[Dynamics of free-radical oxidation in suppurative otitis media]. / Dinamika svobodno-radikal'nogo okisleniia pri gnoino-vospalitel'nykh zabolevaniiakh srednego ukha.

The dynamics of indicators of free-radical oxidation in various purulent-inflammatory diseases of the middle ear in 280 patients was studied. Significant violations of the processes of free radical oxidation in the serum of blood were taken for the patients, the degree of which depends entirely on the severity and nature of the inflammation. A complex treatment with the use of antioxidant therapy in 280 patients was carried out. Positive results were obtained, confirmed by clinical and laboratory indicators. The pathogenetic significance and dependence of the course of purulent-inflammatory diseases of the middle ear on the processes of free radical oxidation was established, which was reflected in the determination of the duration of treatment of various forms of both acute and chronic otitis media.

[The alteration of the parameters of lipid peroxidation and the antioxidative system in the children presenting with pyo-inflammatory diseases of the middle ear].

The objective of the present study was to determine the amount of products of lipid peroxidation (dienic conjugates and malonic dialdehydes) together with antioxidative enzymes (catalase and superoxide dismutase) in 47 children presenting with acute pyo-inflammatory otitis media (APIOM) and 125 patients with chronic suppurative mesotympanitis (CSMT). In addition, 49 children presenting with chronic suppurative epitympanitis (CSET) and chronic suppurative epimesotympanitis (CSEMT) were included in the study. The data obtained were analysed and compared with the similar characteristics of 28 healthy children of the same age comprising the control group. The age of the children in different groups varied between 2 and 14 years. The study revealed the increase in the amount of lipid peroxidation products in all the groups with the simultaneous reduction of the activity of antioxidative enzymes; moreover, these values changed depending on the form and clinical course of the disease.

Polymorphisms in Toll-like receptors 2 and 4 genes and their expression in chronic suppurative otitis media.

OBJECTIVE: Toll-like receptors (TLRs) have a prominent role in inducing innate immune response. It has been suggested that regulation of TLRs is involved in the pathogenesis of chronic otitis media. TLR 2 and TLR 4 polymorphisms were connected with susceptibility to acute otitis and chronic otitis with effusion. The objective of this study was to establish expression of TLR 2 and 4 on middle ear mucosa in different types of chronic suppurative otitis media (CSOM), and the influence of gene polymorphisms TLR 2 Arg753Gln and TLR 4 Thr399Ile and Asp299Gly to susceptibility to CSOM. MATERIAL AND METHODS: Middle ear mucosa and full blood samples were obtained from 85 patients with chronic suppurative otitis media with and without cholesteatoma. Control group for mucosal TLR expression consisted of 71 samples of middle ear mucosa taken from patients with otosclerosis, and control group for DNA polymorphism consisted of 100 full blood samples in healthy subjects. DNA polymorphism detection was done with restriction fragment length polymorphism in RT PCR. Expression of TLR 2 and 4 was determined with immunohistochemical staining. RESULTS: TLR 2 and TLR 4 expression on the middle ear mucosa was not influenced by age of the patients with chronic otitis media. Incidence of TLR 2 Arg753Gln polymorphism was significantly higher in patients with chronic otitis media, compared to control group. Significant association between TLR 2 Arg753Gln polymorphism and different types of mucosal changes in patients with chronic otitis media was established. TLR 2 and 4 expression on experimental group mucosa was significantly different compared to control group, where there was no expression (p=0.000). Strong dependence of TLR 2 and TLR 4 expression on middle ear mucosa with different mucosal changes and immunohistochemical activity after staining was detected. CONCLUSION: Certain polymorphisms in TLR genes could be indicative for susceptibility to chronic otitis media. Expression of TLR 2 and 4 on middle ear mucosa was more dependable on different types of mucosal changes and type of CSOM than on bacteria found in the specimens. This can indicate that the type of mucosal changes are closely correlated with TLRs activity in middle ear.

The role of gastric pepsin in the inflammatory cascade of pediatric otitis media.

IMPORTANCE: Otitis media is characterized as an ongoing inflammation with accumulation of an effusion in the middle ear cleft. The molecular mechanisms underlying the pathogenesis, particularly the inflammatory response, remain largely unknown. We hypothesize that aspiration of gastric contents into the nasopharynx may be responsible for the initiation of the inflammatory process or aggravate a preexisting condition. OBJECTIVE: To investigate the correlation of gastric pepsin A with inflammatory cytokines, bacterial infection, and clinical outcomes. DESIGN, SETTING, AND PARTICIPANTS: Prospective study of 129 pediatric patients undergoing myringotomy with tube placement for otitis media at a tertiary care pediatric hospital. MAIN OUTCOMES AND MEASURES: Ear samples were tested for pepsin A; cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor; and bacterial culture inoculation. Data were analyzed by descriptive statistics and regression analysis to identify risk factors for the presence of pepsin A and to correlate pepsin A levels with cytokine levels, infection status, and clinical outcomes. RESULTS: Of the 129 patients, 199 ear samples were obtained; 82 samples (41%) and 64 patients (50%) were positive for pepsin A as measured by immunoassay. Pepsin A positivity correlated with age younger than 3.0 years (mean [SD], 2.3 [2.1] years in the positive group vs 3.3 [3.0] years in the negative group) and with all 3 cytokine levels (mean [SD] tumor necrosis factor, 29.5 [45.9] pg/mL in the positive group vs 13.2 [21.6] pg/mL in the negative group; IL-6, 6791.7 [9389.1] pg/mL in the positive group vs 2849.9 [4066.3] pg/mL in the negative group; and IL-8, 6828.2 [8122.3] pg/mL in the positive group vs 2925.1 [3364.5] pg/mL in the negative group [all P < .05]); however, logistic regression analysis showed that only IL-8 (odds ratio, 3.96; 95% CI, 1.3-12.0; P = .02) and age (odds ratio, 3.83; 95% CI, 1.2-12.7; P = .03) were significant independent variables. No statistically significant association was found with other parameters. Multiple linear regressions revealed that the levels of pepsin A were correlated with IL-8 levels (R2 = 0.248; P < .001) and the need for second or third tubes 6 to 12 months after the first (R2 = 0.102; P = .006). The presence of pepsin A in the middle ear was not associated with increased bacterial infection. Interleukin 8 was independent and significantly associated with both pepsin A levels and bacterial infection (R2 = 0.144 and 0.263, respectively; P = .001 for both). CONCLUSIONS AND RELEVANCE: Extraesophageal reflux as indicated by the presence of pepsin A is closely involved in the middle ear inflammatory process and may worsen the disease in some children; however, a proof of cause and effect between extraesophageal reflux and middle ear inflammation requires further investigation.

Permeability of the round window membrane to middle-sized molecules in purulent otitis media.

The effect of pneumococcal otitis media on the permeability of the round window membrane was evaluated using tetraethylammonium ions as a tracer. Round window membrane permeability is reduced significantly at resolved stages of purulent otitis media. In contrast, measurements of round window membrane permeability indicate that acute purulent otitis media has a tendency to facilitate such permeability. Moreover, histologic observations of the round window membrane following bacterial inoculation further support the evidence of functional changes in round window membrane permeability. These findings indicate that the round window membrane in resolved stages of purulent otitis media plays a protective role in preventing the penetration of harmful substances into the inner ear.

Permeability of the round window membrane to horseradish peroxidase in experimental otitis media.

The permeability of the round window membrane (RWM) was studied by using horseradish peroxidase (HRP) in the different stages of otitis media developed in guinea pigs, and it was compared with normal RWM under transmission electron microscopic examination. The experimental model of otitis media was developed by inoculation of a Pseudomonas aeruginosa suspension. When HRP could penetrate into the inner layer of a normal RWM, the duration of HRP instillation into the round window niche was 10 minutes. The permeability of the RWM in otitis media was as follows: 3-day animals showed penetration of HRP into the middle layer. In 1-week animals, HRP had penetrated into the inner layer in all 10 ears. In 2-week animals, HRP had penetrated into the inner layer in 6 of 11 ears. In 4-week animals, however, HRP was not seen in 6 of 7 ears. HRP had penetrated through the damaged focus of the epithelial cells by inflammation. These results suggest that any derangement of the epithelial linings could increase the penetration of HRP and also ototoxic materials into the inner ear through the RWM.

Tympanostomy tubes and otic suspensions: do they reach the middle ear space?

The treatment of patients with tympanostomy tubes (TTs) and otorrhea with medicated otic suspensions is well known, but confirmation of penetration into the middle ear is difficult. To address this question, we created an in vitro model of the human head and ear and then tested it with 5 different types of liquid exposure: tap water, soapy water, polymyxin B sulfate (Cortisporin), tobramycin and dexamethasone (TobraDex), and ciprofloxacin (Cipro) suspensions. A positive test result corresponded to liquids entering the middle ear through the TT. No positive test result was elicited with tap water (0/20), but soapy water did enter the middle ear (10/40) and was statistically significant (P = 0.0112). Without the use of slight tragal pressure, Cortisporin, TobraDex, and Cipro drops did not consistently pass through the TT (0/20, 1/25, 1/25). By placing the drops with the addition of tragal pressure, a statistically significant difference was obtained for each solution (20/20, 20/20, and 20/20, respectively [P < 0.0001]). We conclude that with a clean external auditory canal, patent TT, and no middle ear fluid, medicated otic suspensions enter the middle ear only when combined with slight tragal pressure.

Arachidonic acid metabolites in experimental otitis media and effects of anti-inflammatory drugs.

Previous studies have shown that arachidonic acid (AA) metabolites are important in the pathogenesis of otitis media with effusion. The AA metabolites in 4 different experimental models for otitis media were analyzed, and the effect of anti-inflammatory drugs was studied. Purulent otitis media was induced in rats by inoculation of Streptococcus pneumoniae in the tympanic bulla, serous otitis media by blocking the tympanal orifice of the eustachian tube, and mucoid otitis media by combining the two procedures. Middle ear effusion was also induced by stimulating the external auditory canal with cold air. Indomethacin and hydrocortisone were used to inhibit AA metabolism in the latter model. Lipoxygenase products dominated in the purulent and cold air otitis media models. Cyclooxygenase products dominated in the mucoid and serous models. Indomethacin inhibited accumulation of middle ear effusion in the cold air otitis media model, whereas hydrocortisone did not. Apart from AA metabolites, other mechanisms and mediators appear to be responsible for the increased vessel permeability observed in the cold air otitis media model, such as interactions between mast cells and nerves in the middle ear mucosa.

Implication of surfactant apoprotein in otitis media with effusion.

A two-site simultaneous immunoassay using monoclonal antibodies against human surfactant apoprotein (SAP) was used to measure SAP in middle ear effusions (MEEs). In 130 MEE samples from children with otitis media with effusion, SAP was detected in 54 samples (SAP-positive cases, 41.5%). In the remainder, the SAP concentration was below the sensitivity of the immunoassay (SAP-negative cases, 58.5%). A significant difference in periods of observation was found between the SAP-positive cases (17.3 +/- 16.8 months) and the SAP-negative cases (26.2 +/- 22.5 months) (p less than .01). The percentage of positive cases was highest in the serous MEE group (81.2%) and decreased in the purulent MEE group (57%), the mucoid MEE group (30%), and the hyperviscous MEE group (13.6%), in that order. In the purulent MEE group and the mucoid MEE group, the period of observation was significantly shorter in the SAP-positive cases (18.3 +/- 20.4 months and 20.2 +/- 19.4 months) than in the SAP-negative cases (35.9 +/- 24.5 months and 25.4 +/- 18.7 months) (p less than .05). These results suggest that SAP is present in the middle ear cleft and may be a good prognostic predictor of otitis media with effusion in children.

Hyaluronan in experimental serous and purulent otitis media.

The concentration of hyaluronan (hyaluronic acid; sodium hyaluronate; HA), a major component of connective tissue, was analyzed by a specific radioassay in tissue samples from normal rat middle ears and in tissue and effusions from middle ears affected by experimentally induced otitis media. In normal ears the pars flaccida of the tympanic membrane contained a considerably higher concentration of HA (60 to 200 micrograms/g) than the pars tensa and areas of the medial wall (2 to 7 micrograms/g). In purulent otitis media the pars flaccida contained less HA than in normal ears. The HA concentrations were lower in purulent (3 to 40 micrograms/mL) than in serous (20 to 190 micrograms/mL) effusions. The weight average molecular weight of HA in serous fluid was high (greater than 10(6] and comparable to that in lymph. It is inferred from the study that the subepithelial tissue and its matrix components, eg, HA, must be considered when attempting to understand the function of the middle ear normally and in otitis media.

Morphologic and biochemical study of vascular permeability of the middle ear mucosa in experimental otitis media.

The vascular permeability of the middle ear mucosa of chinchillas was examined by light and electron microscopy using horseradish peroxidase (HRP). Two kinds of experimental otitis media were used: serous otitis media (SOM) produced by eustachian tube obstruction and purulent otitis media (POM) produced by inoculation with Streptococcus pneumoniae type 7F. Normal mucosa showed no leakage of HRP. In SOM, vessels were dilated and tortuous, but they did not show HRP leakage. In POM, vessel leakage of HRP was extensive. Biochemically, we measured the middle ear effusion-to-serum ratio of both albumin and alpha 2-macroglobulin by an immunodiffusion method. The ratios of the two marker proteins for vascular permeability were significantly higher in POM than in SOM. This suggests that POM increases vascular permeability to a higher degree than SOM, and morphologic findings in the middle ear mucosa correlate with the biochemical composition of the middle ear effusion.

Concentration of nitric oxide metabolites in middle ear effusion.

Free radicals such as nitric oxide (NO) seem to be important in the pathogenesis of otitis media with effusion (OME). NO can be quantitated by measuring its metabolites, nitrate (NO(3)(-)) and nitrite (NO(2)(-)). The purpose of this study is to determine the concentrations of NO in human middle ear effusion (MEE). Samples of human MEE were collected at the time of myringotomy and tympanostomy tube insertions. The type of MEE was classified as serous (SOM), mucoid (MOM) or purulent (POM) at the time of surgery. Samples of MEE were assayed for NO metabolites (nitrate and nitrite) with colorimetric assay (Griess method). Concentrations of NO metabolites were highest in MOM followed by SOM and POM. This study suggests that NO is present in human MEE and may play an important role in the pathogenesis of OME.

Cholesterol crystals in the temporal bone and the paranasal sinuses.

In order to study the manifestation and genesis of cholesterol deposits in the temporal bone and the paranasal sinuses, 137 temporal bones and 3 paranasal sinuses with deposits were examined. For histological study, 1036 specimens were obtained from temporal bones, and 16 specimens were obtained from paranasal sinuses. Several stages of a progressive foreign body reaction, including the appearance of histiocytes, granuloma formation and the development of granulation into scar tissue, were observed depending on the size and form of the crystals. Many large deposits were found in the temporal bones of patients with chronic non-suppurative lesions. Blue eardrum and hemotympanum were seen in the last stage of this foreign body reaction in these patients. Small deposits were found in the temporal bones and the paranasal sinuses of patients with chronic suppurative lesions. Most of these deposits were found in the obstructed parts. The pathogenesis of the cholesterol deposits is suspected to be fatty degeneration of the connective tissue.

Penetration of doxycycline in respiratory mucosa.

In order to study the penetration of doxycycline (Idocyklin) in respiratory mucosa 100 mg tablets were given in standard dosage to 58 patients with longstanding secretory otitis media, the "secretion-in-cavity" model, and to 37 patients with nasal polyposis, the tissue model. Myringotomy with aspiration of the middle ear secretion or polypectomy was performed at different intervals during the course of medication. Blood samples were drawn simultaneously. The concentration in secretion, tissue and serum was assessed by means of a microbiological agar well method. The mean concentration of doxycycline in respiratory mucosa and secretion was found to exceed the MICs of the common respiratory pathogens as soon as 3 hours after the first dose, and remained on a therapeutical level until 24 hours after the last dose. The results are in full agreement with those obtained from penetration studies in other parts of the respiratory tract, thereby lending support to the validity of the models used.

Round window membrane permeability during experimental purulent otitis media: altered Cortisporin ototoxicity.

Round window membrane (RWM) permeability is the most critical factor influencing cochlear function following otitis media. Because otic drops are frequently used during purulent otitis media (POM), we investigated RWM permeability and ototoxicity of Cortisporin otic suspension after inducing experimental POM. Unilateral POM was induced in eight chinchillas by inoculating type 7F Streptococcus pneumoniae into the right ears. Left ears were inoculated with phosphate-buffered saline (control). When POM resolved, the animals were divided into two groups. The round window niches of group 1 were covered with Cortisporin otic suspension. Compound action potentials were measured before and after drug application. The RWM permeability was measured in group 2 by use of tetraethylammonium (TEA) ions as tracers, and the arrival time of TEA and the slope of the potassium-selective microelectrode response were measured. Animals with otitis media exhibited less susceptibility to ototoxicity of Cortisporin otic suspension and reduced RWM permeability to the medium-sized molecule TEA.

Immunologic characteristics of cytokines in otitis media with effusion.

Levels of cytokines, interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated in samples of the middle ear effusions (MEEs) from 144 ears with otitis media with effusion (OME) by enzyme-linked immunosorbent assay, followed by cytologic analysis. Middle ear effusions of the acute purulent type contained a significantly higher concentration of cytokines compared with normal control sera (p < .001). Cytokines were observed at lower levels in MEE in adults than in children. Tests of children at the chronic stage of MEE showed higher levels of TNF than IL-1 and GM-CSF. Meanwhile, IL-1 beta showed significantly higher concentrations in acute purulent types than in serous and mucoid types (p < .01). In cytologic analysis, the mean level of IL-1 beta was significantly higher in the neutrophil-rich group than in other groups (p < .05). Cytokines possess several biologic properties, some of which are associated not only with acute otitis media but also with chronic otitis media. This study showed that cytokines, especially IL-1 beta, contribute to infiltration into the middle ear by inflammatory cells. This implies that the persistent presence of cytokines in MEE could be a factor in prolonged OME.

Cytological and immunochemical characterization of middle ear effusions: an experimental approach.

The aim of the present study was to experimentally provoke well-defined serous and purulent effusion materials and to analyze the fluid using cytological and immunochemical techniques. Using these parameters an attempt was made to find a marker(s) for the degree of inflammation, duration of the disease and possible sequelae.

[A study of concentration of ampicillin into middle ear effusion after administration of bacampicillin].

We studied the concentration of ampicillin (ABPC) into middle ear effusions after administration of bacampicillin (BAPC). Nine patients with acute purulent otitis media were given orally single doses of the drug at a level of 10 mg/kg, and concentrations of ABPC which is the active antibiotic metabolite of BAPC were determined in middle ear effusions periodically after the administration. Bacteria present in effusions were identified, and their ability to produce beta-lactamase was also determined. ABPC concentrations in middle ear effusions were inversely related to the ability of bacteria detected from the intratympanic cavity to produce beta-lactamase. ABPC concentrations in middle ear effusions from which only beta-lactamase negative organisms were detected were higher by 5.2- and 2.3- fold at 60 and 120 minutes after the administration of BAPC, respectively, than those in effusions from which beta-lactamase positive organisms were detected. ABPC concentrations achieved in middle ear effusions in cases where only beta-lactamase negative organisms were detected exceeded MIC80's of the drug against main causative bacteria of acute purulent otitis media such as Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae. Because only 10% or less of these species produces beta-lactamase, BAPC appears to be one of the highly effective drugs in the treatment of this disease.

[Lipoperoxidation in otorrhea of the middle ear as a marker of infection. Clinical application]. / Lipoperoxidación en otorreas del oído medio como marcador de infección. Aplicación clínica.

If the generation of free oxygen radicals is a pathophysiological finding in middle-ear infections, as has been reported, demonstration of the oxidative consequences of free radicals in middle-ear exudates may be a quick, simple, objective and accessible test for quantifying the degree of mucosal injury. Therefore, samples of the discharge fluid from cases of acute otitis media (n = 17), cholesteatoma (n = 17), chronic discharge in the absence of cholesteatoma (n = 10), and secretory otitis media (n = 15) were tested using an enzymatic colorimetric assay for lipid peroxide levels due to superoxide and hydroxyl radicals during infection. Lipid peroxide levels in acute otitis media, cholesteatoma, and chronic discharge without cholesteatoma ranged from 575 to 650 nmol/mg of exudate, and was 67 nmol/mg in secretory otitis media, which were statistically significant findings. Infectious otitis media showed findings secondary to increased oxidative metabolism, but not secretory otitis media. Measurement of this oxidative metabolism could serve to monitor the evolution of episodes, as well as to detect relapse or reinfection, and to evaluate response to new therapeutic options for suppurative otitis, such as topical antioxidants.

[Pathogenetic validation of optimal antioxidant therapy in suppurative inflammatory otic diseases in children]. / Patogeneticheskoe obosnovanie ratsional'noi antioksidantnoi terapii pri gnoino-vospalitel'nykh zablevaniiakh ukha u detei.

The knowledge of cell-molecular characteristics of middle ear inflammation in children allowed the authors to suggest a beneficial effect of antioxidants in this disease. The trial of ascorbic acid, retinol and alpha-tocopherol as components of combined treatment has confirmed this hypophysis in view of high response achieved in children with middle ear purulent inflammation.