Elizabeth M. Keithley

Evidence Linking the 68 Kilodalton Antigen Identified in Progressive Sensorineural Hearing loss Patient Sera with Heat Shock Protein 70

Immunoblotting against bovine inner ear extracts has previously identified a 68 kd antigen reactive with 22% to 58% of sera of patients with rapidly progressive sensorineural hearing loss (PSNHL) of suspected autoimmune causation. Efforts to purify and characterize this diagnostic antigen suggest that it is ubiquitous rather than inner ear-specific, and may represent the highly inducible heat shock protein (hsp) 70. The antigens identified by PSNHL sera and anti-hsp 70 monoclonal antibodies copurify on ion exchange and adenosine triphosphate affinity chromatography, and comigrate on one- and two-dimensional gel electrophoresis. Additionally, immunoblotting with positive patient sera shows dramatically increased expression of the 68 kd antigen by bovine kidney cells following heat shock in culture. Reactivity with stress proteins of various classes has been reported in a number of autoimmune diseases; however, anti—hsp 70 appears uniquely associated with ulcerative colitis and PSNHL.

Assessment of serum antibodies in patients with rapidly progressive sensorineural hearing loss and Menière's disease.

The immunoreactivity of sera from patients with rapidly progressive sensorineural hearing loss (SNHL) or Menieres disease with bovine inner ear material was determined using the Western blot technique. Patients with other otologic conditions, autoimmune disorders, or arthritic disorders and age‐matched randomly chosen patients with no hearing complaints served as controls. Twenty‐two percent of the patients with bilateral rapidly progressive SNHL and 30% of the patients with Menieres disease had antibodies that reacted with a 68 kd antigen in the inner ear material. In the control groups, the incidence of reactivity was 5.0% (P<.001).

Tumor Necrosis Factor-α, an Initiator, and Etanercept, an Inhibitor of Cochlear Inflammation†‡

Objectives/Hypothesis The inner ear rapidly mounts an immune response that can lead to cochlear degeneration and permanent hearing loss. Identification of proinflammatory cytokine expression during the initiation of the response should lead to rational therapeutic strategies that block the response, reducing damaging sequelae.

Age-related hearing loss and the ahl locus in mice.

C57BL/6 (B6) mice experience hearing loss and cochlear degeneration beginning about mid-life, whereas CAST/Ei (CAST) mice retain normal hearing until old age. A locus contributing to the hearing loss of B6 mice, named age-related hearing loss (ahl), was mapped to Chromosome 10. A homozygous, congenic strain of mice (B6.CAST-+ahl ), generated by crossing B6 (ahl/ahl) and CAST (+ahl/+ahl) mice has the same genomic material as the B6 mice except in the region of the ahl locus, which is derived from CAST. In this study, we have determined the extent of the CAST-derived region of Chromosome 10 in the congenic strain and have examined mice of all three strains for hearing loss and cochlear morphology between 9 and 25 months of age. Results for B6 mice were similar to those described previously. CAST mice showed no detectable hearing loss even at 24 months of age; however, they had a small amount of ganglion cell degeneration. B6.CAST-+ahl mice were protected from early onset hearing loss and basal turn degeneration, but older animals did show some hearing loss and ganglion cell degeneration. We conclude that loci in addition to ahl contribute to the differences in hearing loss between B6 and CAST mice. These results illustrate the complex inheritance of age-related hearing loss in mice and may have implications for the study of human presbycusis.

Local Perfusion of the Tumor Necrosis Factor α Blocker Infliximab to the Inner Ear Improves Autoimmune Neurosensory Hearing Loss

Objective: To evaluate the effect of transtympanic administration of tumor necrosis factor α (TNF-α) blockers to patients suffering from autoimmune inner ear disease (AIED). Study Design: Nonrandomized, prospective pilot study. Setting: Tertiary referral center. Patients: 9 patients (4 men and 5 women; aged 51.22 ± 13.11 years) presenting with autoimmune sensorineural hearing loss who responded to oral steroid treatment. Two groups of patients were treated. Group A consisted of 5 patients with AIED who could not be tapered off steroids. Group B consisted of 4 patients who were treated with intratympanic anti-TNF-α antibody therapy alone after a relapse of hearing loss following discontinuation of steroids. Intervention: A Silverstein MicroWickTM local delivery system was placed in the round window niche and the patients were treated for 4 weeks with a weekly infusion of infliximab, a monoclonal antibody against TNF-α. Main Outcome Measure(s): Evaluation of hearing thresholds at 250–8000 Hz was performed before and after implantation of the Silverstein MicroWick and local delivery of the TNF-α blocker. Results: Local administration of the TNF-α blocker allowed methylprednisolone to be tapered off without loss of hearing function in 4/5 steroid-dependent patients. Four additional patients were treated only with anti-TNF-α perfusion to the round window membrane without concomitant systemic administration of methylprednisolone. In 3 of these 4 patients, the pure tone average improved to 22.6 ± 15.7 dB, resulting in hearing recovery comparable to treatment with systemic methylprednisolone. The 7 responding patients showed a significant reduction of recurrence of hearing loss to 0.028 ± 0.072 episodes per month over the 4.3 ± 2.4 months of the post-treatment period compared to 0.84 ± 0.4 recurrences per week seen in the pretreatment period. Conclusions: The results of this pilot trial demonstrate that in patients with AIED, transtympanic delivery of the TNF-α blocker infliximab once weekly for 4 weeks allowed steroids to be tapered off, resulted in hearing improvement and reduced disease relapses. These preliminary efficacy and safety results appear encouraging enough to warrant further follow-up and studies for better determination of the potential clinical utility of local administration of infliximab for autoimmune hearing loss.

Spiral ganglion cell density in young and old gerbils

The Mongolian gerbil, like other mammalian species, has a decreased number of spiral ganglion cells as a function of age. This loss of cells was first seen in 24- to 30-month old animals in the basal end of the ganglion. In the oldest individuals the apical end of the ganglion was also affected. There were approximately 15-25% fewer cells in the affected areas in the 36- to 42-month old animals. In the oldest animals degeneration of the stria vascularis was seen in the apical turn and some degenerative changes in the organ of Corti were seen throughout the length of the cochlear duct. The aging pattern in the gerbil cochlea, is similar to that described for other species. Vacuoles, previously described in the gerbil cochlear nucleus, were also seen in the auditory nerve within the modiolus, but central to the Schwann-glial border in all animals. Vacuoles were not present within the spiral ganglion or the peripheral processes of the ganglion cells. Because the ganglion cell axons should be similar on either side of the Schwann-glial border, but the vacuoles were confined to the central nervous system, it is concluded that the degenerative process affects glial cells as opposed to neurons.

Proinflammatory Cytokine Expression in the Endolymphatic Sac During Inner Ear Inflammation

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3–48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1β, TNFα, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1β and TNFα between 3 and 12 h after KLH injection. On the other hand, TNFα, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFα was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFα, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response.

Cu/Zn superoxide dismutase and age-related hearing loss.

Mice, in which the genetics can be manipulated and the life span is relatively short, enable evaluation of the effects of specific gene expression on cochlear degeneration over time. Antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1) protect cells from toxic, reactive oxygen species and may be involved in age-related degeneration. The effects of SOD1 deletion and over-expression on the cochlea were examined in Sod1-null mice, Sod1 transgenic mice and in age- and genetics-matched controls. Auditory brainstem responses (ABR) were measured and cochleae were histologically examined. The absence of SOD1 resulted in hearing loss at an earlier age than in wildtype or heterozygous mice. The cochleae of the null mice had severe spiral ganglion cell degeneration at 7-9 months of age. The stria vascularis in the aged, null mice was thinner than in the heterozygous or wildtype mice. Over-expression of SOD1 did not protect against hearing loss except at 24 months of age. In conclusion, SOD1 seems important for survival of cochlear neurons and the stria vascularis, however even half the amount is sufficient and an over abundance does not provide much protection from age-related hearing loss.

Immune cell recruitment following acoustic trauma.

Acoustic trauma induces cochlear inflammation. We hypothesized that chemokines are involved in the recruitment of leukocytes as part of a wound healing response. The cochleas of NIH-Swiss mice, exposed to octave-band noise (8-16 kHz, at 118 dB) for 2h, were examined after the termination of exposure. Leukocytes were identified immunohistochemically with antibodies to CD45 and F4/80. Gene array analysis followed by RT-PCR was performed on cochlear tissue to identify up-regulation of chemokine and adhesion molecule mRNA. The expression of the adhesion molecule ICAM-1 was also investigated immunohistochemically. Few CD45- or F4/80-positive leukocytes were observed in the non-exposed cochlea. Following acoustic trauma however, the number of CD45-positive cells was dramatically increased especially after 2 and 4 days, after which time the numbers decreased. F4/80-positive cells also increased in number over the course of a week. Gene array analysis indicated increased expression of monocyte chemoattractant protein 5 (MCP-5), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1beta (MIP-1beta) and ICAM-1. RT-PCR, performed using primers for the individual mRNA sequences, confirmed the increased expression of MCP-1, MCP-5, MIP-1beta, and ICAM-1 relative to non-exposed mice. In the normal cochlea, ICAM-1 immunohistochemical expression was observed in venules, spiral ligament fibrocytes and in endosteal cells of the scala tympani. Expression increased to include more of the spiral ligament and endosteal cells after acoustic trauma. A cochlear inflammatory response is initiated in response to acoustic trauma and involves the recruitment of circulating leukocytes to the inner ear.

Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations

Presbycusis is a histologically and genetically heterogenous group of disorders, which lead to progressive, primarily sensorineural hearing loss with aging. Acquired mitochondrial DNA defects have been proposed as important determinants of aging, particularly in neuro-muscular tissues. The spiral ganglion and membranous labyrinth from archival temporal bones of 5 patients with presbycusis were examined for mutations within the mitochondrially-encoded cytochrome oxidase II gene. When compared to controls, results indicate that mitochondrial mutations in the peripheral auditory system occur commonly with age-related hearing loss, that there is great individual variability in both quantity and location of mutation accumulation, and that at least a proportion of presbycusis patients have a highly significant load of mutations in auditory tissue. This work supports the hypothesis that acquired mitochondrial mutations are a determinant of hearing loss in a subgroup of presbycusis patients.