慢性鼻-鼻窦炎鼻息肉与正常鼻黏膜差异蛋白质的检测及其意义

目的采用蛋白质组学技术筛选慢性鼻-鼻窦炎、鼻息肉和正常鼻黏膜组织之间差异表达的蛋白质,初步鉴定出慢性鼻-鼻窦炎和鼻息肉的候选蛋白质标记物。方法应用固相pH4~7胶条行双向凝胶电泳,胶体考马斯亮蓝染色后,扫描2-DE胶,应用PDquest图像分析软件比较慢性鼻-鼻窦炎、鼻息肉和正常鼻黏膜组织2-DE图谱,得到三组之间差异表达的蛋白质点。经MALDI-TOF-MS鉴定后获得这些差异表达蛋白质的肽质量指纹图谱,用Mascot软件查询NCBInr和SWISS-PROT数据库,得出被测蛋白质的鉴定结果。结果所获双向凝胶电泳图谱分辨率高、重复性好。测得鼻息肉、慢性鼻-鼻窦炎及正常鼻黏膜组织的蛋白质平均斑点数分别为1020±40、1112±10和1008±25;平均匹配率分别为(93±2)%、(95±1)%和(90±3)%。三组之间共计有13个明显差异表达的蛋白质点。初步筛选出角蛋白8和阿朴脂蛋白AI作为鼻息肉的候选标志物,以及PLUNC蛋白、超氧化物歧化酶、自然杀伤细胞促进因子B、垂体腺苷酸环化酶激活肽为慢性鼻-鼻窦炎的候选标志物。结论用蛋白质组学技术能高通量筛选慢性鼻-鼻窦炎、鼻息肉和正常鼻黏膜间存在的差异表达蛋白质,这将为慢性鼻-鼻窦炎、鼻息肉分类、分型分期和预后判断标准寻找新的客观参考指标。     

Cytolysis of eosinophils in nasal secretions

It is still unknown how eosinophils degranulate in nasal mucus. Currently, cytolysis is being reevaluated as the mode of degranulation of eosinophils in allergic nasal mucosa. To examine whether eosinophils migrating to the nasal mucus degranulate by cytolysis, we sampled nasal mucus from 9 patients with nasal allergy and observed it under electron and light microscopes. Both intact and necrotic eosinophils were observed in the nasal mucus. Although the total eosinophil count in the nasal mucus was not correlated with the frequency of sneezes, there was a significant correlation (p = .0025) between the rate of eosinophil lysis and the frequency of sneezes. Whereas extracellular release of eosinophil peroxidase was not detected from the eosinophils with intact cell membranes, large quantities of eosinophil peroxidase were found outside the eosinophils with injured cell membranes. We concluded that eosinophils migrating to the nasal mucus degranulate mainly by cytolysis, and that granular proteins released from the necrotic eosinophils into the nasal mucus are one of the important factors causing hypersensitivity in the nasal mucosa.     

Histamine metabolism in the nasal tissue of human and nasal hypersensitive guinea-pig

Histamine (HA) is the most important mediator of nasal allergy and nasal hypersensitivity. To investigate HA metabolism, HA content and activities of its synthetic enzyme, histidine decarboxylase (HDC) and degrading enzymes, histamine-N-methyltransferase (HMT) and diamine oxidase (DAO) in nasal mucosa of human and toluene diisocyanate (TDI) sensitized guinea-pigs were measured. In human nasal mucosa and nasal polyps, HA content and HDC activity were 80-200 nmol/g tissue, 20-30 fmol/min/mg protein respectively. Among two degrading enzymes, HMT activity was 20-200 times higher than that of DAO. In the nasal mucosa of guinea-pigs, HA content was significantly increased by TDI sensitization, and was decreased immediately after TDI provocation. In 24 hours after provocation, HA content recovered to 80% of pre-provocation level. HDC activity increased by TDI sensitization significantly. Though HMT activity increased slightly by TDI sensitization and provocation, DAO activity was unchanged. The data suggest that, increase in turnover rate of HA is present in allergic nasal mucosa.     

Eosinophil count in nasal secretions of subjects with and without nasal symptoms

The aim of this paper, based on a cross-sectional study of 129 patients with nonallergic chronic nasal symptoms and 40 healthy controls, was to examine the leucocyte differential count in nasal secretions as a diagnostic test. Nasal secretions were collected using preweighed suction glass canulas under controlled conditions (-100Pa, 30 sec). Leucocyte and differential counts were performed using a Thoma hemocytometer and on cytospin slides after May-Grünwald-Giemsa staining. The percentage of eosinophils (Eo) was significantly higher in patients (mean +/- SEM: 15.1 +/- 2.3%) than in controls (5 +/- 2.6%) (p < 0.04). Comparison of the frequency distribution of the percentage of Eo in patients and controls clearly showed a subgroup of patients presenting with nasal secretion hypereosinophilia, and allowed us to set the positivity criterion at Eo = 20%. Diurnal variations in Eo count in 11 controls and 8 patients confirmed the value of the cutoff point. In 28 patients with nasal polyposis who underwent surgery, a correlation was found between secretion and tissue eosinophelia (r = 0.58, p = 0.001). Patients with nasal secretion hypereosinophilia had no more leucocytes in their secretions than healthy controls, the increase in eosinophils being balanced by a decrease in neutrophils. In patients without hypereosinophilia, the number of leucocytes per milligram of secretion was four times higher (8672 +/- 2521) than in the controls (2020 +/- 823) (p = 0.06) (cut-off point = 2500 leu/mg). These data show that the nasal cytogram can be modified either in qualitative or quantitative way, probably depending on the underlying inflammatory process.     

Eosinophilia and cell activation mediators in nasal secretions.

OBJECTIVES/HYPOTHESIS: In rhinologic disorders such as polyposis or rhinitis, nasal cytology allows differentiation between patients according to the degree of eosinophilia in nasal secretions. The egress of eosinophil and/or neutrophil polymorphonuclears from the underlying mucosa might correlate with the release of soluble mediators of cell activation such as the chemokine IL-8, and such molecules of the innate immunity as the LPS-receptor CD14 or lysozyme. We assayed the levels of these three molecules in nasal secretions in correlation with cytologic findings and especially the degree of eosinophilia. STUDY DESIGN: Fifty-four patients from a prospective study of nasal secretions were enrolled in this work. They constituted two groups of 27 patients each, respectively, with or without more than 20% eosinophils in nasal secretions. Nasal secretions were collected by aspiration, weighed and diluted in a fixed amount of buffer. Classic cytologic analyses were performed on the pelleted cells and IL-8, sCD14, and lysozyme levels were assayed in the cell-free supernatants. METHODS: Cytologic analyses included cell-enumeration in Neubauer's chambers, and differentials performed on May-Grünwald Giemsa-stained cytospins. ELISA tests were used to assay the levels of IL-8 and sCD14. Lysozyme concentrations were assayed in immuno-nephelometry. RESULTS: Significantly lower levels of IL-8 and sCD14 were observed in patients with eosinophilia than in patients with a predominance of neutrophils, whereas no difference was observed in lysozyme concentrations. CONCLUSION: These data show that the egress of neutrophils in nasal secretions is associated with high levels of IL-8 and sCD14.     

Immunoglobulins and inflammatory cytokines in nasal secretions in humoral immunodeficiencies

OBJECTIVE: Chronic respiratory tract infections are a common problem in patients with severe humoral immunodeficiency despite intravenous immunoglobulin therapy (IVIG), often presenting as rhinosinusitis. METHODS: Because it is unclear whether IVIG is a good substitute at the mucosal surface, we analyzed immunoglobulin levels and inflammatory cytokines (ECP, IL-8, and TNF-alpha) in nasal secretions of 13 patients with common variable immunodeficiency (CVID) and in 10 patients with IgA deficiency. RESULTS: In patients with CVID, median IgG and IgM levels did not differ significantly from controls, whereas inflammatory cytokines were markedly elevated, reflecting persistent inflammation at the mucosal site. In contrast, patients with IgA deficiency showed significantly raised IgG and IgM levels, whereas ECP and TNF-alpha were only slightly increased. CONCLUSION: Low levels of SIgA might be compensated locally at the mucosal site by high levels of IgM and IgG. Our findings implicate that adequate IVIG is not sufficient to prevent chronic inflammation of the sinuses in patients with severe humoral immunodeficiency.     

Serum amyloid A, properdin, complement 3, and toll-like receptors are expressed locally in human sinonasal tissue

BACKGROUND: There is a growing appreciation of the role that nasal mucosa plays in innate immunity. In this study, the expression of pattern recognition receptors known as toll-like receptors (TLRs) and the effector molecules complement factor 3 (C3), properdin, and serum amyloid A (SAA) were examined in human sinonasal mucosa obtained from control subjects and patients with chronic rhinosinusitis (CRS). METHODS: Sinonasal mucosal specimens were obtained from 20 patients with CRS and 5 control subjects. Messenger RNA (mRNA) was isolated and tested using Taqman real-time polymerase chain reaction with primer and probe sets for C3, complement factor P, and SAA. Standard polymerase chain reaction was performed for the 10 known TLRs. Immunohistochemistry was performed on the microscopic sections using antibodies against C3. RESULTS: Analysis of the sinonasal sample mRNA revealed expression of all 10 TLRs in both CRS samples and in control specimens. Expression of the three effector proteins was detected also, with the levels of mRNA for C3 generally greater than SAA and properdin in CRS patients. No significant differences were found in TLR or innate immune protein expression in normal controls. Immunohistochemical analysis of sinonasal mucosal specimens established C3 staining ranging from 20 to 85% of the epithelium present. CONCLUSION: These studies indicate that sinonasal mucosa expresses genes involved in innate immunity including the TLRs and proteins involved in complement activation. We hypothesize that local production of complement and acute phase proteins by airway epithelium on stimulation of innate immune receptors may play an important role in host defense in the airway and, potentially, in the pathogenesis of CRS.     

Concentrations of chemical mediators in nasal secretions after nasal allergen challenges in atopic patients

By using a microsuction technique, a quantitative determination of chemical mediators in nasal secretions was performed in 18 hay-fever patients and in a control group of 10 healthy volunteers. The authors then compared these quantitative data for mediators with objective nasal findings counting the number of sneezes, passive anterior rhinomanometry (PAR) and nasal inspiratory peak flow. A sampling protocol was designed with a follow-up of 3 days after nasal allergen challenge (NAC) in order to investigate both early and late allergic reactions. Median baseline concentrations of five major mediators were obtained: histamine, 19 ng/g; leukotriene C4 (LTC4), 5.7 ng/g. tryptase, 0; prostaglandin D2 (PGD2), 477 pg/g; eosinophil cationic protein (ECP), 105 ng/g. Significant increases in histamine (214 ng/g) , LTC4 (20 ng/g) and tryptase (28 μU/g) were found, but a significant decrease occurred in ECP (47 ng/g) and PGD (226 pg/g) immediately after NAC in the patients studied. Most ECP concentrations (94%) increased slowly 1 h after NAC and reached a significantly higher level 24 h later. In evaluating nasal symptoms, sneezes were present in a high percentage of cases (76%) during the early phase but were uncommon during the late phase (29%). Total nasal obstruction occurred in 94% during the early phase. In contrast, unilateral nasal obstruction presented in 82% during the late phase, whereas total nasal obstruction was present only in 41%. The most common type of late phase nasal obstruction shown by PAR was alternating nasal obstruction.     

Concentrations of chemical mediators in nasal secretions after nasal allergen challenges in atopic patients

By using a microsuction technique, a quantitative determination of chemical mediators in nasal secretions was performed in 18 hay-fever patients and in a control group of 10 healthy volunteers. The authors then compared these quantitative data for mediators with objective nasal findings counting the number of sneezes, passive anterior rhinomanometry (PAR) and nasal inspiratory peak flow. A sampling protocol was designed with a follow-up of 3 days after nasal allergen challenge (NAC) in order to investigate both early and late allergic reactions. Median baseline concentrations of five major mediators were obtained: histamine, 19 ng/g; leukotriene C4 (LTC4), 5.7 ng/g. tryptase, 0; prostaglandin D2 (PGD2), 477 pg/g; eosinophil cationic protein (ECP), 105 ng/g. Significant increases in histamine (214 ng/g) , LTC4 (20 ng/g) and tryptase (28 μU/g) were found, but a significant decrease occurred in ECP (47 ng/g) and PGD (226 pg/g) immediately after NAC in the patients studied. Most ECP concentrations (94%) increased slowly 1 h after NAC and reached a significantly higher level 24 h later. In evaluating nasal symptoms, sneezes were present in a high percentage of cases (76%) during the early phase but were uncommon during the late phase (29%). Total nasal obstruction occurred in 94% during the early phase. In contrast, unilateral nasal obstruction presented in 82% during the late phase, whereas total nasal obstruction was present only in 41%. The most common type of late phase nasal obstruction shown by PAR was alternating nasal obstruction.     

Eosinophilia in nasal secretions compared to skin prick test and nasal challenge test in the diagnosis of nasal allergy.

This study was aimed to assess the usefulness of eosinophilia measurements in nasal smears (ENS) in the diagnosis of nasal allergy. Nasal smears were taken from 84 patients with histories suggestive of allergic rhinitis. The smears were stained by the Giemsa method and examined by light microscopy. Positive results were demonstrated in 69.2% of the samples. All the 84 patients also had a skin prick test (SPT); the perceniitage of correct correlation between ENS and SPT was 71.4%. Forty-two patients underwent nasal challenge test (NCT) and the percentage of correct correlation between ENS and NCT was 69%. Nine patients had negative SPT, but positive ENS. All were nasally challenged with 4 proving positive. This leaves 5 individuals (5.9% of the 84 studied) in the non-allergic rhinitis with eosinophilia category. Based on these findings, it is suggested that the assessment of eosinophils in nasal smears should be given more relevance and be more commonly used in the diagnosis of nasal allergy.     

Electron microscope study of basophilic cells in allergic nasal secretions

Summary In order to elucidate whether basophilic cells in nasal secretion belong to blood basophil or tissue mast cell, basophilic cells in the blood, nasal secretion, and nasal mucous membrane were electron microscopically observed in patients with house dust nasal allergy. The majority of basophilic cells in the nasal secretion was identical with the blood basophil in structure. The blood basophils pass through the vessels and emigrate in the mucous blanket in allergy.     

Clinical significance of eosinophilic cationic protein levels in nasal secretions of patients with nasal polyposis

Nasal polyps are characterized by eosinophilic infiltration, and frequently coexist with asthma, aspirin intolerance and allergy. Eosinophilic cationic protein (ECP) is a specific eosinophil granule protein released upon activation of eosinophils. We investigated the ECP levels in nasal secretions of patients with nasal polyposis (NP) in order to correlate them with disease severity and associated diseases and to compare ECP levels between patients with and without recurrence of NP after surgical treatment. A total of 78 patients who had surgery for NP were followed up for a minimum of 18 months. The presence of asthma, allergies or aspirin intolerance was noted. Nasal secretions were obtained 1 day before the surgery and during the follow-up period after surgery. Immunoassays were used to quantify ECP in nasal secretions and serum and interleukin (IL)-5 in nasal secretions. ECP levels in nasal secretions were higher in patients with asthma or aspirin intolerance than in patients without asthma or aspirin intolerance, while no significant differences were found between allergic and non-allergic patients. ECP levels in nasal secretions correlated significantly with IL-5 levels in nasal secretions, the degree of tissue eosinophilia and computed tomographic (CT) scores. In total, 30 patients (38%) developed recurrent NP during the follow-up period. Preoperative ECP and IL-5 levels in nasal secretions were significantly higher in patients with recurrence compared to patients without recurrence. During the follow-up period, patients without recurrence demonstrated a significant reduction in the ECP levels in nasal secretions, whereas there was no significant reduction in the ECP levels of patients with recurrence. The results of this study provide evidence that ECP levels in nasal secretions of patients with NP correlate with the presence of asthma or aspirin intolerance and severity of NP determined by CT scores.     

Pulmonary aspiration of nasal secretions in patients with chronic sinusitis and asthma

BACKGROUND: The role of silent aspiration of nasal secretions in the pathogenesis of asthma has often been questioned. OBJECTIVE: To investigate the presence of pulmonary aspiration of nasal secretions during sleep in patients with chronic sinusitis and asthma and in healthy controls. DESIGN: Prospective, controlled trial. SUBJECTS: The study included 13 patients with chronic sinusitis and asthma and 12 healthy controls. The diagnoses were based on history, physical examination findings, radiologic assessments, and pulmonary function test results. INTERVENTIONS: A radioactive tracer was prepared by diluting 10 mCi of technetium 99m-labeled macroaggregated albumin in 10 mL of physiologic saline. At 10 PM, just before the patients went to sleep, the solution was sprayed into their nostrils. The subjects were examined with a gamma camera to obtain views of the thorax at 8 AM the following morning. The average counts of the lungs and background and the actual lung counts (average lung count minus average background count) were determined. RESULTS: The average counts of the lungs were significantly greater than the average counts of the background in both the sinusitis-asthma group (P =.001) and the control group (P =.002). The difference in the actual counts of the lungs was not significant between the 2 groups (P =.79). CONCLUSIONS: The nasal secretions were aspirated into the lungs both in patients with sinusitis and asthma and in healthy adults during sleep, and the relative amounts that were aspirated did not differ significantly between the 2 groups (P =.79). The amount of the aspirated material alone is probably not responsible for the pathogenesis of asthma in patients with chronic sinusitis.