Immunohistochemical detection of serous cells in nasal mucosa with monoclonal antibody against a component in human nasal secretion.

Secreting mechanisms of secretory cells in nasal mucosa and the changes of nasal secretions in chronic inflammatory sinusitis have been studied by the biochemical and histochemical methods. These methods could not clarify the changes of quality and quantity of nasal secretions and secretory cells. In order to obtain the specific marker for the secretions in different cells, we have produced monoclonal antibodies against a component in human nasal discharge. One antibody was selected for further characterization, because it stained submucosal serous cells specifically. This antibody stained the components of serous cells with molecular weight of 14 kD specifically, and was sensitive to periodate oxidation treatment. This antibody will be useful for detecting the subpopulation in secretory cells of human nasal mucosa, and may be serve as a biochemical probe for secretory activity of particular secretory cell types.     

Immunological response to outer membrane vesicles of Haemophilus influenzae in patients with acute sinusitis

The immunological response of 30 patients with acute sinusitis was examined by using an enzyme-linked immunosorbent assay (ELISA) designed to detect antibodies against outer membrane vesicles of Haemophilus influenzae. Using this ELISA, we found that 15 patients had slight increases in specific antibody in their convalescent serum. Maxillary sinus secretions from 15 patients had specific antibodies. IgG and IgA antibodies were detected with equal frequency, but IgA antibody was often found in maxillary sinus secretions while it was absent from serum. Thus it appears that patients with acute sinusitis respond systemically and locally with the specific antibody to H. influenzae.     

Retention fluids of chronic sinusitis induce neutrophil adherence to microvascular endothelial cells.

The adherence of circulating leukocytes to the vascular endothelium is a critical step in the emigration of leukocytes through blood vessel walls to inflammatory lesions. The influence of nasal secretions on the adherence of neutrophils to the vascular endothelium was investigated using monolayers of human mucosal microvascular endothelial cells derived from the inferior turbinate. Preincubation of vascular endothelial cells with retention fluids from the maxillary sinus of the patients with chronic sinusitis showed increased neutrophil adherence. Recombinant IL-1 beta was also tested and found to induce adherence of neutrophils to human mucosal microvascular endothelial cells. However, no adhesive effect was observed with the nasal secretions of nasal allergy. An enzyme-linked immunosorbent assay detected considerable amounts of IL-1 beta in the chronic sinusitis retention fluids, while the amounts of IL-1 alpha and TNF-alpha were very low. The increased adhesion of the neutrophils by the retention fluids of chronic sinusitis was also neutralized by the incubation with anti-IL-1 beta antibody in a dose dependent manner. These findings suggest that IL-1 beta in the paranasal secretion of chronic sinusitis induces the adherence of neutrophils to vascular endothelium and subsequent infiltration of neutrophils in the paranasal sinuses, thus contributing to the persistence of chronic sinusitis.     

Inhibition of bacterial adherence by nasopharyngeal secretions

The role of secretory immunoglobulin (Ig) A in nasopharyngeal secretions in the adherence of Streptococcus pneumoniae and Hemophilus influenzae to nasopharyngeal epithelial cells was investigated in vitro. The adherence was remarkably reduced by treating bacteria with nasopharyngeal secretions, and the antiadhesive activity was significantly greater in nasopharyngeal secretions having secretory IgA antibody activity against bacteria than in those having no activity. Noticeable changes were not observed in the antiadhesive activity caused by absorption of IgG from nasopharyngeal secretions. Results suggest that secretory IgA in nasopharyngeal secretions is related to bacterial adherence and adds to the prevention of nasopharyngeal infections.     

Lysosomal proteases and protease inhibitors in nasal allergy and non-atopic sinusitis

Patterns of protease activity and levels of protease inhibitors were analyzed in both nasal secretions and tissue extracts from patients with nasal allergy and non-atopic sinusitis to investigate the role of proteases in the inflammatory reaction. Protease activity was measured using specific methyl-coumaryl-7-amide substrates. The pattern of protease activity in the nasal secretions of chronic sinusitis patients was similar to that in neutrophil lysate and quite different from that in plasma. Both gluthatione activation testing and inhibition testing using synthetic inhibitors revealed that the majority of proteases in both secretions and tissues are lysosomal thiol proteases such as cathepsins B and L. Neutrophilic elastase is also a major protease in nasal secretions. In acute sinusitis, both protease activity and inhibitor levels were very high, suggesting an interaction between proteases and inhibitors. Cathepsin B and B-like thiol proteases appear to play a key role in prolonging chronic inflammation against the healing process, due to their resistance to plasma inhibitors and the shortage of thiol protease inhibitors. Protease activity in the secretions of nasal allergy patients was very weak, and the reaction between proteases and inhibitors appeared to be weak.     

Neutrophil elastase and its complex with alpha 1-antitrypsin in soluble and insoluble fractions of nasal secretions of chronic sinusitis.

Immunoreactive neutrophil elastase (NE) and its complex with alpha 1-antitrypsin (AT) was measured by double antibody enzyme linked immunosorbent assay (ELISA) in nasal secretions of chronic sinusitis (CS). Nasal secretions were separated into two fractions: PBS-soluble and insoluble fractions. Elastolytic activity was also examined. Mean value of total NE level was 31.0 micrograms/ml in the soluble fraction, which was significantly lower than that in the insoluble fraction (71.9 micrograms/ml, p less than 0.01). On the other hand, the percentage of complexed NE in total NE in the soluble fraction (33.7%) was significantly higher than that in the insoluble fraction (12.1%, p less than 0.01). Elastolytic activity in the soluble fraction (23.4 RFU) was significantly lower than that in the insoluble fraction (170.5 RFU, p less than 0.01). NE with elastolytic activity exists in nasal secretions of CS, and active-free NE in the insoluble fraction could be a major source of enhancement and continuation of mucosal inflammation.     

Humoral mucosal immunity in allergic rhinitis

BACKGROUND: Mucosa-immunologic aspects are gaining an increasing awareness in the pathophysiology of type I allergies. Humoral mucosal immune responses are dominated by secretory IgA, but there is evidence for a relevant role of IgG in nasal mucosa-associated lymphoid tissue. OBJECTIVE: was to measure allergen-specific immunoglobulins (IgA and IgG) in nasal secretions as an expression of a humoral mucosal immune response in allergic rhinitis. For tissue eosinophilia we studied nasal Eosinophilic Cationic Protein (ECP) and for mast cell activation nasal tryptase. METHODS: Nasal secretions of 40 patients suffering from allergic rhinitis were analyzed for allergen-specific IgA, IgG, and IgE, and for ECP and tryptase. Patients were highly sensitized against the major allergens of house dust mites, timothy, and birch pollen. 43 non-atopic individuals served as controls. In order to study possible effects of the actual pollen season on the studied parameter we secondly compared patients allergic to seasonal allergens co- (n = 28) and extra-seasonally (n = 41). In order to determine a possible influence of allergen-specific IgA in eosinophilic degranulation we additionally studied 5 patients after nasal allergen challenge. RESULTS: In allergic rhinitis we found significantly increased levels of allergen-specific immunoglobulins of all studied subclasses and allergens in nasal secretions. Comparison of nasal ECP and tryptase showed significantly increased concentrations in allergic individuals as well. Co-seasonally we found elevated allergen-specific IgE, ECP, and tryptase but lower concentrations of allergen-specific IgA and IgG. There was no association between late phase eosinophilia and IgA concentrations after local allergen challenge. CONCLUSIONS: The occurrence of allergen-specific immunoglobulins in nasal secretions is interpreted as a local humoral mucosal immune response. The physiologic role of local allergen-specific immunoglobulins is not clear to date. Involvement in degranulation of eosinophils or mast cells, like suggested before, seems unlikely.     

A protease inhibitor from human allergic nasal secretions

Protease and antiprotease activities were estimated in nasal secretions from patients with chronic sinusitis and nasal allergy, using [3H]-casein as substrate. In the purulent nasal secretions, strong protease activity was measured, but there was less activity in the allergic nasal secretions. In contrast, trypsin inhibitory activity in allergic nasal secretions was much higher than in nasal secretions from the patients with chronic sinusitis. A protease inhibitor was partially isolated from nasal secretions of the nasal allergic patients by Sephadex G-150 gel chromatography and characterized. This protease inhibitor has an apparent molecular weight of 10,000 D, determined by SDS-polyacrylamidegel electrophoresis. It depresses the activities of bovine pancreatic trypsin, bovine pancreatic chymotrypsin and proteases in nasal purulent secretions, whereas it does not inhibit porcine pancreatic elastase, papain, or human plasmin.     

Monoclonal antibody-detectable carbohydrate epitopes of human nasal secretions are differentially expressed in tissue and diseases

To study the differential carbohydrate expression of airway secretions, we have produced a series of monoclonal antibodies that recognize human nasal secretory cell products. Mice were immunized with purified nasal secretion from patients with chronic sinusitis (CS) and hybridomas were selected by ELISA and immunohistochemical staining of the maxillary sinus mucosa from patients with CS. Eighteen antibodies were obtained. Antibody HCS 18 reacted with epithelial goblet cells, antibody HCS 4, 5, 6, and 16 stained submucosal gland cells, and antibody HCS 13 and 15 reacted with epithelial goblet cells, submucosal gland cells, and endothelial cells of vessels. The other eleven antibodies recognized epithelial goblet cells and submucosal gland cells. Cross-reactivity of these antibodies with secretory cells in other organs and in other species was determined and the different staining pattern was observed between upper and lower airway tissue, suggesting that secretory products from upper and lower airways may be different. Reactivity of the antibodies with nasal secretory cells was also examined in patients with perennial allergic rhinitis (AR) and normal subjects. Antibody HCS 18 weakly reacted with nasal glands in the tissue from CS and AR patients, but minimally reacted with gland cells in normal tissue. Antibody HCS 1 and 7 partially lost their reactivity with nasal epithelium of inferior turbinate from normal subjects and AR patients. These antibodies may be useful to study nasal secretions.     

The role of bacterial adherence in otitis media with effusion

Adherence of nontypable Haemophilus influenzae and Streptococcus pneumoniae to nasopharyngeal epithelial cells was investigated in vitro. Both strains had higher affinity to the epithelial cells of children than to those of adults. In children, the adherence was significantly greater in patients with otitis media with effusion than in normal subjects. Secretory IgA in nasopharyngeal secretions was found to have antibody activity against the bacteria. Adherence of both bacteria was significantly smaller in the group having secretory IgA antibody activity than in the group having no activity. These results suggest that bacterial adherence to the nasopharynx may play an important role in the pathogenesis of otitis media with effusion in children, and that secretory IgA in nasopharyngeal secretions may be related to the decrease of adherence.     

A study on collagenase production of nasal polyp-derived fibroblasts stimulated by nasal secretions of chronic sinusitis

The collagenase produced by mesenchymal cells has been thought to have a great importance in the pathophysiology of connective tissue metabolism and prolongation of chronic inflammation. The factors, such as IL-1 and PMN factor, released by inflammatory cells have been known to induce mesenchymal cells to produce collagenase. In the present study, the collagenase activity of the nasal secretions were estimated using FITC-labelled collagens as substrates. The factor, enhancing the fibroblasts to produce collagenase, was also isolated from nasal secretions and partially characterized. The fibroblasts used in the present study were cultured with explant of the sections of nasal polyp obtained from a patient with chronic sinusitis. The collagenase activity in nasal secretions from patients with chronic sinusitis was high, whereas that of allergic nasal secretions was extremely low. Furthermore, the collagenase productions of nasal polyp-derived fibroblasts were enhanced by the extracts of nasal secretions from patients with chronic sinusitis. Crude extracts of nasal secretions were fractionated by ammonium sulfate precipitation. The active materials precipitated by 50% to 80% ammonium sulfate were further purified by Sephadex G-75 gel chromatography. The molecular weight determination of the active fraction checked by HPLC utilizing for TSK 2,000 SW gel column indicates 20,000 daltons for the active materials. However, the collagenase production of human microvascular endothelial cells derived from nasal mucosa was not enhanced by this factor. Although either the origin or the nature was not confirmed, the factor was considered to relate to the prolongation of chronic inflammation in the nasal and paranasal sinus pathology. Analysis of these factors will expected to establish methods for new therapeutics in chronic inflammation.     

变应性鼻炎鼻分泌物中上皮细胞集落和嗜酸粒细胞的检测

为了观察变应性鼻炎（allesgicrhinitis，AR）患者鼻分泌物中的上皮细胞集落和嗜酸粒细胞，对20例AR患者（AR组）和15例鼻窦炎患者（感染组）鼻分泌物中细胞成分进行了电镜观察，发现AR组鼻分泌物中有大量上皮细胞集落，其特征是数个至数十个上皮细胞成片状脱落，上皮细胞集落的数量和嗜酸粒细胞的数量、嗜酸粒细胞阳离子蛋白（eosinophiliccationicprotein，ECP）的含量呈正相关。感染组中上皮细胞集落和嗜酸粒细胞极少，ECP含量甚微。结果提示嗜酸粒细胞的ECP可能导致AR患者的鼻粘膜上皮呈片状脱落，对上皮结构造成损害。     

ELISA for determination of immunoreactive free elastase and elastase in complex with alpha 1-antitrypsin in nasal secretions with sinusitis

Sensitive double antibody sandwich ELISA methods was developed in order to quantify immunoreactive neutrophil elastase (NE) levels in nasal secretions with chronic sinusitis (CS). Microwell plate as a solid phase was coated with anti-NE antibody. Two different horseradish peroxidase (HRP) labelled antibodies used as the second antibody were anti-NE-HRP for measuring total (free + complexed) NE level and anti-alpha 1-antitrypsin (AT)-HRP for complexed NE level. Mean value of total NE was 31.0 +/- 20.7 micrograms/ml in nasal secretions from adult patients with CS, and the percentage of complexed NE in total NE was 33.7 +/- 21.4%. This sandwich ELISA is a useful method for measuring both total and complexed NE levels in nasal secretions.     

Role of local immunoglobulin E specific for Alternaria alternata in the pathogenesis of nasal polyposis

OBJECTIVE/HYPOTHESIS: The role of fungal pathogens in the etiology of nasal polyposis remains unclear. The aim of this study was to determine whether there was a correlation between the presence of Alternaria-specific immunoglobulin (Ig)E antibodies, eosinophilic inflammation, and the development of nasal polyps. STUDY DESIGN: Prospective study. METHODS: Serum and nasal tissue homogenates from 21 patients with manifestations of chronic sinusitis with nasal polyps were compared with specimens from 13 chronic sinusitis patients without polyps and 8 healthy controls. The Phadia ImmunoCAP and enzyme-linked immunosorbent assay were used to quantify levels of total IgE and Alternaria-specific (IgE, IgG, and IgA) antibodies. Eosinophil cationic protein (ECP) and tryptase levels were measured in tissue homogenates, whereas the inflammatory response was evaluated using tissue eosinophil counts in tissue samples. RESULTS: Serum analysis revealed no difference in the levels of total IgE and Alternaria-specific IgE, IgG, and IgA antibodies between the study groups. In contrast, the levels of Alternaria-specific IgE in tissue with polyps were significantly higher than in nonpolyp tissue. Increases in total tissue IgE paralleled increased levels of Alternaria-specific IgG and IgA antibodies in chronic sinusitis with nasal polyps as compared with control groups. A positive correlation was found between Alternaria-specific IgE and ECP in tissue. Increased mean levels of ECP corresponded to increased eosinophil counts in the group of patients with polyps. CONCLUSIONS: Alternaria-specific IgE and eosinophilic inflammation in nasal tissue correlates with the incidence of nasal polyps irrespective of specific IgE antibodies in serum. Together, the correlation between the local immune responses and the eosinophilic inflammation in nasal polyps suggests a possible role of Alternaria in the pathogenesis of nasal polyposis.     

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.     

Antibodies specific to outer membrane antigens of Moraxella catarrhalis in sera and middle ear effusions from children with otitis media with effusion

OBJECTIVE: Recent studies have shown that bacterial DNA is present in a significant percentage of middle ear effusions, suggesting that persistent bacterial infection may be more important in pathogenesis and recurrence of otitis media with effusion (OME) than previously considered. Although Moraxella (M.) catarrhalis is one of the most common pathogens of otitis media, relatively little is known about immune response to the organism. The objective of the present study is to investigate how systemic and local immune activities against M. catarrhalis may be associated with severity of OME. METHODS: The antibody levels specific to outer membrane antigens of M. catarrhalis in sera and middle ear effusions (MEEs) from 59 children with OME were measured by enzyme-linked immunosorbent assay. Their ages ranged from 1 to 12 years with a median 5.0 years. The children were followed 1 year prospectively and classified into two groups with or without recurrent/persistent OME according to severity of OME during the follow-up 1 year. RESULTS: Serum IgG, IgM, and IgA antibodies specific to outer membrane antigens of M. catarrhalis were detected in all samples and the median levels were 35, 0.93, and 1.2 microg/ml respectively. The MEE IgG, IgM, IgA, and secretory IgA antibodies were detected in over 95% samples tested and the median levels were 371, 158, 20, and 50 ng/mg total protein respectively. A comparison between acute and subacute/chronic phases revealed that the median levels of MEE IgG and IgM antibodies were higher at the acute phase (692 vs. 340, P = 0.06; 35 vs. 10, P = 0.02, respectively); while the MEE secretory IgA antibody level was increased at the subacute/chronic phase (74 vs. 35, P = 0.02). Either serum or MEE IgG antibody level was significantly lower in recurrent/persistent OME group than that in nonrecurrent/non-persistent OME group (13 vs. 43 ,microg/ml, P = 0.009; 238 vs. 577 ng/mg protein, P = 0.006, respectively). CONCLUSIONS: These data provide additional information on the immunologic aspects of children with OME. Decreased serum and MEE IgG antibody levels specific to outer membrane antigens of M. catarrhalis may lead to failure to eliminate this organism, resulting in persistent and/or recurrent appearance of MEE.     

Immunoglobulins in nasal secretions of patients with allergic rhinitis and chronic rhinosinusitis

Allergic rhinitis and chronic rhinosinusitis are the most frequently encountered inflammatory reactions of the sinonasal mucosa. Nasal-associated lymphoid tissue has been suggested as an inductive site for humoral and cellular immune responses in the upper respiratory tract. Immunoglobulins are important elements in human adaptive immune responses and deficiencies of serum immunoglobulins may be associated with recurrent or refractory infections. However, the local humoral immune response to offending antigens in the nasal environment has not been well elucidated. To determine the levels of IgA and IgG subclasses antibodies in the nasal secretions of patients with allergic rhinitis and chronic rhinosinusitis, 25 patients with allergic rhinitis and 20 with chronic rhinosinusitis were included and their nasal secretions were collected to measure the levels of secretary IgA (sIgA), total IgA (tIgA), and IgG subclasses antibodies. There was a significant elevation of IgG₃ in the nasal secretions of patients with chronic rhinosinusitis. No difference was noted in the levels of sIgA, tIgA, IgG₁, IgG₂ and IgG₄ among the three groups. The local defense mechanism of nose reacts to microorganisms and pathogenic antigens by inducing the adaptive humoral immune response to increase the amount of immunoglobulins, with IgG₃ being the major up-regulated antibody.     

Bacteria in chronic maxillary sinusitis.

Sixty-one chronically inflamed maxillary sinuses produced 131 bacterial strains from mucosal pieces that were taken during a Caldwell-Luc operation and cultured aerobically and anaerobically. Sinus secretions showed only 62 and nasal secretions 106 bacterial strains. Fourteen mucosal strains, including 11 Haemophilus influenzae, grew heavily. None of 24 mucosal anaerobes showed heavy growth. Of 52 antral mucosae with culturable bacteria, 37 disclosed mixed and 15 pure growth. The bacteriological characteristics of the diseased sinus and the nose did not correlate. The duration or extent of the disease, the macroscopic appearance of the diseased sinus, or the presence or absence of allergy were unrelated to bacteriological findings, except that H influenzae was concentrated in purulent sinuses. Intraoperative culture of antral mucosa seems to give the most reliable picture of the bacteriological condition in chronic maxillary sinusitis.     

Quantitative Study of Nasal Secretory Cells in Normal Subjects and Patients With Chronic Sinusitis

The contribution of nasal secretory cells to mucus hypersecretion in chronic sinusitis was investigated. The mucosae of the inferior turbinate were obtained from 18 normal control subjects and 65 patients with chronic sinusitis. Histochemical quantitation showed that there was no significant difference in the number of goblet cells between normal controls and chronic sinusitis. On the other hand, the number of submucosal acinar cells in chronic sinusitis was significantly higher than that in normal controls ( P < 0.01). The area occupied by the acini in lamina propria was also increased in chronic sinusitis ( P < 0.01). There was no significant difference in the distribution of the intra-acinar glycoproteins between normal control subjects and patients with chronic sinusitis. Results suggest that hyperplasia and hypertrophy of nasal acinar cells may have an important role in mucus hypersecretion in chronic sinusitis.     

变应性因素对鼻内镜手术疗效的影响

目的：探讨变应性因素对慢性鼻窦炎伴鼻息肉患者行鼻窦内窥镜手术疗效的影响。方法对我院2011～2012年入院手术的II型慢性鼻窦炎伴鼻息肉患者,术前根据其是否伴有变应性鼻炎症状和体征、是否伴有过敏性疾病史及支气管哮喘病史、术前鼻腔分泌物涂片嗜酸粒细胞检查、变应原皮肤试验、血清特异性IgE检查、过敏性鼻炎,筛选出伴有变应性因素的慢性鼻窦炎伴鼻息肉患者为试验组。选择同期无变应性因素的单纯慢性鼻窦炎伴鼻息肉患者作为对照组。比较两组患者手术后疗效,并同时比较各种变应性因素对手术疗效的影响。结果伴有变应性因素的慢性鼻窦炎伴鼻息肉患者,手术疗效为80.95%（136/168）,同期单纯慢性鼻窦炎伴鼻息肉患者,手术疗效为91.75%（189/206）,两组间差异有显著性（P〈0.01）。在各种变应性因素中,伴有变应性鼻炎症状和体征、嗜酸粒细胞阳性、变应原皮肤试验阳性、血清特异性IgE、过敏性鼻炎的慢性鼻窦炎伴鼻息肉患者,与无变应性因素的慢性鼻窦炎伴鼻息肉患者比较,差异均有显著性（P〈0.05）,伴有过敏性疾病史及伴支气管哮喘病史患者与无变应性因素的慢性鼻窦炎伴鼻息肉患者比较,差异无显著性（P〉0.05）。结论变应性因素与鼻窦内窥镜手术疗效密切相关,是术后复发的重要因素。对慢性鼻窦炎伴鼻息肉患者行鼻窦内窥镜手术时,必须加强围手术期抗变应性治疗。