Inconsistent Skin Prick Tests for Allergy to Birch Homologous Trees May Result from Cross-Reacting Allergens or Technical Errors.

INTRODUCTION: Skin prick tests (SPTs) are the gold standard for diagnosis of allergic rhinitis (AR). A decrease in the number of allergens included in standard SPT panels has recently been debated - particularly regarding the cross-reacting homologous pollen from birch, alder, and hazel trees - but has not yet been implemented in clinical guidelines. METHODS: A subgroup of patients with AR (n = 69) who showed inconsistent SPT results among birch, alder, and hazel was investigated in detail. Beyond SPT, patient workup included assessment of clinical relevance and various serological parameters (total IgE, and specific IgE to birch, alder, and hazel and to Bet v 1, Bet v 2, and Bet v 4). RESULTS: More than half the study group had negative SPT results for birch but positive results for alder and/or hazel, and 87% of the study group was polysensitized, showing at least one more positive SPT result for other plants. Whereas 30.4% of patients showed serological sensitization to birch pollen extract, only 18.8% displayed positive specific IgE to Bet v 1. Clinical assessment revealed that most patients with AR were polysensitized and had perennial symptoms or symptoms also occurring during times other than tree flowering times. If the SPT panel is limited to testing birch only, 52.2% of patients in this subgroup would have been overlooked. CONCLUSION: Inconsistent SPT results in the birch homologous group may result from cross-reacting allergens or technical errors. If patients report convincing clinical symptoms despite negative results from a reduced SPT panel or inconsistent results for homologous allergens, SPT should be repeated, and molecular markers should be added to achieve a correct diagnosis.

Channelrhodopsin fluorescent tag replacement for clinical translation of optogenetic hearing restoration.

Sensory restoration by optogenetic neurostimulation provides a promising future alternative to current electrical stimulation approaches. So far, channelrhodopsins (ChRs) typically contain a C-terminal fluorescent protein (FP) tag for visualization that potentially poses an additional risk for clinical translation. Previous work indicated a reduction of optogenetic stimulation efficacy upon FP removal. Here, we further optimized the fast-gating, red-light-activated ChR f-Chrimson to achieve efficient optogenetic stimulation in the absence of the C-terminal FP. Upon FP removal, we observed a massive amplitude reduction of photocurrents in transfected cells in vitro and of optogenetically evoked activity of the adeno-associated virus (AAV) vector-transduced auditory nerve in mice in vivo. Increasing the AAV vector dose restored optogenetically evoked auditory nerve activity but was confounded by neural loss. Of various C-terminal modifications, we found the replacement of the FP by the Kir2.1 trafficking sequence (TSKir2.1) to best restore both photocurrents and optogenetically evoked auditory nerve activity with only mild neural loss few months after dosing. In conclusion, we consider f-Chrimson-TSKir2.1 to be a promising candidate for clinical translation of optogenetic neurostimulation such as by future optical cochlear implants.

Performance and self-perceived hearing impairment after cochlear implantation in Menière's disease.

OBJECTIVE: Evaluation of the self-perceived hearing impairment and performance after cochlear implantation in patients with definite Menière's disease (MD). PATIENTS AND METHODS: Seventeen unilaterally or bilaterally profoundly hearing-impaired patients suffering from MD who received a cochlear implantat (CI) were eligible for inclusion in this study. Their self-perceived hearing impairment using the short Speech Spatial and Qualities of Hearing Scale (SSQ12) as well as their performance in speech perception (German language Freiburger mono- and multisyllable test, Oldenburger sentence test) were compared with a best-matched control group of non-MD patients up to 24 months of follow-up. RESULTS: MD patients improved significantly in perception of monosyllables presented at 65 dBSPL, from preoperatively best aided 18.2% [2.4, 34.0] to 51.7% [39.4, 63.9] 1 year after cochlear implantation (mean [95% confidence interval]). Their performance approached the matched controls with 63.2% [55.7, 70.8]. Monosyllables presented at a lower intensity of 55 dBSPL revealed a significant underperformance of the MD patients (21.1% [12.6, 29.6]) in contrast to the non-MD controls (39.1% [30.9, 47.4]) 12 months post-CI. Self-assessed hearing disability was significantly more pronounced in MD patients with a mean total SSQ12 score of 3.6 [2.4, 4.9] in comparison to 6.1 [5.4, 6.8] of the matched non-MD controls after 12 months of cochlear implantation. CONCLUSION: Cochlear implantation substantially improves hearing capabilities in profoundly hearing-impaired patients with MD, but they tend to underperform in comparison to non-MD patients at least at lower sound pressure levels. This is likely one reason for the poorer self-assessed hearing function of cochlear implanted MD patients. LEVEL OF EVIDENCE: 3, retrospective, nonrandomized follow-up study.

[Optimized fitting of a midface implant to anchor a magnetic nasal prosthesis using 3D printing]. / 3-D-Druck-optimierte Anpassung eines Mittelgesichtsimplantats zur magnetgetragenen nasalen Epithesenversorgung.

BACKGROUND: Plate-based anchorage systems for craniofacial prostheses offer advantages over extraoral solitary titanium implants in terms of the flexible choice of mounting points and higher stability. Disadvantages become apparent in the complex individual intraoperative adaptation of the plate-based systems to the usually poorly accessible bone. The current article presents a method to overcome these disadvantages and make greater use of the advantages of plate-based systems. MATERIALS AND METHODS: The bony midface of a patient who had undergone rhinectomy for cancer of the nasal entrance was reconstructed as a virtual 3D model based on preoperative CT. The open-source software (3D-Slicer) allowed easy and fast reconstruction as well as adaptation for 3D printing using transparent plastic (MED610; stratasys Ltd., MN, USA). RESULTS: A titanium mini-plate (MEDICON) for anchoring the nasal prosthesis could be fitted extremely precisely on the midface 3D print. Important anatomical structures were spared, and screw placement was selected according to the individual bone thickness. Implantation of the in-advance fitted titanium plate was performed without complications and without further adjustments. CONCLUSION: In-advance fitting of plate-based systems for anchorage of craniofacial prostheses using 3D printing of the midface overcomes their disadvantages of time-consuming and possibly imprecise individual adaptation. This method further exploits the advantages of higher stability through more possible mounting points, even in thinner bone, to prevent loosening. In addition, in-advance fitting of titanium plates on the 3D model enables better identification and protection of important anatomical structures and shortens operative time.

Taste-strip gustometry in cochlear implanted patients.

OBJECTIVE: Investigation of the gustatory function in a large cohort of cochlear implanted patients using lateralized taste-strip tests. PATIENTS AND METHODS: One hundred and seven unilaterally or bilaterally profoundly hearing impaired or deaf patients who received cochlear implants (n = 113) were included in this study. Data on gustometry, subjective gustatory dysfunction, and the detailed surgical procedure were acquired retrospectively. Gustatory function, assessed using lateralized taste-strip tests, was performed the day before, 3 days after cochlear implantation, and on the day of the initial CI adjustment (39 days ±7.3 SD). RESULTS: Averaged taste-strip scores of the cohort declined significantly from preoperatively 12.3 [11.8; 12.7] (mean [95% confidence intervals]) to 10.5 [9.7; 11.2] on the implanted side about 6 weeks after surgery. Patients with intraoperatively exposed and rerouted, or a severed, chorda tympani nerve (CTN) showed significantly reduced unilateral postoperative scores (10.1 [8.8; 11.4] and 9.3 [8.1; 10.5], respectively), when compared to not exposing or to leaving a bony layer over the CTN. Total taste-strip test scores showed a significant decline 6 weeks postoperatively in CI-patients expressing a subjective gustatory dysfunction (from 23.6 [21.4; 25.8] to 17.5 [14.2; 20.8]), as opposed to patients with a documented subjectively normal taste. CONCLUSION: We consider postoperative gustatory dysfunction as a relevant side effect post cochlear implantation, at least within the first month. Taste-strip based gustometry is a suitable diagnostic tool to assess taste function in CI patients and is recommended to be performed routinely. LEVEL OF EVIDENCE: 3, retrospective, nonrandomized follow-up study.

Fast-track flipping: flipped classroom framework development with open-source H5P interactive tools.

BACKGROUND: The availability and popularity of laptops, tablet PCs and smartphones in private and work environments offers considerable potential for reasonably integrating blended learning formats into structured medical learning environments. The promising educational principle of the flipped classroom (FC) provides the opportunity to effectively combine e-learning and face-to-face teaching within a single framework. However, similar to most blended learning formats, the FC requires a solid groundwork of structured digitized learning content. As rearranging a whole curriculum is intense and time consuming, physicians occupied simultaneously in clinical practice and teaching may be confronted with a lack of time during this process. METHODS: We developed two straightforward approaches to transforming a pre-existing, lecture-based otolaryngology curriculum into interactive videos within a Moodle learning management system. Special attention was given to reducing individual working time for medical professionals. Thus, while one approach was mainly guided by a medical professional to control the content-related quality of video processing, we investigated an alternative approach outsourcing work to a technician. Afterwards, the working time was analysed and compared. The resulting videos were revised with the H5P plugin for moodle to adjust the content where necessary. RESULTS: We identified a fast-track approach for creating structured e-learning content suitable for flipped-classroom-based lectures, other blended learning formats, or even providing a whole curriculum online. The alternative approach significantly reduced working time for medical professionals but did not impair the content-related quality significantly. CONCLUSIONS: The use of H5P interactive tools via Moodle LMS provides a major procedural benefit by allowing the easy adjustment of pre-existing video material into suitable online content. Reasonably outsourcing work to technicians can significantly reduce the working time of medical professionals without decreasing the quality of learning content. The presented workflow can be used as a flexible approach for flipped classroom frameworks or other blended learning strategies where interactive videos are applicable.

Understanding and treating paediatric hearing impairment.

Sensorineural hearing impairment is the most frequent form of hearing impairment affecting 1-2 in 1000 newborns and another 1 in 1000 adolescents. More than 50% of congenital hearing impairment is of genetic origin and some forms of monogenic deafness are likely targets for future gene therapy. Good progress has been made in clinical phenotyping, genetic diagnostics, and counselling. Disease modelling, e.g. in transgenic mice, has helped elucidate disease mechanisms underlying genetic hearing impairment and informed clinical phenotyping in recent years. Clinical management of paediatric hearing impairment involves hearing aids, cochlear or brainstem implants, signal-to-noise improvement in educational settings, speech therapy, and sign language. Cochlear implants, for example, have much improved the situation of profoundly hearing impaired and deaf children. Nonetheless there remains a major unmet clinical need for improving hearing restoration. Preclinical studies promise that we will witness clinical trials on gene therapy and a next generation of cochlear implants during the coming decade. Moreover, progress in generating sensory hair cells and neurons from stem cells spurs disease modelling, drug screening, and regenerative approaches. This review briefly summarizes the pathophysiology of paediatric hearing impairment and provides an update on the current preclinical development of innovative approaches toward improved hearing restoration.

Access to the Apical Cochlear Modiolus for Possible Stem Cell-based and Gene Therapy of the Auditory Nerve.

OBJECTIVE: Loss of spiral ganglion neurons (SGN) is permanent and responsible for a substantial number of patients suffering from hearing impairment. It can derive from the degeneration of SGNs due to the death of sensory hair cells as well as from auditory neuropathy. Utilizing stem cells to recover lost SGNs increasingly emerges as a possible therapeutic option, but access to human SGNs is difficult due to their protected location within the bony impacted cochlea. Aim of this study was to establish a reliable and practicable approach to access SGNs in the human temporal bone for possible stem cell and gene therapies. METHODS: In seven human temporal bone specimen a transcanal approach was used to carefully drill a cochleostomy in the lateral second turn followed by insertion of a tungsten needle into the apical modiolus to indicate the spot for intramodiolar injections. Subsequent cone beam computed tomography (CBCT) served as evaluation for positioning of the marker and cochleostomy size. RESULTS: The apical modiolus could be exposed in all cases by a cochleostomy (1.6 mm2, standard deviation ±0.23 mm2) in the lateral second turn. 3D reconstructions and analysis of CBCT revealed reliable positioning of the marker in the apical modiolus, deviating on average 0.9 mm (standard deviation ±0.49 mm) from the targeted center of the second cochlear turn. CONCLUSION: We established a reliable, minimally invasive, transcanal surgical approach to the apical cochlear modiolus in the human temporal bone in foresight to stem cell-based and gene therapy of the auditory nerve.

Multichannel optogenetic stimulation of the auditory pathway using microfabricated LED cochlear implants in rodents.

When hearing fails, electrical cochlear implants (eCIs) provide the brain with auditory information. One important bottleneck of CIs is the poor spectral selectivity that results from the wide current spread from each of the electrode contacts. Optical CIs (oCIs) promise to make better use of the tonotopic order of spiral ganglion neurons (SGNs) inside the cochlea by spatially confined stimulation. Here, we established multichannel oCIs based on light-emitting diode (LED) arrays and used them for optical stimulation of channelrhodopsin (ChR)-expressing SGNs in rodents. Power-efficient blue LED chips were integrated onto microfabricated 15-µm-thin polyimide-based carriers comprising interconnecting lines to address individual LEDs by a stationary or mobile driver circuitry. We extensively characterized the optoelectronic, thermal, and mechanical properties of the oCIs and demonstrated stability over weeks in vitro. We then implanted the oCIs into ChR-expressing rats and gerbils, and characterized multichannel optogenetic SGN stimulation by electrophysiological and behavioral experiments. Improved spectral selectivity was directly demonstrated by recordings from the auditory midbrain. Long-term experiments in deafened ChR-expressing rats and in nontreated control animals demonstrated specificity of optogenetic stimulation. Behavioral studies on animals carrying a wireless oCI sound processor revealed auditory percepts. This study demonstrates hearing restoration with improved spectral selectivity by an LED-based multichannel oCI system.

Flipped classroom frameworks improve efficacy in undergraduate practical courses - a quasi-randomized pilot study in otorhinolaryngology.

BACKGROUND: Curriculum design and specific topic selection for on-site practical courses in clinical disciplines with limited teaching time is challenging. An electronic learning supported curriculum based on the flipped classroom principle has a high potential to effectively gain knowledge and education along with improving practical experience. Here, we demonstrate the introduction of a flipped classroom curriculum for practical courses in Otorhinolaryngology (ORL) in real world practice to improve the on-site time management and students' experience. METHODS: Educational aims of our practical curriculum were analysed and rearranged into a flipped classroom (FC) framework. Core knowledge was taught preliminary based on a moodle platform in predominantly interactive formats. Two quasi-randomized groups were formed with 212 participants either receiving or not receiving access to the e-learning program to reduce a potential allocation bias to the e-learning group. All students completed a questionnaire with learning related items. Focusing the study on the intervention group, we investigated if students using the flipped classroom more often felt better prepared for the practical course. RESULTS: The online learning platform was highly accepted and frequently used by 66% of participating students in the e-learning group. Students with frequent use of our e-learning platform significantly felt better prepared for the practical course (p = 0.001). The far majority of all students supports the idea of further development of e-learning. More than 70% were generally interested in ORL. Handouts were the overall most important learning resource and more than 50% relied solely on them. CONCLUSIONS: Flipped classroom curricula can save time and help improving the on-site experience in practical courses especially in smaller surgical disciplines. The acceptance of digital learning is high, and most students rely on handouts for learning ORL, emphasizing the need for guidance by the teacher e.g. through electronic learning. Our results underline the high potential of FC to address teaching challenges for smaller medical disciplines with limited teaching time like ORL.

Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos.

Optogenetic tools, providing non-invasive control over selected cells, have the potential to revolutionize sensory prostheses for humans. Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in cochlear implants. However, most channelrhodopsins do not support the high temporal fidelity pertinent to auditory coding because they require milliseconds to close after light-off. Here, we biophysically characterized the fast channelrhodopsin Chronos and revealed a deactivation time constant of less than a millisecond at body temperature. In order to enhance neural expression, we improved its trafficking to the plasma membrane (Chronos-ES/TS). Following efficient transduction of SGNs using early postnatal injection of the adeno-associated virus AAV-PHPB into the mouse cochlea, fiber-based optical stimulation elicited optical auditory brainstem responses (oABR) with minimal latencies of 1 ms, thresholds of 5 µJ and 100 µs per pulse, and sizable amplitudes even at 1,000 Hz of stimulation. Recordings from single SGNs demonstrated good temporal precision of light-evoked spiking. In conclusion, efficient virus-mediated expression of targeting-optimized Chronos-ES/TS achieves ultrafast optogenetic control of neurons.

Optogenetic stimulation of cochlear neurons activates the auditory pathway and restores auditory-driven behavior in deaf adult gerbils.

Cochlear implants partially restore hearing via direct electrical stimulation of spiral ganglion neurons (SGNs). However, spread of excitation from each electrode limits spectral coding. We explored the use of optogenetics to deliver spatially restricted and cell-specific excitation in the cochlea of adult Mongolian gerbils. Adeno-associated virus carrying the gene encoding the light-sensitive calcium translocating channelrhodopsin (CatCh) was injected into the cochlea of adult gerbils. SGNs in all cochlea turns showed stable and long-lasting CatCh expression, and electrophysiological recording from single SGNs showed that light stimulation up to few hundred Hertz induced neuronal firing. We characterized the light-induced activity in the auditory pathway by electrophysiological and behavioral analysis. Light- and sound-induced auditory brainstem responses showed similar kinetics and amplitude. In normal hearing adult gerbils, optical cochlear implants elicited stable optical auditory brainstem responses over a period of weeks. In normal hearing animals, light stimulation cued avoidance behavior that could be reproduced by subsequent acoustic stimulation, suggesting similar perception of light and acoustic stimuli. Neurons of the primary auditory cortex of normal hearing adult gerbils responded with changes in firing rates with increasing light intensity. In deaf adult gerbils, light stimulation generated auditory responses and cued avoidance behavior indicating partial restoration of auditory function. Our data show that optogenetic cochlear stimulation achieved good temporal fidelity with low light intensities in an adult rodent model, suggesting that optogenetics might be used to develop cochlear implants with improved restorative capabilities.

High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics.

Optogenetics revolutionizes basic research in neuroscience and cell biology and bears potential for medical applications. We develop mutants leading to a unifying concept for the construction of various channelrhodopsins with fast closing kinetics. Due to different absorption maxima these channelrhodopsins allow fast neural photoactivation over the whole range of the visible spectrum. We focus our functional analysis on the fast-switching, red light-activated Chrimson variants, because red light has lower light scattering and marginal phototoxicity in tissues. We show paradigmatically for neurons of the cerebral cortex and the auditory nerve that the fast Chrimson mutants enable neural stimulation with firing frequencies of several hundred Hz. They drive spiking at high rates and temporal fidelity with low thresholds for stimulus intensity and duration. Optical cochlear implants restore auditory nerve activity in deaf mice. This demonstrates that the mutants facilitate neuroscience research and future medical applications such as hearing restoration.

Analysis of SHOX2 methylation as an aid to cytology in lung cancer diagnosis.

BACKGROUND/AIM: The Epi proLung® BL Reflex Assay [short stature homeobox gene two methylation assay (SHOX2 assay)] (Epigenomics AG, Berlin, Germany) utilizes quantitative methylation-sensitive real-time polymerase chain reaction (QMSP) for the quantification of methylated short stature homeobox gene two (SHOX2) DNA. In the present study, the diagnostic utility of the SHOX2 assay was tested with regard to cytology for different cytological diagnostic categories to assess whether it can complement the cytological examination and the DNA methylation marker panel targeting the gene promoters of adenomatous polyposis coli 1A (APC), cyclin-dependent kinase inhibitor-2A (p16(INK4A)) and Ras association domain family protein 1 (RASSF1A) regarding lung cancer detection in bronchial aspirates. MATERIALS AND METHODS: Prospectively collected DNA from 169 patients (cytological diagnosis: 47 tumor-positive, 56 equivocal and 66 tumor-negative) was analyzed for SHOX2 DNA methylation utilizing QMSP. Patients were followed-up for a period of 11 months maximum. RESULTS: When equivocal diagnoses were categorized as tumor-positive, cytology and SHOX2 DNA methylation achieved 72% and 64% sensitivity and 63% and 98% specificity, respectively. SHOX2 DNA methylation identified 66% of the patients with cancer subsequent to a cytological equivocal diagnosis. SHOX2 complements the cytological diagnosis and the methylation marker panel. CONCLUSION: The assay could be of use for the improvement of diagnostic accuracy if applied subsequent to equivocal or negative cytology (sensitivity=69%, specificity=98%). Furthermore, the SHOX2 assay can complement a methylation-based marker panel.

Equivocal cytology in lung cancer diagnosis: improvement of diagnostic accuracy using adjuvant multicolor FISH, DNA-image cytometry, and quantitative promoter hypermethylation analysis.

BACKGROUND: Sometimes, cytological lung cancer diagnosis is challenging because equivocal diagnoses are common. To enhance diagnostic accuracy, fluorescent in situ hybridization (FISH), DNA-image cytometry, and quantitative promoter hypermethylation analysis have been proposed as adjuncts. METHODS: Bronchial washings and/or brushings or transbronchial fine-needle aspiration biopsies were prospectively collected from patients who were clinically suspected of having lung carcinoma. After routine cytological diagnosis, 70 consecutive specimens, each cytologically diagnosed as negative, equivocal, or positive for cancer cells, were investigated with adjuvant methods. Suspicious areas on the smears were restained with the LAVysion multicolor FISH probe set (Abbott Molecular, Des Plaines, Illinois) or according to the Feulgen Staining Method for DNA-image cytometry analysis. DNA was extracted from residual liquid material, and frequencies of aberrant methylation of APC, p16(INK4A) , and RASSF1A gene promoters were determined with quantitative methylation-specific polymerase chain reaction (QMSP) after bisulfite conversion. Clinical and histological follow-up according to a reference standard, defined in advance, were available for 198 of 210 patients. RESULTS: In the whole cohort, cytology, FISH, DNA-image cytometry, and QMSP achieved sensitivities of 83.7%, 78%, 79%, and 49.6%, respectively (specificities of 69.8%, 98.2%, 98.2%, and 98.4%, respectively). Subsequent to cytologically equivocal diagnoses, FISH, DNA-image cytometry, and QMSP definitely identified malignancy in 79%, 83%, and 49%, respectively. With QMSP, 4 of 22 cancer patients with cytologically negative diagnoses were correctly identified. CONCLUSIONS: Thus, adjuvant FISH or DNA-image cytometry in cytologically equivocal diagnoses improves diagnostic accuracy at comparable rates. Adjuvant QMSP in cytologically negative cases with persistent suspicion of lung cancer would enhance sensitivity.