Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear.

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.

Modified titration intratympanic gentamicin injection for unilateral intractable Ménière's disease.

This study looked into the efficacy of a modified titration protocol of intratympanic gentamicin injection (ITG) in the patients with unilateral intractable Ménière's disease (MD). Modified titration protocol of ITG at a low dose (20 mg/mL) was administered to 10 patients with definite unilateral intractable MD. After initial first two fixed ITGs on weekly basis, the patients might or might not be given any more injections, depending on the appearance of unilateral vestibular loss (UVL). ITG was terminated if the patients satisfied the criteria of UVL. All patients were followed-up for at least two years. The effects of ITG on the vertigo attack, functional level scores and postural balance were evaluated. Of the 10 cases, 8 showed the sign of UVL after receiving initial two ITGs and were not given any more intratympanic injections, and the other 2 patients were administered three ITGs. A two-year follow-up revealed that complete and substantial vertigo control was achieved in 9 cases, and limited vertigo control in 1 patient. Hearing level was lowered in 2 patients. The posture stability and functional level scores were improved. Our study showed that the modified titration protocol of ITG at a low dose could effectively control vertigo in patients with unilateral intractable MD.

Bacterial invasion of the inner ear in association with pneumococcal meningitis.

OBJECTIVE: To examine the pathways of bacterial invasion and subsequent spreading in the inner ear during pneumococcal meningitis. STUDY DESIGN: A well-established adult rat model of Streptococcus pneumoniae meningitis was used. METHODS: Thirty rats were inoculated intrathecally with S. pneumoniae serotype 1, 3 or 9 V and received no additional treatment. The rats were sacrificed when reaching terminal illness or on Day 7 and then prepared for serial sectioning and PAS-Alcian blue staining for light microscopy. RESULTS: During the first few days after inoculation, bacteria invade the inner ear through the cochlear aqueduct, into the scala tympani of the cochlea (perilymphatic space). From here, bacteria spreads apically toward the helicotrema and subsequently basally through the scala vestibuli, toward the vestibule and the vestibular system. When the bacteria after 5 to 6 days had reached scala vestibuli of the basal turn of the cochlea, hematogenous spreading occurred to the spiral ligament and into the cochlear endolymph, subsequently to the vestibular endolymph. We found no evidence of alternative routes for bacterial invasion in the inner ear. Several internal barriers to bacterial spreading were found within the inner ear. Bacterial elimination was evidenced by engulfment by macrophages within the inner ear. CONCLUSION: From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament capillary bed occur at later stages. Although internal barriers exist within the inner ear, the spreading of bacteria occurs via the natural pathways of the fluid compartments. Bacterial elimination occurs by local macrophage engulfment.

Microbial flora of cochlear implants by gene pyrosequencing.

OBJECTIVE: To describe the microbial flora associated with cochlear implants (CIs) removed for infectious and noninfectious indications. STUDY DESIGN: Prospective, controlled. SETTING: Academic, tertiary medical center. PATIENTS: All patients undergoing CI removal. INTERVENTION: CIs were removed with aseptic technique and processed for microbial identification. MAIN OUTCOME MEASURE: CI microbes were identified with routine culture and speciation and 16s deoxyribonucleic acid 454-pyrosequencing. RESULTS: All CIs had evidence of microbes. Propionibacterium acnes and Acidovorax facilis were more common on noninfected CIs (p = 0.005, 0.031). Staphylococcus aureus was more common on infected CIs (p = 0.003). The microbial profiles associated with CI infection were different from, but overlapped with those of noninfected CIs. Microbial culture with selective media identified pathogens not identified on pyrosequencing. CONCLUSION: Bacteria are present on all CIs, both with and without evidence of clinical infection, but species differ with clinical status. Empiric therapy for CI infections should include coverage for S. aureus. Gene pyrosequencing and selective culture techniques may yield complementary results that may impact the management of CI infections.

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media.

BACKGROUND: Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation. METHODS: Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2. RESULTS: Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the in vitro data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation in vivo. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs. CONCLUSIONS: Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.

Effect of lipooligosaccharide mutations of Haemophilus influenzae on the middle and inner ears.

OBJECTIVE: The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear. RESULTS: Fifteen chinchillas were divided into three equal groups and their bullas inoculated bilaterally with 0.5 ml of 10(2)CFU/ml of parent NTHi 2019, B29 or DK1 mutant strains. Two days after inoculation all animals had otitis media and inflamed middle ear mucosa. There was a trend of greater thickness and infiltration of the round window membrane in animals inoculated with the wild-type NTHi strain compared to the mutant strains and a significant increase in both inflammatory cell infiltration and bacteria presence in the scala tympani area of the inner ear. Strial edema was only observed in the wild-type-inoculated group. CONCLUSIONS: LOS mutants of NTHi appear to have a reduced ability to pass through the round window membrane resulting in less inner ear inflammation and pathological changes.

Toll-like receptor 2-dependent NF-kappaB activation is involved in nontypeable Haemophilus influenzae-induced monocyte chemotactic protein 1 up-regulation in the spiral ligament fibrocytes of the inner ear.

Inner ear dysfunction secondary to chronic otitis media (OM), including high-frequency sensorineural hearing loss or vertigo, is not uncommon. Although chronic middle ear inflammation is believed to cause inner ear dysfunction by entry of OM pathogen components or cytokines from the middle ear into the inner ear, the underlying mechanisms are not well understood. Previously, we demonstrated that the spiral ligament fibrocyte (SLF) cell line up-regulates monocyte chemotactic protein 1 (MCP-1) expression after treatment with nontypeable Haemophilus influenzae (NTHI), one of the most common OM pathogens. We hypothesized that the SLF-derived MCP-1 plays a role in inner ear inflammation secondary to OM that is responsible for hearing loss and dizziness. The purpose of this study was to investigate the signaling pathway involved in NTHI-induced MCP-1 up-regulation in SLFs. Here we show for the first time that NTHI induces MCP-1 up-regulation in the SLFs via Toll-like receptor 2 (TLR2)-dependent activation of NF-kappaB. TLR2(-/-)- and MyD88(-/-)-derived SLFs revealed involvement of TLR2 and MyD88 in NTHI-induced MCP-1 up-regulation. Studies using chemical inhibitors and dominant-negative constructs demonstrated that it is mediated by the IkappaKbeta-dependent IkappaBalpha phosphorylation and NTHI-induced NF-kappaB nuclear translocation. Furthermore, we demonstrated that the binding of NF-kappaB to the enhancer region of MCP-1 is involved in this up-regulation. In addition, we have identified a potential NF-kappaB motif that is responsive and specific to certain NTHI molecules or ligands. Further studies are necessary to reveal specific ligands of NTHI that activate host receptors. These results may provide us with new therapeutic strategies for prevention of inner ear dysfunction secondary to chronic middle ear inflammation.

Pseudomonas aeruginosa infection in the hypoventilated middle ear: an experimental model.

CONCLUSION: This is a suitable model for studying different aspects of the pathophysiology of chronic suppurative otitis media. OBJECTIVE: To analyze the methodological features of an animal model of chronic suppurative otitis media induced by intratympanic inoculation of Pseudomonas aeruginosa. MATERIAL AND METHODS: Otitis media was induced by inoculation of P. aeruginosa through the inferior aspect of the rat bulla and cauterization of the Eustachian tube via a transpalatal approach. Inspection of the tympanic membrane, culturing of middle ear effusion and processing of the temporal bones for light microscopy were performed. RESULTS: Abnormal otomicroscopic findings and persistence of infection were correlated with the histopathological changes found in middle ear tissues.

Outbreak of otitis media caused by Burkholderia gladioli infection in immunocompromised mice.

An athymic nude mouse with severe head tilt due to otitis media was identified. Within weeks of identification of this first case, immune-deficient mice of various genotypes from the same facility were similarly affected, and cases from other facilities were found within two months. Culture of ear exudate specimens from affected mice yielded bacteria that were initially identified as Burkholderia cepacia, a plant pathogen considered an important opportunistic pathogen in persons with cystic fibrosis or chronic granulomatous disease. Several of these isolates, however, were subsequently identified as B. gladioli on the basis of results of biochemical analysis and a species-specific polymerase chain reaction (PCR) assay. Genotyping analysis revealed clonality among the isolates, indicating a shared strain among affected mice. A 16S rDNA-based PCR assay specific for the genera Burkholderia and Ralstonia, and a selective culture medium were used in efforts to characterize the epidemiology of this outbreak. In addition to culture of specimens from the oropharyngeal cavity of affected mice, samples were obtained from the environment, feces, sipper tubes, drinking water, and soiled bedding from cages of affected individuals. Burkholderia gladioli was most consistently detected in oropharyngeal swab specimens from affected mice. The PCR assay was equivalent to selective culture in identifying mice in the carrier state that did not have clinical signs of infection. However, neither detection method had sufficient sensitivity to reliably identify all carrier mice, causing the organism to persist at low levels unless entire colonies of immune-deficient mice were removed. The organism was highly resistant to antibiotic therapy. The source and epidemiology of this organism remain unknown. This epizootic serves as an important reminder that immunocompromised rodent colonies may harbor important human opportunistic pathogens.

Interrelations between the middle and inner ear in otitis media.

This article reviews the importance of the round-window membrane in exposing the labyrinth to or protecting it from the toxic effects of otitis media. Characteristics of the immune system in the human middle ear and middle-ear mechanisms against bacteria are explained. The role of bacteria and bacterial products in inner-ear damage is detailed, and related pathological events are described. The hypothetical role of inflammatory mediators in bacteria-induced inner-ear toxicity is particularly emphasized. Clinical conditions causing these events are detailed, and the most frequently involved microorganisms are mentioned. Finally, round-window membrane macroscopic and microscopic anatomy is discussed, and considerations about the exact role of membrane inflammation--protection versus damage of the inner ear--are expressed.

Optimal dose of amoxicillin in treatment of otitis media caused by a penicillin-resistant pneumococcus strain in the gerbil model.

Amoxicillin at doses of 0.2 to 5 mg/kg of body weight was administered for the treatment of pneumococcal otitis media in a gerbil model. Doses greater than or equal to 2.5 mg/kg, which resulted in concentrations in middle ear fluid of > or = 1.4 microg/ml and concentrations in serum higher than the MIC (1 microg/ml) for > or = 14% of the dosing interval, were both clinically and bacteriologically effective.

Identification of beta-actin as a candidate autoantigen in autoimmune inner ear disease.

It has been shown that sera from patients with autoimmune inner ear disease contain antibodies to several inner ear antigens. We report here the characterization of the 42-43 kDa protein against which a significant number of patients' sera react strongly. After separation of inner ear proteins from guinea-pig cochleas by SDS-PAGE, the band corresponding to the 42-43 kDa protein was digested with trypsin and the peptide fragments were separated by high-performance liquid chromatography. Two fractions were then subjected to amino acid sequencing by the classical automated Edman degradation. The sequence of a stretch of 15 amino acids of the first fragment was identical to that of amino acids 148-162 of beta-actin. The sequence of the 10 amino acids of the second fragment was also identical to beta-actin. On Western blots, monoclonal antibody directed against beta-actin reacted with the inner ear 42-43 kDa proteins. The serum samples from the patients and the monoclonal antibody reacted with the non-muscle actin used as antigen in Western blotting. Immunoblot analysis of inner ear proteins after two-dimensional gel electrophoresis showed a spot, corresponding to the region of the 43 kDa as compared to the protein standards. On the basis of these data it is concluded that the target 42-43 kDa protein for antibodies in sera of patients with autoimmune inner ear disease is beta-actin, a molecule, which has important and numerous functions inside cells. This is the first report to identify the cytoskeletal protein beta-actin as a candidate autoantigen in autoimmune inner ear disease.

Clinicomicrobiologic evaluation of active tubotympanic type chronic suppurative otitis media.

OBJECTIVES: This prospective study was conducted to determine the spectrum of micro-organisms encountered in patients with active-stage chronic suppurative otitis media (CSOM) (tubotympanic type) and to see whether prescribing an antibiotic after culture sensitivity was more beneficial as compared to initial treatment without cultures. DESIGN: Prospective randomized study of 110 patients of active CSOM (tubotympanic type) divided into two groups of 55 cases each. SETTING: Departments of Ear, Nose and Throat and Microbiology of a tertiary care hospital. METHODS: The patients in group A were prescribed an antibiotic according to the culture and sensitivity, whereas in group B, culture was not done at the first visit, and a broad-spectrum antimicrobial, namely, co-trimoxazole, was prescribed blindly for a maximum period of 2 weeks. The cases that still had ear discharge were then subjected to culture and sensitivity and the antibiotic was prescribed accordingly. MAIN OUTCOME MEASURES: All patients in group A were subjected to bacterial culture and sensitivity and fungal culture. Only failed cases in group B were subjected to the same. RESULTS: In group A, 47 patients (85.50%) had positive bacterial culture and 20 patients had positive fungal culture. Pseudomonas aeruginosa was the most common bacterial isolate. All of these 47 patients had a dry ear with a maximum 2 weeks of antibiotic therapy. Among the remaining 8 patients who had negative bacterial culture, 5 patients (9.0%) showed fungal isolates on culture and responded to topical antifungal treatment. The remaining 3 failed cases (5.5%) responded to daily dry mopping alone. In group B, 41 patients (74.54%) attained a dry ear. Bacterial culture and sensitivity were done in the remaining 14 (25.46%) failed cases. The culture was positive in 11 patients (20.0%) and sterile in 3 patients (5.5%). In the latter group, only 1 patient had fungus on culture and the remaining 2 patients responded to daily dry mopping alone, which was done at a maximum for a week only. The most common fungal pathogen isolated was Aspergillus flavus. CONCLUSIONS: Pseudomonas aeruginosa was the most common bacteria and Aspergillus flavus the most common fungus isolated in this study. In group A patients, the failed cases were less as compared to the control group B, but the p value was .2. Hence, there is no definite role of culture and sensitivity in the initial management plan of all cases of CSOM. Ideally, every such case should be prescribed a broad-spectrum antibiotic and only in failed cases should culture and sensitivity be done.

Congenital malformation of the inner ear associated with recurrent meningitis.

Congenital deformities of the labyrinth of the inner ear can be associated with meningitis and varying degrees of hearing loss or deafness. A recurrence of meningitis is due to the development of a fistulous communication between the subarachnoid space and the middle ear cavity, and can prove lethal. An illustrative case of a 4-year-old Japanese girl with bilateral severe hearing loss, recurrent meningitis and malformations of the inner ear and stapes footplate is presented. Removal of the stapes during tympanotomy provoked a gush of cerebrospinal fluid. The defect was repaired successfully, and there has been no further episodes of meningitis to date.

Interactions between the middle ear and the inner ear: bacterial products.

The round-window membrane (RWM) is extremely thin and is the only soft-tissue barrier between the middle ear and the inner ear. Under inflammatory conditions of the middle ear the various layers of the triple-layered RWM undergo characteristic changes parallel to the changes of the middle-ear mucosa. Several studies report that bacterial products, exo- and endotoxins, from bacteria invading the middle ear may result in profound inflammatory changes in the inner ear, followed by severe damage to the inner-ear function. The present review, in which we summarized experimental and clinical observations, on bacterial products in interactions between the middle and inner ear, focused on: 1. Bacteria and bacterial products in an inflamed middle ear that may influence inner-ear function. 2. RWM structure and RWM permeability under the influence of bacteria and bacterial products. 3. Morphological and functional inner-ear effects of bacterial infection of the middle ear, and the possible mechanisms involved. 4. Future studies to be directed in this field.

Acute labyrinthitis associated with systemic Candida albicans infection in ageing mice.

The yeast Candida albicans is an important opportunistic pathogen that has been associated with disease of the inner ear. This study describes the histopathology of acute labyrinthitis caused by systemic infection with C. albicans in aging inbred mice. Within four days after infection, yeast and hyphal forms of C.albicans were found in the membranous labyrinth. The utricle and the adjacent parts of the ampullary regions of the semicircular canals were most severely affected, but damage was also seen in the scala media, the scala tympani, the saccule, and the scala vestibuli. In the utricle, the lining epithelium of the membranous labyrinth was disrupted, and the lining cells of the vestibular membrane showed foci in which the membrane was disrupted. The data suggest that age may represent a risk factor for fungal labyrinthitis.

Auditory brain stem response changes after application of endotoxin to the round window membrane in experimental otitis media.

The effects of endotoxin (purified Escherichia coli lipopolysaccharide 0111:B4) on cochlear function in normal and otitis media animals were evaluated. Two types of experimental otitis media models were developed in guinea pigs: eustachian tube obstruction and intratympanic injection of endotoxin. In normal animals, three different concentrations (0.01, 0.1, or 1.0 mg/ml) of endotoxin were applied onto the round window membrane, and auditory brain stem responses were recorded at 1, 3, 6, and 12 hours and 1, 2, 3, and 14 days after the application of endotoxin. Concentrations of 0.01 and 0.1 mg/ml of endotoxin did not affect the auditory brain stem response thresholds, whereas a concentration of 1.0 mg/ml resulted in elevation of the auditory brain stem response thresholds. Alteration of the auditory brain stem response threshold began at 3 hours, reached a peak at 24 or 48 hours, and returned to a normal level 2 weeks after the application of endotoxin. However, when the same concentration (1.0 mg/ml) of endotoxin was applied to the round window membranes of animals that underwent eustachian tube obstruction or intratympanic injection of endotoxin, the endotoxin did not cause any alteration of the auditory brain stem response threshold compared with normal animals.

Spiral modiolar vein: its importance in viral load of the inner ear.

Guinea pig-specific cytomegalovirus and Sendai virus were inoculated into the cochleas of seronegative guinea pigs to study the route of entry of cells participating in inner ear inflammation. Inflammatory cells accumulated around the spiral modiolar vein and appeared to be streaming from this vein into the scala tympani via a collecting venule. Inactivated virus inoculated into the cochlea and normal control cochlea failed to show inflammatory cell infiltrates. The spiral modiolar vein appears to play an important role in the movement of cells from the systemic circulation into the inner ear as part of the host's normal defense against invading pathogens such as viruses.

Differential diagnosis of virus-like particles in the human inner ear.

The entire endolymphatic duct and sac as well as the vestibular epithelia were obtained from four patients with Meniere's disease during translabyrinthine (TL) eighth nerve section and from 12 patients undergoing TL resection of acoustic schwannomas. After these specimens were processed for routine transmission electron microscopy (TEM), they were studied for morphologic evidence of viral infection. Although no virus particles were identified, numerous regularly occurring cell components and artifacts were found to morphologically mimic viruses. An atlas of these structures is presented.