Gastric pepsin in middle ear fluid of children with otitis media: clinical implications.

Gastroesophageal reflux and extraesophageal reflux have been postulated to be involved in the pathogenesis of otitis media. This is supported by recent studies revealing the presence of gastric pepsin in the middle ear space of children with otitis media but not in control patients without otitis media. Reflux's role in otitis media appears to be most pronounced in younger children and those with purulent effusions.

Detection of gastric pepsin in middle ear fluid of children with otitis media.

OBJECTIVE: We sought to confirm the finding of pepsin/pepsinogen in the middle ear fluid of children with otitis media in a larger sample size using a sensitive and specific pepsin assay. STUDY DESIGN AND SETTING: We evaluated 152 children (225 ear samples) in a prospective study at a tertiary care children's hospital. The presence of pepsin in middle ear aspirates was determined using enzymatic assay. RESULTS: Of the patients, 14.4 percent (22 of 152) had detectable pepsin activity in one or both of the ear samples with no pepsin activity detected in control serum. Average pepsin concentration in the samples was 96.6 +/- 170.8 ng/ml, ranging from 13 to 687 ng/ml. Pepsin concentration in the middle ear of children younger than 1.0 year was significantly higher than in older age groups. CONCLUSION AND SIGNIFICANCE: Results indicate that pepsin/pepsinogen is present in the middle ears of children with otitis media, although not at the high rate previously reported. Gastric reflux may be one causative factor in the pathogenesis of otitis media.

Alteration of adenoid tissue alkaline and acid phosphatase in children with secretory otitis media.

OBJECTIVE: The role of pharyngeal lymphoid tissue in etiopathogenesis of secretory otitis is not yet defined. The influence of tonsillar and adenoid mass, weight, obstruction of naspharyngeal orrifitium, bacterial reservoire or some immunological events are of scientific interest. Tissue nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) are enzymes detected in lymphoid tissue, TNAP as characteristic of B cells, ACP as a characteristic of macrophages and folucullardentritic cells. These enzymes interfere in cell metabolism by removing 5' phosphate group from nucleotides and proteins. Specific activity and kinetic properties were studied in palatinal tonsils and adenoids of children with secretory otitis (OME) and compared with children with recurrent tonsillitis without ear involvement. METHOD: Adenoid and tonsillar tissue of l7 children with OME and 30 children with recurrent tonsillitis were subjected to biochemical investigation using method of releasing of p-nitrophenol from p-nitrophenylphosphate (pNPP). Kinetic parameters as Michaelis-Menten constant were calculated by non-linear regression estimation method. RESULTS: Specific activity of adenoid alkaline phosphatase was lower in children with OME in relation to children with recurrent tonsillitis (t=5.733507, p<0.01). Specific activity of adenoid acid phosphatase was also lower in children with OME (t=3.655456, p<0.01). pH optimum for both enzymes was the same in these two groups of children. Michaelis-Menten constant for both enzymes was significantly higher in adenoid of children with OME than in children with recurrent tonsillitis suggesting lower enzyme affinity for the substrate. CONCLUSION: Differences in specific activities and kinetic properties of adenoid alkaline and acid phosphatases between children with OME and children with recurrent tonsillitis without OME were verified in this study. The results of the study are not able to explain the alteration of alkaline and acid phosphatase characteristics but could point to some possible and specific role of nasopharyngeal lymphoid tissue in pathogenesis of secretary otitis.

Pepsinogen identification in the middle ear fluid of children with otitis media with effusion.

OBJECTIVES: This study aims to investigate the presence and concentration of pepsin/pepsinogen in middle ear fluid and to discuss the potential mechanisms involved in the pathogenesis of this condition. PATIENTS AND METHODS: A total of 33 children (21 boys, 12 girls; mean age 5.7±2.4 years; range 3 to 13 years) diagnosed with otitis media with effusion and scheduled for operation were enrolled into the study. Fluids aspirated from the middle ear were assessed for the presence of pepsinogen and albumin and blood samples were drawn simultaneously for comparison. RESULTS: Mean pepsinogen concentration was statistically significantly higher in middle ear fluids compared with serum samples (262.4 ng/mL [range: 211.7 ng/mL - 301.1 ng/mL] versus 102.6 ng/mL [range: 80.7 ng/mL - 134.5 ng/mL], respectively) (p<0.001). On the other hand, mean albumin concentration was significantly lower (1.1 g/dL [range: 0.01 g/dL - 9.5 g/dL] versus 5.8 g/dL [range: 0.9 - 9.5 g/dL], respectively) (p<0.001). The highest pepsinogen concentration was detected in patients with purulent effusion (275.3 ng/mL). CONCLUSION: Our findings support the theory of gastro-esophageal reflux related pepsinogen transition to the middle ear and indicate that pepsinogen may a reliable biochemical marker for the assessment of gastro-esophageal reflux.

Cathepsin gene expression profile in rat acute pneumococcal otitis media.

OBJECTIVES/HYPOTHESIS: Acute otitis media, often caused by infection with Streptococcus pneumoniae, is characterized by inflammation of the middle ear mucosa. A prominent feature of the host response to bacterial infection of the middle ear mucosa is an influx of inflammatory cells that contributes to the local pool of inflammatory mediators by releasing additional inflammatory chemicals, which in turn cause further tissue injury. The objective was to identify candidate effector and signaling molecules involved in acute otitis media pathogenesis caused by S pneumoniae infection. STUDY DESIGN: Male Sprague-Dawley rats were randomly assigned to 1 of 5 groups, including 1 control group without treatment, 2 placebo groups (12 and 48 hours) and 2 infected groups (12 and 48 hours). The rat middle ear was bilaterally inoculated with either 25 microL of tryptic soy broth (TSB group) or 25 microL of TSB containing approximately 1.24 x 10(9) cfu/mL of S pneumonias type 6A (SP group). Rats were killed at 12 and 48 hours after inoculation and the middle ear mucosa was collected. Total RNA was extracted and pooled from each group for gene expression assays. METHODS: Gene expression profiles for rat middle ear mucosa at 12 and 48 hours after S pneumoniae or placebo inoculation were constructed using microarray technology (Clontech Atlas Rat 1.2 Array, 1176 cDNAs). Genes of interest were further validated by real-time polymerize chain reaction. RESULTS: Middle ear mucosa expression of a gene cluster encoding the lysosomal cysteine proteases, cathepsins B (Ctsb), L (Ctsl), and K (Ctsk), was modified after S pneumoniae challenge. Specifically, at 12 hours, Ctsk and Ctsl messenger RNA that was abundantly expressed in the normal middle ear mucosa was decreased, whereas Ctsb transcript was induced. The changes in Ctsb and Ctsk gene expression were sustained at 48 hours. CONCLUSION: The constitutive expression of Ctsk and Ctsl messenger RNA in normal middle ear mucosa supports a function in the maintenance of middle ear mucosa homeostasis, and their downregulation as an early event in acute otitis media may reflect a disruption in that function. The induction of Ctsb messenger RNA in the infected middle ear mucosa suggests a role in early tissue injury; thus, Ctsb may represent a potential target for molecular diagnostics and/or rational intervention during the development of acute otitis media.

Alpha 1-antitrypsin and ilomastat inhibit inflammatory proteases present in human middle ear effusions.

OBJECTIVES: Proteases of both the serine and metalloproteinase families have been shown to play a role in the pathogenesis of otitis media (OM). Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions, but their use has not been studied in OM. The purpose of this study was to assess the activity of the protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat on inflammatory proteases present in human middle ear effusions (MEEs), with a view to their potential utility in the treatment of OM. STUDY DESIGN: Prospective and ex vivo. METHODS: MEEs were collected from 100 patients presenting for middle ear surgery, most commonly tympanostomy tube placement or treatment of acute posttympanostomy otorrhea (APTO). MEEs were analyzed for the presence of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE) and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP levels were highest in APTO, and HNE was highest in chronic suppurative OM and APTO. High levels of MMP and HNE (>3 mAU/min) were found in 52% and 37% of MEEs, respectively. Ilomastat and rAAT demonstrated significant inhibition of MMP and HNE activity (>30% reduction), respectively, in 80% and 82% of MEEs with high levels of activity. CONCLUSIONS: Proteases are commonly found in OM. Ilomastat and rAAT are potent inhibitors of proteases in MEEs across a wide range of OM in humans. Investigation into the potential therapeutic benefits of these protease inhibitors is warranted.

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.

Hydrolase activity in middle ear effusions. Effect of antibiotic therapy.

Hydrolytic enzymes have been shown to be present in middle ear effusions recovered from children with both persistent and acute otitis media. In the present study, we investigated the effect of ampicillin therapy on the expression of hydrolytic enzyme activity in acute middle ear effusions using the chinchilla animal model. The median values of enzyme activities were lower for the ampicillin-treated animals when compared with the nontreated control animals. For the ampicillin-treated animals, eight of 12 assayed activities were characterized by a time-dependent decay of enzymatic activity. For the untreated animals, the majority of assayed activities (seven of 12) showed an increase in activity with time. These results show that sterilization of the middle ear cleft and elimination of the hydrolytic enzyme activity may be benefits of antimicrobial therapy and prerequisite to the healing of the inflamed mucosa.

Leukocyte esterase activity in effusion fluid of patients with otitis media.

Fluid obtained during myringotomy and tube placement in 20 patients with middle ear effusions was assayed for leukocyte esterase activity using a quantitative spectrophotometric assay. This quantitative assay used the synthetic substrate, N-tosyl indoxyl alaninate. Seven of the 20 samples showed no measurable enzyme activity (8 U/ml or less). The remaining samples demonstrated activity ranging from 20 to 1600 units. Although enzyme activity did not correlate well with the physical appearance of the fluid, it did correlate with clinical history, suggesting the presence of a purulent exudate rather than serous effusion. Leukocyte esterase activity in the fluid appears to hold promise as an indicator for the presence of chronic middle ear infection. The enzyme can be assayed by a simple and fast diagnostic strip test, with results available almost immediately.

Middle ear fluid lysozyme source in experimental pneumococcal otitis media.

Middle ear infection with Streptococcus pneumoniae is important in the pathogenesis of acute and chronic otitis media, and lysozyme in middle ear fluid (MEF) is an important inflammatory mediator in this disease. To determine the source of lysozyme during the early period of acute pneumococcal otitis media, chinchillas were irradiated to induce neutropenia, and their middle ears were inoculated with heat-killed, encapsulated pneumococci. The number of inflammatory cells and concentration of lysozyme were measured in MEF between 6 and 72 hours after inoculation. In pneumococcus-inoculated ears, the mean number of inflammatory cells but not lysozyme was significantly lower in MEF from irradiated animals than that from nonirradiated animals at 6 hours. Since lysozyme accumulated in MEF before the influx of inflammatory cells in irradiated animals, the initial release of this inflammatory mediator is most likely not from inflammatory cells; and mucosal epithelial cells, the only other known source of lysozyme in the middle ear environment, were the probable source induced by the direct stimulation of pneumococci. Inflammatory cells may contribute lysozyme later in the inflammatory response, since cellular and lysozyme concentrations in irradiated and nonirradiated animals were similar between 24 and 72 hours. These results suggest that future therapeutic interventions to limit middle ear inflammation in acute otitis media may need to recognize the direct action of pneumococcal cells or their envelope components on middle ear epithelium.

Neuraminidase activity in middle ear effusions.

Analyses of Streptococcus pneumoniae culture filtrates and middle ear effusions (MEE) containing S pneumoniae for various hydrolytic enzymes have demonstrated substantial levels of neuraminidase activity when measured employing a sensitive fluorometric assay. S pneumoniae neuraminidase exhibits optimum activity near neutral pH (6.0 to 6.5), and catalyzes the cleavage of sialic acid residues from glycoproteins, gangliosides and mucopolysaccharides. S pneumoniae begins secreting large amounts of neutral neuraminidase (mean [means] = 43.3 units/mL culture filtrate) when cells enter the stationary phase. Nearly all (96%) human chronic MEEs yielding positive cultures for S pneumoniae contain neuraminidase activity (means = 0.200 units/mg protein), while only 21.1% to 45.5% of all other effusions contain the enzyme. Middle ear effusions obtained from S pneumoniae infected-chinchillas contained large amounts of neuraminidase activity (approximately 200 units/mL), which decayed exponentially in vivo with an apparent half-life of 8 1/2 days. Three neuraminidase isoenzymes (designated I-III) were identified in S pneumoniae culture filtrates, as well as in MEEs from chinchillas infected with the organism, using a combination of ion-exchange and gel filtration chromatography. With 4-methylumbelliferyl-N-acetylneuraminic acid serving as substrate, preparation I from both culture filtrates and MEEs was characterized by a high Michaelis constant (Km), while forms II and III had low Km values. Preferred substrates were orosomucoid and neuramin-lactose; gangliosides, thyroglobulin, and bovine submaxillary mucin were poorer substrates.

Role of superoxide dismutase in otitis media with effusion.

The concentration of superoxide dismutase (SOD) in middle ear effusion (MEE) was measured by the electron spin resonance trapping method in order to clarify the role of SOD in otitis media with effusion (OME) in children. The SOD levels in mucoid MEEs were significantly higher than those in serous and purulent MEEs. There was no significant difference in the levels of SOD between neutrophil-dominant MEEs and mononuclear cell-dominant MEEs, and the levels were negatively correlated with the number of neutrophils in the MEEs. Moreover, the levels were significantly increased in patients having recurrence of MEE within 3 months compared with patients without recurrence of MEE. Although it is known that SOD plays an important role in protecting the host from oxygen radicals, the findings in this study suggest that SOD might be related to the chronicity of OME.

Hydrolase activity in acute otitis media with effusion.

Biochemical studies of middle ear effusions (MEE) from patients with chronic or recurrent otitis media with effusion (OME) have demonstrated the presence of significant levels of certain hydrolytic and oxidative enzymes. We have examined MEE from patients with acute OME for the content of a number of lysosomal hydrolases and find no significant differences in the mean values for acid phosphatase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, hexosaminidase, and neuraminidase between purulent and serous effusions. In every case, the mean activities of these enzymes were greater in culture-positive than in culture-negative effusions although this difference was significant only in the case of neuraminidase. Neuraminidase activity was detected in 78% of those MEEs from which Streptococcus pneumoniae could be cultured and in only 32% to 64% of all other effusions. No correlation was observed between the level of neuraminidase released into the extracellular growth medium and the infectivity of various strains of S pneumoniae.

Experimental alteration of chinchilla middle ear mucosae by bacterial neuraminidase.

Streptococcus pneumoniae secretes a variety of extracellular glycosidase including a neuraminidase which has been found in middle ear effusion from patients with both acute and chronic otitis media. This enzyme cleaves sialic acid from membrane glycoproteins, thereby exposing galactose residues, the penultimate sugar. The ability of partially purified neuraminidase to alter the middle ear mucosa was investigated in the chinchilla. After incubation with neuraminidase, chinchilla middle ears were removed and exposed to galactose residues labeled with tritium. Membrane glycoproteins were solubilized and separated according to molecular weight by sodium dodecylsulfate electrophoresis. Increases in tritium incorporation, when compared to control incubations, indicated that galactose residues had been exposed and sialic acid residues removed from glycoproteins of both high and low molecular weight. Such membrane destruction could contribute significantly to the pathology of otitis media with effusion.

Endotoxin and lysosomal protease activity in acute and chronic otitis media with effusion.

Endotoxin levels and lysosomal protease (collagenase, cathepsin B, and lysozyme) activity were measured in 104 middle ear effusions (MEEs) from patients with otitis media with effusion (OME). The MEE samples were classified into four groups: pediatric serous, mucoid, and acute, and adult serous. Endotoxin levels and lysosomal protease activity in MEEs were significantly different in the following order: adult less than serous less than mucoid less than acute groups, indicating that both endotoxin and lysosomal proteases are more closely related to the pathogenesis of pediatric chronic OME than to adult OME. In pediatric serous and mucoid effusions, endotoxin level had a significant correlation with activity of the lysosomal proteases. In conclusion, endotoxin enhances leukocyte infiltration into the middle ear, and lysosomal proteases released from leukocytes damage the middle ear mucosa and thereby prolong mucosal inflammation, which may be responsible for delayed recovery from acute OME.

Proteinase activity in effusions from children with otitis media with effusion.

Proteinase activity has been demonstrated in middle ear effusions from patients suffering from otitis media with effusion. Proteinase activity was characterized by various biochemical, chemical and physical parameters. Chelators and sulfhydryl group reacting substances reduced the enzyme activity. Enzyme activity was positively correlated to the number of granulocytes present in the effusion. No correlation to bacteriological findings or to tympanic membrane status was seen and the proteinase activity showed wide ranges within different categories of middle ear effusions.

Bactericidal activity of middle ear effusion on a single isolate of non-typable Haemophilus influenzae.

Bactericidal activity of middle ear effusion (MEE) from children with otitis media with effusion (OME) on one strain of non-typable Haemophilus influenzae was measured and was compared with that of normal human serum. H. influenzae culture negative MEEs had a bactericidal activity on one strain of non-typable H. influenzae (P less than 0.01), and the activity of serous effusions was significantly stronger than that of mucoid ones (P less than 0.01). The bactericidal activity of 20% pooled MEE was almost equal to that of 4% normal human serum. Heat treatment (56 degrees C, 30 min) abolished this activity of both pooled MEE and normal human serum. Addition of fresh guinea pig serum (GPS) as a complement source to the heat-treated pooled MEE restored this activity, while its addition to the heat-treated normal human serum failed to do so. These results suggest that complement factor is important in the bactericidal activity of MEE.

Expression of mast cell tryptase in pediatric otitis media with effusion.

OBJECTIVE: To evaluate the possible relationship between allergy and otitis media with effusion (OME), we investigated the presence and level of tryptase, which reflect mast cell activity. MATERIALS AND METHODS: Twenty-five children with intractable OME were studied. Thirteen patients were confirmed allergy positive by multiallergosorbent test. Twelve patients were allergy negative. Evidence for mast cell tryptase presence and degree of activation in the middle ear effusion (MEE) were measured by using Western blotting and radioimmunoassay (RIA). RESULTS: The typical single band of 32 kDa tryptase was found in the MEE. And it was more strongly expressed in the MEE with allergy positive than negative. RIA demonstrated that mean tryptase level was significantly higher in the MEE with allergy positive than negative. CONCLUSION: These results suggest that allergy may be one of the contributing factors in the persistence of intractable OME.

Collagenase activity in middle ear effusions.

Collagenolytic enzyme activity has been demonstrated in middle ear effusions from patients suffering from otitis media with effusion. Collagenase activity was characterized using different biochemical techniques. Various chemical and physical parameters were titrated for optimal enzyme activity. Bivalent cations activated the enzyme with Ca2+ as the most potent activator. Chelators such as EDTA reduced the enzyme at low concentrations. The enzyme was found to have a higher specific activity in mucoid effusions than in serous and had characteristics similar to granulocyte derived collagenase from human leukocytes. Possible relationships between the presence of collagenase activity and tissue destruction in the middle ear after otitis media with effusion are discussed. The collagenase activity showed wide ranges within different categories of middle ear effusions.

Superoxide dismutase in middle ear fluid from children with secretory otitis media.

Superoxide dismutase (SOD) constitutes an important enzymatic defense against the destructive biochemical events associated with inflammation. To examine the presence of SOD in the middle ear fluid (MEF) in secretory otitis media (SOM) the content of SOD was measured in the MEF from 66 children with SOM. The applied method was based on light emission associated with reduction of the chemiluminescence of luminol reacting with superoxide anions (O2-). From the inhibition exerted by the addition of standard SOD the concentration of SOD in the samples was extrapolated. The findings divided the MEF's into two significantly different groups: 48% with undetectable SOD, 52% with a SOD range of 200-12,000 ng/ml, median: 3,333 ng/ml, 95% confidence limits: 2,000-5,000 ng/ml (p < 0.0005). It is suggested that the concentration of superoxide dismutase in the middle ear may play an essential role for the outcome of secretory otitis media and the effect of insertion of ventilation tubes.