Determination of lipid A in human middle ear effusions with the competitive ELISA technique

Lipid A is a covalently bound component of gram-negative bacterial lipopolysaccharides and is the biologically active component in endotoxin. In order to detect and quantitate lipid A in middle ear effusions (MEE) from patients with otitis media with effusion (OME), antisera against lipid A were produced in mice by repeated immunization with conjugates using lipid A from Salmonella minnesota Re595 and bovine serum albumin. As a result, a competitive enzyme-linked immunosorbent assay (ELISA) was developed that was specific for lipid A. From the ELISA results, 10 out of 15 samples from the serous type MEE (mean, 13.57 +/- 2.17 micrograms/ml) and 5 out of 6 from the mucoid type MEE (17.73 +/- 4.34 micrograms/ml) were positive. These findings indicate that lipid A may contribute to the pathogenesis of both types of human OME.     

Quantitative cytologic and histologic changes in the middle ear after the injection of nontypable Hemophilus influenzae endotoxin

This study was undertaken to investigate and quantify the morphologic changes in the middle ear mucosa and connective tissue after the inoculation of graded doses (0.001 to 100 micrograms) of endotoxin prepared from Hemophilus influenzae. Histopathologic changes were observed in the middle ear mucosa in all animals. Marked bleeding and new bone formation in the submucosa were prominent at days 4 through 14. These findings indicate that the endotoxin from nontypable H influenzae is capable of inducing inflammation or pathologic changes in the middle ear mucosa and may play an important role in the pathogenesis of otitis media with effusion and its sequelae.     

Biochemical evidence of platelet-activating factor (paf) in human middle ear effusions

Platelet-activating factor (PAF) is one of the most potent biological lipid mediators. This is especially true in relation to inflammation. In order to characterize the biochemical features of otitis media with effusion, the authors characterized and determined the concentration of the PAF present in human middle ear effusions obtained from 23 patients with otitis media with effusion. Each sample of middle ear effusion was divided into two groups: serous (n = 12) and mucoid effusions (n = 11). The platelet-activating factor activity was found mainly in mucoid middle ear effusions, and the amounts of PAF were higher in mucoid type (3.55 ± 1.19 ng/g, mean ± standard deviation [SD]) than in the serous type (0.44 ± 0.19 ng/g). Phospholipids obtained from the middle ear effusions contained a large amount of lyso-platelet-activating factor, the biologically inactive precursor or breakdown product of platelet-activating factor. Based on these findings, it is suggested that platelet-activating factor may play an important role as a mediator of the inflammatory responses in the pathogenesis of otitis media with effusion.     

Quantitative analysis of the bacterial findings in otitis media

Qualitative and quantitative bacterial analysis of 200 samples of middle ear effusions collected from patients with current otitis media was performed. When middle ear pathogens (S. pneumoniae, H. influenzae and B. catarrhalis) were found during current acute otitis media or otitis media with effusion infection, the quantity of these bacteria was of the magnitude 10(6)-10(8)/ml and 0-5 x 10(5)/ml effusion material, respectively. Mucopurulent effusion material contained 6 x 10(5)-10(8) bacteria per ml whereas effusion from chronically discharging ears exceeded 10(9) bacteria per ml. Serous effusions did not harbour middle ear pathogens. The appearance of the effusion material was dependent on the number of bacteria involved. Quantification of bacteria in various middle ear effusions offers opportunities to make the diagnosis of various otitis media infections more accurate and readily comparable.     

Experimental background for the use of tympanostomy tubes

Already within 24 h, splitting of the soft palate in rats caused production of effusion in the attic space. During the next couple of days the effusion material filled the meso- and hypotympanon. The production of a serous fluid in the attic space appeared concomitant with an elevated histamine content in the middle ear cavity, irrespective of whether the ears were ventilated or not. In the artificially ventilated middle ears no effusion appeared in the meso- and hypotympanon and the hypotympanal mucosal lining remained normal. The serous effusion filling the middle ear cavities with the intact tympanic membranes was replaced at about 7 days by a purulent effusion material containing mainly Gram-negative enteric rods. Thus it seems that a serous otitis media and a purulent otitis media occur in a continuum. The change to a purulent infection could be prevented by artificial ventilation, but as soon as the myringotomies were healed or the tympanostomy tubes got clogged a flocculent, pus-like effusion also appeared in these middle ear cavities.     

Compositional difference in middle ear effusion: mucous versus serous.

OBJECTIVES: Serous otitis media is usually responsive to medical treatment, whereas mucoid otitis media is not. The present study was undertaken to elucidate the compositional difference between serous and mucoid effusion and to investigate whether MUC5AC acts as a major mucin in the middle ear mucosa with mucoid otitis media. STUDY DESIGN: This study involved a chemical analysis of middle ear effusion and immunostaining of the middle ear mucosa. METHODS: Middle ear effusion samples were collected from 27 patients with mucoid otitis media and 18 patients with serous otitis media. The levels of mucin, lysozyme, secretory immunoglobulin A, and interleukin-8 were measured by dot blotting or enzyme-linked immunosorbent assay. Periodic acid-Schiff and immunohistochemical staining with monoclonal anti-MUC5AC antibody were performed on the serial sections of middle ear mucosa with mucoid otitis media. RESULTS: Mucoid effusions contained higher levels of mucin, lysozyme, secretory immunoglobulin A, and interleukin-8 than did serous effusions. Immunohistological study revealed that MUC5AC mucin was expressed in only a small portion of the goblet cells of middle ear mucosa with mucoid otitis media. CONCLUSIONS: The study suggests that both serous secretions and mucin might make the middle ear effusion more viscous and that mucins other than MUC5AC might have a major role in the viscosity of middle ear effusion. Further study is necessary to identify the major mucins in the middle ear effusion of otitis media with effusion.     

肺表面活性物质对豚鼠分泌性中耳炎的治疗作用

目的:研究肺表面活性物质(PS)对豚鼠分泌性中耳炎(OME)的治疗作用,为OME的治疗探讨新途径。方法:将50只豚鼠随机分为对照组(10只)和实验组(40只),实验组豚鼠采用灭活的肺炎链球菌(KHP)鼓室内注射,建立OME的动物模型(听力下降,中耳积液);模型建立后再将其分为造模组(20耳)、造模对照组(10耳)、治疗组(24耳,向中耳腔注射0.05mlPS)和未治组(23耳,不做任何治疗)。造模5d后,耳内镜下观察鼓膜及鼓室积液的情况,检测各组豚鼠听性脑干反应(ABR)Ⅰ、Ⅲ波潜伏期及反应阈。结果:造模组豚鼠鼓室内注射KHP悬浮液5d后,鼓室出现积液,鼓膜浑浊,光锥消失;ABR反应阈由对照组的(14.00±3.08)dB提高至(45.00±5.67)dB,其差异有统计学意义(P<0.01);治疗组鼓室内注射PS7d后,鼓室积液减少或消失,反应阈由(45.00±5.67)dB降低至(23.53±6.32)dB,分别与造模组及未治组比较,差异有统计学意义(均P<0.01);未治组鼓室积液无变化,鼓膜更加浑浊,反应阈由(45.00±5.67)dB提高至(65.50±6.85)dB。结论:PS对豚鼠OME有治疗作用。     

Otitis media with effusion following inoculation of Haemophilus influenzae type b endotoxin

Lipopolysaccharide endotoxin (LPS) was extracted from Haemophilus influenzae type b by using Westphal's phenol water method. The ears of 40 adult male guinea pigs were subsequently inoculated with 10 micrograms/ml solutions of LPS by transmeatal injections. Groups of animals were then sacrificed from day 2 to day 24 after the injections to observe the pathological changes produced. Massive serous effusions filled the tympanic bullae on days 2 and 4, after which the amount of fluid present gradually decreased so that it could hardly be seen on day 11. Pathological changes found in the mucosa included marked interstitial edema, dilated capillaries, as well as elevated and thickened epithelium with intracellular edema. These findings gradually subsided by day 24. We believe that the major pathogenetic factors present were due to the transudation and injury of the middle ear epithelium disturbing mucociliary transport activity, with increased secretions participating somewhat in inducing the effusion. We further suggest that H. influenzae endotoxin may play an active role in the clinical development of otitis media with effusion.     

Mucociliary disease of the middle ear during experimental otitis media with effusion induced by bacterial endotoxin

Lipopolysaccharide (10 micrograms/mL) derived from Klebsiella pneumoniae was injected into the middle ear of guinea pigs. The animals were killed painlessly on days 1, 3, and 7 after inoculation, and the mucosal samples from two sites within the tympanic cavity, close to the tympanic orifice and distal to the orifice, were examined for ciliary activity and epithelial morphology. At day 1 and day 3 serous effusion was observed and deterioration of ciliary activity and morphologic changes were observed. No effusion was recognized at day 7, when the ciliary activity in the distal mucosa was still diminished and that in the proximal mucosa had recovered to a normal level. Our data have shown that lipopolysaccharide extracted from K pneumoniae can produce otitis media with effusion in laboratory animals, and dysfunction of cilia due to lipopolysaccharide probably is responsible for the accumulation of middle ear effusion. The mucociliary system is indeed an important defense system and failure of such a system, especially in the mucosa close to the tympanic orifice, can cause the buildup of effusions.     

A rat model for pneumococcal otitis media

The middle ears of 29 male Sprague-Dawley rats were injected with viable pneumococci, type 3 or 6A, and changes were monitored by otomicroscopy and analysis of bacterial samples from middle ear effusions, blood, and the nasopharynx. Depending on the type of pneumococci and its concentration, three responses were noted: otitis media with purulent effusion, otitis media with serous effusion, or no reaction. The mortality rate was low and the animals recovered without permanent deterioration or otomicroscopically discernable change. The results of this study show the rat to be a suitable animal model for the study of bacterial otitis media.     

Bacterial quantification--a necessary complement for the comprehension of middle ear inflammations.

Quantification of bacteria in various types of middle ear effusion (MEE) obtained during current acute otitis media (AOM), otitis media with effusion (OME) and chronic suppurative otitis media (COM) was performed. The bacteria were stained with acridine orange and their number per ml effusion evaluated under the fluorescence microscope according to a method described in detail elsewhere. During AOM, 53% of the MEE samples were culture-positive and contained 10(6)-10(8) bacteria per ml (median value 10(7) per ml). During OME, serous effusion and 78% of the mucoid effusions contained no bacteria whatsoever, whereas the remaining mucoid effusions contained 10(4)-5 x 10(5) bacteria per ml (median value 10(4) per ml). Mucopurulent effusions contained 6 x 10(5)-10(8) bacteria per ml (median value 5 x 10(6) per ml). During COM, purulent MEE had 6 x 10(6)-10(9) bacteria per ml (median value 10(8) per ml). Quantification of bacteria involved in middle ear diseases provides further information about the etiopathogenesis and appropriate management of various pathological conditions of the middle ear.     

Detection of fungal DNA in effusion associated with acute and serous otitis media

OBJECTIVES/HYPOTHESIS: Routine bacterial and viral cultures of middle ear fluid are often negative, suggesting that other infectious agents may be involved. Because of the similarities between the paranasal sinuses and middle ear space and the recent recognition of fungi as important pathogens in inflammation of the paranasal sinuses, we investigated the potential role of fungi in acute otitis media and serous otitis media using culture and polymerase chain reaction techniques. STUDY DESIGN: Prospective study. METHODS: Middle ear effusions of 29 patients who underwent myringotomy and pressure equalization tube placement for persistent serous otitis media or recurrent acute otitis media were collected. Fungal culture of the effusion samples was performed on potato flake agar. DNA from the effusion was isolated using standard techniques. Polymerase chain reaction, using radiolabeled universal fungus primer for internal transcribed spacer of 5.8s ribosomal DNA, was performed to detect the presence of any fungal DNA in the samples. RESULTS: Culture of middle ear effusions showed no evidence of fungal growth. Polymerase chain reaction analysis was able to detect the constituent ribosomal DNA of a single fungal genome. Fungal DNA was present in 34% of middle ear effusion samples. CONCLUSIONS: Fungal DNA is present in recurrent acute otitis media and serous otitis media suggesting that it may play an etiological role in serous otitis media and acute otitis media. However, additional studies are necessary to delineate the role of fungi in the pathogenesis of otitis media.     

Different structural changes in membrana Shrapnelli in serous and purulent otitis media. An experimental study in the rat

In an animal model, production of serous and purulent effusion material was induced by blocking the Eustachian tube and cleaving the soft palate, respectively. Two and 6 weeks after the different surgical procedures, animals were sacrificed and the membrana Shrapnelli was analysed by light- and electronmicroscopy. The two types of middle ear effusion were associated with various structural changes of the membrana Shrapnelli. In serous otitis media the pars flaccida appeared fibrotic, with large dilated vessels. Sometimes its inner layer, facing the attic space, was contiguous with the investing layer of the ossicles. In purulent otitis media the pars flaccida was thickened and the inner epithelial lining was replaced by a pseudostratified squamous epithelium containing ciliated and secretory cells. Ciliated cells lining the pars flaccida appeared pathognomonic for a longstanding middle ear infection. It is inferred that the structure of membrana Shrapnelli changes under the influence of inflammatory conditions in the middle ear cavity, changes which specifically reflect the different types of otitis media.     

Factors Affecting the Extrusion Rate of Ventilation Tubes

Objectives

The objective of this study was to determine the various factors that affect the extrusion rate of ventilation tubes (VTs), including the nature of the middle ear effusion.

Methods

A retrospective chart review of 82 pediatric patients (177 ears) who received VT insertion surgery under general anesthesia was carried out to evaluate the relationship between various factors and the VT extrusion rate. The factors we analyzed included age, gender, the adenoid size, the amount and content of the middle ear effusion after myringotomy, bleeding events, associated adenoidectomy and the findings of the tympanic membrane status, the tympanometry and the audiometry of the air bone gap.

Results

The mean extrusion time was 254 days (range, 11 to 809 days). The patients with no history of previous VT insertion had a longer extrusion time (mean, 279 days) than did the patients who had undergone previous VT insertion (mean, 203 days). The patients with serous effusion had the shortest extrusion time (mean, 190 days) as compared to those patients with glue (273 days) and pus (295 days) effusions. Other factors had no statistical significant relationship with the extrusion time.

Conclusion

The mean VT extrusion time was 254 days. The VT extrusion time was significantly related to the characteristics of the middle ear effusion and a history of previous VT insertion. Thus, the nature of middle ear effusion can provide a clinical clue to predict the VT extrusion time.     

Determination of lipid A in human middle ear effusions with the competitive ELISA technique

Summary Lipid A is a covalently bound component of gram-negative bacterial lipopolysaccharides and is the biologically active component in endotoxin. In order to detect and quantitate lipid A in middle ear effusions (MEE) from patients with otitis media with effusion (OME), antisera against lipid A were produced in mice by repeated immunization with conjugates using lipid A from Salmonella minnesota Re595 and bovine serum albumin. As a result, a competitive enzyme-linked immunosorbent assay (ELISA) was developed that was specific for lipid A. From the ELISA results, 10 out of 15 samples from the serous type MEE (mean, 13.57±2.17 g/ml) and 5 out of 6 from the mucoid type MEE (17.73±4.34 g/ml) were positive. These findings indicate that lipid A may contribute to the pathogenesis of both types of human OME.     

Predictors for postoperative otorrhea following tympanostomy tube insertion.

This controlled prospective study was designed to identify predictors for postoperative otorrhea among 157 children with chronic otitis media with effusion undergoing myringotomy and tympanostomy tube placement (intubation). Ear canal disinfection with 70% alcohol or povidone-iodine did not significantly alter ear canal or middle ear effusion bacteriology, or the frequency of otorrhea during the first 7 days after surgery. However, the risk of otorrhea on the second postoperative day was significantly increased by the presence of a bacterial pathogen in the ear canal (relative risk, 2.4), or in the middle ear effusion (relative risk, 1.9), and the presence of inflamed middle ear mucosa at surgery (relative risk, 1.7) after controlling for age, preoperative antibiotics, and postoperative ototopical cortisporin treatment. The use of systemic antimicrobial treatment in children with inflamed middle ear mucosa at surgery or whose ear canal or middle ear effusion cultures are positive for bacterial pathogens might reduce the incidence of post-operative otorrhea in children undergoing intubation for chronic otitis media with effusion.     

Histo-morphological changes of eustachian tube of guinea pigs with effusion after being treated by pulmonary surfactant]

OBJECTIVE: To study the effect of pulmonary surfactant on otitis media with effusion in guinea pigs to find a new way to manage otitis media with effusion. METHODS: Nonviable heat-killed pneumococci (HKP) solution was inoculated into the middle ear cavity in guinea pigs via a transeardrum approach to set up a model of otitis media with effusion in guinea pigs. Seven days after being injected with pulmonary surfactant (PS) by transeardrum approach, ABR threshold and histomorphological changes of eustachian tube mucosa of guinea pigs were examined by light microscopy and scanning microscopy. RESULTS: Five days following inoculation of HKB serous effusion were present in the middle ear cavity of guinea pigs, but disappearance of light cone. Response (mean +/- s) threshold raised from (14.0 +/- 3.1) dB to (45.0 +/- 5.7) dB. The eustachian tube mucosa was thickened and lined eosin-stained structureless matter over mucosa, while cilia of eustachian tube mucosa irregularly arranged. Seven days after being treated by PS, serous effusion of tympanum was reduced or disappeared, and response threshold decreased from (45.0 +/- 5.7) dB to (23.5 +/- 6.3) dB. There was significantly difference between them (P < 0.001). Eustachian tube mucosa was thinned, Cilia of eustachian tube mucosa regularly arranged to the nasopharynx. CONCLUSION: Pulmonary surfactant plays a important role in otitis media with effusion of guinea pigs.     

细菌生物膜在急性中耳炎大鼠中耳腔的形成特点及意义

目的 通过观察细菌生物膜在急性中耳炎大鼠中耳腔的形成特点,分析其与急性中耳炎的关系,并探讨该中耳炎模型用于细菌生物膜研究的可行性.方法 30只健康雄性SD大鼠,采用随机数字表法分为实验组(24只)和对照组(6只).麻醉后将50μl肺炎链球菌悬液[1×108菌落形成单位(colony forming unit,CFU)/...     

Lysozyme levels in middle ear effusion and serum in otitis media

Lysozyme concentrations in middle ear effusion and serum were determined in patients with otitis media with effusion. Lysozyme concentrations in middle ear effusion were significantly higher than in serum. Children with mucoid otitis media showed significantly higher levels of lysozyme in middle ear effusion than children with serous otitis media and adults with otitis media with effusion. Higher levels of lysozyme were observed in the group of children younger than 5 years old compared with the age group of 6- to 10-year-olds. Lysozyme concentrations of middle ear effusion in adults were significantly lower than those of mucoid otitis media in children. These results indicate that lysozyme plays an important role in the disease process of otitis media.     

Vascular events in experimental otitis media models: a comparative study

The vascular leakage and components of middle ear effusion (MEE) were determined and compared in various experimental otitis media models in the rat: purulent otitis media (POM); serous otitis media (SOM); mucoid-like otitis media (MOM); stimulating the external auditory canal with a 14 degrees C airstream and vagotomy. When the Evans blue technique was used, all otitis media models exhibited a significant increase in middle ear vessel permeability. The MEEs contained numerous PMNLs, except for animals stimulated with a 14 degrees C airstream, in which the MEE was free of cells. The IgG/SIgA ratio in MEEs indicated that the MEE components are derived from serum in the early phases of fluid accumulation, in these otitis media models. In POM, MEE components appeared to be derived from local synthesis by the middle ear mucosa as well.