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.

Diagnosis of extraesophageal reflux in children with chronic otitis media with effusion using Peptest.

INTRODUCTION: The aim of the study was to investigate whether Peptest, an immunoassay used to detect pepsin, could be used to diagnose extraesophageal reflux (EER) in children with chronic otitis media with effusion (OME). The results obtained using this fast, simple and non-expensive method were compared with the results of previous studies. METHODS: Children 1-7 years old who had been diagnosed with OME and who were undergoing myringotomy with insertion of a ventilation tube were included in the prospective study. Middle ear fluid obtained during myringotomy was analyzed with Peptest to determine the presence of pepsin, and hence EER. RESULTS: Bilateral and unilateral myringotomy was performed in 15/44 (34.1%) and 29/44 (65.9%) children, respectively. Pepsin in the middle ear was detected in 14/44 (31.8%) children and in 19/59 (32.2%) middle ear specimens. Serous and mucous samples were positive for pepsin in 11/32 (34.4%) and 6/27 (22.2%) cases, respectively. Pepsin in the middle ear was detected in 3/7 children (42.9%) with bronchial asthma (p=0.662). CONCLUSIONS: Pepsin was detected in 1/3 of middle ear specimens of patients with OME. These patients probably suffer from more severe reflux and therefore would be potential candidates for antireflux therapy. However, this has to be confirmed in further studies.

C-Jun N-terminal kinase (JNK) isoforms play differing roles in otitis media.

BACKGROUND: Innate immunity and tissue proliferation play important roles in otitis media (OM), the most common disease of childhood. CJUN terminal kinase (JNK) is potentially involved in both processes. RESULTS: Genes involved in both innate immune and growth factor activation of JNK are upregulated during OM, while expression of both positive and negative JNK regulatory genes is altered. When compared to wildtypes (WTs), C57BL/6 mice deficient in JNK1 exhibit enhanced mucosal thickening, with delayed recovery, enhanced neutrophil recruitment early in OM, and delayed bacterial clearance. In contrast, JNK2-/- mice exhibit delayed mucosal hyperplasia that eventually exceeds that of WTs and is slow to recover, delayed recruitment of neutrophils, and failure of bacterial clearance. CONCLUSIONS: The results suggest that JNK1 and JNK2 play primarily opposing roles in mucosal hyperplasia and neutrophil recruitment early in OM. However, both isoforms are required for the normal resolution of middle ear infection.

[The effect of middle ear effusion on enzymatic digestion of DNA in middle ear effusions of chronic otitis media with effusion].

OBJECTIVE: To determine whether or not the bacterial DNA which was detected by PCR comes from viable bacteria. METHOD: Observing the affection of middle ear effusion (MEE) on DNA viscosity and enzymatic digestion of DNA. RESULT: The middle ear effusion and DNA are stable and DNase 1 rapidly digests DNA. The effusion does not seem to degrade DNA. The middle ear effusion signficantly inhibits DNase 1. CONCLUSION: Middle ear effusion provides an inhibition of the enzymatic digestion of purified DNA. Thus any DNA found in effusion by PCR techniques could well be fossilized remains and chronic otitis media with effusion may not be the bacterial infection.

Mature middle and inner ears express Chd7 and exhibit distinctive pathologies in a mouse model of CHARGE syndrome.

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears.

TNFA deletion alters apoptosis as well as caspase 3 and 4 expression during otitis media.

BACKGROUND: Tumor necrosis factor (TNFA) is the canonical member of the TNF superfamily, which plays a major role in both inflammation and apoptosis. To evaluate the role of TNFs in otitis media (OM), the most common disease of childhood, we evaluated middle ear (ME) expression of genes encoding the TNF and TNF receptor superfamilies during bacterial OM in the mouse, characterized OM in TNFA-deficient mice, and assessed apoptosis during OM in normal versus TNF-deficient MEs. RESULTS: TNFs and TNF receptors were broadly regulated during OM, with TNFA showing the highest level of up-regulation. TNF deficient mice exhibited mucosal hyperplasia even in the absence of infection and exuberant growth of the mucosa during OM, including the formation of mucosal polyps. Mucosal recovery during OM was also delayed, in parallel with a delay in mucosal apoptosis and reduced caspase gene expression. CONCLUSIONS: The TNF and TNF receptor superfamilies mediate both inflammation and apoptosis during OM. TNF appears to be critical for the maintenance of mucosal architecture in both the normal and infected ME, since excessive accumulation of mucosal tissue is seen in TNFA-/- MEs both before and after bacterial inoculation of the ME. TNFA is also required for appropriate regulation of caspase genes.

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.

Long-term hearing loss in gerbils with bacterial meningitis treated with superoxide dismutase.

HYPOTHESIS: The treatment of superoxide dismutase (SOD) in gerbils with bacterial meningitis will not only prevent cochlear fibrosis and neo-ossification but also reduce hearing loss. BACKGROUND: SOD an O2-scavenger, has been shown to prevent cochlear fibrosis and neo-ossification in gerbils infected with bacterial meningitis when injected intrathecally. The objective of this study is to investigate the effects of SOD on long-term hearing loss in gerbils infected with bacterial meningitis and to assess the relationship between hearing results and the amount of fibrosis. The effectiveness of middle ear infusion of SOD will also be examined. METHODS: Meningitis was induced in 3 groups of 10 gerbils with injection of Streptococcus pneumoniae into the cisterna magna. Group 1 received intrathecal SOD, group 2 received a middle ear infusion of SOD, and group 3, the control group, received no SOD. Histologic data and auditory brainstem responses were obtained from each gerbil. RESULTS: In the intrathecal SOD group, the average deterioration in pure tone thresholds between the preoperative baseline and 15 weeks after induction of meningitis at 4, 8, 16, and 32 kHz was significantly less than that of the middle ear SOD and the control group (p = 0.001). There was no significant difference between the middle ear SOD and the control group. There was no fibrosis in the intrathecal SOD group, 15% of the gerbils developed an average of 11% fibrosis in the middle ear SOD group, and 20% of the gerbils developed an average of 15% in the control group. CONCLUSION: Intrathecal infusion of SOD not only prevented cochlear fibrosis and neo-ossification after bacterial meningitis but also decreased subsequent hearing loss.

Expressions of cyclooxygenase 1 and 2 in endotoxin-induced otitis media with effusion in the rat.

OBJECTIVE: Recently, a selective COX-2 inhibitor was developed and used for reducing the levels of inflammation-inducing prostaglandin (PG) whilst not inhibiting the release of protective PG by COX-1. COX-1 may be the critical isoform required for the production of PG with a homeostatic function, whereas COX-2 may be the main contributor to PG production in inflammation. The purpose of this study was to investigate COX-1 and 2 expressions in experimental endotoxin-induced OME in rats and to quantify their temporal expressions. METHODS: In a rat model, lipopolysaccharides (LPS) were inoculated into the middle ear cavity. Middle ear mucosa and temporal bone were samples at 0, 1, 3, 6, and 12h, and on days 1, 3 and 7 after instilling either LPS or sterile PBS. RT-PCR, Western blotting and immunohistochemical staining were performed to determine the expressions of COX-1 and COX-2. RESULTS: COX-1 mRNA and protein were detected in normal middle ear mucosa but their levels did not change after endotoxin instillation. However, COX-2 was not identified in normal middle ear mucosa, but COX-2 mRNA was maximally increased at 6h after endotoxin instillation and COX-2 protein was maximally increased at 12h. COX-2 expression, by immunohistochemical staining, was identified only at 12h after endotoxin injection. CONCLUSIONS: In this study, the basal expressions of COX-1 and COX-2 mRNA and protein in middle ear mucosa, as well as their regulations by endotoxin were investigated. COX-1 was not induced in middle ear mucosa by endotoxin whereas COX-2 was induced within 12h of stimulation. Our findings indicate that COX-2 inhibitor administration for the relief of inflammation should be considered within 12h of the initiation of an inflammatory process. These findings may provide an understanding of the mechanisms regulating PG formation in infection of the middle ear cavity.

Expression of phospholipase C-gamma1 in experimental cholesteatoma using Mongolian gerbils.

Phospholipase C (PLC)-gamma1 is known to play a central role in ligand-mediated signal transduction for cell proliferation. The purpose of this study was to elucidate the distribution of PLC-gamma1 in deep meatal skin, retroauricular skin and cholesteatoma matrix of experimental animals. We induced canal ligation cholesteatoma using Mongolian gerbils and investigated the expression of PLC-gamma1 in this model by using Western blot analysis and immunohistochemistry. By Western blot analysis, considerably higher levels of PLC-gamma1 protein were detectable in experimental cholesteatoma. On immunohistochemistry, experimental cholesteatoma showed more intense immunolabeling of PLC-gamma1 protein than deep meatal skin and retroauricular skin. In conclusion, overexpression of PLC-gamma1 may in part contribute to abnormal proliferation and differentiation of experimental cholesteatoma in Mongolian gerbils.

In vivo adenoviral transduction of the neonatal rat cochlea and middle ear.

Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a beta-galactosidase (beta-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of beta-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of beta-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of beta-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express beta-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.

An immunohistochemical study of inducible nitric oxide synthase in the rat middle ear, with reference to tympanosclerosis.

Tympanosclerosis and myringosclerosis are well-known sequelae after acute and chronic otitis media and are also often seen after treatment of secretory otitis media with ventilation tubes. They sometimes cause serious hearing disability. There is no successful treatment for these conditions. There might be factors triggering an immunological or autoimmune chain reaction, which leads to tympanosclerosis. Intervention with the aim of abolishing this type of response might be possible if an interruption of the chain reaction can be found. Nitric oxide is a radical molecule with the ability to kill pathogens and is produced by the enzyme nitric oxide synthase. Expression of inducible nitric oxide synthase (iNOS) was analysed immunohistochemically in a rat model of acute otitis media. In rats sacrificed at days 3 and 6 after inoculation. iNOS was also strongly expressed in the middle ear mucosa and in the tympanic membrane as well as in the inner ear. In control specimens as well as in infected ones. iNOS was expressed in the tissue of the external ear canal. In rats sacrificed at day 10 and after 3 months, iNOS was expressed at the same locations, although less frequently. These data indicate that iNOS expression is induced during acute otitis media and suggest that nitric oxide may be important in the host defence against ear infections.

Inhibition of nitric oxide synthase blocks osteoclastic bone resorption in adaptive bone modeling.

In this study, the auditory bulla of the gerbil was pressurized, leading to active modeling of the bone of the bulla wall with a significant increase in osteoclast surface and mineral apposition rate. Systemic infusion of L-N(G)-nitro-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), inhibited this modeling process. The percentage osteoclast surface (Oc.S/BS) on the inner surface bulla wall was significantly reduced in the L-NAME-treated animals when compared with pressurized saline-treated bullae. Fluorescent bone surface (BSf) mineral apposition rates (MAR) and bone formation rate (BFR) were not significantly different in the pressurized bullae when the L-NAME group was compared with the control (vehicle only) group. However, L-NAME significantly suppressed BSf in the unpressurized bullae. Therefore, it is likely that nitric oxide is a mediator of osteoclastic resorption due to adaptive bone modeling through one or more of the isoforms of NOS.

The presence of platelet-activating factor-acetylhydrolase in human middle ear effusions.

Factors related to inflammation, including platelet-activating factor (PAF), apparently have a role in chronic otitis media with effusion. PAF is metabolized to the biologically inactive lyso-PAF by the enzyme PAF-acetylhydrolase (PAF-AH). We have obtained evidence that PAF-AH activity is present in human middle ear effusions in patients with chronic otitis media with effusion. The present study revealed the enzyme in human middle ear effusions to be the plasma type PAF-AH. We suggest that PAF-AH may be involved in regulating inflammation in the middle ear by inactivating PAF, the potent proinflammatory autacoid.

Matrix metalloproteinase-1 in cholesteatoma, middle ear granulations and deep meatal skin: a comparative analysis.

Nine cholesteatomas, seven middle ear granulations and five deep meatal skin specimens were analysed for gelatinase activity at molecular weights corresponding to those of matrix metalloproteinase-1 (MMP-1) using SDS PAGE zymography. Gelatinase activity at 41-43 kDa and 45-47 kDa was investigated. Western blotting was employed using a primary monoclonal antibody to MMP-1 to provide a qualitative assessment of MMP-1. Western blotting was also used with a monoclonal antibody to MMP-3 to discover if MMP-3 gelatinase activity occurring around the molecular weight of MMP-1 may have contributed to the results. A significantly higher expression of activity was recorded in cholesteatoma and middle ear granulations at 45-47 kDa in comparison with deep meatal skin. Western blotting indices were to be present in all of the cholesteatoma specimens tested. Only one of the specimens (cholesteatoma) tested showed any MMP-3 presence.

Experimental otitis media induced by nonviable Moraxella catarrhalis in the guinea pig model.

Moraxella catarrhalis is a normal resident of the human nasopharyngeal flora, but it is also isolated from middle ear fluid of acute otitis media and otitis media with effusion patients. To determine whether M. catarrhalis has direct pathogenicity in the middle ear, heat-killed M. catarrhalis was inoculated into the middle ear bullae of guinea pigs, and the inflammatory response was investigated. Middle ear mucosal histopathology observed in M. catarrhalis-inoculated ears included subepithelial edema, capillary dilatation, thickening of lamina propria mucosa, inflammatory cell and erythrocyte infiltration into the lamina propria mucosa. Inflammatory cell numbers, lysozyme and myeloperoxidase concentrations in the middle ear washing suspensions of M. catarrhalis-inoculated ears were significantly higher than control ears throughout the experiment. Therefore, nonviable M. catarrhalis induced middle ear inflammation and mucoperiosteal histopathology, which might be caused by direct injury of the nonviable bacteria (e.g. lipooligosaccharide or outer membrane proteins) and metabolic products of inflammatory cells.

Middle ear catalase distribution in an animal model of otitis media.

Increasing evidence implicates free radicals in the pathogenesis of inflammatory disease, including otitis media. The anti-oxidant enzymes catalase, glutathione peroxidase and superoxide dismutase protect tissues from the destructive effects of free radicals. Our previous work has shown depressed levels of superoxide dismutase in the infected middle ears of a guinea pig model of otitis media in comparison with normal control ears. We studied the distribution and relative abundance of catalase in the middle ear of this animal model in an effort to elucidate the role free radicals play in the pathogenesis of otitis media. Catalase distribution was mapped immunohistochemically in the middle ears of guinea pigs with induced streptococcus otitis media, and compared with normal control ears. In the control ears, catalase was localized to the epithelium of the middle ear mucosa, with scant distribution in the submucosa. The infected ears demonstrated inflammatory cell invasion with hyperemia and submucosal edema. Catalase was localized to the epithelium and had scant distribution in the submucosa. This distribution was similar to that found previously with superoxide dismutase. Enzyme-linked immunosorbent assay of catalase demonstrated a mean value of 1.00 +/- 0.06 microgram/mg protein in the control ears, and 1.06 +/- 0.12 microgram/mg in the infected ears, but these two values were not statistically different.

Neutrophil elastase-alpha 1-antitrypsin in middle ear fluid in chronic otitis media with effusion.

Neutrophil elastase-alpha 1-antitrypsin was quantified in samples taken from middle ear effusions collected at operation from 17 children attending for elective myringotomy and grommet insertion. At the time of surgery the effusion was classified as serous or mucoid. Children with a recent history of infection or antimicrobial therapy were excluded. The quantification of immunoreactive neutrophil elastase was by means of enzyme-linked immunosorbant assay (ELISA). The mean value of neutrophil elastase-alpha 1-antitrypsin was 50.6 +/- 38.3 (SD) micrograms/ml in mucoid effusions, which was significantly higher (P < 0.05) than that in serous effusions (5.3 +/- 4.8 micrograms/ml). These results indicate that a mucoid effusion may reflect a more severe inflammatory response and that persistence of neutrophil activity in the middle ear mucosa may contribute to the persistence of at least one group of middle ear effusions.

Superoxide dismutase in an animal model of otitis media.

Cu, Zn superoxide dismutase (SOD) is a metalloprotein that catalyzes the dismutation of the superoxide anion into O2- and H2O2, and therefore functions to maintain a low intracellular concentration of an otherwise toxic metabolite of oxygen. SOD protects living tissue from the destructive effects of free radicals. Increasing evidence implicates free radicals, including the superoxide radical (O2-), in the pathogenesis of disease, including otitis media. In an effort to elucidate the role free radicals play in the pathogenesis of otitis media, SOD was localized immunocytochemically to determine its cellular distribution in specimens of guinea pig middle ear. In normal ears, SOD was found concentrated in the epithelium of the middle ear mucosa. Low quantities were characteristic of connective tissue, bone, and cartilage. In streptococcus-infected ears, SOD localized similarly, concentrating in the epithelium. The infected ears had extensive submucosal edema which stained poorly and appeared to have less SOD than did normal ears. This was confirmed by an assay using laser densitometry of Western blots to quantify the amount of SOD in the mucosa of normal versus infected middle ears. This demonstrated a value of SOD in normal mucosa of 1.77 +/- 0.48 micrograms/mg of protein compared with 1.02 +/- 0.28 micrograms/mg in the infected mucosa. The two groups were significantly different at P < 0.05. These findings are discussed, and suggestions for future experimentation addressed.

Determining otitis media severity from middle ear fluid analysis.

Otitis media has a complex multifactorial pathogenesis, and the middle ear inflammatory response is typified by the accumulation of cellular and chemical mediators in middle ear effusion. However, specific biochemical and immunochemical factors that may be responsible for the severity or chronicity of otitis media have not been identified. Identification of factors involved in chronicity appears to be an essential step in the treatment and ultimate prevention of chronic otitis media. We analyzed 70 effusion samples from patients 1 to 10 years of age who had chronic otitis media with effusion for two cytokines (interleukin-1 beta and tumor necrosis factor alpha) and total collagenase. The highest concentrations of all three inflammatory mediators were found in purulent otitis media, and concentrations were higher in younger than in older patients. Mediator concentrations were similar in samples obtained from patients having their first myringotomy for otitis media with effusion and in those who had had multiple previous myringotomies. The multiresponse star, which incorporates several biochemical parameters in one graphic illustration, may best characterize the complex nature of middle ear inflammation.