Diversity of the cochlear and vestibular pathologies in human temporal bones of newborns infected with cytomegalovirus.

Hypothesis: Human temporal bones of newborns with congenital cytomegalovirus (cCMV) infection can be characterized by diverse cochlear and vestibular histopathologies associated with the variability in sensorineural hearing loss (SNHL) and vestibular dysfunction in these newborns. Background: Only a small number of studies on the cochlear and vestibular pathologies in human temporal bones with cCMV infection have been previously reported. Methods: Cochleovestibular histopathologies were evaluated in 4 temporal bones from 3 infants with cCMV infection by light microscopy. Results: In one available temporal bone of the infant in Case 1, no cytomegalic cells were found. Large areas of cellular and non-cellular structures were observed in the scala tympani of the perilymphatic space; however, there was no obvious loss of cochlear or vestibular hair cells. In Case 2, cytomegalic cells, a loss of vestibular hair cells, and a loss of nerve fibers were observed only in the area of dark cells in the vestibular labyrinth of the left temporal bone. No cytomegalic cells were found in the right temporal bone of the same infant; however, there was a loss of outer hair cells in the organ of Corti and hypervascularity in the stria vascularis. The one available temporal bone of the infant in Case 3 showed cytomegalic cells and a loss of hair cells in both cochlear and vestibular parts of the inner ear. Conclusions: Human temporal bones of newborns with cCMV demonstrate diverse cochleovestibular histopathologies. This diversity is consistent with the variable SNHL and vestibular dysfunction reported in infected newborns.

Formations of Host Fibers and Bacteria in Human Temporal Bones With Otitis Media.

HYPOTHESIS: In temporal bones with otitis media, fibrin and neutrophil extracellular traps (NETs) form a fibrous network with bacteria, which is involved in growth of bacterial clusters/biofilms and chronicity of disease. BACKGROUND: NETs and fibrin are important in host defense against pathogens; however, their role in otitis media is not well understood. METHODS: Eight human temporal bones with serous otitis media, 30 with serous-purulent otitis media, 7 with mucoid otitis media, 23 with mucoid-purulent otitis media (OM), 30 with purulent OM, and 30 with chronic otitis media were selected based on histopathologic findings. Fibrous material with bacteria was detected with hematoxylin-eosin, Gram-Weigert, and propidium iodide stains; and its composition was analyzed with immunohistochemistry. RESULTS: Extensive formations of fibrous material with bacteria were observed in 30% of temporal bones with serous-purulent otitis media, 29% with mucoid otitis media, 50% with mucoid-purulent OM, 57% with purulent OM, and 67% of temporal bones with histological evidence of chronic otitis media. Some of these formations showed large bacterial clusters or biofilms. Immunohistochemical analysis showed that fibrous structures were composed of fibrin or NETs. CONCLUSIONS: Formations of fibrous material with bacteria were detected in human temporal bones with different types of otitis media. Inflammatory cells were observed mostly in areas with low presence of fibrous structures. The network of fibrous material seems to prevent clearance of bacteria by phagocytic cells and thus influences growth of bacterial clusters or biofilms. Fibrin and NETs may be important for the recurrences and chronicity of disease, and contribute to clogging of tympanostomy tubes in children.

Cytomegalovirus-induced pathology in human temporal bones with congenital and acquired infection.

OBJECTIVE: Publications on histopathology of human temporal bones with cytomegalovirus (CMV) infection are limited. We aim to determine histopathology of the inner ears and the middle ears in human temporal bones with congenital and acquired CMV infections. METHODS: Temporal bones from 2 infants with congenital and 2 adults with acquired CMV infection were evaluated by light microscopy. RESULTS: Two infants with congenital CMV infection showed striking pathological changes in the inner ear. There was a hypervascularization of the stria vascularis in the cochlea of the first infant, but no obvious loss of outer and inner hair cells was seen in the organ of Corti. However, cytomegalic cells and a loss of outer hair cells were found in the cochlea of the second infant. The vestibular organs of both infants showed cytomegalic cells, mostly located on dark cells. There was a loss of type I and type II hair cells in the macula of the saccule and utricle. Loss of hair cells and degeneration of nerve fibers was also seen in the semicircular canals. Both infants with congenital infection showed abundant inflammatory cells and fibrous structures in the middle ear cavity. No evidence of cytomegalic cells and hair cell loss was found in the cochlea or vestibular labyrinth in acquired CMV infection. CONCLUSIONS: In two infants with congenital CMV infection, the cochlea, vestibule, and middle ear were highly affected. Temporal bones of adult donors with acquired viral infection showed histological findings similar to donors of the same age without ear disease.

Neutrophil Extracellular Traps and Fibrin in Otitis Media: Analysis of Human and Chinchilla Temporal Bones.

Background: Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. Objective: To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Design, Setting, and Participants: Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Main Outcome Measures: Histopatological and imminohistochemical changes owing to otitis media. Results: Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. Conclusions and Relevance: In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

Deterioration of Vestibular Cells in Labyrinthitis.

OBJECTIVE: To quantitatively assess the effect of serous labyrinthitis, suppurative labyrinthitis, and labyrinthitis ossificans on vestibular hair cells, dark cells, and transitional cells. METHODS: We examined human temporal bone specimens with serous labyrinthitis, suppurative labyrinthitis, and labyrinthitis ossificans, then compared them with age-matched control groups without labyrinthitis. We evaluated the density of type I and II vestibular hair cells, dark cells, and transitional cells in the peripheral sensorial organs. RESULTS: The mean density of type I vestibular hair cells in the maculae of the saccule significantly differed between the serous labyrinthitis group and its control group. The loss of type I and II vestibular hair cells in all of the peripheral sensorial organs was significantly higher in the suppurative labyrinthitis group than in its control group. The mean density of dark cells in the lateral and posterior semicircular canals was significantly lower in the suppurative labyrinthitis group than in its control group. The mean density of type I and II vestibular hair cells, dark cells, and transitional cells was significantly lower in the labyrinthitis ossificans group than in its control group. CONCLUSION: The loss of vestibular hair cells and degenerative changes in dark cells and transitional cells could affect vestibular function in patients with labyrinthitis.

A three-dimensional analysis of the endolymph drainage system in Ménière disease.

OBJECTIVES/HYPOTHESIS: To measure the volume of the endolymph drainage system in temporal bone specimens with Ménière disease, as compared with specimens with endolymphatic hydrops without vestibular symptoms and with nondiseased specimens STUDY DESIGN: Comparative human temporal bone analysis. METHODS: We generated three-dimensional models of the vestibular aqueduct, endolymphatic sinus and duct, and intratemporal portion of the endolymphatic sac and calculated the volume of those structures. We also measured the internal and external aperture of the vestibular aqueduct, as well as the opening (if present) of the utriculoendolymphatic (Bast's) valve and compared the measurements in our three study groups. RESULTS: The volume of the vestibular aqueduct and of the endolymphatic sinus, duct, and intratemporal endolymphatic sac was significantly lower in the Ménière disease group than in the endolymphatic hydrops group (P <.05). The external aperture of the vestibular aqueduct was also smaller in the Ménière disease group. Bast's valve was open only in some specimens in the Ménière disease group. CONCLUSIONS: In temporal bones with Ménière disease, the volume of the vestibular aqueduct, endolymphatic duct, and intratemporal endolymphatic sac was lower, and the external aperture of the vestibular aqueduct was smaller as compared with bones from donors who had endolymphatic hydrops without vestibular symptoms and with nondiseased bones. The open status of the Bast's valve in the Ménière disease group could be secondary to higher retrograde endolymph pressures caused by smaller drainage systems. These anatomic findings could correlate with the reason that some patients with hydrops develop clinical symptoms, whereas others do not. LEVEL OF EVIDENCE: N/A Laryngoscope, 127:E170-E175, 2017.

Pathologic Changes of the Peripheral Vestibular System Secondary to Chronic Otitis Media.

OBJECTIVE: To evaluate the histopathologic changes of dark, transitional, and hair cells of the vestibular system in human temporal bones from patients with chronic otitis media. STUDY DESIGN: Comparative human temporal bone study. SETTING: Otopathology laboratory. SUBJECTS AND METHODS: To compare the density of vestibular dark, transitional, and hair cells in temporal bones with and without chronic otitis media, we used differential interference contrast microscopy. RESULTS: In the chronic otitis media group (as compared with the age-matched control group), the density of type I and type II hair cells was significantly decreased in the lateral semicircular canal, saccule, and utricle (P < .05). The density of type I cells was also significantly decreased in the chronic otitis media group in the posterior semicircular canal (P = .005), but that of type II cells was not (P = .168). The mean number of dark cells was significantly decreased in the chronic otitis media group in the lateral semicircular canal (P = .014) and in the posterior semicircular canal (P = .002). We observed no statistically significant difference in the density of transitional cells between the 2 groups (P > .1). CONCLUSION: The findings of our study suggest that the decrease in the number of vestibular sensory cells and dark cells could be the cause of the clinical symptoms of imbalance of some patients with chronic otitis media.

Cochlear changes in serous labyrinthitis associated with silent otitis media: A human temporal bone study.

PURPOSE: To determine histopathological findings in the cochlea of human temporal bones with serous labyrinthitis. MATERIALS AND METHODS: We compared human temporal bones with serous labyrinthitis (20 cases) associated with silent otitis media and without serous labyrinthitis (20 cases) to study location of serous labyrinthitis, the degree of endolymphatic hydrops, number of spiral ganglion cells and hair cells, loss of fibrocytes in the spiral ligament, and areas of the spiral ligament and stria vascularis. RESULTS: The serous labyrinthitis caused significant loss of outer hair cells in the lower basal (P=0.006), upper basal (P=0.005), and lower middle (P=0.011) cochlear turns, and significant increase in the degree of endolymphatic hydrops than the control group (P=0.036). No significant difference was found in the loss of inner hair cells, in the number of spiral ganglion cells and fibrocytes in the spiral ligament, and in areas of the stria vascularis and spiral ligament (P>0.05). CONCLUSIONS: Serous labyrinthitis resulted in significant loss of outer hair cells and significant increase in the degree of endolymphatic hydrops.

Quantitative Assessment of Cochlear Histopathologic Findings in Patients With Suppurative Labyrinthitis.

IMPORTANCE: Better understanding of the effects of suppurative labyrinthitis (SL) on cochlear elements will aid the development of new approaches to treat its sequelae and complications in the ear. OBJECTIVE: To quantitatively evaluate the effects of SL on cochlear elements in humans. DESIGN, SETTING, AND PARTICIPANTS: A comparative study was conducted at a tertiary academic medical center from October 20, 2014, to January 3, 2015, of the histopathologic characteristics of 28 archived human temporal bone samples from 19 deceased patients with SL and 20 temporal bone samples from 14 deceased, age-matched controls. EXPOSURES: Evaluation of archived human temporal bone samples. MAIN OUTCOMES AND MEASURES: The locations of SL in the inner ear and the degree of endolymphatic hydrops were noted; the area of the stria vascularis and the spiral ligament in all turns of the cochlea at the midmodiolar level and in the adjacent 2 sections were measured; and the number of remaining outer and inner hair cells of the cochlea were counted to calculate the loss of both types of cells. To evaluate the loss of fibrocytes in the spiral ligament, a rating scale in each cochlear turn was used. For each segment of the cochlea, the number of spiral ganglion cells was determined. Outcomes between the group with SL and the control group were compared. RESULTS: Of the 28 temporal bone samples from the 19 deceased patients (16 men and 3 women; mean [SD] age, 23.1 [24.6] years) with SL, all showed SL in the scala tympani of the basal turn. In the group with SL vs the control group, the mean (SD) loss of outer hair cells was significantly higher in the lower (28.6% [11.4%] vs 12.4% [6.2%]; P = .02) and upper (22.3% [9.7%] vs 8.8% [3.2%]; P = .01) basal cochlear turn, the mean (SD) loss of inner hair cells was significantly higher in the lower (15.4% [6.7%] vs 2.6% [1.1%]; P = .02) and upper (10.6% [4.6%] vs 2.2% [0.7%]; P = .03) basal cochlear turn, the mean (SD) total number of spiral ganglion cells (28,132 [2068] vs 30,358 [2036]; P = .001) and the mean (SD) number of spiral ganglion cells in segment I (3554 [847] vs 4223 [649]; P = .003) was significantly decreased, the mean (SD) degree of atrophy of the stria vascularis in the lower (8455 [924] vs 9368 [1049] µm2; P = .003) and upper (7911 [837] vs 8474 [813] µm2; P = .02) basal cochlear turn was significantly greater, and the degree of endolymphatic hydrops was significantly greater (10 bone samples [36%] vs 1 [5%]; P = .006). No significant differences were found between the 2 groups in the number of fibrocytes and in the presence of atrophy of the spiral ligament in any cochlear turn. CONCLUSIONS AND RELEVANCE: This study demonstrates that SL can lead to cochlear damage, especially in the basal turn of the cochlea. These pathological observations have formed the basis for clinical findings of hearing loss and tinnitus detected in those patients with SL.

Pneumococcal PspA and PspC proteins: potential vaccine candidates for experimental otitis media.

OBJECTIVE: Otitis media is the most commonly diagnosed disease in ambulatory care and Streptococcuspneumoniae continues to be the most common bacterial agent. Bacterial resistance to antibiotics underscores the need for better vaccines. Current pneumococcal conjugate vaccines are modestly protective against otitis media; however, limited serotype coverage and serotype replacement have led to the investigation of pneumococcal proteins as potential vaccine candidates. Two proteins, pneumococcal surface proteins A (PspA) and C (PspC) are important virulence factors, expressed by virtually all strains. Although a number of pneumococcal proteins have been investigated in other infection sites, these proteins can have diverse organ-specific effects. In this study, we investigated the viability and virulence of single (PspA(-) and PspC(-)) and double (PspA(-)/PspC(-)) mutants of pneumococcal PspA and PspC proteins in the chinchilla middle ear. METHODS: Bullae of 24 chinchillas were inoculated with 0.5 ml of 10(6) colony forming units (CFUs)/ml bacteria: 6 with wild-type D39 strain; 6 with PspA(-); 6 with PspC(-); and 6 with PspA(-)/PspC(-) isogenic mutant strains. Bacterial CFU levels in middle ear effusions and light microscopic analysis of the number of inflammatory cells in the round window membrane (RWM) were compared 48 h after inoculation. RESULTS: At 48 h, CFUs in middle ears were increased for wild-type and PspC(-) strains compared to inoculum levels; however, they were significantly less for the group inoculated with the PspC(-) strain compared to wild-type strain. No bacteria were detected in the PspA(-) and PspA(-)/PspC(-) groups. The number of inflammatory cells in the RWM was significantly higher in wild-type compared to the PspA(-), PspC(-), and PspA(-)/PspC(-) groups. No significant difference in number of inflammatory cells was observed between any pairs of groups inoculated with mutant strains. CONCLUSION: Viability and virulence of the PspC(-) strain were similar to the wild-type strain. The single PspA(-) and double PspA(-)/PspC(-) mutants were highly attenuated in the ear. Bacterial clearance of the PspA(-)/PspC(-) double mutant was indistinguishable from that of the PspA mutant. These studies provide no reason to exclude PspC from a multi-component protein vaccine containing PspA.

The pathology of silent otitis media: a predecessor to tympanogenic meningitis in infants.

OBJECTIVE: To determine the association of bacteria embedded within a fibrous matrix in the middle and inner ear in infants with tympanogenic meningitis. METHODS: Thirty-one cases with meningitis from the human temporal bone collection at the University of Minnesota were screened to select those with tympanogenic meningitis. Inclusion criteria for tympanogenic meningitis were acute meningitis with histopathological evidence of chronic otitis media, and no other source of infection. The presence of labyrinthitis and pathologic changes such as granulation tissue, fibrosis, cholesterol granuloma, cholesteatoma, tympanic membrane perforation, tympanosclerosis, and the type of effusion were noted. The extent and location of bacteria embedded in a fibrous matrix were also explored. RESULTS: Seventeen temporal bones, from nine cases that included two females and seven males, ranging in age from five to twenty-three months, met our criteria of tympanogenic meningitis. Eighty two percent of these temporal bones had bacteria within the fibrous matrices (BFM). BFM were located in one anatomical region in one temporal bone and multiple anatomic regions in sixteen temporal bones. The most common locations were the areas near the oval and round windows. They were also commonly seen in the epitympanum, facial recess, and supratubal recess. BFM within the inner ear were observed in the scala tympani and modiolus in the middle and basal turns of the cochleae of nine temporal bones. In one of these temporal bones, BFM were seen in the internal auditory canal. Labyrinthitis was seen in all ears. The tympanic membrane was intact in all cases. BFM were not seen in three temporal bones from two patients. In one case only one side was available for study. CONCLUSIONS: Our findings show an association between the presence of BFM in the ear with chronic pathologic changes and tympanogenic meningitis. Potential pathways of bacteria from the middle ear include hematogeous spread and/or direct spread to dura through the tympanic tegmen, and/or to the inner ear through the oval and round windows, and from there to the modiolus and the meninges. Chronic pathologic changes in the middle ear behind an intact tympanic membrane and the lack of ear symptoms may result in potentially serious sequelae and complications in infant age groups. There should be a heightened awareness of this condition.

Viability and virulence of pneumolysin, pneumococcal surface protein A, and pneumolysin/pneumococcal surface protein A mutants in the ear.

IMPORTANCE: Understanding how pneumococcal proteins affect the pathology of the middle ear and inner ear is important for the development of new approaches to prevent otitis media and its complications. OBJECTIVES: To determine the viability and virulence of Streptococcus pneumoniae mutants deficient in pneumolysin (Ply-) and pneumococcal surface protein A (PspA-) in the chinchilla middle ear. DESIGN: Bullae of chinchillas were inoculated bilaterally with wild-type (Wt), Ply-, PspA-, and Ply-/PspA- strains. Bacterial colony-forming units (CFUs) in middle ear effusions were counted at 48 hours. The CFUs of the PspA- group were also counted at 6 to 36 hours after inoculation. Temporal bone histopathological results were compared. SETTING AND PARTICIPANTS: Twenty-seven chinchillas in an academic research laboratory. EXPOSURE: Chinchilla middle ears were inoculated with S pneumoniae to produce sufficient volumes of effusions and noticeable histopathological changes in the ears. MAIN OUTCOMES AND MEASURES: The CFU counts in the middle ear effusions and histopathological changes were compared to determine the effect of pneumococcal protein mutations on chinchilla ears. RESULTS: At 48 hours, CFUs in middle ears were increased for the Wt and Ply-/PspA- strains, but Ply- remained near inoculum level. No bacteria were detected in the PspA- group. The CFUs of PspA- decreased over time to a low level at 30 to 36 hours. In vitro, PspA- in Todd-Hewitt broth showed an increase in bacterial growth of 2 logs at 43 hours, indicating PspA- susceptibility to host defenses in vivo. The PspA- and Ply- groups had fewer pathologic findings than the Wt or Ply-/PspA- groups. Histopathological analysis showed significant differences in the number of bacteria in the scala tympani in the Wt group compared with the Ply-, PspA-, and Ply-/PspA- groups. The PspA- strain was the least virulent. CONCLUSIONS AND RELEVANCE: The PspA- mutant was much less viable and less virulent in the ear than the Wt, Ply-, and Ply-/PspA- strains. There was no significant attenuation in the viability and virulence of the Ply-/PspA- mutant compared with the Wt or single mutants. The viability and virulence of pneumococcal mutants seemed to be protein and organ specific.

Effect of apolactoferrin on experimental pneumococcal otitis media.

OBJECTIVE: To find the effect of apolactoferrin administration on the middle and inner ears after experimentally induced pneumococcal otitis media. DESIGN: Histopathologic and morphometric analysis of the middle and inner ears. SETTING: University of Minnesota, Minneapolis. SUBJECTS: Ten chinchillas. INTERVENTIONS: The middle ear cavities of chinchillas were inoculated bilaterally with type 2 wild-type Streptococcus pneumoniae. Twenty-four hours later, the ears of 5 of the animals were injected with phosphate-buffered saline (PBS) and the other 5 with human apolactoferrin. The animals were killed 24 hours after the last injection. Bacterial counts were made of the middle ear effusions, and the cochleae were processed for histologic analysis. The thickness of the round window membranes and bacterial and inflammatory cell infiltration of the round window membranes, and scala tympani and damage of the hair cells and stria vascularis were compared for these 2 groups of animals. MAIN OUTCOME MEASURES: Comparison of inflammatory and bacterial cells in the middle and inner ears, and damage to inner ear structures. RESULTS: Bacterial plate counts of middle ear effusions (P  = .005) and the number of inflammatory cells in the round window membrane (P  = .047) were significantly lower in the apolactoferrin group compared with the group treated with PBS. CONCLUSION: Further investigation of apolactoferrin as a nonantibiotic approach for the treatment of otitis media and its complications is needed to confirm its safety and efficacy.

Cochlear pathology in human temporal bones with otitis media.

CONCLUSION: Middle and inner ear interactions in otitis media can lead to cochlear pathology. More severe pathological changes observed in the basal turn of the cochlea are consistent with prevalence of sensorineural hearing loss at higher frequencies in patients with otitis media. METHODS: Of 614 temporal bones with otitis media, 47 with chronic and 35 with purulent otitis media were selected following strict exclusion of subjects with a history of acoustic trauma, head trauma, ototoxic drugs, and other diseases affecting the cochlear labyrinth. Temporal bones with labyrinthine inflammatory changes were further evaluated for loss of hair cells and other histopathologic changes compared to age-matched controls. RESULTS: In all, 19% of temporal bones with chronic and 9% with purulent otitis media showed labyrinthine inflammatory changes. In chronic otitis media, inflammatory changes were: 56% localized purulent, 22% localized serous, 11% generalized seropurulent, and 11% generalized serous. Inflammatory changes in temporal bones with purulent otitis media included 67% localized purulent and 33% were generalized seropurulent. Pathological findings included: serofibrinous precipitates and inflammatory cells in scala tympani of basal turn and cochlear aqueduct, significant loss of outer and inner hair cells, and significant decrease in area of stria vascularis in the basal turn of the cochlea, as compared to controls.

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.

Virulence of pneumococcal proteins on the inner ear.

OBJECTIVE: To investigate the effects of the virulence characteristics of specific pneumococcal proteins on the inner ear. MAIN OUTCOME MEASURES: A histologic comparison of inflammatory cell infiltration and pathologic changes in the round window membrane and inner ear. RESULTS: Most of the animals inoculated with high-dose pneumolysin or wild-type bacteria showed severe pathologic changes of the inner ears. The inner ears of most animals inoculated with surface protein A or surface antigen A-deficient bacteria appeared normal. CONCLUSIONS: Pneumococcal surface protein A and pneumococcal surface antigen A are 2 important virulence factors in inner ear damage secondary to pneumococcal otitis media. Mutation of these virulence factors results in less inner ear damage.

Role of pneumococcal proteins in sensorineural hearing loss due to otitis media.

HYPOTHESIS: Two Streptococcus pneumoniae proteins, pneumococcal surface protein A (PspA) and pneumolysin (Ply), have functional and histopathologic effects on the inner ear. BACKGROUND: Temporary or permanent sensorineural hearing loss is known to be a sequela of pneumococcal otitis media. Several pneumococcal proteins such as PspA and Ply have been shown to contribute to the pathogenesis of the middle ear; however, effects of these proteins on the inner ear and hearing loss are unknown. METHODS: Middle ears of chinchillas were inoculated with either wild-type S. pneumoniae or its mutants, deficient in PspA or Ply proteins. After 28 days, auditory brainstem response of animals was tested, and their bullae were processed for histopathologic analysis by light microscopy. RESULTS: Twenty-eight days after instillation of 20 colony-forming units of wild-type pneumococci, auditory brainstem response test showed threshold changes of 10 to 15 dB for 4 to 32 kHz and more than 20 dB for 1 to 2 kHz. No significant hearing loss was observed after instillation of the same or even higher doses of isogenic S. pneumoniae mutants of PspA or Ply proteins, or saline injection, after the same period. Histologic analysis showed no fluid, inflammatory cells, or bacteria in the middle ear, indicating that hearing loss was sensorineural. Inner ear morphology showed pathologic changes in the stria vascularis, suggesting it as the target of otitis media-induced damage, which may lead to sensorineural hearing loss. CONCLUSION: The virulence PspA and Ply proteins of S. pneumoniae affect the inner ear and auditory function.

The round window membrane in otitis media: effect of pneumococcal proteins.

OBJECTIVE: To determine whether mutants of Streptococcus pneumoniae that are deficient in pneumococcal surface protein A (PspA), pneumococcal surface antigen A (PsaA), or pneumolysin (Ply) are less virulent and less likely to penetrate the round window membrane (RWM). DESIGN: Histopathologic comparison of wild-type S. pneumoniae and its mutants deficient in PspA, PsaA, and Ply. SETTING: Otopathology Laboratory, Department of Otolaryngology, University of Minnesota Medical School, Minneapolis. PARTICIPANTS: Forty young chinchillas (weight, 250-350 g) with normal external auditory canals and tympanic membranes. INTERVENTION: Animals were divided into 3 groups and bullae inoculated with wild-type S. pneumoniae serotype 2, strain D39, or its mutants deficient in PspA, PsaA, or Ply. Two days after inoculation, bullae were processed for light microscopy and transmission electron microscopy. MAIN OUTCOME MEASURES: Comparison of inflammatory cell infiltration and penetration of bacteria into the round window membrane and adjacent scala tympani. RESULTS: Histopathologic findings using wild-type S. pneumoniae and Ply(-) mutant were similar and included otitis media and the presence of inflammatory cells and damage to and passage of bacteria through the RWM. Although otitis media was seen with the PspA(-) and PsaA(-) mutants, we observed no passage of bacteria through the RWM. CONCLUSIONS: Both PspA and PsaA affect the ability of S. pneumoniae to penetrate the RWM. Understanding the role of S. pneumoniae virulence proteins in the pathogenesis of the middle ear, RWM, and inner ear will provide new strategies for the prevention and treatment of otitis media and its complications.

Up-regulation of macrophage migration inhibitory factor induced by endotoxin in experimental otitis media with effusion in mice.

CONCLUSION: Injection of endotoxin into the middle ear causes production of macrophage migration inhibitory factor (MIF) in an experimental mouse model of otitis media with effusion (OME). Down-regulation of MIF may become a new approach for the management of OME. OBJECTIVE: To determine the role of MIF in OME. MATERIALS AND METHODS: Mice were divided into two groups and their middle ears were injected with either endotoxin or phosphate-buffered saline (PBS). Mice were sacrificed at 6 h, 12 h, or 1, 3, 7, or 14 days after injection and concentrations of MIF, interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in middle ear effusions were measured by enzyme-linked immunosorbent assay. RESULTS: Concentrations of MIF in the endotoxin group at 1 day and 3 days were significantly higher than in the PBS control group. Concentrations of IL-1beta in the endotoxin group at 6 h, 12 h, 1 day, and 3 days were significantly higher than in controls. Concentrations of TNF-alpha in the endotoxin group at 1 day and 3 days were significantly higher than in controls. Concentration of MIF in the endotoxin group was positively correlated with that of IL-1beta and TNF-alpha.

Specificity of aminoacylase III-mediated deacetylation of mercapturic acids.

Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-l-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate beta-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V(max) value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-l-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of beta-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto- and nephrotoxicity.