Autoimmune inner ear disease patient-associated 28-kDa proinflammatory IL-1ß fragment results from caspase-7-mediated cleavage in vitro.

Interleukin-1ß (IL-1ß) is a key proinflammatory cytokine involved in the progression of many autoinflammatory and autoimmune diseases, including autoimmune inner ear disease (AIED). IL-1ß inhibition has been shown to result in clinical hearing improvement in a small cohort of corticosteroid-resistant patients with AIED. Canonical processing of pro-IL-1ß by caspase-1 generates an active 17-kDa fragment, capable of instigating a proinflammatory microenvironment. However, in response to LPS, PBMCs from patients with AIED uniquely express a 28-kDa IL-1ß fragment, as compared with PBMCs from control subjects. We synthesized and compared the biologic activity of the 28-kDa fragment to the 17-kDa IL-1ß product and the pro-IL-1 31-kDa protein. The 28-kDa IL-1ß fragment induces IL-6, TNF-α, and CCL3 in PBMCs. Uniquely, only caspase-7 treatment showed a dose- and time-dependent increase in 28-kDa band generation. Mass spectrometry confirmed the putative caspase-7 cleavage site of pro-IL-1ß, which was used to generate the 28-kDa fragment used for PBMC stimulation studies. Collectively, these results provide insight into the function of a poorly understood, processed 28-kDa form of IL-1ß in patients with AIED that is uniquely generated by caspase-7 and is capable of activating further downstream proinflammatory cytokines. Further investigation may provide novel pharmacologic targets for the treatment of this rare disease.

Expression of aquaporins in inner ear disease.

The inner ear is responsible for hearing and balance and consists of a membranous labyrinth within a bony labyrinth. The balance structure is divided into the otolith organ that recognizes linear acceleration and the semicircular canal that is responsible for rotational movement. The cochlea is the hearing organ. The external and middle ear are covered with skin and mucosa, respectively, and the space is filled with air, whereas the inner ear is composed of endolymph and perilymph. The inner ear is a fluid-filled sensory organ composed of hair cells with cilia on the upper part of the cells that convert changes in sound energy and balance into electric energy through the hair cells to transmit signals to the auditory nerve through synapses. Aquaporins (AQPs) are a family of transmembrane proteins present in all species that can be roughly divided into three subfamilies according to structure and function: 1) classical AQP, 2) aquaglyceroporin, and 3) superaquaporin. Currently, the subfamily of mammalian species is known to include 13 AQP members (AQP0-AQP12). AQPs have a variety of functions depending on their structure and are related to inner ear diseases such as Meniere's disease, sensorineural hearing loss, and presbycusis. Additional studies on the relationship between the inner ear and AQPs may be helpful in the diagnosis and treatment of inner ear disease. Laryngoscope, 130:1532-1539, 2020.

The Balance of Tissue Inhibitor of Metalloproteinase-1 and Matrix Metalloproteinase-9 in the Autoimmune Inner Ear Disease Patients.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a protein implicated in the control of inflammation in a number of autoimmune diseases. We hypothesized that the balance of TIMP-1 and matrix metalloproteinase-9 (MMP-9) may influence the control or perpetuation of inflammation in corticosteroid-responsive (RES) and corticosteroid-resistant (NR) autoimmune inner ear disease (AIED) patients. In the present study, we observed that plasma from AIED patients exhibited greater levels of TIMP-1 values compared with normal healthy controls. TIMP-1 abrogates lipopolysaccharide-mediated interleukin (IL)-1ß release from peripheral blood mononuclear cells in a dose-dependent manner. RES AIED patients have higher basal TIMP-1 levels and produce more TIMP-1 in response to IL-1ß. Conversely, consistent with our previous studies, we found that NR patients have higher basal MMP-9 levels and produce more MMP-9 levels in response to IL-1ß.

FoxO3a plays a key role in the protective effects of pomegranate peel extract against amikacin-induced ototoxicity.

The use of amikacin (AMK) in present treatment strategies results in severe ototoxicity; however, the underlying molecular mechanisms of this toxicity remain unclear. In this study, we investigated the effectiveness of orally administered pomegranate peel extract (PPE), a strong antioxidant, as a protective agent against AMK-induced ototoxicity. To this end, PPE was orally administered to adult BALB/c mice for 5 days, and the mice were then concurrently treated with AMK (500 mg/kg/day for 15 consecutive days). Auditory threshold shifts induced by AMK were significantly attenuated. The results of immunohistochemical staining and western blot analysis revealed that PPE exerted its protective effects by by downregulating the phosphorylation of Forkhead box O3a (FoxO3a), an important transcription factor which is involved in the responses to oxidative stress. The results also showed that PPE treatment inhibited mitogen-activated protein kinase phosphorylation, prevented the activation of pro-apoptotic protein caspase-3, decreased the levels of apoptosis-inducing Bax protein, and increased the levels of the anti-apoptotic mediator, Bcl-2, induced by AMK in the mouse cochlea. Taken together, our experimental findings suggest that phosphorylated FoxO3a mediates AMK-induced apoptosis in BALB/c mice cochlea. PPE effectively attenuated oxidative stress and ototoxicity by regulating FoxO3a, and may thus prove to be beneficial in protecting auditory cells from ototoxic drugs.

NOX3-TARGETED THERAPIES FOR INNER EAR PATHOLOGIES.

Inner ear pathologies are associated with major morbidity and loss of life quality in affected patients. In many of these conditions, production of reactive oxygen-species (ROS) is thought to be a key pathological mechanism. While the sources of ROS are complex (including for example mitochondria), there is increasing evidence that activation of NOX enzymes, in particular NOX3, plays a key role. NOX3 is a multi-subunit NADPH oxidase, functionally and structurally closely related to NOX1 and NOX2. In both the vestibular and the cochlear compartments of the inner ear, high levels of NOX3 mRNA are expressed. In NOX3 mutant mice, the vestibular function is perturbed due to a lack of otoconia, while only minor alterations of hearing have been documented. However, there is increasing evidence that activation of NOX3 through drugs, noise and probably also aging, leads to hearing loss. Thus, NOX3 is an interesting target to treat and prevent inner ear pathologies and a few first animal models based on drug - or molecular therapy have been reported. So far however, there are no specific NOX3 inhibitors with a documented penetration into the inner ear. Nevertheless, certain antioxidants and non-specific NOX inhibitors diminish hearing loss in animal models. Development of small molecules inhibitors or molecular strategies against NOX3 could improve specificity and efficiency of redox-targeted treatments. In this review, we will discuss arguments for the involvement of NOX3 in inner ear pathologies and therapeutic approaches to target NOX3 activity.

A prediction model of drug-induced ototoxicity developed by an optimal support vector machine (SVM) method.

Drug-induced ototoxicity, as a toxic side effect, is an important issue needed to be considered in drug discovery. Nevertheless, current experimental methods used to evaluate drug-induced ototoxicity are often time-consuming and expensive, indicating that they are not suitable for a large-scale evaluation of drug-induced ototoxicity in the early stage of drug discovery. We thus, in this investigation, established an effective computational prediction model of drug-induced ototoxicity using an optimal support vector machine (SVM) method, GA-CG-SVM. Three GA-CG-SVM models were developed based on three training sets containing agents bearing different risk levels of drug-induced ototoxicity. For comparison, models based on naïve Bayesian (NB) and recursive partitioning (RP) methods were also used on the same training sets. Among all the prediction models, the GA-CG-SVM model II showed the best performance, which offered prediction accuracies of 85.33% and 83.05% for two independent test sets, respectively. Overall, the good performance of the GA-CG-SVM model II indicates that it could be used for the prediction of drug-induced ototoxicity in the early stage of drug discovery.

Auditory and vestibular hair cell stereocilia: relationship between functionality and inner ear disease.

The stereocilia of the inner ear are unique cellular structures which correlate anatomically with distinct cochlear functions, including mechanoelectrical transduction, cochlear amplification, adaptation, frequency selectivity and tuning. Their function is impaired by inner ear stressors, by various types of hereditary deafness, syndromic hearing loss and inner ear disease (e.g. Ménière's disease). The anatomical and physiological characteristics of stereocilia are discussed in relation to inner ear malfunctions.

The performance of Cochlin-tomoprotein detection test in the diagnosis of perilymphatic fistula.

BACKGROUND: Perilymphatic fistula (PLF), defined as an abnormal communication between the inner and middle ear, presents with a symptomatology of hearing loss and vestibular disorder that is indistinguishable from a number of other inner ear diseases. Methods of diagnosis remain controversial. We have previously shown that Cochlin-tomoprotein (CTP) is selectively detected in the perilymph. To establish a definite diagnostic test for PLF using CTP as a biochemical marker, we examined the diagnostic performance of the CTP detection test. METHODS: CTP detection test was performed by Western blot using recombinant human CTP (rhCTP) as a spiked standard. We evaluated the specificity of the CTP detection test by testing non-PLF cases. To describe the limitations of the test, we tested samples from patients with middle ear infection. We also studied the stability of CTP protein by storing the samples at room temperature (25 degrees C) or 4 degrees C for 55 days. The effects of repeated freezing and thawing were also evaluated. Serially diluted perilymph was tested to find out the detection limit of CTP. FINDINGS: We have established a standardized CTP detection test using high (0.27 ng) and low (0.13 ng) spiked standards of rhCTP in Western blotting. Middle ear lavages (MEL) from 54 of 55 non-PLF cases were negative in the CTP detection test, i.e. the specificity of the test is 98.2%. MEL from 43 out of 46 cases with chronic suppurative otitis media or middle ear cholesteatoma were negative for CTP. CTP is a stable protein and detection was not affected by the storage, or freezing and thawing. The detection limit of perilymph was 0.161 microl/lane in an average of 5 samples. INTERPRETATION: CTP is a stable perilymph-specific protein, and this CTP detection could be the first clinically established diagnostic tool to detect PLF with a high specificity. PLF is surgically correctable by sealing the fistula. Appropriate recognition and treatment of PLF can improve hearing and balance in afflicted patients.

Potassium ion movement in the inner ear: insights from genetic disease and mouse models.

Sensory transduction in the cochlea and vestibular labyrinth depends on fluid movements that deflect the hair bundles of mechanosensitive hair cells. Mechanosensitive transducer channels at the tip of the hair cell stereocilia allow K(+) to flow into cells. This unusual process relies on ionic gradients unique to the inner ear. Linking genes to deafness in humans and mice has been instrumental in identifying the ion transport machinery important for hearing and balance. Morphological analysis is difficult in patients, but mouse models have helped to investigate phenotypes at different developmental time points. This review focuses on cellular ion transport mechanisms in the stria vascularis that generate the major electrochemical gradients for sensory transduction.

Cochlin-tomoprotein: a novel perilymph-specific protein and a potential marker for the diagnosis of perilymphatic fistula.

BACKGROUND: Perilymphatic fistula (PLF) is an abnormal connection between the inner and middle ear. A procedure for obtaining definite proof of a PLF remains elusive, and methods of diagnosis remain controversial. To date, there is no clinically relevant biochemical marker for perilymph leakage. Using proteomic analysis of inner ear proteins, we have previously found unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms (p63s, p44s and p40s) in the inner ear tissue and a short 16-kDa isoform of cochlin-tomoprotein (CTP) in the perilymph. Since cochlin was found to be highly specific to the inner ear, we speculated that CTP might also be specific to the perilymph. The aim of this study was to determine whether CTP, a novel perilymph-specific protein, could be used as a marker for the diagnosis of PLF. METHODS: By Western blotting, we investigated the specificity of CTP expression in a range of body fluids that included perilymph, serum, saliva and cerebrospinal fluid. To elucidate the detection limit of CTP, serially diluted recombinant human (rh)CTP as well as human perilymph was tested. RESULTS: CTP was selectively expressed in all 20 perilymph samples tested, but not in 77 samples of the other body fluids. The detection limit of rhCTP was 0.27 ng or 0.022 microl of perilymph per well on Western blot analysis. CONCLUSION: The results strongly suggest that CTP can be a specific marker of perilymph leakage. Moreover, CTP has the potential to be a biochemical marker that allows a definitive diagnosis of the etiology of PLF-related hearing loss and vestibular disorders.

The possible role of proton pump inhibitors of the homeostasis of the inner ear.

The possibility of a benefit in some cases of inner ear sufferance by using proton pump inhibitors has been considered after a casual observation. The hypothesis is advanced considering the adverse effect of reflux on the eustachian tube function, the possible influence of the latter on inner ear symptoms and, from a more general point of view, the trigger effect which a gastric dysfunction requiring proton pump inhibitors could exert on the sympathetic system. These considerations, deserving a further study, seem to be based on logical assessment and therefore in our opinion deserve to be kept in mind in trying to define inner ear disorders of uncertain origin.

Activating Fgfr3 Y367C mutation causes hearing loss and inner ear defect in a mouse model of chondrodysplasia.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal development and activating mutations in FGFR3 cause skeletal dysplasias, including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The introduction of the Y367C mutation corresponding to the human Y373C thanatophoric dysplasia type I (TDI) mutation into the mouse genome, resulted in dwarfism with a skeletal phenotype remarkably similar to that of human chondrodysplasia. To investigate the role of the activating Fgfr3 Y367C mutation in auditory function, the middle and inner ear of the heterozygous mutant Fgfr3(Y367C/+) mice were examined. The mutant Fgfr3(Y367C/+) mice exhibit fully penetrant deafness with a significantly elevated auditory brainstem response threshold for all frequencies tested. The inner ear defect is mainly associated with an increased number of pillar cells or modified supporting cells in the organ of Corti. Hearing loss in the Fgfr3(Y367C/+) mouse model demonstrates the crucial role of Fgfr3 in the development of the inner ear and provides novel insight on the biological consequences of FGFR3 mutations in chondrodysplasia.

Quantitative analysis of mRNA in human temporal bones.

CONCLUSION: Well-preserved mRNA could be extracted from frozen human inner ears. Therefore, this study demonstrates that analysis of mRNA could be performed to study the molecular mechanisms of inner ear disorders using human specimens. OBJECTIVES: Analysis of RNA as well DNA is requisite to study the molecular mechanisms of inner ear disorders. Methods of isolating RNA from experimental animals have been established, while isolation of RNA from human inner ears is much more challenging. In the present study, we demonstrate a method by which messenger RNA (mRNA) was extracted from human inner ears and quantitatively analyzed. MATERIALS AND METHODS: COCH mRNA as well as GAPDH mRNA was extracted from membranous labyrinths dissected from three formalin-fixed and three frozen human temporal bones, removed at autopsy. The length of COCH mRNA and quantity of GAPDH mRNA was compared between the two groups by quantitative RT-PCR. RESULTS: COCH mRNA could be amplified as much as 976 bp in all three frozen specimens. By contrast, it was amplified to 249 bp in two of the three formalin-fixed specimens, with no amplification observed in the remaining. The quantity of amplifiable GAPDH mRNA in the formalin specimens was only 1% of that of the frozen specimens.

Correlation between dizziness and impaired glucose metabolism.

INTRODUCTION: Impaired glucose metabolism is characterized by conditions of hypo and hyperglycemia. AIM: The objective of the present study was to asses whether or not there is a relationship between impaired glucose metabolism and dizziness. In the clinical laboratory settings, patients were examined using vectoelectronystagmography in association with glycemic levels. METHODS: 33 patients were divided in 3 groups: diabetics; patients with dizziness and a control group. RESULTS: 65% of the patients with dizziness showed impaired glucose metabolism. 40% of the patients with dizziness had alterations in their vectoelectronystagmography results. CONCLUSION: Dizziness is a good indicator of glucose metabolism alterations and these may be a good indicator of alterations in vectoelectronystagmography responses. The study of glycemic levels after glucose overexposure is a good prognosis factor to evaluate dizziness and shows the same results as insulin level studies after glucose overexposure.

[Expression of aquaporin-1 in a guinea pig model of labyrinth destruction].

OBJECTIVE: To study the expression of aquaporin-1(AQP-1) in the cochlea and endolymphatic sac in guinea pigs with labyrinth destruction. METHODS: Chloroform was injected into the tympanum to establish the animal model of labyrinth destruction in guinea pigs, and two-step immunohistochemical method was used to examine the expression of AQP-1 in the cochlea and endolymphatic sac at different time points. RESULTS: AQP-1 expression was fluctuant in accordance with the morphological changes of the spiral ligament fibrocytes in the cochlea: destruction of the spiral ligament cells was followed by AQP-1 expression down-regulation, and AQP-1 up-regulation occurred with the cell regeneration. But no such changes were observed in the endolymphatic sac. CONCLUSION: AQP-1 may take part in the maintenance of the structural stability of the spiral ligament.

Correlação entre tontura e disfunções do metabolismo da glicose / Correlation between dizziness and impaired glucose metabolism

INTRODUÇÃO: as alterações do metabolismo da glicose são caracterizadas por estados de hipoglicemia e hiperglicemia. OBJETIVO: A proposta deste trabalho é verificar a associação entre as alterações do metabolismo da glicose, por glicemia de jejum e teste de tolerância à glicose e à tontura, avaliada por sua queixa e exames clínicos e subsidiários. MÉTODO: O estudo foi efetivado num grupo de 33 pacientes divididos em 3 subgrupos: pacientes com queixa de tontura, pacientes diabéticos e pacientes assintomáticos. RESULTADOS: O grupo de pacientes com queixa espontânea ou questionada de tontura apresentava alterações no metabolismo da glicose em 65 por cento dos casos. Já entre os pacientes dos 3 grupos sem queixa de tontura, 30 por cento apresentavam alterações do metabolismo da glicose. 40 por cento dos pacientes que apresentaram queixas de tonturas tinham o exame vestibular clínico e a vectoeletronistagmografia alterados, enquanto que entre os assintomáticos 7,5 por cento apresentaram as alterações vestibulares referidas. CONCLUSÃO: A tontura é um bom indicador de alteração do metabolismo da glicose e a alteração do metabolismo da glicose é um bom indicador de alteração do exame vestibular. O estudo do metabolismo da glicose a partir dos níveis glicêmicos é eficaz e tem resultados próximos dos observados nos estudos que mensuram os níveis insulinêmicos.

INTRODUCTION: Impaired glucose metabolism is characterized by conditions of hypo and hyperglycemia. AIM: The objective of the present study was to asses whether or not there is a relationship between impaired glucose metabolism and dizziness. In the clinical laboratory settings, patients were examined using vectoelectronystagmography in association with glycemic levels. METHODS: 33 patients were divided in 3 groups: diabetics; patients with dizziness and a control group. RESULTS: 65 percent of the patients with dizziness showed impaired glucose metabolism. 40 percent of the patients with dizziness had alterations in their vectoelectronystagmography results. CONCLUSION: Dizziness is a good indicator of glucose metabolism alterations and these may be a good indicator of alterations in vectoelectronystagmography responses. The study of glycemic levels after glucose overexposure is a good prognosis factor to evaluate dizziness and shows the same results as insulin level studies after glucose overexposure.

Is there a role for atrial peptides in the labyrinthine "disease"?

The important role of atrial natriuretic peptides (ANP) in regulating blood pressure and changing vascular permeability has been widely studied and assessed during the last ten years. Considering the characteristics of this mechanism, which is responsible for a hypotensive and hypovolemic effect, and the possible role of hypotension associated with a default of autoregulatory sympathetic reactions in inner ear unexplained disorders, it seems reasonable to hypothesize a possible involvement of ANP system in the genesis of such disorders. As a matter of fact, the presence of specific receptors for ANP in the inner ear has been widely reported in studies concerning both rat and human inner ear, although their precise role in the labyrinthine homeostasis has not been satisfactory explained until now. Some aspects concerning vascular and fluid regulation of the inner ear under different conditions still remain not totally clear, and consequently a detailed explanation to the possible mechanism causing inner ear disorders of functional origin is lacking; from this point of view, an investigation on the serum level of ANP in subjects with labyrinthine affections of uncertain origin could be of some utility in contributing to assess the role of this system in the inner ear fluid regulation and in the inner ear perfusion and to investigate on the possible influence of an abnormal ANP release in some kind of inner ear damage.

[Apoptosis and apoptosis-related genes in experimental autoimmune inner ear disease].

OBJECTIVE: To investigate the protein and mRNA expression patterns of apoptosis-related genes, together with evidence of apoptosis, in relation to experimental autoimmune inner ear disease (AIED). METHODS: Male C57BL/6 mice at 4 weeks age (n = 80) were randomly assigned to one of the five group (n = 16). The inbred mice were given a single subcutaneous injection of diluted solution of pertussis and an emulsion containing equal parts of complete Freund adjuvant (CFA) and inner ear antigens (IEAg) extracted form guinea pig. The animals were sacrificed for inner ear examination at a defined time after the immunization (7, 14, 21 or 28 days). An autoimmune inner ear diseases model was established. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick end-laying (TUNEL) method. Using immunohistochemical techniques and reverse transcriptase polymerase chain reaction to clarify the profile of Fas, FasL, and bcl-2. RESULTS: Under normal conditions, no TUNEL-positive cell was observed in the cochlea except for a few positive cells in the supporting cells of Corti's organ and macula sacculi. Inner ear antigens administration induced TUNEL-positive reactions in a wide variety of cells such as inner hair cells, supporting cells, stria vascularis and spiral ligament fibrocytes. No positive staining was evident in outer hair cells, spiral ganglion cells and Scarpa's ganglion cells during the whole period. Fas proteins were expressed in a wide range of cells in inner ear. The levels of Fas mRNA were no significant differences between normal and AIED mice. FasL and bcl-2 proteins could be detected in spiral ganglion cells and Scarpa's ganglion cells both in normal and AIED mice. FasL positive cells increased in number in inner ear of AIED mice. bcl-2 positive cells were not detectable in inner hair cells, stria vascularis and spiral ligament both in normal and AIED mice. The mRNA of three kinds of apoptosis-related genes was detectable in the normal and AIED mice. FasL mRNA was expressed at low levels in normal, being maximal at 14 d post inoculation and decreased gradually to steady levels by 2 weeks. The levels of bcl-2 mRNA increased significantly during the period of AIED. CONCLUSION: Apoptosis mediated by Fas/FasL signal system may play a role in the initiation and maintenance of AIED. bcl-2 has a crucial role in the regulation of the process of apoptosis in the inner ear of AIED mice.

Mitochondrial ultrastructure and apoptotic protein expression in the vestibular nucleus complex following unilateral labyrinthectomy.

We hypothesized that peripheral vestibular disorders might affect mitochondria in the vestibular nucleus complex (VNC). We tested this using unilateral labyrinthectomy (UL) as a model for the effects of vestibular damage on the VNC and used Western blotting and electron microscopy to analyze mitochondria. In rats receiving UL we did not find any changes in mitochondrial ultrastructure in the medial vestibular nucleus following UL, and there was no change in the expression or activation of the apoptosis effector caspase-3 in the whole VNC following UL. However, we did detect a small but statistically significant upregulation of the anti-apoptotic protein Bcl-2 in the contralateral VNC at 10 h post-UL.

[The expression and significance of VIP and SP in the cochlea of rats].

OBJECTIVE: To investigate the expression of vasoactive intestinal peptide (VIP) and substance P (SP) in the cochlea of spontaneously hypertensive rat (SHR) and normal rat, and to evaluate the function of VIP and SP in the cochlea following the damage of hypertension. METHOD: The expression of VIP and SP in the cochlea of SHR was studied by immunohistochemical staining, and the mean optical density (OD) values of the positive fields were quantitatively examined by image analysis system. RESULT: The number of spiral ganglion cells at base turn in the hypertension group was significantly less than the normal (P < 0.01). VIP was expressed in the spiral ganglion cell plasm and stria vascularis of rats, so did SP. In spiral ganglion cell plasm the expression of VIP and SP had no significant difference between the two groups (P > 0.05); in stria vascularis the expression of VIP of the hypertension group was higher than the normal (P < 0.05). However, no significant difference in SP was found between the two groups. CONCLUSION: VIP not only contributes to regulate the cochlea microcirculation, but also acts as the neurotransmitter in the pathway of the auditory system. However, SP may be only a neurotransmitter in the pathway of the auditory system.