Role of transepithelial ion transport as a determinant of mucus viscoelasticity in chronic inflammation of the maxillary sinus.

Chronic sinusitis is a chronic inflammatory disease with fluid retention, most frequently in maxillary sinus. We measured both viscoelasticity of maxillary sinus fluid and bioelectric properties of maxillary mucosa in an attempt to correlate the mucus viscoelasticity with bioelectric properties. During sinus surgery, maxillary sinus fluid and maxillary mucosa were obtained from 15 patients with chronic sinusitis. Viscoelasticity was measured with an oscillating sphere magnetic rheometer within 24 h after sampling. Potential difference and short circuit current of maxillary mucosa mounted in Ussing chambers were measured before and after exposure of mucosal surface to amiloride. Both elasticity and viscosity showed significant positive correlation with percent inhibition of short-circuit current by amiloride. Our results indicate that the ion transport process plays a role in the determination of viscoelasticity of maxillary sinus fluid in chronic sinusitis.     

An antimicrobial peptide modulates epithelial responses to bacterial products

INTRODUCTION: Changes in the respiratory epithelium and chronic and recurrent infections are thought to play a central role in the pathogenesis of otitis media and sinusitis. The airway epithelium is the primary defense system of the respiratory tract. Bacterial cell membrane components like lipopolysaccharide (LPS) and lipoteichoic acid (LTA) can affect the mucociliary clearance function of the respiratory epithelium. P60.4-Ac is a synthetic antimicrobial peptide based on the structure of the cathelicidin LL-37 that neutralizes the pro-inflammatory activity of LPS and LTA. MATERIALS AND METHODS: Normal respiratory sinus epithelium was cultured at the air liquid interface. The cells were incubated with LPS or LTA in the presence or absence of P60.4-Ac. RESULTS: P60.4-Ac neutralized the LPS- and LTA- induced effect on air-liquid interface cultured epithelial cells. P60.4-Ac significantly inhibited the increase in the epithelial layer caused by LPS or LTA. CONCLUSION: These data demonstrate that P60.4-Ac might be of clinical benefit in the management of otitis media with effusion and sinusitis.     

人鼻粘膜上皮细胞Na^＋通道的初步研究

明确人鼻粘膜上皮细胞Ｎａ＾＋通道的特性，为研究Ｎａ＾＋通道在鼻粘膜病理性改变及治疗中的作用奠定理论基础。方法利用膜片钳技术在经无血清气－液面培养的鼻源性鼾症患者手术切除下鼻甲标本的鼻上皮细胞进行Ｎａ＾＋通道基本特性研究。     

Murine tracheal and nasal septal epithelium for air-liquid interface cultures: a comparative study

BACKGROUND: Air-liquid interface cultures using murine tracheal respiratory epithelium have revolutionized the in vitro study of airway diseases. However, these cultures often are impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. The ability to study ciliary physiology in vitro is of utmost importance in the research of chronic rhinosinusitis (CRS). Our hypothesis is that the murine nasal septum is a better source of ciliated respiratory epithelium to develop respiratory epithelial air-liquid interface models. METHODS: Nasal septa and tracheas were harvested from 10 BALB/c mice. The nasal septa were harvested by using a simple and straightforward novel technique. Scanning electron microscopy was performed on all specimens. Cell counts of ciliated respiratory epithelial cells were performed at one standard magnification (1535x). Comparative analysis of proximal and distal trachea, midanterior and midposterior nasal septal epithelium, was performed. RESULTS: Independent cell counts revealed highly significant differences in the proportion of cell populations (p < 0.00001). Ciliated cell counts for the trachea (106.9 +/- 28) were an average of 38.7% of the total cell population. Nasal septal ciliated epithelial cells (277.5 +/- 16) comprised 90.1% of the total cell population. CONCLUSION: To increase the yield of respiratory epithelial cells harvested from mice, we have found that the nasal septum is a far superior source when compared with the trachea. The greater surface area and increased concentration of ciliated epithelial cells has the potential to provide an eightfold increase in epithelial cells for the development of air-liquid interface cultures.     

Nicotine-induced endocytosis of amiloride-sensitive sodium channels in human nasal epithelium

In previous studies we developed and introduced a method to examine the transport mechanisms of ions in primary cell cultures of human nasal epithelium. In the current study, substances, especially nicotine, that influence these mechanisms are investigated. Specimens of nasal and paranasal epithelium of patients treated by endonasal surgery because of chronic sinusitis (n = 217) were used as primary cell cultures. Cell cultures of smokers (n = 83) and non-smokers (n = 134) were differentiated. Transepithelial Ussing chamber measurements were performed to examine sodium channel functions and to evaluate the influence of nicotine. These examinations were accompanied by simultaneous continuous capacitance measurements. Whereas transepithelial parameters, such as short-circuit current, (Isc), potential (Vt) and resistance (Rt), in tissues derived from smokers and non-smokers showed no difference, the transepithelial conductance was reduced immediately in cell cultures with apical application of nicotine (2 mM). This decrease was accompanied by a marked reduction of epithelial surface area. In the presence of nicotine, amiloride (100 microM) completely lost its inhibitory capacity. Amiloride-insensitive sodium channels were unaffected by nicotine, as proved by Na+ substitution. Furthermore, the Na+ channel blocker was accompanied by an increase in intracellular Ca2+. We conclude that the nicotine-induced increase in intracellular calcium (Ca2+) has stimulated Ca2+-dependent protein kinase (PKC). PKC promotes endocytosis removing amiloride-sensitive Na+ channels from the cell membrane into the cell by means of vesicular transport.     

Effects of macrolides on interleukin-8 secretion from human nasal epithelial cells

Low-dose, long-term macrolide treatment has recently been reported to be very effective in patients with chronic airway diseases. We examined the in vivo and in vitro effects of 14-membered macrolide antibiotics erythromycin (EM) and clarithromycin (CAM) on interleukin (IL)-8 secretion from human nasal epithelial cells. Fifteen patients with chronic sinusitis received macrolide treatment (CAM 400 mg/day) for 1 to 3 months. The number of infiltrated neutrophils and IL-8 concentrations in the nasal discharges of these patients decreased significantly at 1 to 2 months after the treatment. In vitro effects of EM and CAM on IL-8 secretion were examined in nasal epithelial cells cultured at the air-liquid interface. After 14-day culture in the air-liquid interface, macrolide antibiotics were added in medium for 24 h. EM and CAM at concentrations of 10^–4 M did not affect spontaneous secretions or IL-1β-induced secretions of IL-8 either apically or basolaterally. When cells were preincubated with 10^–4 M CAM for 7 days, the IL-1β-induced secretion of IL-8 decreased significantly. However, no difference was observed between the effects of 10^–4 M CAM and 10^–4 M josamycin, a 16-membered macrolide. These results suggest that macrolide treatment inhibits neutrophil infiltration and IL-8 secretion in nasal epithelium in vivo and that these clinical effects depend on a mechanism other than the direct action of macrolide on nasal epithelial cells. Received: 30 December 1998 / Accepted: 8 July 1999     

mRNA for genes associated with antigen presentation are expressed by human middle meatal epithelial cells in culture

OBJECTIVES/HYPOTHESIS: Although the mechanisms underlying the initiation and maintenance of inflammation in chronic rhinosinusitis are poorly understood, the activation of memory T cells within the nasal mucosa is thought to play an important role. T-cell activation requires specialized antigen processing and presentation of antigen by immunocompetent cells in the context of cell surface immune molecules. The purpose of this study was to investigate the expression of such molecules by human sinonasal epithelial cells grown in culture at the air-liquid interface (ALI). METHODS: Middle meatal epithelium was obtained from six patients undergoing endoscopic sinus surgery. Dissociated epithelial cells were grown to confluence in serum-free, defined medium and transferred to filter inserts for culture at the ALI. Cells were harvested at 2 and 21 days of growth at the ALI and processed for real-time polymerase chain reaction (PCR). The presence and relative abundance of constitutively expressed mRNA for human leukocyte antigen (HLA)-B, HLA-DR, B7 to 1, B7 to 2, B7-H2, B7-H3, and cathepsin D were assessed. RESULTS: After 2 days at the ALI, middle meatal epithelial cells demonstrated expression of genes for each of the antigen processing associated genes tested. The expression of HLA-B and HLA-DR increased significantly with cellular maturation at the ALI. Expression of HLA-DR and B7 to 1 increased with cytokine stimulation. CONCLUSIONS: Primary human epithelial cells obtained from the middle meatus express genes associated with antigen presentation function. The pattern of gene expression is modulated by cytokine stimulation and changes as the cells differentiate at the ALI. These findings suggest that mature middle meatal epithelial cells have the cellular machinery to interact with T cells and therefore may be direct participants in the modulation of T-cell activity in chronic sinusitis.     

The microbiology of ethmoid and maxillary sinuses in patients with chronic sinusitis

PURPOSE: To investigate aerob-anaerob microorganisms growth in maxillary and ethmoid sinuses by evaluating aspiration materials from patients with chronic sinusitis. Patients and Methods: The study was performed prospectively, and there were 31 patients (23 men, 8 women; mean age, 31.4+/-14.15, between 18-65 years) who had endoscopic sinus surgery because of chronic sinusitis. During the operation, when the maxillary sinus ostium and ethmoid sinus were opened, readily aspirated materials from sinuses were evaluated regarding aerob and anaerob bacteria. Nose and throat swap samples were collected preoperatively to determine the upper respiratory tract flora and also to understand the relationship between the flora and the microorganisms aspirated from sinuses. RESULTS: Total aerob bacteria count, which was isolated from preoperative nasal swab cultures, was 36, and aerob-anaerob bacteria count that included cultures taken from maxillary and ethmoid sinuses during the functional endoscopic sinus surgery was 42. For each 2 samples, the most common isolated aerob bacteria were coagulase (-) staphylococci. Microorganisms were isolated in 87.0% of 27 patients, in which cultures taken from maxillary and ethmoid sinuses during the functional endoscopic sinus surgery were included. It is determined that the isolated aerob bacteria rate was 90.4%, and the isolated anaerob bacteria rate was 14.2%. All of the 6 samples in which anaerob bacteria isolated were all maxillary sinus aspiration materials. Microorganisms that isolated from the nose and the sinuses were similar with the rate of 25.8%, and microorganisms that isolated from the throat cultures and sinuses were similar with the rate of 22.5%. CONCLUSIONS: This study reveals the aerob and anaerob microbiology of the maxillary and ethmoid sinuses so the treatment of chronic sinusitis will be easier.     

Secretory cell differentiation and mucus secretion in cultures of human nasal epithelial cells: use of a monoclonal antibody to study human nasal mucin

We have developed an air-liquid interface culture system for human nasal epithelial cells that differentiate into mucociliary phenotypes in a defined serum-free medium. Dissociated cells obtained from nasal polyps were cultured on a collagen gel substrate. At confluence, the cells lost characteristics of differentiated cells, and secretory cell and ciliated cell differentiation appeared after 7 days in an air-liquid interface. After 21 days, about half of the epithelial cells were stained with Alcian blue-periodic acid-Schiff stain or monoclonal antibody HCS18, which was directed against human nasal mucin specific for epithelial secretory (goblet) cells. The quantitative examination using the antibody HCS18 revealed that the antibody-reactive nasal mucin was secreted only on the apical side of the cultures, and interleukin-1beta and tumor necrosis factor alpha stimulated these mucus secretions. The culture system with an antimucin monoclonal antibody developed in this study should be useful for studying polarized mucus secretion from human nasal epithelial cells.     

诱生型一氧化氮合酶在慢性上颌窦炎黏膜的表达

目的 观察诱生型一氧化氮合酶(inducible nitric oxide synthase，iNOS)在慢性鼻窦炎病人鼻窦粘膜中的表达。方法 将实验分为两组：对照组为正常成人，无鼻窦病变。实验组为确诊为慢性上颌窦炎的病人。在局麻鼻内镜下行鼻窦黏膜活检，石蜡包埋、切片，用免疫组织化学的方法观察iNOS在两组上颌窦黏膜中的表达。结果 iNOS在正常上颌窦炎黏膜呈阴性表达，在慢性上颌窦炎黏膜呈阳性表达，阳性区域主要在黏膜上皮细胞。结论 iNOS在慢性上颌窦炎黏膜呈阳性表达，提示一氧化氮在慢性上颌窦炎的发病机制中起重要作用。     

K+ and Na+ absorption by outer sulcus epithelial cells.

Transduction of sound into nerve impulses by hair cells depends on modulation of a current carried primarily by K+ into the cell across apical transduction channels that are permeable to cations. The cochlear function thus depends on active secretion of K+ accompanied by absorption of Na+ by epithelial cells enclosing the cochlear duct. The para-sensory cells which participate in the absorption of Na+ (down to the uniquely low level of 1 mM) were previously unidentified and the existence of a para-sensory pathway which actively absorbs K+ was previously unknown. A relative short circuit current (Isc,probe, measured as the extracellular current density with a vibrating electrode) was directed into the apical side of the outer sulcus epithelium, decreased by ouabain (1 mM), an inhibitor of Na+, K(+)-ATPase, and found to depend on bath Na+ and K+ but on neither Ca2+ nor Cl-. Isc,probe was shown to be an active current by its sensitivity to ouabain. On-cell patch clamp recordings of the apical membrane of outer sulcus cells displayed a channel activity, which carried inward currents under conditions identical to those used to measure Isc,probe. Both Isc,probe and non-selective cation channels (27.4+/-0.6 ps, n = 22) in excised outside-out patches from the apical membrane were inhibited by Gd3+ (1 mM). Ics,prob was also inhibited by 5 mM lidocaine, 1 mM quinine and 500 microM amiloride but not by 10 microM amiloride. These results demonstrate that outer sulcus epithelial cells contribute to the homeostasis of endolymph by actively absorbing Na+ and K+. An entry pathway in the apical membrane was shown to be through non-selective cation channels that were sensitive to Gd3+.     

人鼻粘膜上皮细胞Na+通道的初步研究

目的　明确人鼻粘膜上皮细胞Na 通道的特性 ,为研究Na 通道在鼻粘膜病理性改变及治疗中的作用奠定理论基础。方法　利用膜片钳技术对经无血清气 液界面培养的鼻源性鼾症患者手术切除下鼻甲标本的鼻上皮细胞进行Na 通道基本特性研究。结果　在细胞贴附式膜片上 ,可记录到典型的单通道电流 ,其电导为 2 1 0 9pS ,反转电位为 - 50 96mV ,且 77 78%反转电位 <- 4 0mV ,离子通透性比率PNa/PK>5 80。在Na 通道抑制剂 10 - 4 mmol/LAmiloride存在于电极液内时 ,Na 通道发生率从 2 6 7%减少到 5 13 % (P <0 0 5) ,表明大部分Na 通道可被Amiloride抑制。 10 - 3mmol/LCa2 存在于浴夜时 ,Na 通道的发生率无明显增加 (P >0 0 5) ,而电压对开放概率无明显影响。结论　在细胞贴附式膜片上 ,人鼻粘膜上皮细胞具有Amiloride敏感型及Amiloride非敏感型的Na 通道 ,且大部分是Na 选择性通道 ,细胞外液的Ca2 不能激活Na 通道的开放 ,通道的开放概率是非电压依赖性的。     

Inflammation and remodeling of the sinus mucosa in children and adults with chronic sinusitis

OBJECTIVES/HYPOTHESIS: The sinus mucosal inflammatory response in adult patients with chronic sinusitis is well documented in the literature. In contrast, little is known about the pathogenesis of this condition in children. The objective of the study was to compare the inflammatory cell profile and the extent of tissue remodeling in the sinus mucosa of children and adults with chronic sinusitis. STUDY DESIGN: Prospective design. METHODS: Children (n = 7) and adult patients (n = 7) with chronic sinusitis undergoing functional endoscopic sinus surgery were recruited for the study. Patients with no evidence of sinus disease (n = 6) were used as control subjects. Using immunohistochemical analysis, sinus mucosal specimens were assessed for the presence of T lymphocytes, eosinophils, basophils, mast cells, and neutrophils. The extent of submucosal collagen deposition was evaluated in histological sections using van Gieson stain. RESULTS: The number of T lymphocytes, eosinophils, and basophils and the amount of subepithelial collagen deposition are significantly higher in the mucosa of both adults and children with chronic sinusitis compared with normal control subjects (P <.01). The number of mast cells is significantly higher in the mucosa of children with chronic sinusitis compared with normal control subjects (P <.01). The number of eosinophils and neutrophils and the amount of subepithelial collagen deposition are significantly greater in adults compared with children with chronic sinusitis (P <.01). CONCLUSIONS: The sinus mucosal inflammatory profile is similar in adults and children with chronic sinusitis. However, the degree of tissue eosinophilia and remodeling is significantly greater in adult sinus specimens when compared with those of children with chronic sinusitis.     

Bacterial location in chronic sinusitis

BACKGROUND: In chronic nonallergic sinusitis, it is often assumed that bacteria invade the sinus mucosa where the inflammatory condition begins and is maintained. However, the bacterial presence in a normal or moderately damaged epithelial layer has never been proved in biopsy studies. METHODS: In this study, mucosal samples from six consecutive patients with chronic sinusitis were examined. Transmission electron microscopy was used and the presence of bacterial invasion and formation of phagosomes containing bacteria as a marker of host response were studied. RESULTS: Phagocytosis of bacteria was observed in the sinus mucosa in samples from only one patient. In the other five patients, no signs of phagocytosis were seen. CONCLUSION: Based on these results, we concluded that in chronic sinusitis, bacterial invasion in sinus mucosa is not an obligatory phenomenon.     

Immunohistochemical analyses of MUC5AC mucin expression in sinus mucosa of children with sinusitis and controls

OBJECTIVES: The purpose of this study was to analyze MUC5AC protein expression in sinus mucosal specimens of children with and without chronic sinusitis. METHODS: Morphometric, histologic, and immunohistochemical analyses were carried out on sinus mucosa of 7 children with chronic sinusitis and 6 children without sinusitis. RESULTS: MUC5AC protein was expressed in a subset of goblet cells in the surface epithelium, but not in the submucosal glands in either pediatric population. The number of goblet cells that expressed MUC5AC mucin was not significantly different in patients with and without chronic sinusitis. All specimens had similar numbers of goblet cells in the surface epithelium. CONCLUSIONS: The data demonstrate that neither goblet cell hyperplasia nor increased MUC5AC expression occurs in the sinus mucosa of children with chronic sinusitis. This suggests that in contrast to asthma, in which goblet cell hyperplasia is present in the lower respiratory tract, mucus hypersecretion in pediatric chronic sinusitis may involve other secretory cells, eg, submucosal glandular cells, and mucins secreted by these glandular cells.     

Therapeutic effect of roxithromycin on chronic sinusitis with nasal -- polyps clinical,computed tomography,and electron microscopy analysis

To evaluate the therapeutic effect of Roxithromycin (RXM), we studied 56 chronic sinusitis patients with nasal polyps using computed tomography (CT) and electron microscopy in addition to conventional clinical assessment. The paranasal sinus of subjects was observed clinically before and after daily administration of RXM at 300 mg for 3 months all underwent allergy testing for possible complications of allergic rhinitis based on subjective symptoms and objective findings. Improvement after RXM treatment was seen in 50.3% based on subjective symptoms and 59.1% based on objective findings. Overall improvement was seen in 53.6%. In 41 cases (73.2%) of all patients with chronic sinusitis and complications of allergic rhinitis, no significant difference was seen between patients with and without complications (53.7% in those with complications and 53.3% in those without). In CT analysis the paranasal sinus in 51.8% of all posttreated patients showed obvious improvement. In electron microscopy in chronic sinusitis patients with complications of allergic rhinitis, pretreated ethmoidal sinus tissues showed high mucous epithelial cell apoptosis in addition to common histological lesions, while posttreatment patients showed only eosinophil apoptosis in the interstitium and no apoptotic epithelial cells. We divided ethmoidal sinus lesions in patients without complications into 3 types and evaluated them as follows: In type 1, pretreated ethmoidal sinus tissues showed plasma cell infiltration and posttreatment cell apoptosis. In type 2, pretreated tissues showed lymphocyte and plasma cell infiltration and posttreated showed only some lymphocytes and no plasma cells. In type 3, proliferation of fibroblasts, most of which showed apoptosis, was seen in addition to apoptotic epithelial cells before treatment, while after treatment, these lesions remained with some apoptotic bodies phagocytosed by macrophages. In type 3 patients relapsed after surgery. Our findings indicate that RXM treatment had a significant therapeutic effect on chronic sinusitis with nasal polyps with and without complications of allergic rhinitis. We clarify the morphological mechanism of therapeutic effect of RXM on each type of ethmoidal sinus lesion divided by light and electron microscopy.     

慢性筛窦炎黏膜超微结构变化

目的观察慢性筛窦炎（chronic ethmoiditis）患者筛窦黏膜的超微结构。方法15例慢性筛窦炎患者和4例单纯鼻中隔偏曲患者在接受鼻内镜手术时钳取筛窦黏膜,通过扫描电镜、透射电镜观察其黏膜表面形态及炎细胞浸润的超微结构改变。结果慢性筛窦炎患者筛窦黏膜可观察到黏液颗粒呈串珠样或团片状附着在残存纤毛上：纤毛稀疏、排列凌乱、缠结：细胞连接分离,细胞间隙增宽;基底细胞受损：肥大细胞、嗜酸性粒细胞颗粒释放,浆细胞粗面内质网扩张等超微结构的改变。结论慢性筛窦炎患者筛窦黏膜超微结构的改变是筛窦炎慢性迁延的病理学基础。