Nasal Epithelial Cells Of Patients Research Articles

Pharmacologic therapy for stop mutations: how much CFTR activity is enough?

The purpose of this review is to summarize the recent approaches using mutation-specific therapy to correct the genetic defect according to the molecular mechanism by which the mutation causes the defects in cystic fibrosis transmembrane conductance regulator (CFTR). Premature stop mutations (class I mutations) account for 5 to 10% of the total mutant alleles in cystic fibrosis patients, and in certain subpopulations the incidence is much higher. The aminoglycoside antibiotics can suppress premature termination codons by permitting translation to continue to the normal termination of the transcript. The susceptibility to suppression by aminoglycosides depends on the stop codon itself and on the sequence context surrounding it. In vitro studies in cell lines expressing stop mutations and in mice have shown that aminoglycosides caused a dose-dependent increase in CFTR expression and restored functional CFTR to the apical membrane. Clinical studies also provided evidence that the aminoglycoside gentamicin can suppress these CFTR premature stop mutations in affected patients. A recent double-blind, placebo-controlled, crossover study has demonstrated restoration of CFTR function by topical application of gentamicin to the nasal epithelium of cystic fibrosis patients carrying stop mutations. In 21% of the patients there was a complete normalization of all the electrophysiologic abnormalities caused by the CFTR defect, and in 68% there was restoration of either chloride or sodium transport. Furthermore, immunohistochemical staining to the C-terminal part of the CFTR was demonstrated via peripheral staining for CFTR in scraped nasal epithelial cells of patients carrying stop mutations. Inconsistent results were reported regarding the required level of corrected CFTR that has to be reached to achieve normal function. Achieving CFTR activity of 10 to 35% might be needed to prevent significant pulmonary morbidity. It is as yet unknown how much corrected mutant CFTR must reach the apical membrane to induce a clinically relevant beneficial effect. The future goal is to maximize the effect of stop-codon supressors on CFTR while minimizing side effects, but further studies must be performed to find a safer compound that may be administered in small children from the time of diagnosis.

Tumor necrosis factor-alpha and interferon-gamma modulate in vitro expression of nitric oxide synthase in human nasal epithelial cells

Interest in the physiological, pathological and therapeutic implications of nitric oxide (NO) have grown exponentially, with human nasal cavity and paranasal sinuses considered a dominant source of NO, indicating that this molecule possesses the diversity of biological effects in the regulation of airway clearance and nonspecific cellular immunity. We previously observed differences in NO synthase (NOS) isoform constitutively expressed in nasal epithelial cells (NECs) from allergic and normal subjects. We extended the previous work to determine whether in vitro stimulation with proinflammatory cytokines influences levels of different NOS isoform expression. Nasal epithelial cells were sampled from the inferior turbinate in a group of 16 healthy normal controls and 11 patients with perennial allergic rhinitis against house dust (HD) mite antigen. 1 x 10(5) cells were incubated in conditioned medium for 24 hours. Human recombinant interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), or both (cytomix) were added to a final concentration of 10 ng/ml. Cells were then fixed with 4% paraformaldehyde and processed for fluorescence immunocytochemistry. Immunoreactivity for 2 NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), was studied by laser scanning confocal microscopy and fluorescence intensity was assessed quantitatively. We observed constitutive eNOS expression in epithelial cells of all subjects. Different treatments with cytokines did not affect eNOS expression. Cytokine treatment, however, significantly augmented iNOS expression in the control group. The average increase induced with IFN-gamma, TNF-alpha, and cytomix was 1.8, 2.33, and 2.31-fold. Nasal epithelial cells in the HD group showed elevated steady-state iNOS expression even in untreated. Cytokine treatment did not affect the degree of iNOS expression in this group. These results confirm our previous findings that nasal epithelial cells in patients with allergic rhinitis produce higher levels of NO through the concomitant expression of different NOS isoforms. We also demonstrated that nasal epithelial cells have the potential to express iNOS protein spontaneously or upon stimulation with inflammatory cytokines, such as IFN-gamma and TNF-alpha. Because the high level of exhaled NO is considered a potential marker of allergic airway inflammation, preserving the iNOS gene from its unregulated induction may be important for maintenance of nasal homeostasis and may offer a tool for therapeutic intervention.

The potential of active metabolites of antihistamines in the management of allergic disease.

Since the discovery of histamine in the ®rst decade of the 20th century, there has been increasing evidence that this biogenic amine, synthesized predominantly in mast cells and basophils, is released in in ammatory processes and plays a key role in the pathogenesis of urticaria and allergic airway diseases, including asthma, rhinitis, and urticaria (1±3). Studies have demonstrated that histamine levels are signi®cantly increased in the bronchoalveolar lavage (BAL) of symptomatic asthmatics, compared with asymptomatic asthmatics (4, 5), and that allergen challenge leads to a marked increase in the levels of histamine in asthmatics (6, 7). Similarly, studies have demonstrated that allergen challenge also leads to signi®cant increases in the levels of histamine in the nasal secretions of patients with allergic rhinitis (8±10). A comparison of patients with chronic urticaria and healthy subjects has also demonstrated that the former release greater amounts of histamine, both spontaneously and after antigen provocation (11±13). In addition, some studies have demonstrated that the responsiveness of the skin to histamine is slightly increased in patients with urticaria (11). Mechanistic studies have demonstrated that the action of histamine arises from its interaction with one of at least three speci®c histamine receptors, H1, H2, and H3, located on various histamine-responsive target tissues. Interaction of histamine with the H1 receptor leads primarily to smooth-muscle contraction, vasodilatation and hypotension, increased vascular permeability with formation of tissue oedema, increased respiratory mucus secretion, and parasympathetic nerve stimulation (14, 15). These effects can subsequently lead to bronchial obstruction in asthma; nasal blockage, sneezing, itching, and discharge in rhinitis; and itchy skin wheals/ ares in urticaria. At the cellular level, histamine may also act on in ammatory cells and lead to release of mediators, such as leukotrienes and cytokines, and increased expression of class II antigens (HLA-DR) and ICAM-1 on epithelial cells (16, 17), effects which could, in turn, exacerbate the pathology and symptoms of allergic disease. In contrast, effects attributable to H2 receptors include augmentation of gastric acid secretion and increases in gastric and respiratory mucus secretion (14). H3 receptors have been implicated in autocrine regulation of histamine synthesis and release from nerve tissue (14).

Increased expression of inducible nitric oxide synthase in nasal epithelial cells in patients with allergic rhinitis.

Although ciliated epithelial cells of human nose and paranasal sinuses have recently been reported to be the major source of locally detected nitric oxide (NO), changes to the NO production by these cells and their functional roles remain uncertain in relation to allergic rhinitis. The objective of this study is to investigate differences in the ability of induction of nitric oxide synthase (NOS) isoforms by nasal epithelial cells. Epithelial cells of the inferior turbinate taken from 12 normal subjects and 12 allergic patients against house dust mite were used. Samples from the house dust group were taken both before and after antigen provocation. Immunoreactivity for two NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), was examined by the laser scanning confocal microscope. The labeled cells were processed into digital images, and the fluorescence intensity was assessed quantitatively. The degree of iNOS expression of the epithelial cells was significantly elevated in the house dust group compared with that of the control group. The expression appeared identical both before and after antigen provocation in the house dust group. On the other hand, there was no significant difference in eNOS expression between the two groups. We assume that the increased iNOS expression of the epithelial cells in the house dust group might result from stimulated secretion of proinflammatory cytokines during allergic responses. This further suggests profound contribution of nasal epithelial cells to modifying the airway clearance through the production of high levels of NO.

Effect of fexofenadine on eosinophil-induced changes in epithelial permeability and cytokine release from nasal epithelial cells of patients with seasonal allergic rhinitis

Recent studies have suggested that antihistamines, widely used in the treatment of symptoms of patients with allergic rhinitis, may also possess antiinflammatory properties. The mechanisms underlying this property, however, are not clearly understood. We have cultured epithelial cells from nasal biopsy specimens from patients with seasonal allergic rhinitis outside the pollen season and studied the effect of 0 to 10 -3 mol/L fexofenadine, the main active metabolite of terfenadine, on eosinophil-induced changes in electrical resistance (measure of permeability) and release of proinflammatory mediators from these cells. Additionally, we have studied the effect of this drug on eosinophil chemotaxis and adherence to endothelial cells induced by conditioned medium from these human nasal epithelial cell (HNEC) cultures. Incubation of HNEC in the presence of eosinophils treated with opsonized latex beads significantly decreased the electrical resistance of these cultures, an effect that was abrogated by treatment of the cultures with 10 -9 to 10 -3 mol/L fexofenadine. Similarly, incubation of HNEC in the presence of eosinophils treated with latex beads also significantly increased the basal release of the chemokine “regulated upon activation, normal T cell expressed and secreted” (RANTES) (from 96.0 to 613.0 fg/μg cellular protein; p < 0.05), IL-8 (from 42.0 to 198.5 pg/μg cellular protein; p < 0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF) (from 0.54 to 3.4 pg/μg cellular protein; p < 0.05), and soluble intercellular adhesion molecule-1 (sICAM-1) (from 7.8 to 18.4 pg/μg cellular protein; p < 0.05) from HNEC. The eosinophil-induced release of IL-8, GM-CSF, and sICAM-1 from the HNEC was significantly attenuated by treatment with fexofenadine. Analysis of the effects of conditioned medium from HNEC demonstrated that this significantly increased both eosinophil chemotaxis and adherence to endothelial cells. Addition of 10 -6 to 10 -3 mol/L fexofenadine to the conditioned medium significantly attenuated eosinophil chemotaxis and adherence to endothelial cells. These results suggest that fexofenadine may reduce nasal inflammation by modulating the release of proinflammatory mediators and adhesion molecules from HNEC. (J Allergy Clin Immunol 1998;101:410-20.)