The ototoxic effects of radiotherapy have been poorly characterized. We examined adult survivors of childhood cancer who were treated with radiotherapy, which included the head, before the age of 22 years and between 1952 and 2016. Those who received platinum chemotherapy were excluded. Demographic, diagnosis, and treatment outcomes were captured. Audiograms were graded using the Chang and International Society of Paediatric Oncology ototoxicity (SIOP) scales. Among 276 patients with a history of radiation to sites that included the brain, orbit, nasopharynx, and total body irradiation, the median age at treatment was 10.1 years and 59% were male. Of 51 survivors who had post-treatment audiograms, 19 demonstrated severe hearing impairment according to both the Chang and SIOP scales after a median follow-up of 16.6 years. Of those with severe impairment, 10 were using hearing aids. Among the 23 patients with more than one audiogram, five had normal hearing on the first audiogram but hearing loss upon subsequent study. Ototoxic effects of radiotherapy are present in a significant portion of survivors, but impairment may present over time, and our results suggest that many are not being screened. Further, among patients with severe hearing loss, use of hearing aids is not universal. Expansion of access to audiology testing and hearing interventions may be warranted. While cancer mortality has declined in recent decades, late effects of treatment impact the long-term health and quality of life for cancer survivors ,2. HeariRadiotherapy-induced hearing loss can present as conductive, sensorineural or mixed hearing loss ,5,6. TheThe Adult Long-Term Follow-Up (ALTFU) program at Memorial Sloan Kettering Cancer Center provides risk-based care to adult survivors of childhood and young adult cancer. A retrospective chart review was conducted for patients seen in the ALTFU Program between September 2005 and January 2017. Memorial Sloan Kettering Institutional Review Board approval was obtained for this research (#16-1523); because of the retrospective nature, the requirement for consent was waived. Eligibility criteria included at least one clinical visit at ALTFU, cancer diagnosis before 22 years of age, and RT to fields that included the cochlea or ear. Radiotherapy fields included the brain, posterior fossa, craniospinal, nasopharynx, skull, face, orbit, total body irradiation, sinus, and the pineal gland. Exclusion criteria included receipt of platinum chemotherapy. Aminoglycoside and occupational exposures were noted when annotated in the audiology report.We collected information on degree of hearing impairment and use of hearing aids. The RT field and dose were also abstracted. Consistent with the Children’s Oncology Group guidelines for ototoxicity evaluation, we defined low dose RT as <30 Grey (Gy) and high dose RT as ≥30 Gy ,19.Two authors evaluated pure tone audiograms performed post-treatment. Bone conduction thresholds were used to determine the presence of sensorineural hearing loss when patients demonstrated mixed or conductive hearing loss. In audiograms with normal tympanograms, air conduction thresholds were used where bone conduction was unavailable. The audiologic evaluation included audiologic history, air and bone conduction thresholds (250–8000 Hz and 250–4000 Hz respectively), speech audiometry, immittance audiometry, and distortion product otoacoustic emissions when applicable. A mixed or conductive hearing loss was designated by an air/bone gap greater or equal to 15 dB and/or flat tympanograms. When a mixed hearing loss was observed, bone conduction thresholds were measured to evaluate sensorineural loss.Audiometric data were graded according to both the Chang Ototoxicity Scale  and the We found 276 survivors with a history of RT and without a history of platinum-based chemotherapy who were seen in the ALTFU program. Approximately one in five  underwent post-treatment audiology testing at a median of 3.0 years  after the end of treatment. Patients with follow-up audiograms had a younger median age at diagnosis, greater proportion of CNS tumor diagnoses, more equitable gender distribution, greater proportion of hearing aid use, and greater proportion with a history of cranial surgery, compared to those without audiogram . Median Among the 51 patients with a history of RT who did not receive platinum chemotherapy but had follow-up audiogram, one had Fanconi’s anemia, one was diagnosed with neurofibromatosis 2 and had associated schwannoma, and one had congenital choleastoma and ear surgery. Aminoglycoside treatment was noted in audiology reports and progress notes for three patients; however, many patients treated for infections and transplant treatment may have also received aminoglycoside treatment.Among these 51 patients, 19 were found to have severe hearing impairment. Survivors with hearing impairment had a median age at diagnosis of 8.0 years , and primarily included leukemia and CNS tumor survivors. Cumulative radiation dose exceeded 30 Gy for 13 patients. However, six patients had RT doses less than 30 Gy, a threshold below which patients are not typically screened for hearing loss. Six patients received RT to more than one field. The cumulative RT dose range was from 1350–10,800 Gy.International Society of Paediatric Oncology and Chang ototoxicity grading scales were used to grade 119 post-treatment audiograms. A single audiologist performed 108 audiology evaluations at Memorial Sloan Kettering; 11 audiology evaluations were performed at outside institutions. On first post-treatment audiogram, 27/51 (53%) of patients had hearing within normal limits. Amongst the total of 119 audiograms collected, sensorineural hearing loss was observed on 64 (54%), hearing within normal limits was seen on 29 (24%) of the audiograms, and the remaining showed mixed loss in 15 (13%) and conductive hearing loss in 11 (9%).Fifteen of 51 patients exhibited hearing thresholds consistent with both Chang grade 2b or above and SIOP grade 3 or above on their first post-treatment audiogram. Within this group, four patients had characteristics noted on their audiology report that may affect auditory outcomes: one developed severe otitis media secondary to treatment; one developed acoustic neuroma after treatment; one received RT for an acoustic neuroma; and one received RT for a pediatric and adult cancer.Changes in ototoxicity between post-treatment audiograms were evaluated for 23 patients with more than one audiogram, starting a median of two years after treatment . Nine of 23 (39%) patients demonstrated severe hearing impairment on their first post-treatment audiogram, while an additional five patients (22%) demonstrated severe hearing impairment upon subsequent testing. Therefore, severe hearing impairment emerged over the course of follow-up for a substantial proportion of patients.Importantly, of 19 patients with a history of RT and severe hearing impairment, only 10 wear hearing aids. All 10 patients wearing hearing aids also demonstrated high-grade ototoxicity on audiologic evaluation. Reasons for not using hearing aids, in spite of eligibility, were not uniformly noted in the chart but included cost and discomfort.In this evaluation of adult survivors of childhood and young adult cancer exposed to ototoxic therapy, 19 of 51 patients with a history of RT, but without a history of treatment with platinum-based chemotherapy, were found to have severe hearing impairment. Amongst 19 patients with severe impairment, 15 presented with severe impairment on their first post-treatment audiogram, and four had developed severe impairment by the time of subsequent audiograms. Of those with severe impairment, 10 patients used hearing aids.Our findings are consistent with those of past studies of survivors of childhood and young adult cancers, suggesting that RT-induced ototoxicity may present several years later than hearing loss secondary to platinum therapy, and that many survivors do not have regular audiograms ,7,16. FuOur work expands upon what has been demonstrated with regards to RT and hearing loss. Prior studies have shown that RT doses to the cochlea greater than 32 Gy are ototoxic when present with CSF shunts, while RT doses greater than 40–45 Gy are independently ototoxic ,12. The Audiologic testing can detect hearing loss that may not be detected subjectively by patients or clinically by health care providers ,5, whileOur study has several limitations. Only patients seen in the ALTFU survivorship program were included, and a small sample of that population had post-treatment audiograms. Post-treatment audiograms that were performed outside the institution may not have been captured. In addition, the reasons for referral to audiology were not always noted. The incidence of aminoglycoside exposure and otitis media was not consistently documented, and therefore, the effects of these comorbidities are unknown. Treatment dates ranged from 1952 to 2016, and for many patients in this study, cochlear dose was unavailable at the time of their treatment. However, all radiation fields where the cochlea may have been inadvertently targeted were included. Further, in 47 of 51 patients with follow-up, there were no indications in clinical notes or audiology reports that hearing loss preceded treatment. Pre-treatment hearing loss may have been present due to congenital choleastoma in two patients, pre-radiation ear surgery in one patient, and acoustic neuroma associated with neurofibromatosis in another patient. Even so, we have included these results in our findings, as these types of patients may be seen in the survivorship setting. Further, we presume in our analysis that normal tympanograms can exclude conductive hearing loss due to middle ear fluid and negative pressure, but these findings do not preclude ossicular conductive hearing loss. Thus, there may be a greater component of conductive hearing loss than identified using tympanograms. Finally, intervals at which audiometric tests were performed were inconsistent and duration of follow-up varied. Thus, it is possible that hearing loss may have been detected earlier if more routine testing was performed.In contrast to treatment related hearing loss in adult survivors of childhood cancer, aging and noise exposure are the most common causes of hearing loss in the general population. No noise exposures were documented in clinical reports for the patient population. Additionally, all patients but one received their first post-treatment audiogram between ages 4 and 35 years, and 18 of 19 patients who presented with severe hearing loss did so before 40 years of age. Therefore, we conclude that age-related sensorineural hearing loss was not a factor for 18 of the 19 patients with severe impairment.Ten patients in our study population used hearing aids, and all of these patients exhibited severe impairment on audiogram; other patients with severe impairment on audiogram may also benefit from hearing aids, although an audiological consultation may be needed to make that determination . BarrierHearing loss presents unique challenges to adult survivors of childhood cancer. Hearing loss has been associated with decreased academic performance, lower prevalence of independent living and employment, and decreased psychosocial outcomes in cancer survivors during critical adolescent and young adult years ,26,27. TIn conclusion, our study supports the use of audiologic testing in long-term follow-up for patients with CNS and non-CNS tumors who received RT to fields involving the cochlea. We support the guidelines for audiologic monitoring after high-dose RT, but suggest that routine monitoring may be warranted at RT doses less than 30 Gy and at more routine intervals. Additionally, in light of the social consequences of impaired hearing, further research is needed to determine outcomes for hearing impaired patients and whether those who may benefit from hearing aids have access to them.


Bacillus thuringiensis and Pseudomonasentomophila in the red flour beetle Tribolium castaneum, which was previously allowed to coevolve with the generalist entomopathogenic fungus Beauveria bassiana. We combine survival and gene expression assays upon infection to test for cross-resistance and underlying mechanisms. We show that larvae of T. castaneum that evolved with B. bassiana under coevolutionary conditions were positively cross-resistant to the bacterium B. thuringiensis, but not P. entomophila. Positive cross-resistance was mirrored at the gene expression level with markers that were representative of the oral route of infection being upregulated upon B. bassiana exposure. We find that positive cross-resistance towards B. thuringiensis evolved in T. castaneum as a consequence of its coevolutionary interactions with B. bassiana. This cross-resistance appears to be a consequence of resistance to oral toxicity. The fact that coevolution with B. bassiana results in resistance to B. thuringiensis, but not P. entomophila implies that B. thuringiensis and B. bassiana may share mechanisms of infection or toxicity not shared by P. entomophila. This supports previous suggestions that B. bassiana may possess Cry-like toxins, similar to those found in B. thuringiensis, which allow it to infect orally.Host-parasite coevolution can influence interactions of the host and parasite with the wider ecological community. One way that this may manifest is in cross-resistance towards other parasites, which has been observed to occur in some host-parasite evolution experiments. In this paper, we test for cross-resistance towards  Drosophila melanogaster evolved with the parasitoid Asobara tabida displayed reduced competitive ability under high competition [In nature, hosts are likely to exist within a complex community and interact with multiple parasites ,2,3. Clopetition . Howeverpetition .Drosophila melanogaster with the bacterium Pseudomonas entomophila (A) results in the flies being resistant to P. putida (B). Alternatively, since mounting an immune response and developing resistance can come with associated life-history costs [Drosophila C virus (DCV)) as compared to their control counterparts. It was proposed that the higher survival of flies evolved to P. entomophila upon infection with P. putida comes at a cost which is manifested in the form of hyper-susceptibility to viral infections (C) [D. melanogaster was allowed to adapt to DCV for 20 generations, a genome-wide analysis of the evolved flies revealed that their cross-resistance to the cricket paralysis virus (CrPV) and the FHV are mediated by the gene Pastrel and two other loci, namely Ubc-E2H and CG8492 [Regiella insecticola increases host resistant to fungal parasites Pandora aphididis [Zoophthora occidentalis [Acryrthosiphon pisum. The male-killing endosymbionts of D. melanogaster, Spiroplasma, and Wolbachia, protect flies by reducing the survival of parasitoid wasps [Cross-resistance is a host defence mechanism that develops as a response to parasite infection, whereby host evolutionary interactions with one parasite (A) leads to either host resistance, tolerance, or hyper-susceptibility to another parasite (B or C) . When evry costs ,25, evolry costs . Martinsry costs  also obsions (C) . In anotd CG8492 . The autd CG8492 . Howeverphididis  and Zoopdentalis  in the pid wasps .D. melanogaster evolved to DCV when exposed to CrPV and FHV, as discussed before [Anopheles gambiae to the microsporidian parasite Vavria culicis (A) results in the mosquitoes being more resistant to Plasmodium berghei (B), when compared to control mosquitoes, due to an enhanced melanisation response [Cross-resistance can occur at the evolutionary (manifested across generation) or ecological (manifested within generation) level . At the d before . At the d before . For insresponse . Within D. melanogaster experimentally evolved with the parasitoid wasp Leptopilina boulardi demonstrated positive cross-resistance towards L. heterotoma, but no difference in resistance towards A. tabida. Kraaijeveld et al. [D. melanogaster experimentally evolved to A. tabida exhibited no change in resistance towards either the fungus Beauveria bassiana or microsporidian Tubulinosema kingi. Bentz, and colleagues [D. melanogaster’s resistance towards Drosophila sigma virus after experimental evolution with Bacillus cereus. Similarly, the greater wax moth Galleria mellonela evolved with the fungus Beauveria bassiana, displayed no difference in resistance to the fungus Metarhizium anisopliae [To date, only a handful of studies have tested whether, under simple experimental evolution conditions, host resistance leads to general cross-resistance effects upon exposure to different parasites ,11,23,29D. melanogaster that evolved with P. entomophila by oral infection, Martins et al. [P. putida was observed only upon oral infection and not when the flies were infected systemically (i.e. cuticular breaching); indicating that route of infection might be an important factor in cross-resistance. Adaptations to different routes have been shown to have different genetic underpinnings. In a study by Behrens et al. [Tribolium castaneum has different gene expression profiles upon oral and systemic infection by the same parasite.The few studies reporting cross-resistance have tried to understand the underlying mechanisms at play, such as specificity of route of infection  or the gs et al.  observeds et al. , it was T. castaneum, which had evolved under conditions allowing for host-parasite coevolution with the fungal parasite B. bassiana [B. bassiana was present in the environment of the beetles, thereby, allowing for infection to occur naturally. We performed survival assays with the entomopathogenic bacteria Bacillus thuringiensis bv. tenebrionis  and P. entomophila (Gram negative), and additionally with non-evolved B. bassiana and tested the gene expression profiles of T. castaneum evolved with B. bassiana upon exposure to B. thuringiensis, P. entomophila and non-evolved B. bassiana. B. thuringiensis and P. entomophila are phylogenetically distinct entomopathogenic bacteria, belonging to distinct clades of the bacterial phylogenetic tree [B. thuringiensis as a consequence of coevolution with B. bassiana and that cross resistance is potentially due to a shared route of infection between B. bassiana and B. thuringiensis.Although evolutionary studies of cross-resistance have been carried out, cross-resistance in a host arising out of experimental host-parasite coevolution  has so bassiana ,33 was tbassiana ,33, B. btic tree . Our resThe red flour beetle T. castaneum  in a 5% w/w mixture of brewer’s yeast and organic wheat flour . The flour-mix serves as the beetles’ immediate environment, as well as food resource. Throughout this paper, we refer to the hosts as ‘Control’ for beetles originating from populations evolved without the presence of B. bassiana and ‘Coevolved’ for those originating from populations forced to evolve with B. bassiana in the environment. Both ‘Control’ and ‘Coevolved’ selection regimes were simultaneously performed on seven replicate populations.pulation ) were userations . B. bass fitness . Controllour-mix  to minimlour-mix . A schemlour-mix , Figure B. thuringiensis was cultured strictly as per the protocol in Milutinovic et al. [Cry toxins. P. entomophila was grown overnight in LB medium (Carl-Roth) in a 250 mL culture flask at 30 °C and under shaking conditions of 200 RPM. The overnight culture of P. entomophila was centrifuged at 3200 G-force to obtain bacterial pellets while the culture supernatant was discarded. The pellets thus obtained were suspended in Phosphate Saline Buffer (pH = 7) prior to use in survival assay. Non-evolved B. bassiana was plated on Potato-Dextrose agar (Carl-Roth) and stored at room temperature for 2–3 weeks prior to spore collection. All of the parasites were cultivated from their respective glycerol stocks  stored at −80 °C. c et al. , allowinOf the seven replicates beetle populations per selection regime, five populations from the ‘Control’ and ‘Coevolved’ regimes were used for the survival assays. F1 adult beetles belonging to generation 13 of the coevolution experiment  were setB. thuringiensis survival assay was performed in a 96-well plate (Greiner Bio-one) setup with a spore concentration of 5 × 109 spores·mL−1 of flour-mix & Phosphate buffer saline  solution, in line with Milutinovic et al. [B. thuringiensis spore suspension or flour-mix with sterile Phosphate Buffer solution  were prepared, which were dried overnight at 50 °C. The next day, one larva per well was placed in the dried 96-well plates, with each plate containing two replicate populations per treatment. Finally, each plate was sealed with transparent sticky tape and three holes using board pins were punctured on top of each well to allow air circulation. These were then put in plastic boxes that were stored under standard rearing conditions. Each 96-well plate that was set up contained only one treatment and larval survival was observed for seven days on a daily basis. c et al. . On the P. entomophila survival assay, larvae were infected systemically the protocol by Roth et al. [9 spores·mL−1 of P. entomophila spore suspension or sterile PBS, for INFECTION and CONTROL treatments, respectively. Post pricking, larvae were individualised in 96-well plates containing 40 µL of flour-mix & PBS solution dried overnight at 50 °C in a manner similar to that for B. thuringiensis survival assay. Survival was recorded every day for a period of 10 days. For the h et al. . IndividB. bassiana survival assay, larvae were placed individually in small glass vials  with 0.17 g mixture of flour-mix containing 108 of B. bassiana spores·g−1 for INFECTION treatment and vials were capped with cotton wool stoppers (Carl-Roth). For CONTROL, larvae were placed in glass vials containing just 0.17 g of flour-mix. Since B. bassiana is a slow killer of T. castaneum, survival was monitored every alternate day for a period of 30 days  at the end of the assay, for investigating gene expression as logistically we could not test all populations. Adults were set up for mating and egg laying as mentioned before and the larvae could develop. Following this, larvae were infected as per the methods that are described in the survival assay with B. thuringiensis, P. entomophila and B. bassiana individually. For CONTROL, the coevolved larvae were not exposed to parasites but handled similarly. Post treatment, the larvae were placed under standard rearing conditions for beetles. For each parasite, larvae were sampled from both CONTROL and INFECTION treatments 12 and 24 h post exposure. These two time-points were chosen based on previous transcriptomic studies using B. thuringiensis [B. bassiana [Since there was no difference between the different replicate populations within each treatment, we selected two replicate ngiensis  and B. bbassiana . After c260/A280  and A260/A230 (nucleic acid purity) ~ 2 containing two sharp bands representing 23S and 18S rRNA were used for cDNA synthesis, in accordance with MIQE guidelines [18 primers and the reagents from the First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific) were used in accordance with the manufacturer’s protocol. The resulting cDNA was diluted to a working concentration of 10 ng·μL−1, and stored in separate 1 mL Eppendorf tubes at −80 °C until used.RNA was extracted from pools of 15 larvae (per treatment and time-point). The samples were homogenized (2 × 30 s) in 2 mL Eppendorf tubes with one 5 mm diameter stainless steel bead  per tube. Each tube contained 400 µL of TRI reagent (Sigma-Aldrich (Merck), Darmstadt, Germany), using Tissue–lyser II (Qiagen) at 30 Hz for 2 × 30 s. Further treatment for RNA extraction was performed on this homogenate using Direct-zol™ RNA MiniPrep kit  as per the specifications in the manufacturer’s protocol. RNA concentration and purity were determined spectrophotometrically  and quality was checked using agarose gel electrophoresis with 1× TAE buffer. RNA samples with a quality ratio of Aidelines . UnsatisT. castaneum genes expressed post infection and decided on representative candidate genes enlisted in http://www.genelink.com/tools/gl-oe.asp). All primers were designed to have 19–23 nucleotides with a Tm ~60 °C and amplification products of length 70–150 base-pairs (bp). The volume of the reaction was 10 μL, containiing 5 μL Power SYBR Green PCR Master Mix , 1 μL (10 ng) of cDNA and specific forward and reverse primer different concentrations , with remaining volume scaled up with water in order to determine the optimum forward to reverse primer ratios.Primers were tested on cDNA prepared from stock CRO1 T. castaneum in a melt curve assay to determine the optimum forward to reverse primer ratio, as mentioned in 2R ≥ 0.98 and a single sharp melt curve peak corresponding to specific amplification were used for RT-qPCR experiments. Additionally, all primers were tested with water and stock CRO1 mRNA as template to check for primer dimers and unspecific amplifications, respectively . Only tTM optical adhesive film (Thermo Fischer Scientific). The total reaction volume of 10 μL contained 5 μL Power SYBR Green PCR Master Mix (Applied Biosystems), 1 μL (10 ng) of cDNA, specific forward and reverse primer concentrations  using optical 96-well plates (Applied Biosystems) and covered with MicroAmptrations  with remAll data were statistically analysed using R software version 3.2.3  for statistical programming .Since the survival data did not meet the assumptions of normality, we performed non-parametric Kaplan Meier analysis using the package ‘survival’ in R . MultiplGene expression data from RT-qPCR  were anaB. thuringiensis, P. entomophila, and B. bassiana, respectively. Survival of F2 larvae of generation 13 beetles from the coevolution experiment was recorded for a period of 7, 10, and 30 days post exposure to, B. thuringiensis assay, survival of groups within INFECTION and CONTROL treatments were significantly different from each other  = 3, p < 0.001). Pairwise comparison of Kaplan-Meier curves (p = 0.05), and highly differ in INFECTION treatment (p < 0.001). Here, higher survival of coevolved beetles upon INFECTION, in comparison to their control counterparts indicates positive cross-resistance to B. thuringiensis upon oral infection. In the r curves  show thaP. entomophila assay, survival of groups in INFECTION and CONTROL treatments were significantly different from each other . Pairwise comparison of Kaplan-Meier curves (p = 0.19) or INFECTION (p = 0.80). This indicates that B. bassiana coevolved beetles are neither positively or negatively cross-resistant to P. entomophila upon systemic infection.In the r curves . show thB. bassiana assay, survival of groups in INFECTION and CONTROL treatments were significantly different from each other . Pairwise comparison of Kaplan-Meier curves (p = 0.004), but not within INFECTION treatment (p = 0.397), with the ‘Control’ group surviving better than ‘Coevolved’ group.In the r curves  revealedB. bassiana, B. thuringiensis and P. entomophila on F2 larvae of generation 12 beetles evolved with B. bassiana under conditions allowing for coevolution. The larvae were sampled for RT-qPCR experiments 12 and 24 h post exposure. Most of the candidate genes were up-regulated 12 h post B. thuringiensis exposure with markers for stress, Hsp90 (3 fold) and p450 (6.6 fold) being prominent  and p450 (1.65 fold) were less up-regulated. Additionally, the innate immunity marker, Lysozyme was up-regulated (P. entomophila exposure Attacin-2 (411 fold) and Thaumatin (34 fold) were highly up-regulated  and ObpC-12 (~8 fold) were up-regulated but not Thaumatin  and treatment (CONTROL vs. INFECTION) are summarized in Changes in gene expression upon T. castaneum, as a consequence of coevolution with B. bassiana, which is shown towards B. thuringiensis upon oral exposure, but not upon systemic exposure with P. entomophila. This observation is mirrored in expression pattern of host genes related to resistance to oral toxicity upon B. bassiana exposure. It has been reported that oral infection with B. thuringiensis and exposure to coleopteran specific Cry-III toxins leads to the expression of several odorant-binding proteins [T. castaneum. We observed an upregulation in Apo-III and ObpC-12, our markers for oral infection, upon B. bassiana exposure. Infection with Gram-negative P. entomophila caused no variation in coevolved T. castaneum larvae neither in survival assay nor qPCR. The up-regulation of Atta-2 is consistent with reports that show the expression of this anti-microbial peptide upon Gram-negative bacteria infection [B. bassiana during coevolution [T. castaneum ingesting B. bassiana spores. Furthermore, there is some evidence that B. bassiana may be able to infect orally [T. castaneum adapting to oral infection. This is consistent with previous findings that the coevolved beetles have more flexible PO responses that vary depending on infection route, indicating that during the course of coevolution, B. Bassiana adapted to infecting the host orally [B. bassiana and B. thuringiensis exposure imply that similar defence mechanisms are effective against both of the parasites. Here, it is imperative to mention that the overserved response of the coevolved hosts could have been influenced by carried over maternal effects, due to constant exposure of the previous generations to parasites in the environment. However, immune priming has not been consistently observed in insects exposed to B. bassiana [T. castaneum has been over a single generation [B. bassiana is unlikely to be an adequate explanation to our results.Here, we present the first report of evolutionary positive cross-resistance in proteins ,52 and Aproteins ,42 in T.nfection . Due to volution , there wt orally ,54, altht orally . Togethebassiana  and to dneration ,37,56. TB. thuringiensis is mediated by the production of Cry toxins that solubilise in the insect midgut due to a change in pH and disrupt peritrophic membrane integrity [B. bassiana is known to infect by germination of spores on the insect cuticle followed by cuticular breaching through hyphae [B. bassiana possesses the ability to infect orally has been a matter of speculation with some experimental evidence [Solenopsis invicta, B. bassiana was shown to successfully infect via oral ingestion of conidia [B. bassiana possess Cry-like toxin coding regions [B. thuringiensis upon coevolution with B. bassiana coupled with the expression of oral infection marker upon B. bassiana exposure, suggest that B. bassiana and B. thuringiensis share a similar infection route.Positive cross-resistance is more likely to occur if the defence mechanisms employed by the host towards the two parasites are similar or shared, owing to a similarity in the route of parasite entry and/or mechanism of infection . Oral tontegrity ,58. B. bh hyphae ,59. Thatevidence ,60. For  conidia . Further regions . Our finT. castaneum larvae differs based on the natural  and artificial (systemic) routes via which B. thuringiensis infects the beetle. Experimental evolution of D. melanogaster to P. entomophila via oral and systemic routes, separately, revealed that adaptation to different routes was specific; flies adapted to one infection route were not resistant to P. entomophila infection via the route that they had not evolved to [B. bassiana infection coupled with the fact that genomic analyses of B. bassiana reveals potential oral toxicity [B. bassiana coevolved beetles are positively cross-resistant to B. thuringiensis supports the argument that B. bassiana is able to infect orally [Behrens et al.  showed tolved to . Indeed,toxicity  and thatt orally ,61.B. bassiana provides contrasting results in terms of evolution of host resistance. While increase in resistance to B. bassiana was reported for G. melonella [D. melanogaster displayed no change in resistance towards B. bassiana [T. castaneum larvae showed no difference in survival following exposure to B. bassiana when compared to ‘Control’ larvae. They do, however, buffer their fitness across evolutionary time by maintaining, or in the highest infection load treatment increasing pupae numbers when challenged with B. bassiana during coevolution and show infection route specific changes in phenoloxidase activity [B. bassiana. The increased survival we observe here following B. thuringiensis infection adds support to the hypothesis that oral infection by B. bassiana drove selection specifically against oral infection.Experimental evolution of insect hosts with elonella , evolvedbassiana . In our activity , implyinB. bassiana spore suspensions and B. thuringiensis strains specific to different pest insect orders are widely used as biological control agents [B. bassiana could potentially lead to positive ecological cross-resistance in pest insects when exposed to B. thuringiensis strains and vice-versa, with implications in sustainable pest management. Positive cross-resistance can be beneficially applied by providing controlled doses of a parasite that protects against attacks by a more harmful parasite. This is similar to the application of probiotics in the culture of insects for food and feed [Our results also have wider ecological and applied implications. l agents ,64. Prioand feed . FurtherT. castaneum beetles coevolved with B. bassiana towards B. thuringiensis. We propose that this observation could be based on similarity in the route of entry and/or mechanism of infection between the two parasites. Supporting this hypothesis, RT-qPCR experiments performed in this study indicate that B. bassiana can induce expression of host genes that are related to oral toxicity. Adaptations of beetles to oral infection by B. bassiana may have led to positive cross-resistance in the coevolved beetles upon infection with B. thuringiensis. We thereby support the fact that the route of infection is highly important in host-parasite interactions and the physiological response of the host, as well as that of the parasite warrant more research.We observe positive cross resistance of


In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells. Cyclic nucleotides such as cyclic adenosine monophosphate (cAMP) act as second messengers, transducing extracellular stimuli into intracellular signals and leading to various effects inside the cell. In general, the cAMP concentration is assumed to be homogeneous throughout the entire cell body. However, more recent findings suggest that second messenger signaling displays an intriguing complexity requiring a more complex intracellular distribution to achieve signaling specificity. These observations gave rise to the concept of cAMP compartmentation—intracellular compartments with different concentrations of cAMP . Key rolβ-adrenergic agonist isoproterenol. Interestingly, this “shielding” of the cAMP sensor from cAMP by the adjacent PDE molecule is partially persistent in diluted, crude cytosolic preparations. This renders cAMP diffusion, PDE activity, and PDE clustering as the most prominent and sufficient mechanisms to account for cAMP compartmentation by PDEs.Although first reports on cAMP compartments in single living cells have been published several decades ago –19, the To establish a theoretical framework based on these experimental data, we develop analytically-solvable, diffusion-reaction equations to describe cAMP nanocompartments biophysically. By deriving estimates for the interrelation of diffusive mobility of cAMP and cAMP degradation rates, the interdisciplinary experimental and modeling approach applied here narrows down the possible mechanisms for cAMP compartmentation to three most important factors, i.e. restricted cAMP diffusion, PDE catalytic activity, and, to some extent, PDE clustering. Taken together, our data indicate that cAMP diffusion must be significantly slower or more heterogeneous than previously reported to allow for the observation of cAMP nanocompartments around a single PDE molecule in intact cells.Live single-cell FRET imaging was carried out in HEK-TsA cells as described previously . In brieβ-adrenergic agonist isoproterenol (1μM) and the PDE4-specific inhibitor rolipram (1μM). Ratiometric FRET imaging was performed using an upright epifluorescence microscope  equipped with a water-immersion objective , a xenon lamp coupled to a monochromator , filters for CFP  and YFP  excitation, a beam splitter  with a 505LP dichroic mirror and emission filters for CFP (480/30) and YFP (535/40), and an electron-multiplied charge coupled device (EMCCD) camera . CFP and YFP images upon CFP excitation were captured every 5s with 50ms illumination time. FRET was monitored in real-time with the MetaFluor 5.0 software (Molecular Devices) as the ratio between YFP and CFP emission. The YFP emission was corrected for direct excitation of YFP at 436nm and the bleedthrough of CFP emission into the YFP channel as previously described [48 h after transfection, cells were mounted in an imaging chamber and FRET ratios were measured in single cells in real time before and after the addition of the escribed . Images 7 cells were resuspended in 300μl of 10mM TRIS-HCl, 10mM MgCl2 buffer (pH7.4) containing 1mM PMSF and protease inhibitors (20μg/mL soybean trypsin and 60μg/mL benzamidin). Cells were broken by two 10s bursts of an Ultraturrax device. Cell debris and nuclei were removed by centrifugation  and the supernatant was centrifuged again  to yield the cytosolic fraction. For FRET experiments, 80 − 120μl of the cytosol were diluted with buffer ad 600μl. Fluorescent spectra of the cytosolic fractions between 460nm and 550nm were recorded upon illumination with 436nm before and after addition of increasing concentrations of cAMP using the LS50B spectrometer . To ensure equal sensor concentration during measurements, all cytosol preparations were adjusted to the same YFP emission intensity (535nm upon direct illumination at 500nm). To quantify the effects of global PDE activity, two cytosolic fractions expressing either Epac1-camps or PDE4A1 were mixed in a manner that recapitulates the expression of Epac1-camps-PDE4A1. The amount of the Epac1-camps cytosol was adjusted to the same YFP emission intensity measured in cytosolic fractions of Epac1-camps-PDE4A1. The amount of the PDE4A1 cytosol was then adjusted to the same PDE activity as measured with Epac1-camps-PDE4A1. Concentration-effect curves were generated by calculating the 535nm/480nm FRET emission ratios at different cAMP concentrations. Data points were fitted with a three-parameter logistic equation using Graph Pad Prism 6.0 . Data were then normalized to the lower  and upper plateau  of the concentration-effect curve.HEK-TsA cells were transfected with the cDNAs encoding either Epac1-camps, or the fusion proteins Epac1-camps-PDE4A1, or Epac1-camps-PDE4A1 (D352A), or PDE4A1 using calcium phosphate precipitation. 48h after transfection cells were harvested and ca. 1 × 10β-receptors upon isoproterenol stimulation induced a nearly maximal decrease in the FRET ratio in cells expressing only the cAMP FRET-sensor Epac1-camps. No further decrease in the FRET ratio was recorded upon inhibition of endogenous PDE4s with rolipram. The protection of Epac1-camps seen in the presence of an active PDE is specific to the local PDE activity because first, it was partially lost upon expression of a fusion protein containing a catalytically-impaired PDE mutant (Epac1-camps-PDE4A1(D352A)), and second, the protection was completely lost, if the PDE was blocked by the PDE4 specific inhibitor rolipram. These experiments indicate that the cAMP concentration in direct vicinity of an active PDE is below the detection limit of the sensor [To measure the cAMP concentration in direct vicinity of a single PDE molecule, we expressed fusion proteins of the cAMP FRET-sensor Epac-1-camps  and PDE4e sensor . The effTo check if a local cAMP minimum around a PDE persists even upon destruction of the cellular architecture, we conducted biochemical in vitro FRET experiments in cytosolic fractions of transfected HEK-TsA cells . In contS of the sensor molecule. This ratio depends on the cAMP concentration at the sensor molecule,KD is the dissociation constant of the sensor protein, RPDE the radius of the spherical assumed PDE, and, hence, RPDE + d is the position of the sensor molecule, when the center of the PDE is considered as reference.Based upon the experimental data presented here, we develop a biophysical model for cAMP degradation on a single molecule level . The senρ remains when expressed in spherical coordinates D and diffusive flow density r, i.e. jr = j. We also consider solely steady state conditions, i.e. the cAMP concentration is stationary ∂tρ = 0, and, hence,kcat is the maximum degradation rate of a single PDE molecule and Km the Michaelis-Menten constant which is defined as the substrate concentration at which the degradation rate is half of kcat. As we consider only one reaction center, the cAMP concentration reaches a constant asymptotic value far away from the centerA is determined from the boundary condition on the protein surface—describing the enzyme kinetics of the PDE )≡const..This is →∞ρ(r)=ρ0From Eqs→ c0 see . From thδ is the distance from the center of absorption, at which cAMP concentration reaches the average of its maximum c0 = c(∞) and minimum c(RPDE) value,γ as the ratio between the minimum and maximum concentration of cAMP,γ = 1 when there is no concentration gradient, and γ = 0 when there is no cAMP near the PDE molecule. The width of the nanocompartment depends on the spatial extension of the absorbing enzyme (δ = 2RPDE), which implies that a single molecule can only generate a nanocompartment of roughly twice the size of the absorbing enzyme. The depth γ depends on the absorptive action η, and, which is important, on the concentration c0 of the surrounding cAMP (see c0 → 0 and vice versa the compartments disappear (γ → 1) for high values of c0 → ∞. This is due to the saturation of PDE activity that starts at concentrations higher than the Michaelis-Menten constant ρcAMP = Km, i.e. c0 = 1. Once the PDE approaches its highest performance, the compartment is filled and the depth decreases when further increasing the cAMP concentration.To quantify the capability of a PDE molecule to generate a nanocompartment we define its depth and width as follows. The width cAMP see . The deec0 → 0, one getsc → ∞c1/2, at which the compartment is half way flooded, i.e. when η → 0) this concentrations approaches the Michaelis-Menten constant as c0 → 1. Literature values of the kinetic data required in our model vary depending on the PDE subtype as well as methodical choices made in the experiments. For the diffusive mobility D values have been reported in the order of from D = 136μm2/s [kcat ≈ 5s−1 and the Michaelis-Menten constant Km ≈ 2.41μM [RPDE = 2.5nmThe above mentioned relationships may be quantified see . In the 136μm2/s , 32 whil≈ 2.41μM , 34. The≈ 2.41μM , 36 to bη1 = 7.7 ⋅ 10−4. Inserting this value into γ ≈ 1, which implies that the absorptive action of a single PDE molecule is much (more than 100-fold) too small to lead to any significant nanocompartment. This is in strong disagreement with experiments (s. above) [Based on these data the absorptive action of the PDE is estimated as . above) , in whickcat that have been reported for other PDE families. For example [kcat = 20s−1 for the PDE2 family, which yields η1 = 30.8 ⋅ 10−4.Even though we used values that are typical for the PDE4A1 subtype used in our experiments, this result is also true if we assume higher values for  example  reportedη of the PDE. The dependence of the sensor signal intensity as a function of the cAMP concentration at the sensor position is revealed by c0 = ρ0/Km and inter-spatial PDE-sensor distance RPDE + d asS was determined for cAMP concentrations ranging from ρ0 = 10−9M to ρ0 = 10−3M. In control experiments the free sensor protein Epac1-camps  was measured, i.e. η = 0. Note that in this case the concentration-effect curve η = 0, d → 0 and then RPDE → 0. Fitting this simplified concentration-response curve to KD of the sensor as KD = 7.3 ± 0.66μM, with χ2 = 3.2, p = 0.66 indicating good fit results. In the other experiments the sensor was directly fused to the PDE molecule, i.e. the intermolecular distance d was close to zero, where the overall PDE concentration was the same as in the experiments with the free sensor. The shift of the FRET signal curve to the right when the sensor is fused directly to the PDE . This simplifies the absorption rate of a single PDE molecule . In the steady state one obtains for the cAMP concentration in-, and around the spherical clusterr → s = r/R•. So for this cluster geometry ζ is analogous to the absorptive action η in our model of the single PDE molecule in the way that it is a dimensionless parameter that characterizes the shape of the microcompartment.The PDE molecules are assumed to be aggregated within a spherical cluster with radius cule see  to4πRPDNPDE and radius R•. As shown above we obtain very different values of the absorptive action η in the microscopic model—depending on the approach used to determine it. Therefore we also obtain very different values for the absorptive action ζ in the mesoscopic model of the spherical cluster.With η1 = 7.7 ⋅ 10−4 (as derived from the kinetic data found in literature) we obtainη2 = 6.1 ± 1.4  yieldsFor γ as defined by γR• and depth γ. If we set R• = 100nm and η1 = 9.2 ⋅ 10−4 (from theory) or NPDE = 8.5 for η2 = 6.1 ± 1.4 (from our experiments).In this section we derive conditions under which a spherical cluster of PDE can lead to a significant decrease in the local cAMP concentration. For the depth of the compartment NPDE,total = 5000 [η1 = 9.2 ⋅ 10−4 as derived from theory.The total number of PDE in e.g. a pulmonary microvascular endothelial cell is estimated to be about l = 5000 . This woη2 = 6.1 ± 1.4 as derived from our experiments.In χ > 0 and the delta distribution δ(r − R°) restricting absorption is to the shell surface. The steady state solution of this equation is given byχ to the number of the absorbing centers NPDE on the surface of the spherical shellη the absoprtive action of a single molecule of PDE in the microscopic model. Of particular interest is the relationship between the number of absorbing PDE molecules on the shell surface and the “depth” of the compartment γ. In the case of a spherical shell it depends on the ratio NPDE/R° as can be derived from Eqs (γ and of radius R° the required number of PDE is η2 = 6.1 ± 1.4 (as obtained from our experiments)—for example γ = 0.1, R° = 100nm yields: NPDE ≈ 25, while assuming η1 = 7.7 ⋅ 10−4 (as obtained from theory) yields NPDE ≈ 39 ⋅ 104 for the same compartment . Such clusters would need to be formed by stabilizing proteins. In line with this, compartmentation of PDEs by A-kinase anchoring proteins (AKAPs) has been reported by other authors –41. HoweSeveral other groups have reported mathematical models of cAMP degradation on a large scale , 7 as weμm2/s) slower than previously anticipated [η in our models. However it is yet unclear, whether areas of highly impeded diffusion coincide with the localization of PDE enzymes.Therefore we suggest that cAMP diffusion within the nanodomain must be restricted. Recent studies have provided first evidence that cAMP diffusion within cells may be 1 order of magnitude  and Epac1-camps-PDE4A1 (b) obtained in a 10mM TRIS − HCl/10mM MgCl(TIF)Click here for additional data file.S2 Fig50) of Epac1-camps-PDE4A1 (D352A) is 5.56 ± 0.08(= 2.7μM) and thereby not different from Epac1-camps (see manuscript text). Data are means ± s.e.m. of three independent experiments carried out with 2-3 repetitions.Concentration-effect curve of cAMP-induced changes of the FRET ratio of the cAMP sensor Epac1-camps-PDE4A1 (D352A) in cytosolic preparations of transiently transfected HEK-TsA cells (black curve). The concentration-effect curves of Epac1-camps (grey) and Epac1-camps-PDE4A1 (red) are shown for comparison. The apparent affinity (pEC(TIF)Click here for additional data file.S3 FigμM cAMP (red arrow) leads to a decrease in FRET (YFP/CFP) ratio due to binding of cAMP to the sensors. (a) In case of Epac1-camps-PDE4A1 the FRET change is transient and increases to the basal FRET ratio after ≈350s due to PDE activity. (b) At the same expression level the FRET change is not transient in cytosolic preparations only expressing Epac1-camps indicating that endogenous PDE activity is negligible. (c) Separate expression of Epac1-camps and PDE4A1: the amount of PDE4A1 cytosol was adjusted to the same catalytic activity (Δτ as surrogate parameter) as measured with Epac1-camps-PDE4A1. (d) Δτ values in cytosolic preparations expressing Epac1-camps-PDE4A1 (red) or Epac1-camps + PDE4A1 (blue) are not significantly different . Data in (d) are means ± s.e.m. of 4 independent experiments, representatives of which are shown in (a) and (c).(a-c) Representative real-time, in vitro FRET measurements of cytosolic preparations of HEK-TsA cells transiently expressing the indicated constructs. Addition of 100(TIF)Click here for additional data file.


Dear Colleagues,in the long history of the German Society of Oto-Rhino-Laryngology, Head & Neck Surgery, it was now for the second time that our annual meeting took place in Erfurt . Nine key note lectures and written contributions reflected the main topics dealing with diagnostics and therapy of hearing disorders as well as vertigo, tympanoplasty, idiopathic sudden hearing loss, the treatment of vestibular schwannomas, cochlear implantations, and the state-of-the-art of hearing aid provision.At this point, I want to extend my special thanks to all the authors and co-authors of the articles. I am fully aware of how much work and effort was necessary to create “manuals” on specific topics in a highly effective way. You may all be very proud of your contributions.Another focus of this year’s congress was the special program for medical assistants in ENT practices and nurses working in ENT departments and hospitals. Particular emphasis was placed on practice-related topics with independent application.Furthermore, at our “days of the private practitioner”, we discussed in cooperation with and under the responsibility of the Professional Association of Otolaryngologists various topics which had been chosen as closely related to everyday issues of the private practitioner.The high number of submitted abstracts showed how multifaceted our discipline is. It also showed the high relevance of the sensory organs we have to examine and treat. Hearing, smelling, tasting, and the preservation or restitution of the senses of balance and hearing are important aspects of private and professional life. ENT-specific oncology, traumatology, and dysphagia were further main topics of the meeting program.th Annual Meeting represented a first milestone. The main topics that are highly relevant for the future of our discipline were in the focus of numerous invited keynote lectures and round table discussions.From my personal point of view, this meeting also summarized the year of my presidency. On the one hand we further expanded our German Study Center for Otorhinolaryngology, Head & Neck Surgery together with the Professional Association of Otolaryngologists; on the other hand we achieved a close relationship between our scientific society and the Professional Association. Based on the identification of key topics we will work on the so-called Agenda 2025 together with the Professional Association during the next years. In this context, the 88A high number of internationally renowned experts provided an excellent basis for specific discussions on the highest level.th Annual Meeting with regard to scientific and practical contents that became obvious in the high number of participants, the industrial exhibition, and finally in a lively scientific exchange and intensive discussions – and visits of the state capital of Thuringia.I want to express my personal thanks to all authors of the scientific contributions. You all contributed to the great success of the 88Prof. Dr. med. Dirk Eßer President of the German Society of Oto-Rhino-Laryngology, Head & Neck SurgeryFigure 1


The correlation between hearing and cognition is well established in dementia,but not in mild cognitive impairment (MCI).The aim of the present study was to define the prevalence of hearingimpairment in elderly patients with MCI and in controls.Twenty-nine patients with MCI and 24 control subjects were analyzed. Weevaluated memory and hearing impairments through clinical tests, includingthe Mini Mental Status Examination, Clinical Dementia Rating (CDR) andHearing Handicap Inventory for the Elderly Screening (HHIE-S). Audiometrieswere performed in 22 patients with MCI and 19 subjects in a controlgroup.MCI patients showed more hearing complaints (68.9%) compared to the controlgroup (25%) (p=0.001). No differences in the intensity of hearingcomplaints, measured by the HHIE-S, were detected. Nonetheless, differencesbetween mean hearing threshold (MCI group=23.4±11.3dB and controlgroup=16.0±10.1dB) (p=0.03) were identified.There is a significant association between MCI and hearing impairment.Hearing impairment in MCI patients may be a contributory factor to cognitivedecline. This may however be related to the same neuropathological process,due to lesions of cortical areas related to hearing. The early diagnosis ofhearing impairment in MCI patients may offer a more appropriate approach tothis disease. A link has also beenestablished among hearing loss, depression and functional decline,3 especially evident in individuals withhearing threshold >35 dB.4The relationship between hearing impairment and cognitive decline has previously beendemonstrated,5 Although thisrelationship between cognition and hearing has been well studied in patients withdementia, the association in those with mild cognitive impairment (MCI) has not yet beenassessed. MCI is a clinical entity, first described in the 1990s,6 and involves an intermediate statebetween normal cognitive aging and dementia.7 To date, no studies published in the English language have evaluatedthe prevalence of hearing impairment in such patients compared to normal control groupsfrom a cognitive standpoint.More than 90% of patients with Alzheimer disease (AD) have some kind of hearingloss.This paper aimed to analyze the prevalence of hearing impairment and its quantitativecharacteristics in elderly patients with MCI compared with normal elderly subjects froma control group, according to the cognitive standpoint – through a comparativetransversal study.Twenty-nine patients with subjective memory complaints (MCI group) and 24control-subjects without cognitive complaints (control group) were studied. Subjectswere recruited from May to November of 2005. The MCI group was formed throughassessment of subjects attending a memory clinic at the Geriatrics Department ofHospital das Clínicas – São Paulo University Medical School(SG-HC/FMUSP). The control group was formed through assessment of subjects followedby a multidisciplinary care group from SG-HC/FMUSP in 2003 and 2004. The study wasapproved by the Ethics Commission of the Institution. The inclusion criteriawere:(a) age of 60 years or over;(b) Clinical Dementia Rating (CDR) of 0.5 in patients with cognitivecomplaints and CDR of 0 in controls;(c) acceptance of an informed written consent.The exclusion criteria were:(a) presence of memory complaints among controls;(b) presence of clinical signs of dementia, as well as scores below thoseexpected for schooling level in the Mini Mental Status Examination(MMSE) as established in the following section;(c) presence of any central nervous system disease, psychiatric diseases,such as anxiety and depression, severe inflammatory systemic diseases,as well as hypothyroidism, deficiency of vitamin B12, syphilis, renaland liver insufficiency;(d) use of drugs that act on the central nervous system, mainlybenzodiazepines, antidepressives and anticholinergic drugs;(e) current or previous use of acetylcholinesterase inhibitor;(f) current or previous use of hearing aids, or presence of visual andhearing losses severe enough to prevent clinical interview or proposedtests from being conducted.8, CDR,9, CAMCOG,a structured interview based on the cognitive section of Cambridge Examination forMental Disorders of the Elderly10and validated for the Brazilian population,11 Rivermead Behavioral Memory Test, which is divided intoRivermead 1 for scores of standardized profile and Rivermead 2 for scores ofscreening,12 Digit Span,which is divided into Digit Span 1 for direct-order numbers and Digit Span 2 forreverse-order numbers,13 WhisperedTest14 and HearingHandicap Inventory for the Elderly Screening – HHIE-S.15. Of the total subjects included in this study, 19from the control group and 22 from the MCI group were submitted to an auditoryexamination, tonal audiometry and determination of speech reception threshold andpercentage rate of speech recognition tests.All subjects were investigated for schooling level, comorbidities, medications beingused, and hearing and memory complaints. Moreover, the following medicalquestionnaires were used: the MMSE,16 considering a cut off point of <28 forsubjects with more than 8 years at school, <24 for subjects who studied from4 to 7 years, <23 for subjects who studied between 1 and 3 years, and <19for illiterate subjects.MMSE was assessed according to schooling level of the subject, using the criteriasuggested for Brazilian population by Herrera et al.,15Subjects were investigated for hearing complaints and analyzed according to theHHIE-S, a scale that puts questions regarding the auditory performance inhabitual situations. It is self applicable and assesses the degree of auditoryloss, although is not capable of quantifying the level of the losses.All subjects were submitted to the Whispered Test and auditory examination.Twenty-two subjects from the MCI group and 19 from the control group wererandomly selected and submitted to tonal audiometry. All tests were executed bythe same examiner.17 The result of the best ear wasconsidered, in order to eliminate any confounding factor caused by unilateralhearing losses.18 Subjects whohad a cerumen plug during otoscopy underwent removal before audiometry.Considering levels of severity of hearing loss, the threshold up to 25 dB isusually defined as normal, between 25 and 40 dB indicative of mild loss, between40 and 65 dB as moderate loss, and over 65 dB as severe loss.19 We also calculated meanhearing thresholds at high frequencies , as well as thespeech reception threshold and percentage rate of speech recognition.Tonal audiometry was performed by the MIDIMATE 622 audiometer, employing AZ 7 andZodiac 901 middle-ear analyzers, studying air and bone conduction, in anacoustically isolated environment. The hearing threshold for frequencies from250 to 8000 Hz was determined, as well as for frequencies of 500, 1000 and 2000Hz, where means of these values for each ear were calculated.Both groups were compared regarding average age, schooling, comorbidities,medications used and scores on the tests applied. In addition, prevalence ofhearing complaints and positivity of Whispered Test were compared as wereaverages of the hearing threshold obtained through audiometry. Continuousvariables were compared using the Student t test while categorical variableswere compared using the chi-square test, determining the p values. Proportionswere compared using Fisher’s Exact Test. Binary logistic regressions wereperformed to assess risk ratios (RR). For calculations, the statistical programMINI TAB 14 was used.Of 135 subjects initially considered, 53 were included in this study –29 of whom werepart of the MCI group and 24 of the control group. The main causes of exclusion areshown in The MCI group was older (75.0±5.6 years of age) than the control group(70.3±5.4 years of age), p<0.05. In addition, a greater number ofcomorbidities was observed in the control group (4.1±1.7) compared to the MCIgroup (3.0±1.4), p<0.05. The most common diagnoses were similar for bothgroups: systemic arterial hypertension (54.1% and 58.6%), dyslipidemia (50% and51.7%), and osteoarticular diseases (45.8% and 41.3%) respectively. The most commondrugs used by both groups were also similar: calcium carbonate (50% and 44.8%),statins (41.6% and 44.8%) and angiotensin-converting enzyme inhibitors (41.6% and37.9%) respectively.Regarding other clinical features, no statistical differences between the groups wereobserved, nor in relation to the score on the MMSE and Digit Span.However, scores of the two groups were different on the CAMCOG  and in theirsubscale of memory , aswere scores on the Rivermead Behavioral Test .The MCI group presented more hearing complaints . The risk ratio (RR) for patients with MCIpresenting hearing complaints was 6.6. No significant differences in proportions ofnormal otoscopies were detected between the two groups. The Whispered Test wasabnormal in 12.5% of control group subjects and 41.3% of the MCI group (p<0.05).The risk ratio (RR) of a patient with MCI being positive on the Whispered Test was4.9. No differences in the severity of hearing complaints were detected, accordingto the HHIE-S. The mean hearing threshold of the best ear assessed was statisticallydifferent between the two groups . Nevertheless, these thresholds lie within the range ofaudiometric normality. When we analyzed hearing means, classifying them individuallyas normal (up to 25 dB) or abnormal (>25dB), we observed that 10.5% of thecontrol group subjects showed abnormal thresholds, while this was the case in 31.8%of the MCI group (p=0.09).The analysis of hearing thresholds at high frequencies (4-8 KHz) revealed differencesbetween control (33.2±15.4dB) and MCI groups (46.2±20.1dB), p<0.05.No significant differences between speech reception threshold and percentage ratesof speech recognition were detected. 6Nonetheless, other authors have determined differences between subjects with CDR of0.5 and with MCI, particularly if the criteria defined by Petersen areconsidered.20 However,these were reviewed recently by the European Alzheimer disease Consortium.21 Thus, diagnostic elements for MCIwere initially defined as the presence of memory complaints reported by the patientand/or relatives, with decline of cognitive function in the last year, cognitivealterations in a medical assessment, along with impairment of memory or othercognitive domains, without effect on daily life, and in the absence of dementia.According to these criteria, specific tests for assessment of cognitive losses werenot devised, nor were performance deficits that classify the disease. Given theabsence of neuropsychological assessments in this paper, the criterion of CDR of 0.5was chosen. All patients diagnosised as MCI cases were of the mnestic type.Some papers have used a CDR score of 0.5 as a criterion for MCI, formally known as“mild dementia status”.22 suggesting that the rate of 68.9%of hearing complaints in patients with MCI found in this study, was not aconsequence of age but associated with cognitive impairment. In the general elderlypopulation, the prevalence of hearing impairment varies from 30-60% and may reach79% in the demented.23The fact that the MCI group was more aged than the control group may be explained bythe fact that these patients at greater risk of developing dementia syndromes, whereage represents a risk factor. As this study was not conducted following thecase-control standard-paired according to age, we tried to detect markers in thesample of our population in order to ensure equality between the studied groups soas to facilitate comparison. Since the main objective of this study was related tohearing complaints, most likely intensified by the aging process, we alsoinvestigated the frequency of hearing complaints in the medical literature. Amongnormal subjects, 33% of them aged between 64 and 74 years, and 45% of those agedbetween 75 and 84 years, tend to present hearing impairment,The only variable different between groups was the number of comorbidities. Despitetheir advanced age, patients with MCI presented a lower number of diseases comparedto the control group. This data emphasizes the fact that hearing impairment seen inthis group was not associated with worse general clinical condition.24 analyzed 41subjects, controls were detected with normal memory and MMSE scores >28, CAMCOGmean of 91±2 whereas patients with MCI scored a mean of 82±4 (mean ageof 73.05 years and schooling level of 5.61 years) – in line with that seen in oursample.In assessment with CAMCOG, we observed that even though none of the groups had scoresthat suggested dementia features, values differed from each other. Similarly, whenBottinoOn the memory subscale of CAMCOG, scores also differed, showing loss of memory inpatients with MCI. In the Rivermead Behavioral Test, the MCI group obtained lowerscores with a statistically significant difference.Alterations of memory observed were not a consequence of attention disorders, assuggested by the Digit Span 1 score (direct-order). The absence of differences inreverse-order of repetition of digits (Digit Span 2) suggested that operationalmemory (very short term) was similar in both groups. Furthermore, long-term memoryis expected to be compromised in the MCI group, justifying the worse performance incognitive tests.25demonstrated that the Handicap does not correlate correctly with scores ofself-perception of hearing losses. Nonetheless, there is the possibility thatpatients with MCI have a certain loss of critical ability in relation to their ownhearing deficiency.In spite of the higher frequency of hearing complaints in patients with MCI, theseverity of complaints was not different between both groups, demonstrated by scoreson the HHIE-S. Vesterager17 associated withpresbyacusis. In presbyacusis there is bilateral and gradual loss of hearingsensitivity for high-frequency sounds26, mainly in noisy environments. In the analysis of highfrequencies (4-8 KHz), we observed differences between the groups, with mild loss inthe control group and moderate in the MCI group, suggesting that MCI patients havemore severe presbyacusis, which may be caused by a cognitive worsening since thiscontributes to poor sound comprehension, but may also be a consequence of theneurodegenerative process related to memory losses. Another possibility is that thisphenomenon is an indicator of severity of the process of cognitive loss and thathearing complaints of patients with MCI are related to alterations in centralauditory processing. Central auditory dysfunction was evident in studies on patientswith mild AD, compared to controls of the same age. The peripheral auditory systemhowever, seems to be similar in normal and demented patients.27There was difference between mean hearing thresholds obtained through audiometrybetween the frequencies of 500 and 2000 Hz, often used to define the level ofhypoacusis. However, the thresholds observed in both groups remained within therange of normality. Speech reception threshold and percentage rate of speechrecognition were also similar, suggesting that hearing complaints presented by thesubjects cannot be accounted for by the differences detected in frequencies from500-2000 Hz. Among the elderly, neurosensory losses are common in 90% ofcases,28 formerly called“central presbyacusis”.29 Hearingloss among patients with dementia is not limited to peripheral alterations, but alsoinvolves reduction in speed of auditory processing.30 Thus, hearing losses may not be properlycharacterized by audiometric analysis, where assessment of the central auditoryfunction is also required.28Patients with AD show evidence of degeneration of structures related to auditoryprocessing, including the colliculus, medial temporal lobes, and auditory cortex,where neuritic plaques and neurofibrillar tangles have been detected in theseareas.31 Furthermore, hereis histopathological evidence of impairment of the medial geniculate body andinferior colliculus in patients with AD.32 Moreover, prefrontal cortex is affected more precociouslyby amyloid-beta plaques in animal models,33 which may reduce the response of auditory cortical neuronsgiven the direct connections between these two areas.34 Prefrontal cortex lesions are associated with anincrease in amplitude of evoked potentials generated in the auditorycortex.35Presbyacusis may be accompanied by degeneration of central auditory structures andauditory cortex,17 Pekkonen36 investigated how auditory processing is correlated with damagein superior cortical functions through the use magnetoencephalography techniques.Besides, he observed that inter-hemispheric auditory processing in AD is slower onthe same side of the brain stimulated by sounds. In addition, he observedneurodegenerative alterations in primary auditory cortex, as well as in thalamus andinferior colliculus.37 WhenGolob38 assessed motorreaction time after an auditory stimulus, he detected alterations in modulation ofauditory cortex of subjects with MCI resulting from neuropathological alterations inassociative cortical areas.Central auditory dysfunction has previously been characterized through responses ofbrainstem in patients with mild AD, who did not show higher rates of peripheralhearing impairment compared to controls.17Studies employing positron emission tomography (PET) have showed reduction ofmetabolism of glucose in the temporo-parietal region of patients with dementia,including the auditory cortex.39However, in another study, subjects with CDR of 0.5 showed signs of central, and notperipheral, hearing deficiency.The most important limitation of this study is the absence of grouping for age, whichaffects the conclusions. Future studies should evaluate larger groups, pairedaccording to age, while addressing peripheral and central auditory losses.Early diagnosis of patients with MCI and the study of its epidemiologicalcharacteristics may allow an earlier clinical approach, particularly when riskfactors are closely associated to the development of dementia, as is the case ofhearing impairment.


Sudden sensorineural hearing loss (SSHL) is a disease, which severely affects the patient’s social and relational life. The underlying pathomechanisms have not been finally clarified yet and outcome is not predictable.We conducted a retrospective study in order to identify parameters that influence hearing recovery. The data base contains results of basic otoneurological tests and clinical parameters of 198 patients with idiopathic SSHL of at least 60 dB in at least four frequencies, diagnosed and treated at the University Hospital of Münster, Germany, between 1999 and 2015. Hearing recovery was measured by pure tone audiometry.Multivariate linear and logistic regression analyses indicate that the chance as well as the magnitude of hearing recovery is higher for patients with normal caloric testing than for patients with pathological caloric testing. However, for the subgroup of patients who attained a hearing recovery, the caloric testing result was not found to influence the magnitude. Instead, the magnitude was noticeably lower for patients within this subgroup who had a previous hearing loss. Furthermore, we found indications that the magnitude is higher for men than for women and that receiving a high-dose steroid therapy is associated with a higher chance and magnitude of a hearing recovery.We conclude that SSHL associated with disorders of the vestibular system or previous hearing loss represent special sub-entities of SSHL that may be caused by unique pathophysiological mechanisms and are associated with worse outcome. Furthermore, our data support the importance of elevated dosage of steroids in SSHL therapy. Sudden, sensorineural hearing loss (SSHL) is an acute, mostly unilateral dysfunction of the inner ear that is characterized by sudden onset and potential progression to complete deafness. Elderly people are primarily affected, but, it can occur at every age. While the estimated incidence is 10-20/100.000/year, the true incidence is probably much higher since it increases in industrialized countries and many cases are often misdiagnosed or regarded as age-related unavoidable fate . HearingIn general, no effective treatment option is available for the majority of SSHL patients and many patients do not completely recover. The most important innovations in SSHL therapy in the last few decades are apheresis of fibrinogen and low density lipoproteins (LDL) and administration of steroids via intratympanic routes , 20. NewThe outcome of SSHL, especially the idiopathic form, is not predictable. In the last few decades, numerous studies have attempted to establish a relationship between certain accompanying symptoms or findings, e.g. in the otoneurological or general examination, and the degree of hearing improvement with, however, often different results –48.Therefore, the present retrospective study was initiated to further explore the impact of basic clinical parameter on chance and magnitude of hearing recovery after SSHL in order to detect potential variables that predict outcome.In a retrospective approach we identified patients with an acute, unilateral SSHL ≥60 decibel (dB) in at least 4 frequencies between 125 and 8000Hz compared to the healthy ear. All patients were diagnosed and treated at the Department of Otorhinolaryngology, Head and Neck Surgery at the University Hospital of Münster, Germany between 1999 and 2015. By entering appropriate ICD-10 codes into a digital hospital information system , all eligible patients could be identified. Next, each individual patient record was reviewed for the requirements of this study. If they fulfilled the inclusion criteria, further evaluation was started. In all cases hearing loss was measured by pure-tone audiometry. Pure tone air and bone conduction threshold audiometry (with and without masking of both ears) was performed by certified and experienced audiometrists during the patients’ first visit of the outpatient care unit. Accompanying tinnitus was characterized audiometrically by determination of tinnitus loudness and frequency. Hearing recovery was calculated by comparing the pure-tone thresholds of the initial visit and the last investigation . Hearing improvement was registered in dB in six frequencies: 250Hz, 500Hz, 1000Hz, 2000Hz, 3000Hz and 4000Hz. Possible underlying pathologies were tried to identify by otorhinolaryngological examination including ear microscopy, magnetic resonance imaging and serological blood investigations for herpes simplex virus (HSV), varizella zoster virus (VZV) and borrelia burgdorferi infections. All patients were asked for possible anamnesis of familial deafness, chronic otological history, trauma, previous ear surgery, or prior sudden deafness. Vestibular examination recorded spontaneous nystagmus, gaze nystagmus, positional nystagmus, caloric vestibular test and perilymph fistula test. Patients’ medical chart was evaluated for basic clinical parameters.The dataset contains the results of basic otoneurological tests and several clinical parameters of 198 patients. The patient’s overall magnitude of hearing improvement (in dB) is expressed as the weighted mean of the hearing improvement in the six frequencies 250Hz, 500Hz, 1000Hz, 2000Hz, 3000Hz and 4000Hz, where the weights are 0.1, 0.2, 0.2, 0.2, 0.2 and 0.1.  According to Siegel’s criteria, a hearing recovery is defined as an overall magnitude of hearing improvement of at least 15 dB .Furthermore, the dataset includes several clinical parameters that are investigated w.r.t an influence on the chance and the magnitude of a hearing recovery: age at diagnosis (in years), sex, therapy , exploratory tympanotomy with sealing of the round and oval window niche (yes/no), tinnitus (yes/no), vertigo (yes/no), spontaneous nystagmus (yes/no), caloric test result , positional nystagmus (yes/no), nicotine abuse (yes/no), objectively identifiable perilymph fistula (yes/no), previous hearing loss (yes/no), and fibrinogen level .2-test were used to assess the relationship between two categorical variables (e.g. between sex and hearing recovery), where Fisher’s exact test was used for two binary variables [p-values, were intended to be exploratory (hypotheses-generating), not confirmatory. Therefore, p-values are to be interpreted in Fisher’s sense, representing the metric weight of evidence. Since a global level of significance was not controlled, p-values smaller than 5% are termed noticeable instead of significant.Descriptive statistical analyses were carried out computing medians and interquartile ranges (IQR), absolute and relative frequencies, and Spearman’s correlation coefficient . Fisher’ariables . The relariables . Multivaariables . Coefficariables . Please By critical chart review we were able to identify 198 patients with hearing loss in accordance to our inclusion criteria. The time delay between onset of symptoms and start of the therapy was one to seven days. Some of the examined factors could not be evaluated in all 198 patients  patients were offered an explorative tympanotomy. During this procedure the round and oval window were screened for the presence of a PLF and were covered with soft tissue regardless of a visible defect. An exploratory tympanotomy was conducted in 147 of the 198 included patients (74.2%), whereby only in 7.2% of these patients a PLF could reliably be detected by the surgeon.p-value were the tympanotomy (OR(yes vs. no) = 2.06) and the caloric test result  = 2.90). Furthermore, the p-values suggest that the type of therapy and the presence/absence of vertigo might possibly be predictors of a hearing recovery.Table p = 0.022), with an estimated OR  of approximately 3.4. Yet the model also indicates that the chance of a hearing recovery is possibly higher for male patients and for patients who received a high-dose steroid therapy, compared to patients with a pentoxifyllin application (data not shown).Starting from a multivariate logistic regression model that includes all these variables as covariates, the AIC-based backward variable selection delivered a model that includes the three covariates caloric test result, sex, and type of therapy. Here the caloric test result was the only noticeable predictor .The influence of the considered clinical parameters on the magnitude of a hearing recovery was investigated by means of boxplots, see Fig. In order to investigate the noticeable variables simultaneously as to differences of the magnitude, they are taken as possible covariates of a multivariate linear regression model. Since almost all patients who received a tympanotomy also received a cortisone therapy (i.e. low-dose or high-dose steroids), the variable tympanotomy was also considered as possible covariate of the model. The AIC-based backward selection delivered a model that includes the four covariates sex, therapy, vertigo, and caloric test result. The estimated model coefficients equate to the differences in magnitude between the categories, e.g. between men and women. The differences and the referring 95% CIs are depicted in Fig. p = 0.036 and p = 0.039). The magnitude was estimated to be around 6 dB higher for men and around 8 dB lower for patients with pathologic caloric test result, in comparison to the respective counterpart. The model also suggests that the magnitude is possibly lower for patients with vertigo and that the high-dose therapy possibly leads to a better outcome than the pentoxifyllin application: the magnitude was estimated to be around 9 dB higher for patients with high-dose steroid therapy .Here the variables sex and caloric test result were noticeable predictors  in order to find possible predictors for the magnitude of a hearing recovery . The model also indicates that patients who received a high-dose steroid therapy possibly attain a higher magnitude of hearing recovery than patients with a pentoxifyllin application; the magnitude was estimated to be approximately 13 dB higher for patients with a high-dose steroid therapy .For this subgroup, an influence of the caloric test result on the magnitude of hearing recovery could not be observed. Instead, the magnitude was noticeably higher if no hearing loss had occurred before. The multivariate linear regression model that results from AIC-based backward selection includes the three covariates therapy, vertigo, and previous hearing loss. The model coefficients and the 95% CIs are shown in Fig. The etiology and mechanisms that cause SSHL are still unknown. Even the natural course of SSHL in patients has been still a matter of discussion in the last decade since the rate of spontaneous recovery has been estimated between 30% and 60% –26. MoreThe aim of this study was to investigate possible positive or negative predictors of hearing recovery in patients with severe SSHL. We could demonstrate a clear benefit in hearing recovery for patients missing subjective and objective symptoms of vestibular dysfunction accompanying hearing loss. Regarding all patients, the chance for hearing recovery as well as the magnitude of hearing recovery demonstrated a dependency on such vestibular parameters. While the absence of vertigo or spontaneous nystagmus were only associated with higher magnitude of recovery in univariate analysis, normal caloric testing proofed to be the decisive key variable for a higher chance of hearing recovery as well as for a higher magnitude of recovery in both, univariate and multivariate analysis. However, looking just at the patients with actual recovery, absence of spontaneous nystagmus was associated with a higher magnitude of recovery in univariate analysis and absence of vertigo was associated with a higher magnitude of recovery in univariate and multivariate analysis.In the last few decades many studies analysed possible predictors of hearing recovery in SSHL –45. The Unfortunately, because of insufficient documentation we were not able to do a subgroup analysis on hearing recovery in dependency on time delay from onset of symptoms like it was done by the other study groups. Age was no relevant predictor in the here presented study, as well as in several other studies, but, also in contrast to other studies , 32–37. Generally, the classification of improvement shows a great variation in the above mentioned studies. Many study groups use their own developed classification system, thus reducing comparability between the different studies, if not making it impossible. We used the Siegel’s criteria for classifying hearing recovery, an internationally accepted classification system, which is, nevertheless, not often used , 40, 49.Interestingly, considering all patients studied, a previously occurred hearing loss had no relevant prognostic importance. While in patients with documented improvement the magnitude of hearing recovery was significantly higher if such an event was not reported by the patients, in univariate as well as in multivariate analysis. To be noted, even though 13% of our patients had recurrent hearing loss none of these patients had a history or symptoms for diagnosing Ménière’s disease and a vestibular schwannoma could be ruled out by MRI. Similar findings demonstrating poorer prognosis after recurrent episode of SSHL could also be shown by others , 48. TheHearing loss therapy is an intensively debated field. The vast majority of the patients in this study were treated with intravenous steroids. Despite intensive review of patients’ medical chart we could not identify decision criterion for applying low-dose or high-dose steroids. There was a clear trend towards improved outcome in patients receiving high-dose steroids, but, no significant relationship. The question if steroids or the dose of the administered steroids have important influence on hearing recovery remains controversial , 55–57.Although, nineteen percent of our patients had elevated fibrinogen plasma levels at first presentation none of them received apharesis. The therapeutic value of fibrinogen and LDL apharesis in SSHL patients seems to be comparable to that of standard regimen with glucocorticoids , 21. LatA novel treatment option is application of steroids through intratympanic routes . ReliablMany of the patients included in this study underwent an explorative tympanotomy in order to rule out a PLF. The reason for the surprisingly high number of operative interventions could not be further elucidated despite the intensive research of the patients’ medical charts. A PLF could only be detected in 7%. Comparing both collectives, patients receiving tympanotomy and those who did not, there was no significant difference in hearing recovery between these two groups. To our surprise, even if a PLF could be seen by the surgeon, the coverage of it did not influence hearing recovery. This is in accordance to findings of others .Since we only evaluated test results prior to therapy and the vast majority of our patients initially showed up outside regular working hours we unfortunately were not able to use data from other audiometric tests, e.g. speech audiometry to evaluate hearing recovery .Altogether we were able to demonstrate that the presence of symptoms or objective criteria of vestibular dysfunction in patients with SSHL are associated with worse hearing recovery. We therefore think that patients with hearing loss and additional vestibular dysfunction represent a subentity of SSHL patients. This might indicate a special disease entity with an own pathophysiological background, e.g. viral labyrinthitis. A similar context could exist for hearing loss associated with previous events. Unfortunately, due to the retrospective character of the presented study there is no sufficient data available to allocate the origin of these potential sub-entities to an individual background, namely vascular or inflammatory . Further