells derived from human nasal epithelial stem/progenitor cells (patients with NPs: 9.20 ± 0.56 µm vs control subjects: 5.21 ± 0.37 µm, P < .0001). Ciliary beat frequency was found to be significantly slower in patients with NPs than control subjects in vitro. Both protein and mRNA levels of ciliogenesis-associated markers (centrosomal protein 110 [CP110], forkhead box J1 [Foxj1], and P73 isoform with an N-terminal transactivation domain [TAp73]) were significantly increased in patients with NPs versus those seen in control subjects and were positively correlated with cilia length. CONCLUSION: For the first time, this study demonstrates for that motile cilia impairment is a co-condition of epithelial hyperplasia in patients with NPs, and this impairment of function is a likely cause of chronic mucosal inflammation or infection (eg, biofilm) observed in patients with chronic rhinosinusitis.

Myrtol standardized affects mucociliary clearance.

BACKGROUND: Myrtol standardized (Gelomyrtol forte) has been shown to be effective in controlling nasal symptoms of rhinosinusitis by promoting mucociliary clearance. Our aim was to evaluate the short- and long-term effects of myrtol on ciliated columnar cells and goblet cells in an in-vitro setting. METHODS: Nasal epithelial cells were harvested (42 days) from an air-liquid interface (ALI) culture of human nasal epithelial stem/progenitor cells (hNESPCs), which was derived from biopsies of nasal inferior turbinate mucosa. Myrtol 0.1% was applied to the ALI culture system at 2 different time-points (day 0 and day 35) on progenitor and differentiated cells. Ciliary beat frequency (CBF), supernatant fluid, and ciliated and goblet cell markers were evaluated after short- (7 days) and long-term (42 days) treatment. RESULTS: In the long-term treatment with myrtol, there was an increase in cilia area (type IV ß-tubulin+ , 1.53-fold, p = 0.031) and ciliogenesis-related markers (Foxj1 and CP110) with no change in CBF, as compared with control. In addition, the short-term myrtol treatment group exhibited greater mucin secretion compared with control. CONCLUSION: This study demonstrates, through cellular and molecular mechanisms, that myrtol standardized enhances the mucus production from goblet cells in the short term, and promotes ciliated cell differentiation in the long term.

High-risk HPV genotypes and P16INK4a expression in a cohort of head and neck squamous cell carcinoma patients in Singapore.

Human papillomavirus (HPV), especially HPV16 genotype, is associated with oropharyngeal squamous cell carcinoma (OPSCC). We aim to determine the prevalence and characterize the high-risk (HR)-HPV genotypes in head and neck SCC (HNSCC) in a South-East Asian multi-ethnic society in Singapore and examine its prognostic significance.159 HNSCC archival tissue samples were retrieved and tumour DNA was screened for 18 HR-HPV genotypes using a PCR-based assay (Qiagen, digene HPV genotyping RH test). P16 protein overexpression was identified using immunohistochemistry (IHC). Statistical correlation between clinical outcomes were performed between HPV-positive and negative HNSCC patients.Six HR-HPVs (HPV16, 18, 31, 45, 56, 68) were detected in 90.6% of HNSCC; and 79.9% had multiple HPV genotypes detected. HPV31 and HPV45 were the most prevalent (79.2% and 87.4%, respectively); and HPV16 was predominantly found in OPSCC (p < 0.001). HPV-DNA PCR assay yielded a high sensitivity (96%) but low specificity (11%) when compared to p16 immunohistochemistry as the reference standard.P16-positive HNSCC was predominantly observed in OPSCC (73.7%; p = 0.005); and p16-positive OPSCC exhibited improved overall survival compared to p16-negative OPSCC (p = 0.022). Similarly, smoking and alcohol consumption were poor prognostic factors of overall survival (p = 0.007; p = 0.01) in OPSCC patients.HR-HPVs were identified in 90.6% of HNSCC patients using the HPV-DNA PCR assay. This test had a poor specificity when compared to p16 IHC; making it an unreliable detection technique in selecting patients for radiation dose de-escalation treatment protocol. P16-positive tumor was predominantly found in the oropharynx these patients demonstrated better overall survival than those with p16-negative OPSCC.

Reduced growth and proliferation dynamics of nasal epithelial stem/progenitor cells in nasal polyps in vitro.

Basal cells in nasal epithelium have stemness/progenitor characters and play essential roles in the epithelial remodeling in nasal polyps (NP). We investigate whether the human nasal epithelial stem/progenitor cells (hNESPCs) from patients with NP are inherently distinct from those obtained from healthy controls. Epithelial basal cells were isolated and cultured for four passages from NP tissues and control nasal mucosa. hNESPCs from controls were stained positively with stem cell marker p63 and KRT5 and presented a consistent high Ki67 expression level over four passages. In contrast, hNESPCs from NP patients showed: i). a reduced growth and proliferation rate at each passage by evaluating colony-forming efficiency and doubling time; ii). a lower percentage of Ki67(+) cells among p63(+) cells in the colonies in late passages, which was also confirmed by immunostaining in the NP tissues. Thus reduced growth/proliferation dynamics in hNESPCs from NP could be an important pathological phenomenon in NP development.

The genomic landscape of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC.

Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: a potential strategy for axonal regeneration.

Myelin-derived proteins, such as tenascin-R (TN-R), myelin associate glycoprotein (MAG), and Nogo-A, inhibit the CNS regeneration. By targeting specifically the inhibitory epitopes, we have investigated whether vaccination with a recombinant DNA molecule encoding multiple domains of myelin inhibitors may be useful in CNS repair. We show here that the recombinant DNA vaccine is able to activate the immune system but does not induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Importantly, it promotes axonal regeneration in a spinal cord injury model. Thus, the application of DNA vaccine, encoding multiple specific domains of major inhibitory proteins and/or their receptors, provides another promising approach to overcome the inhibitory barriers during CNS regeneration.

NB-3/Notch1 pathway via Deltex1 promotes neural progenitor cell differentiation into oligodendrocytes.

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases.