Transforming growth factor-β expression in otitis media with effusion

Objective: To characterize the existence and role of transforming growth factor-β (TGF-β) in otitis media with effusion (OME). Study Design: Retrospective. Methods: The levels of two major TGF-β isoforms, TGF-β1 and TGF-β2, in the middle ear effusions (MEEs) of 44 children were evaluated using enzyme-linked immunospecific assays (ELISAs). Forty-eight MEEs were separated into three clinically relevant groups (i.e., serous, mucoid, and purulent), and TGF-β levels were correlated with clinical parameters of disease for these MEEs. Results: Both TGF-β1 and TGF-β2 were present in the samples. Mean levels of TGF-β1 (920.36 ± 437.75 pg/mg total protein) were generally 100-fold greater than those of TGF-β2 (9.65 ± 11.19 pg/mg total protein). TGF-β1 levels were elevated in association with a history of previous tympanostomy tube placements (TTPs) (P = .029) and mucoid effusions (P = .042). TGF-β2 levels were elevated in association with a history of previous TTPs (P = .100) and chronic (i.e., serous or mucoid) effusions (P = .003). Conclusions: TGF-β1 is present in the MEEs of children with OME. Furthermore, TGF-β1 and TGF-β2 levels were elevated differentially in the presence of chronic disease indicators in OME, suggesting that these isoforms may have differing roles in the inflammatory processes that characterize OME.     

Interleukin-8 expression in otitis media

Based on recent studies in the authors' laboratory on the correlation of cytokines and inflammation in otitis media (OM), the authors hypothesized that in chronic otitis media with effusion (COME) interleukin-8 (IL-8) is responsible for 1. the accumulation of leukocytes in the middle ear cleft and 2. in situ leukocyte activation with subsequent tissue damage. Additionally, the authors hypothesized that IL-8 expression is at least in part under the control of interleukin-1 (IL-1) and tumor necrosis factor (TNF). To begin to test this hypothesis, middle ear effusions (MEE) obtained from children ages 2 to 90 months (mean age, 29 months) undergoing tympanostomy tube placement for the presence of these inflammatory cytokines were analyzed. For these studies, IL-8, interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α), and tumor necrosis factor-β (TNF-β) were measured in MEE by radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). IL-8, IL-1β, TNF-α, and TNF-β were present in 92%, 67%, 77%, and 0% of effusions, respectively. The mean (± SEM) values for IL-8, IL-1β, and TNF-α were 4805 (± 913) pg/mg, 4076 (± 1510) pg/mg, and 163 (± 90) pg/mg. Further analysis indicated that levels of IL-8 correlated with IL-1β (R² = .500, P = .000) and TNF-α (R² = .387, P = .023). Thus the authors' studies clearly demonstrate that IL-8 is consistently present in the MEE of children with COME and is strongly correlated with levels of IL-1β and TNF-α, both known inducers of IL-8 production. These results support the authors' hypothesis that IL-1β, TNF-α, and IL-8 are intimately involved in the inflammatory cascade in the middle ear and suggest regulation of these cytokines as possible sites of future therapeutic intervention in otitis media with effusion (OME).     

The immunological role of the outer membrane proteins of non-typableHemophilus influenzae in otitis media with effusion in children

Summary Specific IgG and IgA antibodies against the outer membrane proteins of non-typableHemophilus influenzae were investigated in otitis media with effusion in children. Amounts of these antibodies were determined in middle ear effusions (MEEs) and in sera by enzyme-linked immunosorbent assay. At the same time the amounts of total IgG and IgA antibodies in MEEs in comparison with those in sera were analyzed by laser nephelometry. The amounts of specific and total IgG and IgA in the MEEs were higher than those in the sera. The MEEs/sera ratios of IgG and IgA antibodies in the children with mucoid effusions were higher than those in the children with serous effusions. The exception involved IgG determined by laser nephelometry. These data support the hypothesis that bacterial infections and the subsequent immune response contribute to the prolongation of otitis media with effusion in children, especially when effusions become mucoid.     

The effect of middle ear effusions from children on in vitro ciliary activity

Summary In an attempt to clarify the effect of middle ear effusions (MEEs) on ciliary activity, the ciliary beat frequency (CBF) of nasal epithelium from normal subjects was determined following incubation in Hanks' solution containing effusions from pediatric otitis media with effusion. Both serous and mucoid types of effusions increased CBF 15 min after incubation. The mucoid type of effusion was then found to decrease CBF 6 h after incubation. Since the pH and osmotic pressure of the solution containing the effusions were within the physiological ranges for normal ciliary beating, our findings show that MEEs can influence CBF in vitro. Offprint requests to: Y. Sakakura     

Cytokines in children with otitis media with effusion

We assayed 38 middle ear effusions from 23 children aged 4–13 years (mean 7) undergoing tympanostomy tube placements. All fluid was assayed for tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL-8, and IL-10. Cytokine concentrations were measured by means of an enzyme-linked immunosorbent assay. Detectable levels of IL-1β, IL-8, and IL-10 were found in all of the effusions. TNF-α was detected in 18 of the middle ear effusions (47.4%). The mean concentration of TNF-α, IL-1β, IL-8, and IL-10 was, respectively, 0.423 ± 1.39, 30.58 ± 68.7, 7001.9 ± 6743, and 56 ± 58.7 pg/ml. There was a strong, statistically significant correlation between the concentrations of TNF-α and IL-1β (r = 0.87, P = 0.001) and between IL-1β and IL-8 (r = 0.53, P = 0.001). There was no correlation between the concentrations of IL-10 and other cytokines examined or between tympanic membrane pathology and the concentrations of TNF-α, IL-1β, IL-8, or IL-10. The presence of IL-10 in middle ear effusions may be one of the causes of a lack of clinical features of acute inflammation and may lead to a chronic inflammatory state. Received: 25 August 1999 / Accepted: 5 January 2000     

Viscoelastic properties of middle ear effusions from pediatric otitis media with effusion and their relation to gross appearance

Summary The viscosity and the elasticity were measured in 146 individual middle ear effusions (MEEs) from 93 children with otitis media with effusion (OME). The effusions were classified into serous or mucoid type by their macroscopic appearance. The distribution of elasticity showed two peaks, corresponding to the peaks of serous and mucoid types of effusions. There were a small number of effusions in the intermediate range of elasticity, where the effusions were most effectively transported by the cilia. These findings suggests that the MEEs of pediatric OME are accumulations of rheologically suboptimal fluid in the middle ear cavity.     

Characteristic differences in the lipid composition of middle ear effusions in adult and pediatric patients: phosphatidylethanolamine and phosphatidylserine levels

Summary Middle ear effusions (MEEs) from adult patients with otitis media with effusion are usually serous in nature, but those from pediatric patients younger than 8 years old are frequently mucous in consistency. MEEs contain substances secreted by the epithelial cells of the middle ear and eustachian tube to regulate surface tensions as well as those produced as the result of inflammation. Since the biochemical bases of serous and mucous MEEs have not been clearly established, we analyzed all lipid components of MEEs from seven children and seven adults by thin-layer and gas-liquid chromatography, and also compared them with those from sera. Although no significant difference in the lipid composition was observed between adult and pediatric sera, the relative concentration of phosphatidylethanolamine (PE) in the pediatric MEEs was 26.5%, while that in the adult MEEs was 9.2% and was significantly different. A similar high concentration of phosphatidylserine (PS) was also observed in the pediatric MEE. Since phospholipids are major components of surfactants secreted from the epithelial cells of the middle ear, significantly high concentrations of both PE and PS as charged phospholipids may be responsible in part for the mucoid characteristics seen in pediatric MEEs.This work was supported in part by a Grant-in-Aid for Acute Profound Deafness from the Ministry of Health and Welfare, Japan Offprint requests to: R. Yabe     

Alloiococcus otitidis in acute otitis media

OBJECTIVE: The bacterium Alloiococcus otitidis has been found to be associated with otitis media with effusion (OME). When the culture method is used, its detection rate is low, whereas applying the polymerase chain reaction (PCR) yields significantly higher frequencies. This study was carried out to investigate the incidence of A. otitidis in children with acute otitis media (AOM). METHODS: Multiplex PCR was used to detect A. otitidis together with Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the middle ear effusions (MEEs) of 118 children with AOM. The clinical outcome of AOM and the bacterial findings of MEEs were compared. RESULTS: A. otitidis was detected in 25% (30 of 118) of the tested MEE samples. Children over 2 years of age had significantly more often A. otitidis-positive MEEs (37%; 22 of 59) than younger children (14%; 8 of 59) (chi-square test, P<0.01). There were no significant differences in the duration, clinical failures (after antibiotic treatment), or number of recurrences of AOM between the A. otitidis-positive and A. otitidis-negative children. CONCLUSIONS: A. otitidis is found from the MEEs of AOM. The present data suggest that it has no clinical significance in AOM, and it does not increase the risk of developing OME after AOM.     

Cytokines in experimental otitis media with effusion

Studies in the authors' laboratory have recently demonstrated the presence of potent inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in human middle ear effusions. The clinical significance of this finding has not been fully elucidated because of the limitations of human studies. We hypothesized that the chinchilla model of otitis media may be an appropriate system with which to study the role of cytokines in otitis media with effusion. To begin to investigate this possibility, 30 chinchillas underwent surgical blockage of the eustachian tube (ET) to promote effusion development. After 2 weeks, examination by otoscopy demonstrated 27 ears to have developed an effusion. Next, all middle ear clefts, in random manner, were either injected with heat-killed Streptococcus pneumoniae 1 × 10⁶ in 0.1 mL normal saline, injected with 0.1 mL normal saline alone, or received no injection at all. Middle ear effusions were obtained and analyzed for IL-1β and TNFα by enzyme-linked immunosorbent assay (ELISA). This study demonstrated a significant correlation between IL-1β and the presence of an effusion (P<.001). Additionally, increased TNFα levels correlated with bacterial component presence (P<.001), i.e., mean TNFα level was 108, 10.8, and 0 pg/mL in bacteria, normal saline, and noninjected ears, respectively. These findings would suggest that cytokine expression may relate to specific pathological conditions and that the chinchilla model for otitis media with effusion (OME) could be used to further explore the role of cytokines in OME.     

TH1/TH2 and regulatory cytokines in adults with otitis media with effusion.

OBJECTIVE: Otitis media with effusion is one of the most common and intractable ear diseases. However, the role of Th1, Th2, and immunoregulatory cytokines on the pathogenesis of the disease in adult patients remains to be determined. The aim of this study is to disclose the cytokine expression in middle ear effusions (MEEs) in adults and to compare the profile on the basis of the presence of allergic rhinitis and the type of effusions. STUDY DESIGN: A prospective controlled clinical study. PATIENTS: MEEs were collected from 80 adult subjects. The concentration of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, and interferon (IFN)-gamma in MEEs were determined by using enzyme-linked immunosorbent assay. RESULTS: IL-2, IL-4, IL-5, IL-10, IL-12, and IFN-gamma in MEEs were detected in 60 (75.0%), 33 (41.3%), 42 (52.5%), 14 (17.5%), 80 (100%), and 66 (82.5%) samples, respectively. Among these cytokines, only the concentration of IL-4 in the allergic rhinitis-positive group was significantly higher than that in the allergic rhinitis-negative group. On the other hand, IL-2, IL-12, and IFN-gamma were detected, regardless of the presence of allergic rhinitis, and the concentration of these cytokines correlated with each other. The correlation between the concentration of IL-4 and IL-5 was also detected. In addition, both the incidence rate and the concentration of IL-10 in MEEs were significantly higher in the mucoid type compared with those in the serous type effusions. CONCLUSION: Regardless of allergic status, IL-12 may play a critical role in the pathogenesis of otitis media with effusion by affecting the production of IL-2 and IFN-gamma. In addition, IL-4 may have some impact on the immunologic condition in adults with allergic rhinitis. IL-10 potentially affects the viscosity of MEEs.     

The role of vascular endothelial growth factor in pediatric otitis media with effusion

Objectives

Vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis and vascular permeability, and has been shown to play an important role in inflammatory events, together with hypoxia and inflammatory cytokines. VEGF messenger ribonucleic acid (mRNA) is expressed in the middle ear in an experimental animal model of otitis media with effusion (OME) and in patients with OME. However, the protein levels of VEGF in middle ear effusions (MEEs) are unknown and the role of VEGF in the pathogenesis of OME is unclear. The goals of this study were to determine the VEGF levels in MEEs and to investigate the role of VEGF in production of MEEs by comparing these levels with those of interleukin-8 (IL-8), endotoxin, and albumin.

Methods

Forty-six MEEs obtained from 33 children (24 boys, 9 girls) were used in the study. The mean age of the subjects was 6.3 years old (range, 1-12 years old). The patients underwent myringotomy and/or insertion of a ventilation tube for treatment of OME. After myringotomy, MEEs were collected with a Juhn Tym-Tap. The samples were divided into serous and mucoid types based on observation by the naked eye. After measuring the weight of the MEE, the sample was diluted with phosphate-buffered saline and frozen until use. The concentrations of VEGF and IL-8 in the MEEs were determined by enzyme-linked immunosorbent assays, endotoxin concentrations were measured by the Limulus Amebocyte Lysate test, and albumin levels were determined using an immunoturbidimetric assay.

Results

VEGF, endotoxin, IL-8, and albumin were detected in 100%, 89%, 98%, and 100% of the 46 MEEs, respectively. The concentrations of VEGF, endotoxin, and IL-8 were significantly higher in mucoid MEEs than in serous MEEs (p < 0.01), whereas there was no significant difference in the albumin concentration between mucoid and serous MEEs. The VEGF levels were positively correlated with those of endotoxin (R² = 0.17, p < 0.05) and albumin (R² = 0.65, p < 0.01) in mucoid MEEs, but not in serous MEEs.

Conclusions

Our results suggest that VEGF is produced in response to bacterial components such as endotoxin in the middle ear cavity and is associated with production of mucoid MEEs by increasing serum exudation and mucosal secretion.     

Soluble L-selectin and interleukin-8 in otitis media with effusion

Objective: adhesion of leukocytes to vascular endothelium and their invasion into local tissues are important steps in inflammation. L-selectin plays a crucial role in leukocyte rolling and adhesion on endothelial cell surface. Interleukin-8 (IL-8) is a potent chemotactic substance toward neutrophils and lymphocytes-T and mediate their migration to local inflammation. The levels of soluble L-selectin (sL-selectin) and IL-8 were measured in middle ear effusions (MEE) from children with otitis media (OM) to estimate their role in pathogenesis of inflammation in middle ear. Methods: MEE were collected from 113 ears of 62 children during routine myringotomy. The entire specimen was diluted with phosphate-buffered saline (PBS) to 2 ml and centrifugated at 1500 rpm for 15 min to separate cellular components. Supernatants of MEE were stored frozen at -20 degrees C for sL-selectin and at -80 degrees C for IL-8 assessment. The concentration of sL-selectin and IL-8 were estimated with ELISA and compared with total protein concentration measured by Bradford assay. Results: MEEs from children with chronic mucous otitis media contained significantly higher mean concentrations of IL-8 559.4 pg/mg total protein (TP) (+/-535.6) in comparison with normal serum 17.79 pg/mg TP (+/-10.9), serous OM 40.3 pg/mg TP (+/-28.1) and purulent OM 104.4 pg/mg TP (+/-128.6). High concentration of IL-8 was found in MEE from the children with early recurrence of OMS. The levels of sL-selectin were higher in purulent effusions 77.2 ng/mg TP (+/-67.4) than those of chronic mucoid 27.6 ng/mg TP (+/-36.7) and serous OM 26.3 ng/mg TP (+/-27.1). Conclusion: the results support a hypothesis that IL-8 can be responsible for prolongation of inflammatory process in middle ear, therefore long-term treatment and observation of children with the high levels of IL-8 in MEE may be necessary. Elevated level of sL-selectin in acute OM suggests the involvement of L-selectin in the acute phase of OM.     

Biochemical differences in middle ear effusions between pediatric and adult patients, (II). The protein composition

To clarify the biochemical differences between pediatric and adult middle ear effusions (MEEs) in secretory otitis media with effusion (OME), we analyzed the protein components from MEEs of 3 children and 3 adults by electrophoresis. High molecular protein components (280 and 265 kDa proteins), which contained in sera in the concentrations less than 2.5%, were not detected in MEEs even in trace amounts. The protein components which molecular weights were less than 260 kDa are transferred from sera into MEEs in a same proportion as in sera. Mucin type glycoprotein was comprised about 30% of the total protein in pediatric mucous MEEs and the weight ratio of mucin to albumin in pediatric mucous MEEs was 2.69, but serous MEEs from adult patients did not contain mucin. Immunoglobulin (IgA, IgG and IgM) concentrations in pediatric mucous MEEs were same as in adult serous MEEs. The concentrations of IgA and IgM in MEEs were higher than in sera. Lysozyme concentration in MEEs was also higher than in sera, and that in pediatric mucous MEEs was nearly 2 times higher than serous MEEs from adult patients.     

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.     

Otitis media: the middle ear effusion total white cell count

The middle ear effusion (MEE) total white cell count (TWCC) was correlated with clinical and bacteriologic data in 184 MEEs from 125 patients diagnosed as having acute primary and recurrent otitis media and chronic otitis media with effusion. The MEE total white cell count was classified as high, low, or acellular. Polymorphonuclear leukocytes predominate in the MEE having a high TWCC. The overall incidence of culture-positive MEE was 62.5 percent. A cellular component was found in 81 percent of the MEEs, 63 percent having a neutrophilic-predominant high TWCC. In very young children (up to 2 years old) a neutrophilic-predominant high TWCC was found in 80 percent of MEEs as compared with 28 percent in patients over 5 years old. According to the MEE type, neutrophilic-predominant high TWCCs were found in 89 percent of the purulent effusions, 46 percent of the mucoid, and 35 percent of the serous effusions. The incidence of neutrophilic-predominant high TWCC was 78 percent in acute primary 76 percent in recurrent otitis media and 18 percent in chronic otitis media with effusion. The impact of age on the MEE cellularity was shown to be independent of the chronicity of the disease. The incidence of neutrophilic-predominant high TWCC in MEE in which Streptococcus Pneumoniae or Hemophilus influenzae was identified was 85 percent and 92 percent respectively. The diagnostic value of a high TWCC in predicting a culture-positive MEE was shown by a sensitivity of 83 percent, false positivity of 25 percent, and false negativity of 27.5 percent.     

Bacteria and endotoxin in middle ear fluid and the course of secretory otitis media

To determine the influence of bacteria and bacterial products on the course of secretory otitis media (SOM) following ventilation tube insertion we examined 68 middle ear effusions from 57 children with SOM. Swabs were taken for bacteriological examination and the content of endotoxin was measured. The children were followed up for one year and relapse of SOM was recorded. Bacteria were present in 38% of effusions and endotoxin was detectable in 96%. Relapse occurred in 26% of the patients and was related to the presence of bacteria in the original effusion (P 0.01). The concentration of endotoxin, however, did not correlate with the course of SOM. Keywords bacteria endotoxin secretory otitis media prognosis     

Evidence of T-helper cell 2 cytokine regulation of chronic otitis media with effusion

Conclusions

Cytokine and cellular patterns of effusions may reflect stages of middle ear inflammation. The local interplay between IL-2 and -4 is likely to play a crucial role in the switching of inflammation in the chronic stage. The T-helper cell 2 (Th2) cytokines IL-4, -5 and -13 and the Th2/Th1 cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) regulate the cellular and molecular processes of chronic inflammation in the middle ear and therefore the chronic condition of otitis media with effusion (OME). Early identification of the cytokine and cellular patterns of effusions can be helpful in directing the clinical treatment of OME.

Objective

We hypothesized that IL-2 and the group of Th2 cytokines regulate chronic inflammation in the middle ear and chronic OME.

Material and methods

Effusions from children with persistent OME were analysed to determine the presence of cytokines (the Th1 cytokine IL-2, the Th2 cytokines IL-4, -5 and -13 and the Th1/Th2 cytokine GM-CSF), inflammatory cells (CD4⁺ T cells, eosinophils, macrophages and neutrophils) and mucin. Cytokines were evaluated by means of a quantitative “sandwich”-type ELISA, inflammatory cells by means of alkaline phosphatase–anti-alkaline phosphatase immunocytostaining and mucin by means of a modified periodic acid–Schiff method based on a slot–blot technique.

Results

The cytokine pattern in effusions varied from patient to patient. GM-CSF correlated positively and IL-4 inversely with IL-2 and the increased level of IL-4 may have had an inhibitory effect on IL-2. IL-5 and -13 correlated with IL-4. Inflammatory cells correlated with cytokines as follows: CD4⁺ T cells with IL-2 and -4; macrophages and neutrophils with GM-CSF; and eosinophils with IL-5. Some cytokine–cellular correlations in effusions were reflected at the clinical level. The mucin content of effusions correlated with the concentrations of IL-4 (>10 pg/ml) and -13, suggesting involvement of IL-4 and -13 in upregulation of the middle ear mucin metabolism.     

Characterization of cytokines present in pediatric otitis media with effusion: comparison of allergy positive and negative

To investigate the possible relationship between allergy and otitis media with effusion (OME), we investigated the cytokine level in the middle ear effusion (MEE)s of children with persistent OME. Interleukin (IL)-2, IL-4, IL-6 and tumor necrosis factor (TNF)-alpha in the MEEs were measured by devised sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and compared allergy positive group with allergy negative group. The mean levels of IL-4, IL-6 and TNF-alpha in MEE were significantly higher in allergy positive group than allergy negative group (P<0.05). Elevation of Th-2-driven cytokines (IL-4, IL-6) and TNF-alpha in MEEs may be a contributing factor in the persistence of OME with allergy.     

Prognosis of secretory otitis media in relation to viscoelasticity of effusions in children

Both elasticity (G') and viscosity (eta') of middle ear effusions (MEEs) were measured with an oscillating sphere magnetic rheometer and compared with continuance of fluid retention in 93 ears of 69 children with otitis media with effusion (OME). The ears were divided into four groups according to the viscoelastic properties of MEEs at the first myringotomy. Eight-four percent of the ears in group 2 were free from effusion within 4 months; the difference from the other groups was statistically significant. These results indicate that the mucociliary clearance function plays an important role in the process of recovery from OME.     

Findings at initial surgery for childhood otitis media with effusion and subsequent outcome

The outcome of initial surgical treatment in children with otitis media with effusion (OME) was analysed in a prospective study involving 225 new patients. The presence or absence of effusion in each ear and type of effusion present were recorded at surgery. The outcome measure studied was resolution or recurrence of middle ear effusions necessitating further surgical intervention. The outcome was significantly more favourable in children with unilateral effusions at surgery as opposed to bilateral effusions (P> 0.03) but was unrelated to the type of effusion (serous or mucoid). Patients with unilateral effusions at surgery appear to have a fluctuating form of OME in which effusions are present in either or both ears at different times.