Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound.

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l-lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.

Brain-Specific Angiogenesis Inhibitor 3 Is Expressed in the Cochlea and Is Necessary for Hearing Function in Mice.

Mammalian auditory hair cells transduce sound-evoked traveling waves in the cochlea into nerve stimuli, which are essential for hearing function. Pillar cells located between the inner and outer hair cells are involved in the formation of the tunnel of Corti, which incorporates outer-hair-cell-driven fluid oscillation and basilar membrane movement, leading to the fine-tuned frequency-specific perception of sounds by the inner hair cells. However, the detailed molecular mechanism underlying the development and maintenance of pillar cells remains to be elucidated. In this study, we examined the expression and function of brain-specific angiogenesis inhibitor 3 (Bai3), an adhesion G-protein-coupled receptor, in the cochlea. We found that Bai3 was expressed in hair cells in neonatal mice and pillar cells in adult mice, and, interestingly, Bai3 knockout mice revealed the abnormal formation of pillar cells, with the elevation of the hearing threshold in a frequency-dependent manner. Furthermore, old Bai3 knockout mice showed the degeneration of hair cells and spiral ganglion neurons in the basal turn. The results suggest that Bai3 plays a crucial role in the development and/or maintenance of pillar cells, which, in turn, are necessary for normal hearing function. Our results may contribute to understanding the mechanisms of hearing loss in human patients.

Potential Usefulness of Tracking Head Movement via a Wearable Device for Equilibrium Function Testing at Home.

Many studies have reported the use of wearable devices to acquire biological data for the diagnosis and treatment of various diseases. Balance dysfunction, however, is difficult to evaluate in real time because the equilibrium function is conventionally examined using a stabilometer installed on the ground. Here, we used a wearable accelerometer that measures head motion to evaluate balance and examined whether it performs comparably to a conventional stabilometer. We constructed a simplified physical head-feet model that simultaneously records "head" motion measured using an attached wearable accelerometer and center-of-gravity motion at the "feet", which is measured using an attached stabilometer. Total trajectory length (r = 0.818, p -false discovery rate [FDR] = 0.004) and outer peripheral area (r = 0.691, p -FDR = 0.026) values measured using the wearable device and stabilometer were significantly positively correlated. Root mean square area values were not significantly correlated with wearable device stabilometry but were comparable. These results indicate that wearable, widely available, non-medical devices may be used to assess balance outside the hospital setting, and new approaches for testing balance function should be considered.

Deficiency of large tumor suppressor kinase 1 causes congenital hearing loss associated with cochlear abnormalities in mice.

Mammalian auditory hair cells are not spontaneously replaced. Their number and coordinated polarization are fairly well-maintained and both these factors might be essential for the cochlear amplifier. Cell cycle regulation has critical roles in regulating appropriate cell size and cell number. However, little is known about the physiological roles of the Hippo pathway, which is one of the most important signaling cascades that regulates cell growth, differentiation, and regenerative capacity in the cochlear sensory epithelium. Herein, we investigated the in vivo role of the large tumor suppressor 1 (LATS1), an essential kinase in the Hippo/yes-associated protein pathway, in the cochlea using the LATS1 knockout mice. LATS1 was expressed in hair cells and supporting cells. It was strongly expressed on the surface of the cuticular plate of the organ of Corti. We found that LATS1 knockout caused congenital hearing loss due to the irregular orientation and slightly reduced number of hair cells, whereas the number of supporting cells remained unchanged. On the surface of the hair cells, the kinocilium and stereocilia were dispersed during and after morphogenesis. However, the expression of the receptor-independent polarity regulators, such as Par3 or Gαi, was not affected. We concluded that LATS1 has an indispensable role in the maturation of mammalian auditory hair cells, but not in the development of the supporting cells, and thus, has a role in the hearing acquisition.

Three cases of otitis media caused by Mycobacterium abscessus subsp. abscessus: Importance of medical treatment and efficacy of surgery.

This study aimed to assess the clinical presentation, antibiotic therapy, surgery, and outcomes in patients with otitis media caused by Mycobacterium abscessus subsp. abscessus and discuss the efficacy of surgery. This is a retrospective case review of three patients diagnosed with otomastoiditis caused by M. abscessus subsp. abscessus. All patients had refractory otorrhea. One patient had granulation tissue in the tympanic membrane. They received medical treatment and underwent surgery. Otorrhea was resolved several months after the initiation of long-term multiantibiotic therapy in all cases. The timing of surgery varied among patients. Before initiating antibiotic therapy, mastoidectomy was performed to achieve definitive diagnosis in two patients, and wound dehiscence developed in these patients. Two patients underwent debridement after the initiation of multiantibiotic therapy. After antibiotic administration, tympanoplasty was performed to improve hearing in one patient. All patients achieved culture negativity after treatment, and no recurrences have been noted. From three cases, it is suggested that the mainstay of treatment for M. abscessus subsp. abscessus is long-term multiantibiotic therapy, and surgery itself may have little effect on achieving ear dryness. Thus, in most patients, drug therapy should be prioritized. Considering postoperative complications, surgery before achieving ear dryness should be avoided, except in emergency cases. In addition, if the diagnosis is not confirmed by repeated bacteriological tests, mastoidectomy should be performed to collect specimens. Tympanoplasty for hearing loss or eardrum perforation is recommended after discontinuation of medications.

Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin.

Tissue homeostasis demands regulatory feedback, suggesting that hematopoietic stem cell (HSC) activity is controlled in part by HSC progeny. Yet, cell extrinsic HSC regulation has been well characterized only in niche cells of non-hematopoietic origin. Here we identify feedback regulation of HSCs by megakaryocytes (Mks), which are mature hematopoietic cells, through production of thrombopoietin (Thpo), a cytokine pertinent for HSC maintenance. Induced ablation of Mk cell population in mice perturbed quiescent HSCs in bone marrow (BM). The ablation of Mks resulted in decreased intra-BM Thpo concentration presumably due to Thpo production by Mks. Thpo administration Mk ablated mice restored HSC functions. Overall, our study establishes Mk as an essential cellular component of the HSC niche and delineates cytokine-oriented regulation of HSCs by their own progeny.

[Long-term effects of tinnitus retraining therapy involving monaural noise generators].

We have previously reported on the effects of tinnitus retraining therapy (TRT) involving monaural noise generators (NGs) up to 24 months after the start of treatment (Eur Arch Otorhinolaryngol. 2013 Feb; 270(2) : 443-8.) but very few reports exist about the long-term effects of TRT for periods of over 2 years. The aim of this study was to report the effects of TRT involving monaural NGs more than 24 months after the start of treatment. Thirty-three patients with chronic tinnitus were included in this study. All received directive counseling and monaural NGs without any other combination treatment. Effects were evaluated with the Tinnitus Handicap Inventory (THI) at their final visits to our clinic (average 31 months after the start of treatment). The average THI scores significantly improved from 55.3 +/- 19.7 at baseline to 33.5 +/- 23.3 at their final visits. Seventeen patients (52%) improved by more than 20 points from the baseline. Eleven patients who were treated with TRT for more than 3 years were individually observed in a detailed manner. Some of them experienced aggravation of their symptoms after 2 years' successful treatments. This study suggests that, although TRT seems effective more than 2 years after the start of treatment, the clinical course of each patient can vary and we need to follow them periodically depending on their situations and symptoms.

Generation of an induced pluripotent stem cell line (KEIUi008-A) from a hearing loss patient with an A1555G mutation in mitochondrial DNA.

We report the establishment of a human induced pluripotent stem cell (iPSC) line from a 54-year-old male patient with an A1555G mutation in the mitochondrial 12S ribosomal RNA gene (MTRNR1), associated with sensorineural hearing loss. The established iPSC line expressed stemness markers or undifferentiated state markers. We also demonstrated the capacity of the cells to differentiate into the three germ layers, suggesting its pluripotency and utility in the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Generation of four induced pluripotent stem cell lines (KEIUi004-A, KEIUi005-A, KEIUi006-A, and KEIUi007-A) from patients with sensorineural hearing loss with mutation in EYA4 gene.

Disease-related cells differentiated from patient-derived iPSCs are useful for elucidating the pathophysiological mechanisms underlying these diseases. In this study, four iPSC lines were established from independent patients with sensorineural hearing loss and a mutation in EYA4. These iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes, suggesting that these lines are useful for the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer.

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

Phase I/II Study of a Vascular Endothelial Growth Factor Receptor Vaccine in Patients With NF2-Related Schwannomatosis.

PURPOSE: The humanized antivascular endothelial growth factor (VEGF) antibody bevacizumab (Bev) is efficacious for the treatment of NF2-related schwannomatosis (NF2), previously known as neurofibromatosis type 2. This study evaluated the safety and efficacy of a VEGF receptor (VEGFR) vaccine containing VEGFR1 and VEGFR2 peptides in patients with NF2 with progressive schwannomas (jRCTs031180184). MATERIALS AND METHODS: VEGFR1 and VEGFR2 peptides were injected subcutaneously into infra-axillary and inguinal regions, once a week for 4 weeks and then once a month for 4 months. The primary end point was safety. Secondary end points included tolerability, hearing response, imaging response, and immunologic response. RESULTS: Sixteen patients with NF2 with progressive schwannomas completed treatment and were assessed. No severe vaccine-related adverse events occurred. Among the 13 patients with assessable hearing, word recognition score improved in five patients at 6 months and two at 12 months. Progression of average hearing level of pure tone was 0.168 dB/mo during the year of treatment period, whereas long-term progression was 0.364 dB/mo. Among all 16 patients, a partial response was observed in more than one schwannoma in four (including one in which Bev had not been effective), minor response in 5, and stable disease in 4. Both VEGFR1-specific and VEGFR2-specific cytotoxic T lymphocytes (CTLs) were induced in 11 patients. Two years after vaccination, a radiologic response was achieved in nine of 20 assessable schwannomas. CONCLUSION: This study demonstrated the safety and preliminary efficacy of VEGFR peptide vaccination in patients with NF2. Memory-induced CTLs after VEGFR vaccination may persistently suppress tumor progression.

[Current status and perspectives of the research in Pendred syndrome].

Pendred syndrome is an autosomal recessive disorder characterized by sensorineural hearing loss, goiter, and a partial defect in iodide organification, and is the most common syndromic hearing loss. Hearing loss is congenital in most cases and is accompanied by an enlarged vestibular aqueduct and a Mondini cochlea. Pendred syndrome and autosomal recessive deafness-4 (DFNB4) with enlarged vestibular aqueduct comprise a phenotypic spectrum caused by mutations in SLC26A4. Recently, mutations in FOXI1 and KCNJ10 have also been identified in DFNB4. Molecular mechanism of hearing loss and goiter remains to be elucidated, and therapies which can reverse or prevent the progression of the symptoms are not available. Here, we describe advances in the basic, clinical, and translational studies on Pendred syndrome.

Hydroxyapatite Prostheses in Endoscopic Transcanal Stapes Surgery for Otosclerosis Cases.

OBJECTIVES: Hydroxyapatite is a commonly used material for medical applications due to its excellent biocompatibility. We use hydroxyapatite prosthesis for the reconstruction of the ossicular chain in stapes surgery. In this study, we report a case series of endoscopic ear surgery using a basket-type hydroxyapatite prosthesis. METHODS: We retrospectively examined 8 cases of endoscopic transcanal stapes surgery using hydroxyapatite prostheses. We evaluated the postoperative results and complications. RESULTS: The average postoperative air-bone gaps were within 10 dB in all cases. Postoperative sensorineural hearing loss was not observed in any case. There was an intraoperative complication with the chorda tympani in 1 patient. We were able to preserve the chorda tympani of all patients, including this case. Postoperative transient dizziness and transient taste disorder were observed in 50% of cases. No other complications, including facial nerve palsy, tympanic membrane perforation, or postoperative infection, were observed. CONCLUSIONS: The postoperative results and complications were comparable to those of surgery under a microscope. The hydroxyapatite prosthesis could be a possible alternative for the piston-type titanium or polytetrafluoroethylene prosthesis.

Mapping of Notch signaling in the developing organ of Corti in common marmosets.

Introduction: The well-regulated development of the sensory epithelium is essential for hearing. This process involves the specification of a pro-sensory epithelium containing common progenitors that differentiate into hair and supporting cells. Notch signaling is one of the most critical pathways during these processes, and its modification is thought to be a feasible approach for treating hearing loss. Despite interspecies differences between rodents and primates or humans, most of our current knowledge regarding cochlear development has been obtained from rodent models. Methods: We therefore examined and mapped the expression patterns of Notch signal components in the developing cochlea of the common marmoset (Callithrix jacchus), a small monkey species native to the New World, a primate model animal. Results: In contrast to the preserved expression patterns of the Notch signaling components in the hair cell differentiation between primates and rodents, we unveiled relatively large interspecies differences during the maturation of supporting cells. Discussion: This improved knowledge of Notch signaling during primate cochlear development will facilitate the development of future regenerative therapies.

Apoptosis of type I spiral ganglion neuron cells in Otof-mutant mice.

Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otoftm1a(KOMP)Wtsi allele (Otoftm1a) and analyzed spiral ganglion neurons (SGNs) in Otoftm1a/tm1a mice by immunolabeling type Ⅰ SGNs (SGN-Ⅰ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otoftm1a/tm1a mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otoftm1a/tm1a mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otoftm1a/tm1a mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otoftm1a/tm1a mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otoftm1a/tm1a mice showed a reduction in SGNs accompanied by apoptosis of SGN-Ⅰs even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.

Generation of two induced pluripotent stem cell lines from individuals without auditory disorders.

We report the establishment of two human induced pluripotent stem cell (iPSC) lines from individuals without auditory disorders. Extensive audiometry tests were performed to confirm normal hearing. The generated iPSC lines expressed pluripotency genes and showed differentiation capability into the three germ layers. The iPSC lines will be used as controls for pathological analysis and drug screening for ear disorders.

Correlation between genotype and phenotype with special attention to hearing in 14 Japanese cases of NF2-related schwannomatosis.

NF2-related schwannomatosis (NF2) is an autosomal dominant genetic disorder caused by variants in the NF2 gene. Approximately 50% of NF2 patients inherit pathogenic variants, and the remainder acquire de novo variants. NF2 is characterized by development of bilateral vestibular schwannomas. The genetic background of Japanese NF2 cases has not been fully investigated, and the present report performed a genetic analysis of 14 Japanese NF2 cases and examined genotype-phenotype correlations. DNA samples collected from peripheral blood were analyzed by next-generation sequencing, multiplex ligation-dependent probe amplification analysis, and in vitro electrophoresis. Ten cases had pathogenic or likely pathogenic variants in the NF2 gene, with seven truncating variants and three non-truncating variants. The age of onset in all seven cases with truncating variants was < 20 years. The age of onset significantly differed among cases with truncating NF2 variants, non-truncating NF2 variants, and no NF2 variants. However, the clinical course of tumor growth and hearing deterioration were not predicted only by germline pathogenic NF2 variants. The rate of truncating variants was higher in the present study than that of previous reports. Genotype-phenotype correlations in the age of onset were present in the analyzed Japanese NF2 cases.

Notch signaling alters sensory or neuronal cell fate specification of inner ear stem cells.

Multipotent progenitor cells in the otic placode give rise to the specialized cell types of the inner ear, including neurons, supporting cells, and hair cells. The mechanisms governing acquisition of specific fates by the cells that form the cochleovestibular organs remain poorly characterized. Here we show that whereas blocking Notch signaling with a γ-secretase inhibitor increased the conversion of inner ear stem cells to hair cells by a mechanism that involved the upregulation of bHLH transcription factor, Math1 (mouse Atoh1), differentiation to a neuronal lineage was increased by expression of the Notch intracellular domain. The shift to a neuronal lineage could be attributed in part to continued cell proliferation in cells that did not undergo sensory cell differentiation due to the high Notch signaling, but also involved upregulation of Ngn1. The Notch intracellular domain influenced Ngn1 indirectly by upregulation of Sox2, a transcription factor expressed in many neural progenitor cells, and directly by an interaction with an RBP-J binding site in the Ngn1 promoter/enhancer. The induction of Ngn1 was blocked partially by mutation of the RBP-J site and nearly completely when the mutation was combined with inhibition of Sox2 expression. Thus, Notch signaling had a significant role in the fate specification of neurons and hair cells from inner ear stem cells, and decisions about cell fate were mediated in part by a differential effect of combinatorial signaling by Notch and Sox2 on the expression of bHLH transcription factors.

Pranlukast dry syrup inhibits symptoms of Japanese cedar pollinosis in children using OHIO Chamber.

Pranlukast (PLK) is a leukotriene receptor antagonist (LTRA) that has been approved for treatment of asthma in patients of all ages and allergic rhinitis (AR) in adults but not for AR in children in Japan. This randomized, double-blind, placebo-controlled, crossover study used an artificial exposure chamber (OHIO Chamber) to investigate the efficacy and safety of PLK in children from 10 to 15 years old with seasonal AR (SAR) due to Japanese cedar (JC) pollen. Eighty-four subjects were enrolled and randomized to the treatment arm and 74 were included in the per protocol set. Subjects received either PLK dry syrup (DS) or placebo for 1 week. They were challenged with JC pollen in the OHIO Chamber for 3 hours. Total nasal symptom scores (TNSSs) were recorded every 30 minutes during the exposure. PLK DS treatment suppressed the TNSS changes from baseline significantly when compared with placebo. The difference in the least square means in TNSS between the PLK DS-treated group and placebo group was -0.37 (95% CI, -0.54, -0.20) with a value of p < 0.0001, showing that PLK DS significantly suppressed the nasal symptoms. Regarding specific nasal symptoms, PLK DS significantly suppressed sneezing, nasal discharge, and nasal obstruction. The effect of PLK DS on nasal obstruction was most prominent, with significant improvement relative to placebo beginning 60 minutes after the start of exposure. No serious adverse events were reported during the study. In this study, PLK DS is effective and safe for treatment in children with SAR.

Noninvasive biological evaluation of response to pranlukast treatment in pediatric patients with Japanese cedar pollinosis.

Pranlukast (PLK) is a cysteinyl leukotriene receptor 1 antagonist approved for the treatment of bronchial asthma and allergic rhinitis in Japan. We previously reported that PLK dry syrup (DS) improved the total nasal symptom score, as well as sneezing, nasal discharge, and nasal obstruction scores over placebo. We investigated the efficacy of PLK DS with a noninvasive method in 10- to 15-year-old children with Japanese cedar (JC) pollinosis challenged with pollen allergen using an artificial exposure chamber (OHIO Chamber). Levels of eosinophil cationic protein (ECP) in nasal secretions, nasal obstruction score, and the relationship with nasal obstruction scores were analyzed. The estimated difference of means in ECP levels (PLK DS--placebo) was -22.9 micrograms (95% CI, -45.2 to -0.5), suggesting PLK DS reduced ECP significantly when compared with placebo (p = 0.0454). The difference in the least square means for nasal obstruction between the PLK DS and placebo was -0.25 (95% CI, -0.36 to -0.14) with a value of p < 0.0001. In addition, a statistically significant, although weak, positive correlation between the nasal obstruction score and nasal ECP levels was observed with placebo treatment (correlation coefficient = 0.2394; p = 0.0428). Moreover, the inhibition rate of nasal ECP with PLK DS relative to placebo was statistically significant, although weak, positively correlated with the inhibition rate of nasal obstruction (correlation coefficient = 0.3373; p = 0.0219). PLK DS significantly decreases nasal ECP levels and nasal obstruction score compared with placebo in children with JC pollinosis challenged with pollen allergen. Suppression of mucosal eosinophilic inflammation is one of the pathways by which PLK DS improves pollinosis-induced nasal obstruction.