Marc S. Krug

Antibodies against a 30 kilodalton cochlear protein and type II and IX collagens in the serum of patients with inner ear diseases.

Collagen molecules are major extracellular matrix proteins involved in the development and support of delicate auditory sensory organs. Type II collagen is widely distributed within inner ear tissues, while type IX is found only within the labyrinthine membrane and dense fibers of the tectorial membrane. Antibody specific for type II collagen has been shown to be elevated in some patients with hearing loss due to several presumably autoimmune illnesses (including Menieres disease, otosclerosis, chronic progressive sensorineural hearing loss, and relapsing polychondritis). Purified human type II and LX collagens and an extract of human cochlear tissue were subjected to isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. The sera of 21 patients with inner ear disease were examined for the presence of anticollagen and anticochlear antibodies; the sera were used to probe Western blots of purified human collagens U, LX, and XI, and cochlear protein extract with peroxidase-conjugated goat anti human polyvalent immunoglobulin as the second antibody. Anti-type II collagen antibodies were seen in 12 of 21 (57%) patients, while 13 of 21 (62%) had anti-type IX antibodies detectable by Western blot. A previously unreported 30 kd (probably noncollagen) protein was found by SDS-PAGE of human cochlear tissue extracts, with 3 patients, all with Menieres disease, having antibody activity to this protein detected by Western blot. Anti-type II and anti-type LX antibodies were found in a high percentage of patients with Menieres disease, otosclerosis, and strial atrophy. Six patients (29%), and all control patients, had no detectable antibodies to these proteins by our assay.

Antibodies against Inner-Ear Proteins in the Sera of Patients with Inner-Ear Diseases

Sera from patients with various inner-ear diseases, especially Meniere’s disease, were investigated by Western blot against guinea pig inner-ear proteins. Of 45 patients, 24 (53%) with various inner-e

House dust mite–induced sensitivity in mice

Sensitivity induced by house dust mite (HDM) extract in mice was investigated in this study. Sensitized B10.RIII mice (H-2r background) had T-cell proliferative responses to HDM extract in vitro and an HDM-specific IgE response. When mice were immunized by injection and intranasal inhalation with HDM extract, a histologic study showed eosinophils and mononuclear cell infiltration in the lung tissue and bronchial wall. Tcr alphabeta-positive cells were also found in the cell infiltration area of the lung lesions. In the control mice that were immunized by injection or intranasal inhalation (but not both), we did not observe cell accumulation in the lung tissue or in the bronchial wall. Epitope studies suggest that T cells recognize multiple epitopes. Molecular analysis of these HDM-specific T-cell hybridoma clones suggest that T-cell receptor use is restricted to members of the V alpha 8 and Vbeta 6 subfamilies.

Proto-Oncogene Raf-1 as an Autoantigen in Meniere's Disease

The 28 kd protein extracted from the guinea pig inner ear membranous fraction, which reacted with sera from patients with Menieres disease, has been subjected to microsequencing. Nineteen amino acids were obtained (IVQQFGFQRRASDDGKLTQ). A protein data bank search showed that this sequence corresponded to residues 41 to 60 of human Raf-1 protein. Sera from 16 of 27 patients with Menieres disease showed reactivity to the recombinant purified glutathione-S-transferase–Raf-1 protein. These results support the hypothesis that a subgroup of patients who suffer from Menieres disease, as well as some other kinds of autoimmune inner ear diseases, have an autoantibody against Raf-1 protein.

Molecular Basis of Type II Collagen Autoimmune Disease: Observations of Arthritis, Auricular Chondritis and Tympanitis in Mice

A type II collagen autoimmune response results in arthritis, auricular chondritis and tympanosclerosis in humans and animals. The purpose of this study is to further define the molecular and pathogenic events involved in these lesions in rodents. Type II collagen fragment CB11-specific monoclonal antibodies induced lesions in joints, ear lobes and tympanic membranes. In immunized mice, the thickness of tympanic membranes increased to two- to fourfold normal size. Electron micrography showed that the arrangement of collagen fibers is irregular in both radial and auricular layers, containing fibroblasts, a homogeneous material resembling low-density cholesterol crystals and cell infiltration. The mice with auricular chondritis had lymphocytes expressing V beta-8 T cell receptor (TCR) in arthritic joints and lymphocytes expressing V beta-6 TCR in ear lobe lesions. A monoclonal antibody specific to the TCR V beta-8 subfamily suppressed the onset of arthritis. Sequence analyses of the V beta structure of TCR involved in the lesions confirm the immunohistologic study.

Murine model of autoimmune hearing loss induced by myelin protein P0

Myelin protein P0 has been identified as an autoantigen in inner ear diseases. In order to study autoimmune hearing loss, we performed brain stem auditory-evoked potential (BAEP) studies on P0-sensitized mice. Two P0-sensitized mice showed hunched posture, poor coat, loss of body weight, and abnormal walking with a waddling gait. About 25% of the P0-sensitized mice developed hearing loss. In the BAEP study, peak latencies of waves I, III, and V and the interpeak latency I-III were prolonged in the P0-sensitized hearing loss group of mice. Hearing thresholds were elevated in this group of mice in comparison with the control mice. Inflammatory cell infiltration was observed in the cochlear nerve region, and a reduced number of spiral ganglion cells was also detected. These results suggest that P0-sensitized mice are a useful model for studying autoimmune inflammation of the peripheral portion of the auditory system.

Successful Prevention of Retrocochlear Hearing Loss in Murine Experimental Allergic Encephalomyelitis with T Cell Receptor Vβ8–Specific Antibody

Experimental allergic encephalomyelitis is an animal model of a T cell—mediated autoimmune disease — for example, multiple sclerosis. We demonstrated that mice with experimental allergic encephalomyelitis developed retrocochlear hearing loss, and that the lesion of the auditory pathway might be related to T cell receptor Vß8–expressing T cells. To investigate whether anti-Vß8 antibody could prevent hearing loss, we carried out brain stem auditory evoked potential testing, histologic examinations, and flow cytometry in antibody-treated and control myelin basic protein—immunized B10.PL mice. The antibody was administered just before immunization of myelin basic protein. The disease incidence and severity were significantly reduced in the mice injected with the antibody. The results of brain stem auditory evoked potential testing, histologic examinations, and flow cytometry indicated that the depletion of Vß8-expressing T cells brings the prevention of hearing loss, as well as prevention of other neurologic deficits. The development of T cell receptor—specific antibody therapy might help treat retrocochlear hearing loss in multiple sclerosis.

Brain Stem Auditory-Evoked Potentials of Mice with Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is an experimental model for multiple sclerosis. In order to study autoimmune retrocochlear hearing loss, we performed brain stem auditory-evoked potential (BAEP) studies on EAE mice. The EAE was induced in B 10.PL and (PL/J x SJL)F1 mice. In the BAEP study, all of the peak and interpeak latencies were prolonged significantly in the diseased mice. Hearing thresholds were slightly elevated in the immunized mice during the acute phase. Inflammatory and phagocytic cell infiltration, demyelination, and Vβ8.1, 8.2 T-cell receptor-positive cells were observed in the cochlear nerves or their proximity by histologic study. It is suggested that immunologic reactions identified with EAE also occurred in the cochlear nerve, and that these reactions were responsible for the hearing problems. It appears that EAE is a useful model system for studying autoimmune insults on the neural portion of the auditory system.

Antibodies against inner ear proteins in the sera of patients with inner ear diseases

Sera from patients with various inner-ear diseases, especially Ménières disease, were investigated by Western blot against guinea pig inner-ear proteins. Of 45 patients, 24 (53%) with various inner-ear diseases had antibodies against inner-ear proteins, compared with 0 of 10 (0%) in control subjects without inner-ear diseases. Of the 10 proteins that showed a positive reaction with patient sera, the 28-kD band was unique in that it appeared only in the membranous fraction of the inner ear and was highly positive (28%) in reaction with Ménières disease patient sera. The results in the present study with proteins extracted from guinea pig inner ear were consistent with our previous study using proteins from human inner ear, suggesting that the 28-kD protein may be a candidate for detecting autoimmune inner-ear disease.

Prevention of retrocochlear hearing loss in murine experimental allergic encephalitis with T cell receptor Vβ8-specific antibody

Abstract Experimental allergic encephalomyelitis (EAE) is an animal model of a T cell-mediated autoimmune disease, for example multiple sclerosis (MS). We demonstrated that EAE mice developed retrocochlear hearing loss and the lesion of the auditory pathway might be related to T cell receptor (TCR) Vβ8-expressing T cells. To investigate whether anti-Vβ8 antibody could prevent hearing loss, brainstem auditory evoked potentials (BAEPs) histological examinations were carried out in antibody treated and control myelin basic protein (MBP) immunized B10.PL mice. The antibody was administered just before immunization of MBP. The disease incidence and severity were significantly reduced in the mice injected with the antibody. The results of BAEPs and histological examinations indicated that depletion of Vβ8-expressing T cells causes the prevention of hearing loss as well as prevention of other neurological deficits. The development of TCR specific antibody therapy might help treat retrocochlear hearing loss in MS.